Reports of China’s Basic Research

Gang Pei Editor

Epigenetic
Mechanisms of
Cell Programming
and
Reprogramming
Reports of China’s Basic Research

Editor-in-Chief
Wei Yang, National Natural Science Foundation of China, Beijing, China
Zhejiang University, Hangzhou, Zhejiang, China
The National Natural Science Foundation of China (NSFC) was established on
February 14, 1986. Upon its establishment, NSFC was an institution directly under
the jurisdiction of the State Council, tasked with the administration of the National
Natural Science Fund from the Central Government. In 2018, it became managed
by the Ministry of Science and Technology (MOST) but kept its due independence
in operation. Since its establishment, NSFC has comprehensively introduced and
implemented a rigorous and objective merit-review system to fulfill its mission of
supporting basic research, fostering talented researchers, developing international
cooperation and promoting socioeconomic development.
Featuring science, basics, and advances, the series of Reports of China’s Basic
Research is organized by the NSFC to present the overall level and pattern of China’s
basic research, share innovative achievements, and illustrate excellent breakthroughs
in key fields. It covers various disciplines including but not limited to, computer
science, materials science, life sciences, engineering, environmental sciences, math-
ematics, and physics. The series will show the core contents of the final reports of
the Major Programs and the Major Research Plans funded by NSFC, and will closely
follow the frontiers of basic research developments in China.
Gang Pei
Editor

Epigenetic Mechanisms
of Cell Programming
and Reprogramming
Editor
Gang Pei
Tongji University
Shanghai, China

Supported by the NSFC Major Research Plan “Epigenetic Mechanisms of Cell Programming
and Reprogramming”

ISSN 2731-8907 ISSN 2731-8915 (electronic)

Reports of China’s Basic Research
ISBN 978-981-19-7418-2 ISBN 978-981-19-7419-9 (eBook)
https://doi.org/10.1007/978-981-19-7419-9

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Reports of China’s Basic Research

Editorial Board

Editor-in-Chief
Wei Yang
Associate Editors
Wen Gao
Ruiping Gao
Editors
Yu Han
Changrui Wang
Yonghe Zheng
Zhongwen Zheng
Feng Feng
Yanze Zhou
Tiyu Gao
Weitong Zhu
Qingguo Meng
Yongjun Chen
Shengming Du
Qidong Wang
Ming Li
Yuwen Qin
Ziyou Gao
Erdan Dong
Zhiyong Han
Xinquan Yang
Shengli Ren

v
Preface to the Series

As Lao Tzu said, “A huge tree grows from a tiny seedling; a nine-storied tower
rises from a heap of earth.” Basic research is the fundamental approach to fostering
innovation-driven development, and its level becomes an important yardstick for
measuring the overall scientific and national strength of a country. Since the begin-
ning of the twenty-first century, China’s overall strength in basic research has been
consistently increasing. With respect to input and output, China’s input in basic
research increased by 14.8 times from 5.22 billion yuan in 2001 to 82.29 billion
yuan in 2016, with an average annual increase of 20.2%. In the same period, the
number of China’s scientific papers included in the Science Citation Index (SCI)
increased from lower than 40,000 to 324,000; China rose from the 6th to the 2nd
place in global ranking in terms of the number of published papers. In regard to the
quality of output, in 2016, China ranked No. 2 in the world in terms of citations in
9 disciplines, among which the materials science ranked No. 1; as of October 2017,
China ranked No. 3 in the world in the numbers of both Highly Cited Papers (top
1%) and Hot Papers (top 0.1%), with the latter accounting for 25.1% of the global
total. In talent cultivation, in 2006, China had 175 scientists (136 of whom from the
Chinese mainland) included in Thomson Reuters’ list of Highly Cited Researchers,
ranking 4th globally and 1st in Asia.
Meanwhile, we should also be keenly aware that China’s basic research is still
facing great challenges. First, funding for basic research in China is still far less than
that in developed countries—only about 5% of the R&D funds in China are used
for basic research, a much lower percentage than 15%–20% in developed countries.
Second, competence for original innovation in China is insufficient. Major original
scientific achievements that have global impact are still rare. Most of the scientific
research projects are just a follow-up or imitation of existing research, rather than
groundbreaking research. Third, the development of disciplines is not balanced, and
China’s research level in some disciplines is noticeably lower than the international
level—China’s Field-Weighted Citation Impact (FWCI) in disciplines just reached
0.94 in 2016, lower than the world average of 1.0.

vii
viii Preface to the Series

The Chinese government attaches great importance to basic research. In the 13th
Five-Year Plan (2016–2020), China has established scientific and technological inno-
vation as a priority in all-round innovation and has made strategic arrangements to
strengthen basic research. General Secretary XI Jinping put forward a grand blueprint
of making China into a world-leading power in science and technology in his speech
delivered at the National Conference on Scientific and Technological Innovation in
2016, and emphasized that “we should aim for the frontiers of science and tech-
nology, strengthen basic research and make major breakthroughs in pioneering basic
research and groundbreaking and original innovations” at the 19th CPC National
Congress on October 18, 2017. With more than 30 years of unremitting exploration,
the National Natural Science Foundation of China (NSFC), one of the main chan-
nels for supporting basic research in China, has gradually shaped a funding pattern
covering research, talent, tools and convergence, and has taken action to vigorously
promote basic frontier research and the growth of scientific research talent, rein-
force the building of innovative research teams, deepen regional cooperation and
exchanges, and push forward multidisciplinary convergence. As of 2016, nearly 70%
of China’s published scientific papers were funded by the NSFC, accounting for 1/9
of the total number of published papers all over the world. Facing the new strategic
target of building China into a strong country in science and technology, the NSFC
will conscientiously reinforce forward-looking planning and enhance the efficiency
of evaluation, so as to achieve the strategic goal of making China progressively share
the same level with major innovative countries in research total volume, contribution
and groundbreaking researchers by 2050.
The series of Advances in China’s Basic Research and the series of Reports of
China’s Basic Research proposed and planned by the NSFC emerge against such
a background. Featuring science, basics and advances, the two series are aimed
at sharing innovative achievements, diffusing performances of basic research and
leading breakthroughs in key fields. They closely follow the frontiers of basic research
developments in China and publish excellent innovation achievements funded by the
NSFC. The series of Advances in China’s Basic Research mainly presents the impor-
tant original achievements of the programs funded by the NSFC and demonstrates
the breakthroughs and forward guidance in key research fields; the series of Reports
of China’s Basic Research shows the core contents of the final reports of Major
Programs and Major Research Plans funded by the NSFC to make a systematic
summarization and give a strategic outlook on the achievements in the funding prior-
ities of the NSFC. We hope not only to comprehensively and systematically introduce
backgrounds, scientific significance, discipline layouts, frontier breakthroughs of the
programs and a strategic outlook for the subsequent research, but also to summa-
rize innovative ideas, enhance multidisciplinary convergence, foster the continuous
develop of research in concerned fields and promote original discoveries.
As Hsun Tzu remarked, “When earth piles up into a mountain, wind and rain will
originate thereof. When waters accumulate into a deep pool, dragons will come to
live in it.” The series of Advances in China’s Basic Research and Reports of China’s
Basic Research are expected to become the “historical records” of China’s basic
research. They will provide researchers with abundant scientific research material
Preface to the Series ix

and vitality of innovation and will certainly play an active role in making China’s
basic research prosper and building China’s strength in science and technology.

Academician of the Chinese Academy of Sciences

Beijing, China
Preface

Epigenetics, an emerging discipline since the late 1980s, refers to “the study of
changes in gene function that are heritable but do not entail a change in the DNA
sequence.” How is the selective expression of the same genome determined in
different types of cells within multicellular organisms? How do cells induce and
memorize the gene expression in response to changes in the internal and external
environment of the organism? The answers to these questions can be found with the
epigenetic research.
Epigenetic research started from the epigenetic phenomena observed in plants and
animals, followed by the discoveries of a series of epigenetic modifications, and then
the identification of multiple epigenetic regulators. Gradually, it became to focus on
the regulation of gene expression by chromatin structure. The scientific community
came to understand that epigenetic factors and transcription factors jointly determine
the spatiotemporal expression of genes. In 2006, Japanese scientist Shinya Yamanaka
achieved the somatic cell reprogramming with transcription factors. Chinese scien-
tists quickly realized the importance of epigenetic mechanisms in the processes of
cell programming and reprogramming. Initiated by Pei Gang, Meng Anming, Chen
Runsheng, Shang Yongfeng, Cao Xiaofeng, Sun Fanglin, Xi Zhen and other scien-
tists, departments of Life Sciences, Chemical Sciences and Information Sciences
of the National Natural Science Foundation of China (NSFC) jointly supported the
Major Research Plan Program (MRP) of “Epigenetic Mechanisms of Cell Program-
ming and Reprogramming” in 2008. It funded 156 projects, including the fostering
projects, key projects and integrated projects, with an investment of RMB 190 million
within 8 years. This MRP has actively encouraged forward-looking, original and
systematic research. By the end of 2016, it completed all proposed scientific objec-
tives and made a series of major scientific achievements that attracted worldwide
attention. For example, our scientists have, for the first time in the world, analyzed the
30-nm chromatin structure and pointed out that the tetra-nucleosome is an important
structural and regulatory unit in chromatin; the “seesaw model” of cell reprogram-
ming has been suggested and the system for inducing somatic cell reprogramming
with small chemical molecules has been significantly optimized; the technologies
of haploid embryonic stem cells and “semi-cloned” mice have been successfully

xi
xii Preface

established to generate “artificial spermatids”; the transdifferentiation from fibrob-

lasts to hepatocyte-like cells has been first achieved in the world, which has been
gradually developed toward the application of the bioartificial liver. Series of break-
throughs have also been made in the epigenome research. DNA and histone methy-
lomes in mammalian early embryos have been first established, and the mechanisms
to regulate activities of many important epigenetic modifying enzymes, such as
DNA methylases and demethylases, have been analyzed. This MRP has cultivated
numerous world-class excellent scientists, improved the level of research on epige-
netics and cell fate determination in China and achieved the leapfrog progress from
“follow-up” to “world-leading position.”
The successful completion of this MRP has provided new directions for further
explorations of the roles of epigenetic regulation in cell fate determination. Current
research still focuses on the discovery and functional verification of key epigenetic
factors; however, the understanding on the chromatin dynamic changes in cell repro-
gramming, changes in cell epigenomes and their regulation on transcriptomes, and
mechanisms for determining cell fate transitions remains elusive. Our scientists
should continue to give full play to the interdisciplinary advantages, deepen the
research on epigenetic transcriptomes, single-cell transcriptomes and epigenomes,
epigenome editing, four-dimensional genome and the mechanisms for intergener-
ational inheritance of epigenetics, crack the genetic codes stored in the form of
chromatin and comprehensively analyze the role of epigenetics in cell fate deter-
mination, to make unremitting efforts for the overall improvement and sustainable
development of the innovation ability of China’s epigenetic research.

Gang Pei
Academian of the Chinese Academy
of Sciences, Shanghai, China
Contributors

Yongfeng Shang Hangzhou Normal University, Hangzhou, Zhejiang, China

Fanglin Sun Tongji University, Shanghai, China

Zuoyan Zhu Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan,
Hubei, China
Runsheng Chen Institute of Biophysics, Chinese Academy of Sciences, Beijing,
China
Xiaofeng Cao Institute of Genetics and Developmental Biology, Chinese Academy
of Sciences, Beijing, China
Zhen Xi Nankai University, Tianjin, China

xiii
Contents

1 Project Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Arrangement and Comprehensive Integration of This
MRP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.2 Interdisciplinary Cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Research Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.1 Overall Scientific Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.2 Key Scientific Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Significant Progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Research in China and International Research . . . . . . . . . . . . . . . . . . . . . 7
2.1 Introduction of Epigenetic Research Plans . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Status Quo of the Epigenetic Research . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3 Development Trends of the Epigenetic Research . . . . . . . . . . . . . . . . . 12
2.3.1 Mechanisms of Epigenetic Regulation of Gene
Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.3.2 Discovery, Identification, and Function Studies of New
Epigenetic Modifications, Modifying Enzymes,
and Reader Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.3.3 Mechanisms of Establishment and Maintenance
of Epigenetic Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.4 Epigenetic Regulation on Reproduction
and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.5 Molecular Mechanisms of Epigenetic Regulation on Cell
Programming and Reprogramming . . . . . . . . . . . . . . . . . . . . . . 14
2.3.6 Higher-Order Chromatin Structures and Subnuclear
Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.7 Origins and Evolution of Epigenetic Regulatory
Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3.8 Cancer, Neurodegenerative Diseases, and Other Major
Diseases Related to Epigenetic Regulation . . . . . . . . . . . . . . . . 15

xv
xvi Contents

3 Major Research Achievements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.1 New Epigenetic Regulators and Chromatin Remodeling Factors
as Well as Their Biological Functions and Mechanisms . . . . . . . . . . . 18
3.1.1 New Mechanisms of Interactive Regulation Between
DNA Methylation and Histone Methylation
Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.1.2 New Regulation Mechanisms of DNA Methylase
and Demethylase Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.1.3 New Histone Modification Codes and the Interpretation
Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.4 Higher-Order Structure of 30-Nm Chromatin Fibers
and Their Dynamic Regulation Mechanisms . . . . . . . . . . . . . . 23
3.1.5 Mechanisms of Centromeric Chromatin Establishment
and Nucleosome Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.1.6 Roles of Histone Variants in Chromatin Assembly . . . . . . . . . 25
3.1.7 New Interaction Patterns Between Epigenetic Regulation
and DNA-Damage Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2 Discovery of Epigenetic Regulation Mechanisms for Stem
Cell Self-renewal and Somatic Cell Reprogramming,
and Breakthroughs in Animal Cloning and Reproductive
Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.1 Establishment of ESCs and Semi-cloning Technologies
to Make “Artificial Spermatids” . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.2 First Utilization of the Polar Body Genome Transfer
for Preventing the Transmission of Inherited
Mitochondrial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2.3 The Seesaw Model of Lineage Specifiers Inducing
Somatic Cells to iPSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.2.4 Safety Evaluation of Traditional Methods of iPSC
Induction by Yamanaka Factors . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2.5 Exploration of the Regulation Mechanisms of Somatic
Cell Reprogramming and Discovery of New Regulators . . . . 32
3.2.6 Discovery of New Signals for Maintaining Stem Cell
Self-renewal and Developmental Pluripotency . . . . . . . . . . . . . 33
3.3 Epigenetic Mechanisms Related to Cell Differentiation
and Transdifferentiation, Ontogeny, and Diseases . . . . . . . . . . . . . . . . 34
3.3.1 Discovery of the Key Factors to Promote
the Transdifferentiation of Somatic Cells to Hepatocytes . . . . 34
3.3.2 Discovery of the Epigenetic Mechanisms and Cell
Signaling Pathways Inducing the Differentiation
and Transdifferentiation from Embryonic Stem Cells
or Somatic Cells to Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.3.3 Discovery of Important Disease-related Epigenetic
Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Contents xvii

3.4 Establishment of Epigenetic Maps to Reveal the Characteristics

and Rules of Epigenetic Modifications During the Embryonic
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.4.1 Discovery of Distribution and Change Rules
of Genome-Wide Histone Modifications
in Pre-implantation Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.4.2 Establishment of Single-Cell Transcriptome Sequencing
and Analysis Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.4.3 Establishment of Genome-Wide Methylation Profiles
to Reveal the Inheritance and Evolution Rules
of Epigenetic Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.1 Directions to Be Strengthened in China’s Epigenetic Research . . . . . 46
4.2 Strategic Needs of the Epigenetic Research in China . . . . . . . . . . . . . . 47
4.3 Conceptions and Suggestions for Further Research . . . . . . . . . . . . . . . 47

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Chapter 1
Project Overview

Gang Pei, Yongfeng Shang, Fanglin Sun, Zuoyan Zhu, Runsheng Chen,
Xiaofeng Cao, and Zhen Xi

1.1 Introduction

The Major Research Plan of “Epigenetic Mechanisms of Cell Programming and

Reprogramming” (hereinafter referred to as “this MRP”) was launched by the
National Natural Science Foundation of China (NSFC) during the 11th Five-Year
Plan period, which was started in October 2008 and concluded at the end of 2016. It
has funded 156 projects with a total investment of RMB 190 million, including
68 fostering projects, 23 key projects, and 59 integrated projects, covering the
fields supported by NSFC departments of Life Sciences, Chemical Sciences, and
Information Sciences.
Epigenetics, an emerging discipline since the late 1980s, studies the molecular
mechanisms of changes in cell phenotypes expressed by heritable genes without any

G. Pei (B)
Tongji University, Shanghai, 200000, China
e-mail: [email protected]
Y. Shang
Hangzhou Normal University, Hangzhou, Zhejiang, China
F. Sun
Tongji University, Shanghai, China
Z. Zhu
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China
R. Chen
Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
X. Cao
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
Z. Xi
Nankai University, Tianjin, China

© Zhejiang University Press 2023 1

G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming,
Reports of China’s Basic Research,
https://doi.org/10.1007/978-981-19-7419-9_1
2 G. Pei et al.

alterations of the DNA sequence. The epigenetic regulation mechanism, as a common

way of gene expression regulation in life phenomena, is one of the important mech-
anisms to regulate growth, development, aging, and disease. Especially, epigenetic
regulation plays a decisive role in stem cell maintenance, self-renewal and differen-
tiation, individual aging and dysplasia, development of complex conditions such as
cancer, diabetes, mental illness, and nervous system diseases. The orderly responses
of individual life to environmental factors (including those of nutrition, physical
chemistry, and psychology) largely depend on the effective operation of epigenetic
regulation networks. It also plays an important role in plant development, resistance,
and formation of heterosis.

1.1.1 Arrangement and Comprehensive Integration of This

MRP

Since it was organized and implemented, this MRP has always followed NSFC’s
general principle of “limited goals to enable stable supports, and integrated themes
to realize leapfrog development.” With focuses on the key scientific issues of the
overall scientific objectives, it has funded 68 fostering projects, 23 key projects, and
59 integrated projects.
Epigenetics gradually rose in the late 1980s. After 2000, research in this field has
been widely valued and become hotspots in life sciences. Studies on cell program-
ming and reprogramming cover the basic scientific questions of epigenetics. In 2006,
scientists of the USA and Japan reported the establishment of induced pluripotent
stem cell, symbolizing a new development stage of the research on reprogramming
of somatic cells. Before this MRP was launched, great progress had been made in
epigenetic research worldwide, but scientists’ understanding of epigenetic mech-
anisms was still the tip of the iceberg. Many key problems remained unsolved.
For instance, how do DNA methyltransferases (DNMTs) selectively act on target
genes? How can we clone and identify DNA demethylases? How can we decode the
composition and recognition of the “histone code”? How do the higher-order struc-
tures of chromatin interact with epigenetic information? How does noncoding RNA
participate in the epigenetic regulation? What are the molecular mechanisms of the
epigenetic plasticity and cell reprogramming? What are the relationships between
epigenetic regulation and environment, disease, aging, etc.? What are the features of
the composition, origin, and evolution of epigenetic regulatory networks?
At the beginning of this MRP, the epigenetic research was an emerging field where
there was little gap between Chinese scientists and their international counterparts in
research level and opportunities outweighed challenges. The Advisory Expert Group
enhanced the top-down design and utilized the advantages of our young scientists
engaged in epigenetic research, to focus on the international frontier and carry out
innovative research.
1 Project Overview 3

Centering on the scientific objectives and the key scientific issues, this MRP
initially established 5 research directions:
(1) Molecular mechanisms of establishment and maintenance of epigenetic infor-
mation;
(2) Epigenetic mechanisms of directional differentiation of stem cells;
(3) Epigenetic mechanisms of somatic cell reprogramming;
(4) Epigenetic mechanisms of tissue and organ development and regeneration;
(5) Origins and evolution of epigenetic networks.
Four years after the launch of this MRP, the Advisory Expert Group, based on a
comprehensive investigation on the progress of funded projects, aimed at the cutting-
edge scientific questions and development trends in epigenetics, and condensed the
research directions into three integrated themes, to further fulfill the strategy of
“focusing on goals to make key breakthroughs” of this MRP:
(1) Molecular mechanisms and biological significance of DNA methylation and
demethylation;
(2) Epigenetic mechanisms of cell reprogramming;
(3) Higher-order structures and dynamic changes of nuclear chromatin and
noncoding RNAs during the cell reprogramming.
As a result of the joint efforts of the Advisory Expert Group, the Project Review
Group, and project scientists, this MRP has fully completed the planned scientific
objectives and achieved numerous breakthroughs with great international influences
in the three integrated themes, which has promoted the overall improvement and
leapfrog development of epigenetic research in China.

1.1.2 Interdisciplinary Cooperation

During the implementation of this MRP, the Advisory Expert Group took into consid-
eration both the current situation of epigenetic research in China and the international
frontier and development trends in this field. It vigorously promoted multidisciplinary
convergence among cell biology, biochemistry, developmental biology, structural
biology, bioinformatics, and clinical medicine, and, through various forms of project
support, promptly applied the latest ideas and technologies of related disciplines to
epigenetic research. It thus has made outstanding contributions to the comprehensive
leapfrog development of epigenetic research in China.
Especially, the following measures should be noted.
(1) Cooperation from such disciplines as cell biology, biochemistry, genetics, devel-
opmental biology, genesiology, and evolution was promoted to catapult China
into world’s top ranks of somatic cell reprogramming, nuclear transfer, and
semi-cloned technology. Specially, the number of the papers on haploid stem
cell research published in high-impact journals has exceeded the sum of those
4 G. Pei et al.

published by other countries in the world, suggesting that China is in a leading

position in this area.
(2) The convergence between epigenetics and physics was actively encouraged.
With the introduction of the research means of structural biology, the analysis
on the spatial structures of DNA, RNA, histone modifiers, and higher-order
structures of chromatin was enhanced. It has effectively promoted the discovery
and identification of key molecules and the elucidation of their mechanisms,
making China a leader in this area.
(3) The convergence with mathematics and information science was emphasized.
The means of computational biology were introduced for the massive data anal-
ysis in the epigenetic research, which has effectively promoted the discovery
of key nodes in the interactions of the epigenetic signal networks and the
elucidation of systems biology mechanisms.
Since its implementation, this MRP has followed NSFC’s general principle of
“limited goals to enable stable supports, and integrated themes to realize leapfrog
development.” With focuses on the scientific frontiers of related epigenetic areas
such as somatic cell reprogramming, ontogeny, cell differentiation, and diseases, the
Advisory Expert Group and the Management Group enhanced the top-down design,
refined scientific objectives, and carried out innovative research, which have made
major contributions to the comprehensive upgrade of the original innovation in the
basic research of epigenetics in China.

1.2 Research Overview

1.2.1 Overall Scientific Objectives

The scientific objectives of this MRP were to, with the application of interdisci-
plinary research methods, understand the rules and characteristics for formation,
maintenance, and functions of the epigenetic information during cell programming
and reprogramming, elucidate the mechanisms of the epigenetic regulation in cell
growth, development, and environmental adaptation, and to reveal the mechanisms
of the composition, evolution, and operation of epigenetic networks.

1.2.2 Key Scientific Issues

This MRP aimed to explore the following core scientific themes:

(1) Molecular mechanisms of establishment and maintenance of epigenetic infor-
mation;
(2) Epigenetic mechanisms of directional differentiation of stem cells;
1 Project Overview 5

(3) Epigenetic mechanisms of somatic cell reprogramming;

(4) Epigenetic mechanisms of tissue and organ development and regeneration;
(5) Origins and evolution of epigenetic networks.

1.3 Significant Progress

During its implementation, this MRP has achieved breakthroughs with great interna-
tional influences in the core scientific themes and realized the leapfrog development
of China’s epigenetic research from “follow-up” to “world-leading position.” The
representative achievements are as follows.
(1) New epigenetic regulators and chromatin remodeling factors have been discov-
ered, and their biological functions and mechanisms have been revealed.
(2) The technologies of haploid embryonic stem cells and semi-cloned mice have
been successfully established, and “artificial spermatids” have been made with
the manipulation of imprinted genes. New regulation mechanisms and methods
of somatic cell reprogramming have been found, and the “seesaw model” of
cell reprogramming has been suggested.
(3) Several regulation mechanisms of cell differentiation and trans-differentiation
have been revealed. Especially, the key factors to promote the transdifferentia-
tion of somatic cells to hepatocytes have been discovered, which has provided
a solid foundation for the clinical application of the bioartificial liver. Multiple
disease-related epigenetic modifications have been found.
(4) For the first time in the world, the epigenetic map has been established with
high-throughput data collection to reveal the characteristics of epigenetic modi-
fications during the early embryonic development of different species and the
rules of genetic evolution, which has enriched our understanding of the origins
and evolution of epigenetic networks.
(5) The 30-nm chromatin structure has been first analyzed in the world, and it has
been indicated that the tetra-nucleosome is an important unit of structure and
regulation in chromatin.
(6) The polar body genome transfer has been first used to prevent the transmission
of inherited mitochondrial diseases.
After the completion of this MRP, the development trends in the fields are
compared in Table 1.1.
6 G. Pei et al.

Table 1.1 Comparison of development trends before and after the completion of this MRP
Core scientific Status of China’s Status of international Strengths and gaps of
themes research at the start of research at the end of China’s research at the
this MRP this MRP end of this MRP
Molecular Despite China’s gaps Internationally, rapid The research on the
mechanisms of with the international development has been mechanisms of DNA
establishment and advanced levels, a made in this area, but methylation and
maintenance of group of young China has taken the demethylation,
epigenetic scientists returned to lead in some areas discovery of new DNA
information China and were methylation
gearing up modifications and
their mechanisms, and
higher-order structures
of chromatin has kept
ahead internationally.
The research on other
areas is equivalent to
the
international research
level
Epigenetic Internationally, Europe The international China has ranked top
mechanisms of and America enjoyed research level of the in the research on the
somatic cell the absolute leading mechanisms of haploid stem cells. It
reprogramming positions in the somatic cell has been similar in the
research on the reprogramming has international research
mechanisms of somatic been similar to that in level of the
cell reprogramming, China, but lagged mechanisms of
while China just started behind it in terms of somatic cell
the research on the reprogramming, but
haploid stem cells the number of its
research teams in this
area is still small
Epigenetic There were big gaps Internationally, there China has become a
mechanisms of cell between China and have been many leader in the research
differentiation and international leaders in studies on the on the
transdifferentiation, terms of cell transdifferentiation of transdifferentiation of
tissue differentiation and other types of cells, hepatocytes, and an
and organ transdifferentiation as such as nerve cells, average player in other
development and well as organ myocardial cells, and areas such as the
regeneration development and blood cells transdifferentiation
regeneration of nerve cells
Origins and The research The international China has rose to the
evolution of foundation of this area research advantages top of the research on
epigenetic networks in China was relatively over China is gradually the reprogramming of
poor becoming smaller the epigenetic
modifications during
the early embryonic
development, leaving
the rest of the world
behind
Chapter 2
Research in China and International
Research

Gang Pei, Yongfeng Shang, Fanglin Sun, Zuoyan Zhu, Runsheng Chen,
Xiaofeng Cao, and Zhen Xi

The classic definition of epigenetics is “the study of changes in gene function that are
heritable but do not entail a change in DNA sequence.” The epigenetic phenomenon
was first observed in Drosophila melanogaster in the 1930s. Since the 1970s, a
series of epigenetic markers were found. Subsequently, a large number of epigenetic
regulators were identified, leading to gradual understanding of their biological signif-
icance. With an analysis of the biological mechanisms of several classical epigenetic
phenomena, the focus of epigenetic research evolved to the regulation of chromatin
structure on gene expression. At the beginning of the twenty-first century, scien-
tists learned that the epigenetic regulation mechanism, as a common way of gene
expression regulation in life phenomena, is one of the important mechanisms of
regulating growth, development, aging, and disease. Especially, epigenetic regula-
tion plays a decisive role in stem cell maintenance, self-renewal and differentiation,

G. Pei (B)
Tongji University, Shanghai, 200000, China
e-mail: [email protected]
Y. Shang
Hangzhou Normal University, Hangzhou, Zhejiang, China
F. Sun
Tongji University, Shanghai, China
Z. Zhu
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China
R. Chen
Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
X. Cao
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
Z. Xi
Nankai University, Tianjin, China

© Zhejiang University Press 2023 7

G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming,
Reports of China’s Basic Research,
https://doi.org/10.1007/978-981-19-7419-9_2
8 G. Pei et al.

individual aging and dysplasia, development of complex conditions such as cancer,

diabetes, mental illness, and nervous system diseases. The orderly responses of indi-
vidual life to environmental factors (including those of nutrition, physical chemistry,
and psychology) largely depend on the effective operation of epigenetic regulation
networks. A growing number of epigenetic regulators have become new drug targets
for the treatment of cancer, neurodegenerative diseases, and other major diseases.
Epigenetic regulation also plays an important role in plant development, resistance,
and formation of heterosis. The main epigenetic regulation mechanisms include
DNA methylation modifications, histone modifications, histone variants, noncoding
RNAs, nucleosome positioning, and higher-order structures of chromatin.
Research on cell programming and reprogramming covers the basic scientific
questions of epigenetics and studies classic changes in heritable cell traits without
alterations in the DNA sequence. In 2006, Japanese scientist Shinya Yamanaka first
reported the reprogramming of somatic cells with transcription factors. This achieve-
ment promoted the research of somatic reprogramming into a new stage of devel-
opment, focusing on three main directions: (i) finding new factors and technologies
to improve reprogramming efficiency and biosafety; (ii) exploring the regulation
mechanisms of programming and reprogramming; (iii) developing new treatments
for specific diseases with cell programming and reprogramming technologies. To
study the mechanisms of programming and reprogramming and apply them to clin-
ical and regenerative medicine to produce both economic and social benefits, we
should explore the epigenetic regulation mechanisms in depth. Before this MRP was
launched, great progress had been made in epigenetic research worldwide, but scien-
tists’ understanding of epigenetic mechanisms was still the tip of the iceberg. Many
key problems remained unsolved, including selectivity of DNA methyltransferases
(DNMTs) acting on target genes, cloning and identification of DNA demethylases,
the composition and recognition mechanisms of various histone modifications, inter-
action of higher-order structures of chromatin with epigenetic information, partici-
pation of noncoding RNA in the epigenetic regulation, molecular mechanisms of the
epigenetic plasticity and cell reprogramming, relationships between epigenetic regu-
lation and environment, disease, aging, and the features of the composition, origins,
and evolution of epigenetic regulatory networks.
Since epigenetic phenomena were discovered at the beginning of the twentieth
century, the research themed by epigenetics had almost covered all aspects of growth
and development of organisms, individual health, and environmental response. As
time goes, the research methods and the understanding are constantly improving, and
the research focus of epigenetics is also gradually changing. From the discovery of
classic epigenetic phenomena, to identification of numerous epigenetic modifiers and
regulators, to comprehensive epigenomics, the research on the epigenetic regulation
mechanisms remains a hot area of life sciences, and also one of the most active and
path-breaking fields.
2 Research in China and International Research 9

2.1 Introduction of Epigenetic Research Plans

In April 2003, the Human Genome Project was declared complete. The National
Human Genome Research Institute (NHGRI) of the USA launched the Encyclopedia
of DNA Elements (ENCODE) project in September of the same year, which aimed
to analyze and identify all functional elements encoded in the human genome. The
project is now in the fourth phase. Its first phase of pilot and the second phase
of technology development were carried out simultaneously (2003–2007). There
were 8 research teams participating in the pilot phase, 12 teams in the phase of
technology development, and some teams involved in the comparison, calculation,
and analysis of sequencing data. The total investment of the two phases were about
55 million dollars. In 2007, ENCODE published 29 papers in Nature and Genome
Research to report its achievements in the phases. In the third phase of production
(2007–2017), the project scaled and globalized the research, and established the data
integration center and data analysis center, with an investment of about 130 million
dollars. In September 2012, the findings of this phase were reported in 30 papers
in Nature, Genome Biology, and Genome Research. Starting in February 2017, its
Phase IV funded the first batch of 19 research programs and 2 data centers, with a
total investment of 33 million dollars.
The National Institutes of Health (NIH) of the USA launched the Roadmap Epige-
nomics Project in 2007. It had two goals: (i) to develop comprehensive reference
epigenome maps, i.e., the structure and organization of epigenome; (ii) to develop
new technologies for comprehensive epigenomic analyses, i.e., to deeply under-
stand the functions and significance of epigenome and to discover new epigenome
components. Five initiatives of the project were implemented: (i) 10 million dollars
per year for building the Reference Epigenome Mapping Center; (ii) 1.5 million
dollars per year for each grantee to develop and run the Epigenomics Data Analysis
and Coordination Center which would support the Reference Epigenome Mapping
Center and be responsible for delivering standardized data to the National Center
for Biotechnology (NCBI); (iii) funding the development of new technologies that
would revolutionize epigenetic research approaches; (iv) funding the discovery of
novel epigenetic marks in mammalian cells; (v) supporting research on epigenomic
changes of human health and disease. Based on the Roadmap Epigenomics Project,
the NIH also invested 190 million dollars to the research under International Human
Epigenome Consortium (IHEC) founded in Paris, France in 2010.
In 2015, the NIH announced the launch of the 4D Nucleome Program with the
goal of studying the genome conformation and nuclear organization in an interdisci-
plinary manner and developing improved research approaches with new cutting-edge
technologies. The program planned to support the following six aspects: (i) estab-
lishment of the Nuclear Organization and Function Interdisciplinary Consortium
(NOFIC); (ii) development of new technologies for studying higher-order structures
of chromatin and their interactions; (iii) funding studies on subcellular structures;
(iv) development of high-throughput, high-resolution, and high-content microscopy
10 G. Pei et al.

imaging technologies; (v) establishment of the 4D Nucleome Network Organiza-

tional Hub to promote cooperation and source sharing; (vi) establishment of the
Data Coordination and Integration Center. It funded the first batch of 29 five-year
research projects, with a total investment of 25 million dollars.
To conclude, the international epigenetic research programs, represented by those
of the USA, focus on the research of big data omics with more descriptive results
and the development of new technologies.

2.2 Status Quo of the Epigenetic Research

The early epigenetic research focused on the discovery of classic epigenetic

phenomena, identification of the relationship between chromatin modifications and
gene expression regulation, identification and purification of epigenetic regulators,
and studies of the physiological functions of epigenetic regulators with traditional
methods of genetics and biochemistry. In the 1960s, there were quite prospective
predictions of the functions of various chromatin modifications. Thanks to the devel-
opment of technologies in genetics and molecular biology since the 1990s, the
methods for studying chromatin biology were becoming increasingly mature and
diverse. Meanwhile, the role of epigenetic regulators directly regulating gene expres-
sion was finally confirmed in eukaryotic cells. In 1996, C. David Allis of Rockefeller
University and Stuart Schreiber of Harvard University of the USA respectively cloned
and identified a histone acetyltransferase and a deacetylase, and their findings were
published in Cell and Science of the same year. The progress led people to focus on
the epigenetic modifiers and regulators of various types. The period from the 1990s
to the early twenty-first century saw the blowout development of the research on
epigenetic modifiers and regulators. It was during the studying of their mechanisms
that the epigenetic theories and methods were continuously improved. Recently, with
the advances in genomics and high-throughput sequencing technologies, epigenetics,
which is at the forefront of the life sciences, has entered a new stage of development.
This MRP started in 2008 when the epigenetic research was in full swing inter-
nationally, especially the identification of new epigenetic modifiers and regulators.
Since the technical means were increasingly mature, the international competition
in this field was fierce, with many breakthroughs achieved. Take the research on
histone methylation as an example. In 1999, Michael Stallcup of the University of
Southern California, the USA, cloned the first histone arginine methyltransferase.
In 2000, Thomas Jenuwein of the Research Institute of Molecular Pathology (IMP)
in Vienna, Austria, cloned the first histone lysine methyltransferase. In 2001, the
teams of Thomas Jenuwein and Tony Kouzarides of Cambridge University, the
UK, independently reported the first reader protein binding with histone methy-
lation modification in Nature at the same time. In 2002, various research teams in
the USA and Europe analyzed the crystal structures of histone modification reader
proteins and catalytic domains of histone methyltransferases. Meanwhile, in the keen
world competition in the research area of histone demethylases, Yang Shi of Harvard
2 Research in China and International Research 11

University of the USA discovered the first histone demethylase in 2004; he and Yi
Zhang of the University of North Carolina of the USA independently found a new
class of histone demethylases (containing the JMJ domain). Subsequently, the studies
on histone methylation gradually turned to explore the physiological functions of
known factors.
In 2006, Shinya Yamanaka of Kyoto University in Japan achieved the repro-
gramming of somatic cells with transcription factors, which had set off an upsurge
internationally and promoted the stem cell research into a new stage. With years
of accumulation of the research in genetics and developmental biology, Japan had
many achievements in the field of epigenetics and stem cells. In 2009, Anjana Rao of
Harvard University at that time and Nathaniel Heintz of the Rockefeller University
independently reported in Science of the same issue the discovery of the phenomenon
that methylated DNA could be oxidized in mammalian cells. Then, the research on
the mechanism of oxidative DNA demethylation started.
These achievements, which were attributed to the long-term accumulation and
hard work of epigenetic researchers worldwide, led to a world research pattern domi-
nated by the USA, Europe, and Japan. Epigenetic research institutes and centers were
springing up, and the main force of research was a large number of institutes and
universities, including Johns Hopkins University, Harvard University, the National
Cancer Institute, Cold Spring Harbor Laboratory, University of Southern Cali-
fornia, University of Virginia, Massachusetts Institute of Technology, the Rockefeller
University, New York University, University of California (San Francisco), Univer-
sity of California (Berkeley), University of California (Los Angeles), University of
California (San Diego), Stanford University, University of Massachusetts, University
of Washington (Seattle), Pennsylvania State University, Washington University in St.
Louis, Ohio State University, etc. of the USA; Cambridge University, Oxford Univer-
sity, University of Edinburgh, and John Innes Centre of the UK; Max Planck Insti-
tutes of Germany, Curie Institute of France, and Institute of Physical and Chemical
Research (RIKEN) of Japan. In these institutions and universities, various important
scientists in the field of epigenetics emerged, for example, Shiv Grewal (heterochro-
matin) from National Cancer Institute, Yi Zhang (histone-modifying enzymes) of
Harvard University, Steven Jacobson (DNA methylation in plants) of University
of California (Los Angeles), Job Dekker (higher-order structures of chromatin) of
University of Massachusetts, Bing Ren (higher-order structures of chromatin) of the
University of California (San Diego), Wolf Reik (DNA methylation in mammals) of
Cambridge University, Edith Heard (X chromosome inactivation) of Curie Institute,
and Hiroyuki Sasaki (genomic imprinting) of Kyushu University of Japan.
Before this MRP, China just started the research in epigenetics and kept strength-
ening its research teams. Nevertheless, some of its research work was recognized
by international peers, and relevant results were published in Cell, Nature, and other
international journals. The representative work included the study on a G protein-
coupled receptor (GPCR) of the adrenaline receptor and epigenetic regulation by Pei
Gang’s team of Shanghai Institutes for Biological Sciences of Chinese Academy of
Sciences, with the results published in Cell in 2005; the regulation of a histone and
epigenetic proteins on higher-order chromatin structures by Sun Fanglin’s team of
12 G. Pei et al.

Fig. 2.1 Number of papers on epigenetic research published by major countries and collected in
the Web of Science Core Collection during 2009–2016

Tsinghua University, with the results published in Genes & Development in 2006; the
regulation mechanism of DNA demethylation on gene silencing by Gong Zhizhong’s
team of China Agricultural University, with the results published in Plant Cell in
2006 and EMBO Reports in 2007. On the whole, compared with the international
counterparts, the epigenetic research in China started later with smaller team size.
In terms of the number of papers, there was a total of 12,542 articles and reviews
in epigenetics from 2000 to 2008 that were collected in the Web of Science Core
Collection. Half of them were published by researchers from the USA (46.4%),
while fewer than 5% were from China, ranking the 7th internationally. Since this
MRP was launched, the epigenetic research in China has saw rapid progress. From
2009 to 2016, 6,728 articles and reviews were published, accounting for 13.1% of the
related papers in the world (Fig. 2.1) and exceeding the UK in 2014. From 2014 to
2016, the number of the papers on epigenetics published in academic journals by the
researchers in China was second only to the USA (Fig. 2.2). With support from this
MRP, our scientists have made several major achievements and published a series
of original papers in the world’s top journals, which has won unanimous praise at
home and abroad and has produced great international influences. Eight years after
the launch of this MRP, in the field of epigenetics, China has surpassed Japan in terms
of paper quality and quantity and talent building, and has become another epigenetic
research center on a par with the USA and Europe.

2.3 Development Trends of the Epigenetic Research

The current development trends of the epigenetic research could be summarized as

follows.
2 Research in China and International Research 13

Fig. 2.2 Number of papers on epigenetic research published by major countries and collected in
the Web of Science Core Collection during 2004–2016

2.3.1 Mechanisms of Epigenetic Regulation of Gene

Expression

This area focuses on the interactions and regulation between epigenetic modifiers
and regulators, and the regulation modes (excluding enzymatic reactions) of epige-
netic modifiers on gene expression. It also pays attention to the mechanisms that
cells respond to changes in intracellular and extracellular environment via epige-
netic regulation. At present, the catalytic processes and molecular mechanisms of
most epigenetic modifying enzymes are basically clear. Further studies on the regu-
lation mechanisms of epigenetic factors help to understand why gene expression is
highly spatiotemporally specific and why its roles present complex dynamic changes.

2.3.2 Discovery, Identification, and Function Studies of New

Epigenetic Modifications, Modifying Enzymes,
and Reader Proteins

This area focuses on the interdisciplinary studies of biochemistry and structural

biology to analyze the functions and mechanisms of epigenetic modifying enzymes
and modification reader proteins.
14 G. Pei et al.

2.3.3 Mechanisms of Establishment and Maintenance

of Epigenetic Information

This area focuses on the interdisciplinary research with developmental biology and
systems biology to study the carriers and mechanisms of the transgenerational inher-
itance of epigenetic information in different model organisms. It emphasizes distin-
guishing the contribution of different elements to the establishment and maintenance
of epigenetic information and their regulation mechanisms, and studying the molec-
ular mechanisms of the establishment and maintenance of spatiotemporally specific
epigenetic information in cells.

2.3.4 Epigenetic Regulation on Reproduction

and Development

This area focuses on the mechanisms of establishing and maintaining epigenetic

modifications and their dynamic changes during these processes, as well as the molec-
ular mechanisms of epigenetic factors integrating signal transduction pathways and
regulating cell differentiation.

2.3.5 Molecular Mechanisms of Epigenetic Regulation

on Cell Programming and Reprogramming

This area focuses on the interdisciplinary research with genetics and bioinformatics
to study, from the perspective of systems biology, the dynamic changes and regulation
mechanisms of chromatin modifications and higher-order structures during cell fate
changes, as well as the mechanisms of the heterogeneity of cell changes during these
processes. It also emphasizes improving the efficiency of cell reprogramming in vitro
with regulations on epigenetic factors.

2.3.6 Higher-Order Chromatin Structures and Subnuclear

Structures

This area focuses on the interdisciplinary research with physics and computational
biology, and especially, the development of new technologies to study the composi-
tion and dynamic changes of higher-order chromatin structures. It also emphasizes
the research of general principles for the composition of higher-order chromatin
structures.
2 Research in China and International Research 15

2.3.7 Origins and Evolution of Epigenetic Regulatory

Networks

This area focuses on the interdisciplinary research with bioinformatics to establish

the epigenetic maps during early embryonic development of different species and
study the rules of the inheritance and evolution of epigenetic information.

2.3.8 Cancer, Neurodegenerative Diseases, and Other Major

Diseases Related to Epigenetic Regulation

This area focuses on the regulatory mechanisms of epigenetic factors in disease

occurrence, development, and deterioration. It also emphasizes the interdisciplinary
research with chemical biology to develop potential targeted drugs of small-molecule
compounds on epigenetic factors.
Chapter 3
Major Research Achievements

Gang Pei, Yongfeng Shang, Fanglin Sun, Zuoyan Zhu, Runsheng Chen,
Xiaofeng Cao, and Zhen Xi

The successful implementation of this MRP has promoted the overall improvement
and leapfrog development of the epigenetic research in China. A series of break-
throughs with great international influences have been achieved in discovering new
epigenetic regulators and chromatin remodeling factors and revealing their biolog-
ical functions and mechanisms; analyzing the regulation mechanisms for stem cell
self-renewal and somatic cell reprogramming and developing new cloning methods;
revealing the rules for epigenetic regulation of cell differentiation and transdifferen-
tiation, ontogeny, and disease occurrence and development; and understanding the
composition and operation of epigenetic networks at the whole genome level.

G. Pei (B)
Tongji University, Shanghai, 200000, China
e-mail: [email protected]
Y. Shang
Hangzhou Normal University, Hangzhou, Zhejiang, China
F. Sun
Tongji University, Shanghai, China
Z. Zhu
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China
R. Chen
Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
X. Cao
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
Z. Xi
Nankai University, Tianjin, China

© Zhejiang University Press 2023 17

G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming,
Reports of China’s Basic Research,
https://doi.org/10.1007/978-981-19-7419-9_3
18 G. Pei et al.

3.1 New Epigenetic Regulators and Chromatin Remodeling

Factors as Well as Their Biological Functions
and Mechanisms

Epigenetic regulation is a common way of gene expression regulation in life

phenomena. Its important means include DNA methylation modification, histone
modification, nucleosome assembly, establishment and recognition of the histone
code, as well as formation, maintenance, and transformation of higher-order chro-
matin structures. Therefore, this MRP has supported the research directions of discov-
ering and identifying the new molecules for DNA methylation modification and
histone modifications, exploring the mechanisms for nucleosome assembly and the
composition, recognition, and functions of the histone code, studying the regulation
mechanisms and functions of higher-order chromatin structures and the transforma-
tion between heterochromatin and euchromatin, and clarifying the roles of impor-
tant signal transduction pathways in the establishment and maintenance of epigenetic
information. It has also actively encouraged structural biology research and achieved
a series of breakthroughs.

3.1.1 New Mechanisms of Interactive Regulation Between

DNA Methylation and Histone Methylation
Modifications

DNA methylation can cause changes in chromatin structure, DNA conformation,

DNA stability, and interactions between DNA and proteins, to control gene expres-
sion. The enzymes involved in DNA methylation are mainly DNMT1, DNMT3A,
and DNMT3B. Previous studies have established a connection between DNA methy-
lation and histone modifications and revealed a histone-guided mechanism for the
establishment of DNA methylation. The ATRX-DNMT3-DNMT3L (ADD) domain
of DNMT3A recognizes unmethylated histone H3 (H3K4me0). The histone H3
tail stimulates the enzymatic activity of DNMT3A in vitro, whereas the molecular
mechanism remains elusive.
With the support of this MRP, our scientists have determined the crystal struc-
tures of DNMT3A-DNMT3L (autoinhibitory form of DNMT3A) and DNMT3A-
DNMT3L-H3 (active form) complexes at 3.82 and 2.90 Å resolution, respectively.
Structural and biochemical analyses indicated that the ADD domain of DNMT3A
interacted with and inhibited enzymatic activity of the catalytic domain (CD) through
blocking its DNA-binding affinity. Histone H3 (but not H3K4me3) could disrupt
ADD-CD interaction, induce a large movement of the ADD domain, and thus release
the autoinhibition of DNMT3A. The finding has added another layer of regulation of
DNA methylation to ensure that DNMT3A is mainly activated at proper targeting loci
when unmethylated H3K4 is present, and has strongly supported a negative correla-
tion between H3K4me3 and DNA methylation across the mammalian genome (Guo
3 Major Research Achievements 19

et al. 2015). This study has provided a new insight into an unexpected autoinhibition
and histone H3-induced activation of the de novo DNA methyltransferase after its
initial genomic positioning, and thus has been recommended by the professional
academic review website F1000.
Another study has found the interactive mechanism of methylated H3K9 and
methylated DNA (Liu et al. 2013), and put forward a new model for maintaining
DNA methylation: UHRF1 could bind to DNA replication forks more effectively
through the cooperative binding of hemi-methylated DNA and methylated histone,
and thus recruited DNMT1 for the maintenance of DNA methylation. In the mouse
model, the study further demonstrated that UHRF1-mediated cross-talk between
histone modification and DNA methylation via recognizing H3K9 methylation. It
also indicated that H3K9 methylation plays an auxiliary rather than decisive role
in DNA methylation in mammalian cells. This work has shown that the interaction
between histone modification and DNA methylation in different species differs in
the degree and the mechanism (Zhao et al. 2016a). Meanwhile, our scientists have
discovered the negative regulation of de novo DNA methylation by UHRF1/2, and
proposed that UHRF1/2 promote DNA methylation maintenance and inhibit de novo
DNA methylation, to support the hypothesis of cellular DNA methylation home-
ostasis (Jia et al. 2016). Additionally, another study has found that hemi-methylated
DNA (formed after DNA replication) binds itself to epigenetic regulator UHRF1 and
opens its closed conformation to facilitate its recognition of histone modification,
which ensures UHRF1 to precisely locate itself in specific regions of the genome
and play a role in maintaining DNA methylation (Fang et al. 2016).

3.1.2 New Regulation Mechanisms of DNA Methylase

and Demethylase Activities

In recent years, light-controlled proteins have become a powerful tool for studying the
spatiotemporal regulation of cellular signal transduction. Compared with ultraviolet-
activated probes, two-photon-activatable probes can significantly reduce cytotoxity
and thus have a broad application prospect. Our scientists utilized protein chem-
ical synthesis as a core technique and developed light-controlled protein probes
targeting immune cells. With these probes, they studied the directional migration
of immune cells under precise spatiotemporal stimulation and the activation mech-
anism. Through the development of two-photon-controlled chemokine probes, they
and their collaborators explored the mechanism of directional migration of immune
cells in living tissues and designed and synthesized the first two-photon-activatable
chemokine probe hCCL5** . With its high spatiotemporal resolution, they showed
at the single-cell level that “T cells perceive the directional cue without relying
on PI3K activities, which are nonetheless required for persistent migration” (Chen
et al. 2015b). Additionally, utilizing the good tissue penetration of the two-photon
technique, they and their collaborators realized the directed migration of immune
20 G. Pei et al.

cells in living tissues (ears and lymph nodes of mice). This probe is an ideal molec-
ular tool for understanding and potentially manipulating cell positioning and related
cell biology. Based on the developed light-controlled protein antigens, they revealed
the early signal transduction dynamics of B cell activation. HEL-K96NPE, the first
light-controlled protein antigen for B cell, was developed with light-controlled amino
acid screening and protein chemical synthesis. They found that for hen egg lysozyme
(HEL) and its antibody HyHEL-10 (or B cells from MD4 transgenic mice expressing
HyHEL-10), masking a single site (Lys96 of HEL) was sufficient to block the inter-
action as high as 20 pm with a large interaction surface (1800 Å2 ). Combined with
the high-speed high-resolution live cell imaging, HEL-K96NPE was used to monitor
the early formation of B cell synapse and the periodic responses of calcium influx in
a real-time manner. This probe is a promising tool for in-depth studies on the early
signal transduction dynamics of B cell activation.
5-methylcytosine (5mC), often called “the 5th base,” is a methylated cytosine base
in mammalian genome. TET protein is an oxidase in mammalian cells, which can
function in DNA demethylation. The mammalian TET plays a vital role in critical
life processes, such as epigenetic reprogramming of fertilized eggs, pluripotent stem
cell differentiation, and medullary hematopoiesis, and its inactivation is also closely
related to multiple diseases, especially hematological tumors. The research on the
DNA demethylation centered on TET is one of the most active areas in epigenetics.
Previous studies have shown that during the demethylation, TET oxidizes 5mC into
5-hydroxymethylcytosine (5hmC, the 6th base) and continues to catalyze it into
5-formylcytosine (5fC, the 7th base) and 5-carboxylcytosine (5caC, the 8th base).
5hmC is relatively stable in cells, and its content is much higher than that of 5fC and
5caC. There remained no reasonable biological explanation for this phenomenon. Our
scientists have uncovered the mystery with research methods of structural biology,
biochemistry, computational biology, and other disciplines. Structural analyses indi-
cated that the orientation of 5mC in the TET catalytic cavity made it prone to be
captured by the catalytic domain and oxidized into 5hmC. Due to the existence of
oxygen, 5hmC and 5fC were restrained within the catalytic cavity and were less
prone to further oxidation, resulting in the decrease of TET activity to these two
bases. With such a difference in catalytic efficiency, it was easy for TET to oxidize
5mC into 5hmC. After the generation of 5hmC, however, it was not easy for TET to
further oxidize it into 5fC or 5caC. As a result, cellular 5hmC was relatively stable and
significantly more prevalent than 5fC/5caC. The study has demonstrated the mech-
anism of substrate preference of TET and provided an explanation for the stable
existence of 5hmc in the genome at the molecular level. In specific gene domains,
TETs might be activated by specific regulators to produce TETs with high activity to
iteratively oxidize 5hmC into 5fC and 5caC. This discovery has solved a conundrum
in epigenetics and also provided a new idea and method for revealing the molecular
mechanisms of the stepwise catalysis of other proteins (Hu et al. 2015).
Our scientists have found that the zinc finger protein SALL4A preferentially binds
itself to 5hmC-modified DNA. The Sall4a gene is important during the early embry-
onic development, and its mutation leads to the autosomal dominant condition of
Duane radial ray syndrome. The Sall4a knockout mouse embryos stop developing
3 Major Research Achievements 21

during the peri-implantation and die soon. The study found that in the mouse embry-
onic stem cells, the majority of SALL4A localized at enhancers and its chromatin
association largely depended on TET1. Further analyses on the cytosine modification
at the Sall4a-binding sites in the genome indicated that these sites were depleted of
stable 5hmC, but were enriched for the further oxidized products of 5fC and 5caC,
suggesting a possible role of Sall4a in facilitating further oxidation of 5hmC. As
expected, the researchers found that knockout of Sall4a resulted in elevated 5hmC
at the original Sall4a-binding sites, and reduced the stable binding of TET2 which
is not conducive to the further oxidation of 5hmC (Xiong et al. 2016a). This study
has enriched the understanding of DNA oxidation and demethylation regulated by
TET family proteins and proposed the concept of the cooperative stepwise oxida-
tion of 5mC: SALL4A does not function as a general 5hmC-binding protein like the
role of MeCP2 for 5mC; instead, it is a cell type and genomic region-specific regu-
lator of 5hmC; it recruits TET2 at specific sites and facilitates further oxidation of
5hmC into 5fC and 5caC to fine-tune gene expression regulation. This discovery has
promoted our understanding of the dynamics of DNA methylation and its functional
roles in embryonic stem cells and cell reprogramming. It has been introduced and
recommended on F1000.
Previous studies have shown that USP7 binds itself to DNMT1 and regulates
DNMT1 stability through acetylation and ubiquitination. However, the molecular
mechanism for USP7-mediated stabilization of DNMT1 remains largely unknown.
To answer this question, our scientists determined the crystal structure of human
DNMT1 in complex with USP7 at 2.9 Å resolution. Structural and biochemical
analyses revealed that the interaction between the two proteins was primarily medi-
ated by the KG linker of DNMT1 and a previously uncharacterized acidic pocket
that acted as a substrate-binding site near the C-terminus of USP7. Mutations of
these acidic residues disrupted the interaction between DNMT1 and USP7, leading
to increased turnover of DNMT1. Acetylation of lysine residues of the KG linker
impaired the DNMT1-USP7 interaction and promoted proteasomal degradation of
DNMT1. Treatment with histone deacetylase (HDAC) inhibitors resulted in increased
acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation
was observed in differentiated neuronal cells and pancreatic cancer cells. These
findings have revealed that USP7-mediated stabilization of DNMT1 is regulated by
acetylation and provided a structural basis for the design of inhibitors by targeting
the DNMT1-USP7 interaction surface (Cheng et al. 2015).

3.1.3 New Histone Modification Codes and the Interpretation

Mechanisms

Histone modifications, one of basic mechanisms of epigenetic regulation, are consid-

ered to constitute a type of “histone codes.” They regulate the interpretation of genetic
information at the chromatin level and play an important role in the processes such
22 G. Pei et al.

as gene expression and cell fate determination. In recent years, many new histone
modifications have been found, one of which is histone lysine acylation, such as
acetylation (ac), propionylation (pr), butyrylation (bu), and crotonylation (cr). There
have been various studies on the histone acetylation, whereas the histone crotonyla-
tion is a type of newly discovered modification codes conserved from yeast to human.
It is closely associated with active transcription and gene activation, and regulates
the biological processes such as gene expression and gamete maturation. Since the
histone crotonylation was reported by Zhao Yingming’s team of the University of
Chicago in 2011 (Tan et al. 2011), the research on its generation, elimination, and
identification mechanisms has become a hotspot. The opportunity and challenge that
follows is to discover the specific recognition reader of crotonylation as a direct
interpreter of the “histone code.”
With the support of this MRP, our scientists have discovered new histone crotony-
lation readers. They published three high-quality papers (Xiong et al. 2016b; Li et al.
2016c; Zhao et al. 2016a) and reported two such readers—YEATS and DPF domains.
According to the structural and functional analyses of the YEATS domains of the
epigenetic regulators AF9 and YEATS2 and the DPF domains of MOZ, both domains
have been found to be histone crotonylation readers with preference. These studies
have elucidated the molecular and cellular mechanisms that these domains promote
gene transcription by specifically reading the histone crotonylation codes. Notably,
the important work of YEATS mentioned above has been specially emphasized in
three heavyweight reviews. They believe that this discovery, as a breakthrough in
the area of histone modification regulation, has opened a new direction of metabolic
and epigenetic research and deepened our biological understanding of crotonylation
modification.
The histone methylation is another important type of codes. Our scientists
have discovered and demonstrated that KIAA1718 (KDM7A) is a dual-specificity
histone demethylase for both H3K9 and H3K27 (Huang et al. 2010). The studies
on the histone demethylase ceKDM7A from Caenorhabditis elegans found that it
binds itself to H3K4me3 to demethylate H3K9me2 and H3K27me2, and demon-
strated the mechanism that the methylation associated with transcription repres-
sion (H3K9me2 and H3K27me2) and the methylation associated with transcription
activation (H3K4me3) distribute in a mutually exclusive manner (Lin et al. 2010;
Yang et al. 2010). Six co-crystal structures of ceKDM7A with peptides containing
different histone methylation modifications have been completed to explain its speci-
ficity and substrate recognition mechanisms from the perspective of structure. Our
researchers have found that PHF8 is a histone H3K9 demethylase locating in nucle-
olar region and regulating rRNA transcription (Zhu et al. 2010). The related findings
were published in Cell Research and recommended as a featured article on the cover.
It was commented by experts in the same issue and won “Sanofy-Cell Research
Outstanding Paper Award of 2010.”
Additionally, the 20 different histone lysine demethylases that have been identi-
fied so far can only eliminate the methylation modifications at the 4 lysine sites of
H3K4, H3K9, H3K27, and H3K36. There are many important lysine methylation
sites in histone, including H3K79 and H4K20, and their demethylases are waiting
3 Major Research Achievements 23

to be discovered. Also, the arginine demethylases have not been found yet. With
the support of this MRP, our scientists have established a system for the genome-
wide screening of novel histone demethylases and their methylation regulation, and
utilized it to discover the new demethylase KDM9A/9B of H4K20. This family is a
new histone lysine demethylase that has no conservatism with LSD and JmjC fami-
lies. KDM9A/9B could regulate the methylation of H4K20 at its binding domain,
activate gene transcription by removing H4K20me1 methylation, and activate the
transcription of repetitive sequences by removing H4K20me3 methylation. Related
research is being carried out.

3.1.4 Higher-Order Structure of 30-Nm Chromatin Fibers

and Their Dynamic Regulation Mechanisms

Using the in vitro self-developed chromatin assembly technology and cryogenic

electron microscopy, our scientists have, for the first time in the world, analyzed the
high-resolution cryogenic electron microscopy structures (11 Å) of 30-nm chromatin
fibers. The structures show a left-handed double-helical twist of the repeating tetranu-
cleosomal structural units (Fig. 3.1). With the single-molecule magnetic tweezers,
the study has explored in depth the dynamic processes of establishing and regulating
the 30-nm chromatin fiber structure (Song et al. 2014). The study was specially intro-
duced by Science editors with the title of “Double helix, Double.” In the same issue,
it was evaluated in “The as Perspective” by Professor Andrew Travers of Cambridge
University in the UK, as the origin of DNA double helix structure model, with the
article titled “The 30-nm Fiber Redux.” The findings have been collected in the latest
editions of the world-famous biochemistry textbooks Fundamentals of Biochemistry:
Life at the Molecular Level and Lehninger Principles of Biochemistry. This paper
has been recommended by F1000 as “Exceptional.” The 3D electron microscopic
structure of the 30-nm chromatin fibers has been collected in “A Tale of Chromatin
and Transcription in 100 Structures” (Cramer 2014) by Dr. Patrick Cramer, Director
at the Max Planck Institute for Biophysical Chemistry in Germany. Our scientists
have made breakthroughs in solving the major scientific question of the higher-order
structure of 30-nm chromatin fibers, making China a global leader in the research
area of chromatin structure. The following studies have established and improved the
MNase-Seq technique for mapping the genome-wide chromatin structure—gMNase-
Seq (a method for analyzing the chromatin structure in nucleus). Through modifica-
tions of MNase, the spatial resolution of the MNase-Seq technique was improved.
They further demonstrated the dynamic regulation of the 3D structure of 30-nm chro-
matin fibers. For the first time, they have reported that the “tetranucleosomes-on-a-
string” appears as an important intermediate structure during the folding of 30-nm
chromatin fibers. The histone chaperone FACT negatively regulates the structure of
tetranucleosomal unit during the gene transcription (Li et al. 2016a).
24 G. Pei et al.

Fig. 3.1 Unraveling the 3D organization of 30-nm chromatin fibers by cryo-electron microscopy
(Song et al. 2014)

3.1.5 Mechanisms of Centromeric Chromatin Establishment

and Nucleosome Assembly

With the support of this MRP, our scientists have systematically studied the mech-
anisms of recognition and targeting of the histone variant CENP-A during the
centromeric chromatin establishment as well as the assembly into nucleosomes.
The study analyzed the crystal structure of a centromeric CENP-A-H4 heterodimer
in complex with the histone chaperone HJURP, revealed the specific recognition
mechanism between CENP-A and HJURP, and discovered that the important residue
Ser68 of CENP-A played a critical role in HJURP recognition (Hu et al. 2011). The
following study has found that the phosphorylation of CENP-A at Ser68 regulates
its specific recognition by HJURP, and orchestrates its dynamic assembly during
the cell cycle (Yu et al. 2015). These findings have provided strong evidence for
understanding the centromeric nucleosome assembly. According to the comment on
F1000, “the results have revealed that HJURP can specifically recognize the serine
at site 68 of CENP-A, which will become a focus of related work in the future.” Dr.
Patrick Cramer also collected this work in “A Tale of Chromatin and Transcription
in 100 Structures.”
3 Major Research Achievements 25

3.1.6 Roles of Histone Variants in Chromatin Assembly

The histone variants of H3.1, H3.3, and centromere-specific CenH3 in the histone H3
family remain conserved in both plants and animals (from Drosophila melanogaster
to humans). H3.3 and H3.1 of Arabidopsis differ in only 4 amino acids—amino
acids 31 and 41 in the N-terminal tail and amino acids 87 and 90 in the core domain.
Through the delicate dynamic cell biology analysis of Arabidopsis histone H3.3,
H3.1, and their mutants in nucleolar rDNA, our scientists have proposed and veri-
fied the model in which amino acids 87 and 90 in the core domain of H3.3 guide
nucleosome assembly, whereas amino acids 31 and 41 in the N-terminal tail guide
nucleosome disassembly (Shi et al. 2011).
To study the molecular mechanisms of the recognition and assembly of the histone
variant H3.3, our scientists have analyzed the crystal structure of a DAXX- H3.3-
H4 complex and revealed the molecular mechanism for the recognition of H3.3 by
the complex of the H 3.3-specific chaperone DAXX and HIRA, which has laid the
foundation for outlining the storage pathway of H3.3 and understanding its specific
recognition and assembly mechanisms. Additionally, the study has discovered that
H3.3 and H2A.Z function together to dynamically regulate the chromatin structures
over the enhancer and promoter regions, to maintain the self-renewal of stem cells
and promote their neural differentiation (Chen et al. 2013).

3.1.7 New Interaction Patterns Between Epigenetic

Regulation and DNA-Damage Repair

Homologous recombination (HR) is essential for maintaining genome integrity and

variability. Before HR, chromatin needs to release DNA, and its structure needs to be
reconstructed after HR. The study has shown that depletion of either the nucleosome
assembly protein 1 (NAP1) group of H2A/H2B-type histone chaperones or NAP1-
related protein (NRP) group proteins in Arabidopsis cause a reduction in HR in
plants. The hyperrecombinogenic phenotype caused by the depletion of CAF-1,
the H3/H4-type histone chaperones, relied on NRP, but the telomere shortening
phenotype did not, suggesting that the dependence was specific in HR. The analysis
of DNA lesions, H3K56 acetylation, and expression of DNA repair genes argued for
a role of NAP1/NRP participating in HR via nucleosome disassembly/reassembly.
For the first time, the study has established a crucial function for NAP1 family of
H2A/H2B-type histone chaperones in somatic HR in eukaryotes (Gao et al. 2012).
Histone demethylase KDM5B, a member of the JmjC domain-containing histone
demethylases, becomes enriched in DNA-damage sites due to its poly-ribosylation
and recognition of histone variants. It changes the structures and states of the chro-
matin in the damaged regions with its demethylase activity and recruits Ku70 and
BRCA1, the essential components of nonhomologous end-joining and homologous
26 G. Pei et al.

recombination, respectively, to regulate double-strand DNA-damage repair to main-

tain the genome stability. The results help to analyze the epigenetic roles in the
maintenance of genetic fidelity and to understand the genomic instability related
diseases, such as cancer (Li et al. 2014a).

3.2 Discovery of Epigenetic Regulation Mechanisms

for Stem Cell Self-renewal and Somatic Cell
Reprogramming, and Breakthroughs in Animal
Cloning and Reproductive Technologies

Somatic cell reprogramming remains one of the hotspots in life sciences. It can be
realized through somatic cell nuclear transfer. In 2006, Japanese scientist Shinya
Yamanaka reported to induce somatic cells into pluripotent stem cells, similar to
embryonic stem cells, by introducing four transcription factors, Oct3/4, Sox2, c-
Myc, and Klf4. The establishment and discovery of the induced pluripotent stem
cells (iPSCs) has not only created a new method for somatic cell reprogramming,
but also a milestone in the history of life sciences. Somatic cell reprogramming
plays an important role in regenerative medicine and the development and utilization
of drugs, as well as in the treatment of various genetic and functional diseases of
human beings. After this MRP was launched, the Advisory Expert Group took stock
and believed that somatic cell reprogramming would become a hotspot in the future
frontier research, and provided strong support for the research in this area. As a result,
China has made great achievements in the research of animal cloning, reproductive
technology, and somatic reprogramming mechanisms in a short time and become a
world leader in this research area.

3.2.1 Establishment of ESCs and Semi-cloning Technologies

to Make “Artificial Spermatids”

The haploid stem cell is a new type of artificial cells. With the support of this MRP,
our scientists have made a series of important progress in the establishment and appli-
cation of mammalian haploid stem cells. They have, for the first time, established
the rat and mouse androgenetic haploid embryonic stem cell lines that can replace
sperms to complete the reproductive process and the monkey parthenogenetic haploid
embryonic stem cell lines; developed the genetic screening and modification tech-
nologies based on the haploid embryonic stem cells (ESCs); applied the haploid stem
cells as a new technology and tool to the research of reproductive and developmental
biology which produced the offspring of two female mice; and for the first time
created a new type of cells—mammalian interspecific hybrid allodiploid stem cells.
Their achievements have enriched the theories and systems of the reproductive and
3 Major Research Achievements 27

developmental research, expanded the research scope, and presented the important
application prospects of haploid stem cells for reproductive biology, developmental
biology, genetics, and evolutionary biology. The studies have been published in
Nature, Cell, Cell Stem Cell, and other journals. The number of the papers published
by China in high-impact journals has exceeded the sum of those published by other
countries in the world, suggesting that China is in a leading position in the haploid
stem cell technologies and relevant research fields.
(1) Establishment of mammalian haploid stem cell lines to study their features
and functions
With the nuclear transfer technique, our scientists have established the androgenetic
haploid embryonic stem (ahES) cell lines by transferring a sperm into an enucleated
oocyte to generate androgenetic haploid embryos. The cells possessed the ability to
differentiate into three layers of epiblasts and germ cells.
After injecting into the cytoplasm of M II phase oocytes, they could replace
a sperm to fertilize an egg. The embryo could further develop into a healthy and
fertile organism—a “semi-cloned” mouse. This is called semi-cloned. In the study, a
neomycin-resistant gene neor, controlled by the PGK promoter, was electroporated
into the ahES cell line AHGFP-4, and the transgenic macrohaploid stem cell lines
were established after the G418 drug selection. Three transgenic cell lines were
selected for the intracytoplasmic ahES-cell injection (ICAI). They found that all the
three lines could get due fetuses, and 7 and 1 healthy alive transgenic animals were
obtained from 2 cell lines, respectively. The study has provided a new model for
studying the basic questions such as reproduction and genomic imprinting, a new
method for obtaining transgenic animals, and a new idea for the development of
assisted reproductive technology. The findings were published in Nature in 2012 (Li
et al. 2012a), and selected as one of the Top 10 Scientific Advances in China of the
same year. Another study has established the haploid epiblast stem cells (hEpiSCs)
to provide a new tool for the genetic screening of haploid stem cells and the research
on the developmental mechanisms such as diploid maintenance (Shuai et al. 2015).
(2) Reproductive and developmental studies with haploid stem cells
The mammalian interspecific hybrid allodiploid embryonic stem cells (AdESCs)
have been generated with haploid ESCs. Our scientists have reported the generation
of mouse–rat hybrid AdESCs by fusing androgenetic and parthenogenetic haploid
ESCs of the two species, which avoids the reproductive isolation barrier that the
fusion of the sperm and egg of mice with rats’ cannot develop (Li et al. 2016b).
The AdESCs have the ability to differentiate into all three germ layers as well as
early stage germ cells while maintaining a stable allodiploid genome. The gene
expression analyses have revealed the unique high-parent or low-parent expression
patterns and biological traits in these AdESCs. The analyses on the two types could
lead to effective explorations of the molecular regulation mechanisms of the trait
differences between species. Additionally, rather than the “random inactivation”
model common in mammals, the X chromosomes in the interspecific hybrid cells
follow the model of mouse X chromosome-specific inactivation. With this feature,
28 G. Pei et al.

the study has systematically identified the mouse X inactivation-escaping genes.

This has been the first case of artificially created interspecific hybrid embryonic
stem cells in the form of stable diploid, which provides a new model and tool for the
research of evolutionary biology, developmental biology, and genetics. The results
were published in Cell in 2016.
Our scientists have bred healthy fertile mice by haploid fusing cells and demon-
strated that the interactions between parental genomes before the blastocyst stage are
not necessary for embryo development, which provides a new idea for developmental
biology and the development of assisted reproductive technology (Li et al. 2015).
Precise imprinting modification on the haploid embryonic stem cells can realize the
“homosexual” reproduction (Li et al. 2016c). Imprinted genes are those with different
expression patterns on paternal and maternal chromosomes. The haploid stem cells
are suitable imprinted gene research platforms because they have genetic materials
only from paternal or maternal sources and the advantages of ESCs in gene manipu-
lation. With the parthenogenetic ESCs as the platform, the study had the two paternal
methylated imprinting control regions knocked out. By repairing the imprinted gene
expression pattern of the region, and injecting the knockout parthenogenetic haploid
ESCs into oocytes, the parthenogenetic embryos with two mothers were generated,
and parthenogenetic mice could be produced in relatively high efficiency. The study
has provided a new way for mammalian parthenogenesis, a new idea for animal repro-
duction and breeding research, and also a new research method for the exploration
of imprinting abnormalities and treatment methods.
(3) Genetic screening and modifications with the haploid stem cell technology
The “fertilization” capacity of the haploid cells gradually loses with the passage
of cells. Especially, after gene editing, it is difficult to obtain healthy semi-cloned
mice by injecting them into the oocytes. With the support of this MRP, our scientists
have shown that the spermatid-like haploid cells (artificial spermatids) are produced
by deleting H19-DMR and IG-DMR regulating two paternally imprinted genes, and
they are proved to be able to efficiently support the generation of genetically modified
semi-cloned mice. Importantly, these artificial spermatids carrying a CRISPR-Cas9
library can further produce various mice with different mutations, which provides
technology supports for organism-wide genetic screening and modifications in mice
(Zhong et al. 2015). It has been further demonstrated that the haploid cells from
the oocytes, after deleting H19-DMR and IG-DMR, also have the capacity of the
artificial sperm, which has realized the effective parthenogenetic development in
mammals. Additionally, the parthenogenetic haploid ESCs from human oocytes have
been generated (Zhong et al. 2016).
In haploid stem cells, CRISPR-Cas9 can be used for efficient treatment of genetic
diseases. Generally, the direct injection of the CRISPR-Cas9 system into zygotes
with the genetic defect of cataracts for gene editing could cure such defect in mice.
However, this method had two problems: a low rate of the newborn mice being cured
(about 30%) and a small amount of off-target effects (Wu et al. 2013). To solve the
problems, our scientists produced spermatogonial stem cells (SSCs) carrying the
homozygous mutant gene from the testis of the cataract mice and established a series
3 Major Research Achievements 29

of cell lines from single SSC expansion after CRISPR-Cas9-mediated correction of

the genetic defect. After further analyses, they then transferred the SSCs from the
cell lines carrying the corrected gene without off-target mutations and maintaining
normal epigenetic traits into recipient testes to produce healthy mice. Their findings
have provided a new idea for human gene therapy (Wu et al. 2015). When the paper
was published, it was released globally in Cell and got 7 comments or special reports
in Cell Stem Cell, Nature, Nature Review Genetics, Addgene Blog, Sci Bull, MIT
Technology Review, and on F1000. It is believed that this study has demonstrated
that “the CRISPR-Cas9 technology can be used for treating human genetic diseases,”
starting a craze of gene therapies. Both papers have been ranked in the world’s top
1% in the Percentiles for Papers Published by Field, 2006–2016 of Essential Science
Indicators (ESI).

3.2.2 First Utilization of the Polar Body Genome Transfer

for Preventing the Transmission of Inherited
Mitochondrial Diseases

Our scientists have used polar bodies as donor genomes and placed them in the cyto-
plasm of healthy donor oocytes, to realize the genome transfer—namely mitochon-
drial replacement (Wang et al. 2014b). This study has developed the cell organelle
replacement technology in addition to the stem cell therapy, providing a new strategy
and path for treating refractory diseases. The study was published in Cell and has
been introduced in the articles in Nature and Nature Review Genetics, titled “Assisted
Reproductive Technologies to Prevent Human Mitochondrial Disease Transmission”
and “Clinical Genetics: Mitochondrial Replacement Techniques under the Spotlight”
respectively, stating that the invention “is important to prove the feasibility of polar
body transfer and significantly improve the efficiency of mitochondrial transfer
therapy.” Dr. Herman, President of American College of Medical Genetics and
Genomics (ACMG), has praised the study, believing that it has provided interesting
hypothesis, new discoveries, and advanced intervention means for mitochondrial
disease intervention. The Human Fertilization and Embryology Authority (HFEA) of
the UK released a 45-page Review of the Safety and Efficacy of Polar Body Transfer
to Avoid Mitochondrial Disease as a reference for the British public and parlia-
ment to discuss the amendment of the law to allow “mitochondrial DNA replace-
ment.” According to the review, the polar body transfer (PBT) techniques invented by
Chinese scientists to prevent mitochondrial diseases might offer five advantages over
maternal spindle transfer (MST) and pronuclear transfer (PNT) techniques because
they: may reduce mtDNA carryover; reduce the risk, when compared to MST, of
leaving chromosomes behind (as these are all packaged within the polar body); avoid
the need to use cytoskeletal inhibitors to allow removal of the spindle or pronuclei
from the patient’s oocyte or zygote; involve the use of more conventional micro-
manipulation procedures, which will reduce the chance of damaging the patient’s
30 G. Pei et al.

karyoplast or the donor’s oocyte/embryo and therefore lead to greater efficiency;

raise the possibility of carrying out both PB1T and MST, or PB2T and PNT, to
double the chances of success for each patient cycle.
In the past, China’s research and development of the mitochondrial replacement
techniques, an area of high biological technology with international competition, was
nearly blank. The invention of the polar body transfer for mitochondrial replacement
has made us a global leader in this research area and contributed to technological
innovation. This research has won the second prize of National Science and Tech-
nology Award of China and been selected as one of the Top 10 Scientific Advances
in China 2014.

3.2.3 The Seesaw Model of Lineage Specifiers Inducing

Somatic Cells to iPSC

The traditional induced pluripotent stem cell (iPSC) technology is to establish

pluripotency by introducing pluripotency-related genes into somatic cells. Our scien-
tists have found that lineage specifiers can substitute for pluripotency-associated
factors to induce somatic cells into iPSCs. Based on the new discovery, a “seesaw”
model has been proposed to explain the theory of cell fate determination in a new
prospective. Pluripotency is maintained as a consequence of the balance of different
lineage-specifying forces. There might be a universal principle to induce repro-
gramming by balancing such forces. For the first time, our scientists have reported
that all six members of the GATA transcription factor family, as lineage specifiers,
could substitute for OCT4, a Yamanaka factor. Additionally, they have found that all
members of the GATA family could inhibit the expression of the ectodermal-lineage
genes, which is consistent with the “seesaw” model. They then have determined that
the pluripotency-related gene Sall4 serves as a bridge linking the GATA transcription
factor to the pluripotency circuit (Shu et al. 2015). The dynamic “seesaw” model has
been found in the reprogramming of somatic cells by small-molecular compounds
into chemically-induced pluripotent stem cells (CiPSCs). Different from traditional
transcription factor-induced reprogramming, at the early stage of reprogramming,
small molecules start the XEN-related gene expression to tilt the “seesaw” and make
the cells into XEN-like states; at the later stage of reprogramming, the expression
of Sox2 is induced by switching to the mice ESC culture conditions to restore the
tilted “seesaw” to the balance position, so as to make cell transitions to pluripotency
(Zhao et al. 2015b). These results have indicated that the seesaw model is a universal
model to change cell fate (Fig. 3.2).
3 Major Research Achievements 31

Fig. 3.2 Seesaw model for induction of pluripotency in mouse somatic cells with lineage specifiers
(Shu et al. 2013)

3.2.4 Safety Evaluation of Traditional Methods of iPSC

Induction by Yamanaka Factors

Previous studies on somatic cell nuclear transfer (SCNT) have shown that somatic
cells could be reprogrammed and mice generated by SCNT can be successfully
re-cloned to more than 25 generations through serial nuclear transfer technology.
However, for the iPSC induction, another classic method of the somatic cell repro-
gramming, the number of generations of all-iPSC mice that can be serially produced
using this inducible iPSC system has not been determined. Additionally, since the
introduction of the iPSC technology, scientists have been making comprehensive
evaluations on the safety and feasibility of its clinical application, and assessing
whether there are gene mutations and whether it has an impact on the development
potential. This affects not only the cell quality but also the safety of future appli-
cations. For the first time in the world, our scientists have used a Tet-on system to
establish the OSKM iPSCs for up to six generations. They found that the viability
of the iPSC mice decreased with increasing generations, mainly because mutations
accumulated throughout the sequential reprogramming process. This finding has
further suggested that iPSC induced by traditional methods may have certain risks.
The results were published in Nature Communications in 2015 and cited by many
papers in Cell Stem Cell and other journals.
32 G. Pei et al.

3.2.5 Exploration of the Regulation Mechanisms of Somatic

Cell Reprogramming and Discovery of New Regulators

Despite the wide use of the method of Yamanaka factor-induced iPSC, we know little
about the dynamic binding models of Yamanaka factors in the reprogrammed cells
and the mechanisms of their bindings to regulate transcriptome changes and deter-
mine cell transitions. With the support of this MRP, our scientists have carried out
systematic research on the core pluripotency factor Oct4 with the secondary repro-
gramming system and high-throughput sequencing technologies. They have demon-
strated that at different stages of the somatic cell reprogramming, Oct4 binds itself
to the genome in a hierarchical fashion with primed epigenetic modifications; the
Oct4 binding plays an important role in the hierarchical activation of the pluripotency
circuitry during the reprogramming (Chen et al. 2016a). Additionally, pluripotency
factors, such as Oct4, have been found to bind themselves to ESC-specific promoters
and make ubiquitously expressed genes express stem-cell-specific transcripts (Feng
et al. 2016).
Our scientists have discovered that N(6)-methyladenosine (m6A) RNA methyla-
tion is regulated by microRNAs (miRNAs) and promotes reprogramming to pluripo-
tency. More than 100 types of modifications have been identified in eukaryotic
RNAs so far, among which m6A RNA methylation is one of the most prevalent
modifications of messenger RNAs (mRNAs) in higher-level organisms. The m6A
modification participates in the regulation of splicing, transportation, stability, and
translation efficiency of mRNAs, and it is implicated in obesity, cancer, and other
abnormal physiological functions and human diseases. Our scientists have reported
the m6A modification profiles in the mRNA transcriptomes of mouse ESCs, iPSCs,
neural stem cells (NSCs), and testicular sertoli cells (SCs), and identified the differ-
ence in m6A distribution between pluripotent and differentiated cell types. Bioin-
formatic analyses indicated that the m6A-enriched signature sequences preferred to
complementarily pair with microRNA sequences. Multi-level experiments of cell and
molecular biology demonstrated that microRNAs regulate m6A modification in the
corresponding sites in mRNAs via a sequence pairing mechanism. Increased m6A
modifications enhanced the expressions of Oct4 and other key pluripotent regulatory
genes, to promote the reprogramming of mouse fibroblasts to pluripotent stem cells
(Chen et al. 2015a). The results have revealed a new mechanism of microRNA regu-
lating mRNA methylation via sequence pairing and discovered the important role of
m6A modification, which promotes the reprogramming of somatic cells to pluripotent
stem cells. The study has made groundbreaking achievements in analyzing the site
selection mechanism for m6A modification, expanding new functions of microRNAs,
and discovering new regulators of the cell reprogramming. It has been published as a
cover article in Cell Stem Cell, and selected as the feature report of the issue, “Stem
Cell Highlights from Cell Press,” and one of Abcam Monthly Excellent Epigenetics
Papers.
Our scientists have discovered that E3 ligase RNF20-mediated H2B ubiquitination
regulates chromatin relaxation at the early stage of cell reprogramming, affecting the
3 Major Research Achievements 33

recruitment of gene transcription factors, leading to the initial expressions of pluripo-

tent genes at the early stage of reprogramming. After the depletion of RNF20, the
establishment of the pluripotency gene network at the early stage of cell reprogram-
ming failed. Additionally, in iPSC, RNF20 regulated the DNA-damage responses
during the replication through H2B ubiquitination. After the depletion of RNF20,
the DNA double-strand breaks in iPSC generated during the replication could not be
repaired by homologous recombination, resulting in unstable genomes and finally
apoptosis. A germ cell-specific knockout of RNF20 resulted in abnormal meiosis
and male infertility (Xu et al. 2016).

3.2.6 Discovery of New Signals for Maintaining Stem Cell

Self-renewal and Developmental Pluripotency

Human embryonic stem cells (hESCs) can self-renew and differentiate into all cell
types in vitro and thus have a broad application prospect in organ regeneration and
cell replacement therapy. However, the molecular mechanisms of maintaining hESC
self-renewal and developmental pluripotency remain poorly understood, preventing
safe and effective clinical applications of hESC-differentiated cells. As a result, it is
particularly important to delve into the mechanisms how hESCs maintain their own
features.
Through a genome-wide transcription factor siRNA screen of transcription factors
for hESC self-renewal, our scientists have identified a series of genes that play an
important role in the maintenance of hESC identity. PHB is found to have a unique
role in maintaining the right histone methylation modifications in hESCs, which helps
the hESC self-renewal and the reprogramming of human somatic cells. Further anal-
yses indicated that PHB interacted with histone H3.3 chaperone HIRA complexes
and stabilized the protein levels of HIRA complex components. Additionally, in
hESCs, PHB and HIRA jointly regulated the genome-wide H3.3 enrichment at chro-
matin, especially the enrichment of H3.3 involved in regulation at the promoters
of isocitrate dehydrogenase (IDH) genes and their expressions, thus to control the
production of a-ketoglutarate (a-KG), a key metabolite regulating ESC fate, which
led to the establishment of correct level of histone methylation and the maintenance
of hESC self-renewal and their epigenetic features. Based on the findings, our scien-
tists have proposed an epigenetic-metabolic circuit for the maintenance of hESC
identity (Zhu et al., 2017).
Histone demethylases play a key role in the establishment and maintenance
of developmental pluripotency, but not all of them have been identified, and
their functions during the processes remain unclear. Our scientists have revealed
that Jmjd1c/Kdm3c, H3K9 demethylases, can inhibit the activation of MAPK/Erk
signaling pathway and EMT by regulating the expression of miR-200 and miR-
290/295 family, and then promote ESC self-renewal and the maintenance of devel-
opmental pluripotency. During the somatic cell reprogramming, interference or
34 G. Pei et al.

knockout of Jmjd1c expression significantly reduced the reprogramming efficiency,

while overexpression of miR-200 family could partially recover the reprogram-
ming efficiency of Jmjd1c-null MEF. This has provided a new perspective for the
establishment and maintenance of developmental pluripotency, and laid a theoretical
foundation for the application of pluripotent cells in cell therapy and regenerative
medicine.
BMP signaling, through downstream Smad proteins, upregulates the expression
of phosphatase DUSP9 at the transcriptional level to make ERK protein dephospho-
rylated and deactivated, to sustain low ERK activity, which helps cells to maintain
the state of self-renewal and nondifferentiation. Therefore, under the joint efforts of
BMP and LIF, ERK activity is maintained at an appropriate level to keep mouse ESCs
in self-renewal and undifferentiated state. The results have an important influence on
understanding the molecular mechanism of cell fate determination in mouse ESCs
and lay a foundation for further research in regenerative medicine (Li et al. 2012b).
After the publication, the findings were introduced in Nature Chemical Biology and
commented on F1000.

3.3 Epigenetic Mechanisms Related to Cell Differentiation

and Transdifferentiation, Ontogeny, and Diseases

3.3.1 Discovery of the Key Factors to Promote

the Transdifferentiation of Somatic Cells to Hepatocytes

Our scientists have demonstrated that with a lentiviral system, adult mouse tail-tip
fibroblasts were successfully induced into functional hepatocyte-like (iHep) ells by
overexpression of transcription factors FOXA3, HNFLA, and GATA4, and inactiva-
tion of p19. They discovered that the activation of p53 was the key inhibitory mech-
anism of dedifferentiation reprogramming (Fig. 3.3). iHep cells could be integrated
into the mouse liver and acquire hepatocyte functions in vivo. Additionally, tumors
were not found in Fah − / − mice or nonobese diabetic/ severe combined immunod-
eficient (NOD/SCID) mice with transplanted iHep cells, which has further suggested
the safety of iHep cells (Huang et al. 2011). With the transcription factors FOXA3,
HNF1A, and HNF4A as well as SV40 Large T, human embryonic fibroblasts have
been converted to expandable human hepatocyte-like cells (hiHeps). hiHeps have
the gene expression profile similar to human primary human hepatocytes and display
functions characteristic of hepatocytes in vitro, especially a good biliary excretion
capability, which could be used for the assessment of biliary drug clearance in drug
discovery. The work has been reported in Cell Stem Cell and SciBX, which believe
that hiHeps overcome the hurdle of the proliferation arrest of differentiated cells,
and that the functional hepatocytes obtained via transdifferentiation have made an
exciting step toward the hepatocytes needed for drug research and treatment. It has
also been recommended by F1000 with the comment that using the method in the
3 Major Research Achievements 35

100
HFF1(n=17)
HepG2(n=10)
BAL

Surval rate of acute liver

80
PHH(n=4)
clinical

failure mice (%)

hiHepLT(n=14)
60 ALF mice
40 study
treatment
20
0
initiated
0 20 40 60 80 100
hours post ConA treatment

Lentiviral
infection
block’s medium Empty-BAL treated hiHep-BAL treated
art
p19 -null TTFs iHep cells
Day 0 Day 2 Day 14–21

iHep cells

ALB AAT Merge

HFF1

ALF pig treatment by bio-

hiHep

hiHep cells

Fig. 3.3 iHep generation and application (Huang et al. 2011; Shi et al. 2016)

research, functional hepatocytes can be generated on a large scale, a very important

progress in the whole field. It has been selected as one of “Cell Press Papers of the
Year 2014 China.”
The further research has produced hiHeps at clinical scales and developed a bioar-
tificial liver device implanted with induced human functional hepatocytes to success-
fully treat miniature pigs with acute liver failure (ALF) (Shi et al. 2016). The survival
rate of the treated pigs was significantly higher than that of the control group, and
all the serum biochemical parameters and inflammatory indexes returned to normal
levels, which has provided a solid foundation for the clinical application of hiHeps. In
January 2016, through cooperation with Nanjing Drum Tower Hospital, our scientists
successfully treated the first case of acute liver failure and saved a patient suffering
from hepatitis B for 40 years who had a sudden acute liver failure. After treatment with
a hiHep bioartificial liver, all the liver function indexes were significantly improved
without any adverse effects. This case marks the successful completion of the first
clinical treatment with the novel hiHep bioartificial liver. Promoting the clinical
application of the hiHep bioartificial liver will greatly advance the transformation of
scientific research achievements in China.
36 G. Pei et al.

3.3.2 Discovery of the Epigenetic Mechanisms and Cell

Signaling Pathways Inducing the Differentiation
and Transdifferentiation from Embryonic Stem Cells
or Somatic Cells to Neurons

Histone modification is closely related to gene transcription regulation and plays an

important role in neurogenesis. At the epiblast stage, inhibition of histone deacety-
lase activity can significantly suppress the neural differentiation of embryonic stem
cells and promote mesendoderm differentiation, suggesting an important role of
histone deacetylation in the neurodevelopment. The study has discovered that histone
deacetylase 1 (HDAC1) specifically bound at the Nodal gene, and histone deacety-
lase inhibitors could significantly upregulate the expression of Nodal. The activation
of Nodal signals promoted mesendoderm fate and inhibited neural fate commitment,
which has indicated that HDAC1 ensures neural fate commitment by repressing
Nodal signaling at the epiblast stage (Liu et al. 2015). The acetylation of H3K9 grad-
ually declined during day 0–4 of the neural differentiation of embryonic stem cells
in vitro, and increased during day 4–8. Correspondingly, during the neural differen-
tiation, the enrichment of H3K9 acetylation decreased in the pluripotency-related
genomic locus while increased in the neurodevelopment-related locus. Histone
deacetylase inhibitors can inhibit the activity of HDAC3 to promote the expres-
sion of pluripotency genes and maintain the pluripotency of human embryonic
stem cells. During the neural differentiation, histone deacetylase inhibitor relieves
the inhibitory activities of HDAC1/5/8 and thereby promotes early neurodevelop-
mental gene expression (Qiao et al. 2015a). Histone methylation is also involved
in the regulation of neural differentiation. The study has identified KIAA1718
(KDM7A) as a dual-specificity histone demethylase for H3K9 and H3K27. The
transcription level of Fgf4 gene is positively regulated through the demethylation
of H3K9me2 and H3K27me2, to mediate the neural differentiation of embryonic
stem cells. The enzyme has been demonstrated to be related to the neurodevelop-
ment in chick embryos (Huang et al. 2010). Additionally, DNA demethylation is
found to play an important role in neurogenesis. The study has indicated that AF9
is necessary and sufficient for hESC neural differentiation and neurodevelopmental
gene activation. DNA dioxygenase TET2 could interact with AF9, and they co-
localized in 5-hydroxymethylcytosine (5hmC)-positive neurons. Upon recognizing
AAC-containing motifs and binding at target gene promoters, AF9 recruited TET2 to
occupy their common downstream neurodevelopmental gene loci to direct 5mC-to-
5hmC conversion, which was followed by sequential activation of the expression of
these neurodevelopmental genes and hESC neural commitment (Qiao et al. 2015b).
The treatment of nervous system disorders with neural stem cells is a research
hotspot in the field of regenerative medicine. Inducing the transformation of somatic
cells into neural stem cells indicates that somatic cells can also be directly converted
to neural stem cell-like somatic stem cells without first going through a pluripotent
state, which provides a rich source of cells for cell therapy. Our scientists have
successfully used specific factors (Pax6, Ngn2, Hes1, Id1, Ascl1, Brn2, c-Myc, and
3 Major Research Achievements 37

Klf4) to induce the transdifferentiation of sertoli cells to neural stem cells. These
induced neural stem cells (iNSCs) expressed normal neural stem cell markers, and
the expression of the whole genome was also highly similar to that of normal neural
stem cells. Functionally, iNSCs could maintain self-renewal and differentiate into
neurons with various electrophysiological functions, such as dopaminergic neurons,
γ-Aminobutyric acid neurons, and acetylcholinergic neurons. Importantly, the iNSCs
could survive normally and establish synaptic connections with surrounding neurons,
after they were introduced into the dentate gyrus of hippocampus in the adult brain
of mice. The study has indicated that iNSCs could become a cell resource for clinical
treatment of neurodegenerative diseases and new drug screening (Sheng et al. 2012).
The paper has been selected as one of “The Top 100 Most Cited Chinese Papers
Published in International Journals 2012,” ranking 13th.
It has been found that overexpression of the catalytic domain of demethylase
TET2 in neural progenitor cells (NPCs) could activate the astrogliogenic program
and simultaneously inhibit the neurogenic program. The transcription factor OLIGO2
could directly bind itself to the promoter of TET2 gene to regulate its expression.
During the early stages of embryonic neural development, the transcription factor
Ngn1 could bind itself to the promoter of a brain-enriched microRNA, miR-9, to
suppress the activation of the Jak-Stat pathway, to inhibit astrogliogenesis (Zhao
et al. 2015a).

3.3.3 Discovery of Important Disease-related Epigenetic

Modifications

Rett syndrome (RTT) is caused by mutations in the X-linked Mecp2 gene. The
mouse models are quite different from clinical patients, so it is difficult to use them to
promote the research of pathogenesis and the development of new treatment methods.
Nonhuman primates, such as rhesus and cynomolgus macaques, are ideal experi-
mental animals to study brain development and neurological disorders because they
share many similarities of genetic backgrounds and brain structures with humans.
In early 2014, our scientists, for the first time in the world, reported successful
TALEN-mediated mutagenesis of Mecp2 gene in monkey models. The results have
been published in Cell Stem Cell (Liu et al. 2014). The further study has shown
that unlike rodent models, Mecp2 mutant monkeys have a series of pathological and
behavioral features resembling clinical manifestations of RTT patients. Given their
incomparable advantages over rodents, they would have a profound impact on the
pathogenetic studies as well as development of therapeutic interventions for RTT in
future (Chen et al. 2017).
Our scientists have found that a lymphocyte lineage-restricted transcription factor,
Aiolos, alters the higher-order chromatin structure of the p66Shc gene to disrupt
enhancer–promoter interactions and silence p66Shc transcription, and therefore
promotes cancer cells to bypass anoikis and performs distant metastasis. They have
38 G. Pei et al.

presented a new idea that carcinoma cells “co-opt” a lymphocyte lineage-restricted

transcription factor to mimic the lymphocyte properties of low adhesion and anoikis
resistance to realize distant metastasis. They have also demonstrated the important
role of higher-order chromatin structure as a type of epigenetic regulation in tumori-
genesis (Li et al. 2014b). After its publication, it has been selected as a featured
article. In the same issue, Steven Frisch, a pioneer in the anoikis research, wrote a
preview on the paper and commented that it “provides provocative evidence that lung
cancer cells utilize a hematopoietic transcription factor, Aiolos, to drive both anoikis
resistance and anchorage independence.” The article of “Update in Lung Cancer in
2014” in American Journal of Respiratory and Critical Care Medicine, published by
Professor Powell of ISMMS in the USA, said that “the discovery of the mechanism
of tumor cells using hematopoietic cell signaling opens up a new promising direction
for the study on lung cancer pathogenesis.” Their research has obtained two Chinese
invention patents, one of which was transferred to Beijing Biocytogen Co., Ltd. in
2015 for achievement transformation.
Within the DNA methyltransferase (DNMT) family, DNMT3A and DNMT3B are
responsible for the de novo methylation. While DNMT3A is frequently mutated in
hematological malignancies, DNMT3B is rarely mutated. Our scientists have studied
its role in the acute myeloid leukemia (AML) in an MLL-AF9-induced AML mouse
model. The deletion of DNMT3B accelerated MLL-AF9 leukemia progression by
increasing the percentage of leukemia stem cells (LSCs) and enhancing leukemic cell
cycle progression. Depletion of DNMT3B and DNMT3A synergistically promoted
leukemia development. The study has provided new insights into the roles of DNA
methyltransferases in leukemia development (Zhang et al. 2016).
Enhancers are important regulatory elements controlling gene expression. Our
scientists have found that the enhancers jointly marked by H3K4me3 and H3K27Ac
might be overactive, and the complex containing RACK7 and KDM5C, two potential
tumor suppressors, is a negative regulator of the overactivation. RACK7, a potential
chromatin reader, and KDM5C, a histone demethylase, together act as a “brake” of
active enhancers by controlling the dynamics between H3K4me1 versus H3K4me3
at active enhancers. Loss of such an enhancer surveillance mechanism can lead to
altered cell behaviors, which may contribute to tumorigenesis (Shen et al. 2016).
With the TAB-Seq sequencing method, our scientists have explored the repro-
gramming models and rules of 5mC and 5hmC in ccRCC at single-nucleotide reso-
lution, and found that loss of 5hmC but not 5mC was a sensitive prognostic marker for
kidney cancer. The nude mice experiment demonstrated that restoring 5hmC levels
suppressed tumor growth, indicating that the 5hmC was more likely to be a driving
force for cell reprogramming in kidney cancer than a concomitant phenomenon.
This suggests that 5hmC can function as a novel epigenetic marker for prognostic
monitoring of renal cell carcinoma and a therapeutic target (Chen et al. 2016b).
It has been found that stabilization of histone demethylase PHF8 mediated by the
deubiquitinase USP7 confers cellular resistance to DNA damage and promotes breast
carcinogenesis (Wang et al. 2016). The results have been published in the Journal
of Clinical Investigation and selected in “Editor’s Picks” of the issue. Additionally,
3 Major Research Achievements 39

mutations in PHF8 have been found to play an important role in the diagnosis of
X-linked mental retardation and other diseases (Yu et al. 2010).
Previous studies have found that the transcriptional mediator orchestrates the basal
transcription machinery with RNA polymerase II for precise transcriptional control.
The mediator complex is known to regulate transcription initiation and elongation, but
its roles in histone modifying enzymes and protein post-translational modification
have not been reported. The study has revealed that the mediator subunit Med23
specifically regulates H2B mono-ubiquitination (H2Bub) to control the transcription
of specific genes, which plays an important role during the cell fate determination.
It has demonstrated a new mechanism of transcription elongation regulation and
the new function in cell fate determination. The results have been published in a
well-known international journal as the cover article, and highly praised by peers.
The tumor suppressor FoxO1 is a key protein to induce autophagy. FoxO1 in
the cytoplasm binds itself to the histone deacetylase SIRT2 to stay inactivated. In
response to stress, FoxO1 is acetylated by dissociation from SIRT2 and becomes
activated. The activated FoxO1 specifically binds itself to ATG7, a key autophagy
protein, to induce the autophagy process. Both animal experiments and the study on
the clinical tumor specimens have indicated that cytosolic FoxO1-induced autophagy
may be a critical factor in the anti-neoplastic effect of FoxO1. This finding has linked
the histone deacetylase with epigenetic modifications to autophagy and anticancer
activity. It has been published in Nature Cell Biology (Zhao et al. 2010).
Telomere regulation and the mitochondrial function regulation related to cell
metabolism were long considered as two independent pathways for cell aging. TIN2
is recruited to telomeres and associates with multiple telomere regulators including
TPP1. TPP1 interacts with TIN2N-terminus and controls TIN2 localization. Our
scientists have discovered that the telomeric protein TIN2, besides its important
roles in the telomere functions, is post-translationally processed in mitochondria and
regulates mitochondrial oxidative phosphorylation. Reducing TIN2 expression by
RNAi knockdown inhibited glycolysis and reactive oxygen species (ROS) production
and enhanced ATP levels and oxygen consumption in cells. TIN2 could locate in the
telomeres and the mitochondria, as regulated by TPP1. These results have suggested
a direct link between telomeric proteins and metabolic control, providing another
important mechanism by which telomeric proteins regulate cancer and aging. The
study has been published in Molecular Cell as the cover article, titled “Mitochondrial
Localization of Telomeric Protein TIN2 Links Telomere Regulation to Metabolic
Control.”
Craniofacial bones are derived from the development and differentiation of cranial
neural crest cells, involving the processes of their fate determination, migration,
and differentiation. Abnormalities in any link could result in craniofacial deformity.
Several BMP ligands are expressed in the pharyngeal pouches originated from the
endoderm in the pharyngeal region, and their antagonist Noggin3 is expressed in the
chondrogenic progenitors next to the pouch. The study has found that miR-92a is
expressed in the Zebrafish pharyngeal region and acts to maintain the signaling of
BMP, a protein involved in the pharyngeal cartilage formation, to ensure its normal
development. This work has not only elucidated the important role of miR-92a in the
40 G. Pei et al.

cartilage development, but also further indicated that BMP signals must be strictly
regulated during pharyngeal cartilage formation, and its activity higher or lower
than the physiological level would lead to serious developmental defects (Ning et al.
2013). The discovery has been recommended by F1000 which believes that it has
found a new molecular mechanism leading to craniofacial deformity that is worthy
of writing in textbooks.

3.4 Establishment of Epigenetic Maps to Reveal

the Characteristics and Rules of Epigenetic
Modifications During the Embryonic Development

The epigenetic maps have been established with high-throughput data collection
to reveal the characteristics of epigenetic modifications during the early embry-
onic development of different species and the rules of genetic evolution, which has
enriched our understanding of the origins and evolution of epigenetic networks.

3.4.1 Discovery of Distribution and Change Rules

of Genome-Wide Histone Modifications
in Pre-implantation Embryos

Epigenetic remodeling is critical in the reprogramming of highly specialized gametes

during fertilization and embryo development. The changes in these epigenetic modi-
fications are key points to the activation of the embryonic genome and the first
cell lineage differentiation. The post-transcriptional modification of histone directly
regulates activation or silencing of gene expression. In early studies with immunoflu-
orescent staining methods, most histone modifications underwent obvious changes
during the pre-implantation embryo development, and abnormal expression or dele-
tion of some enzymes regulating histone modification led to abnormal embryonic
development or even death of pre-implantation embryos. These studies have indicated
that the changes in the epigenetic modifications have critical roles during the early
embryonic development. However, it remains poorly understood how the histone
modifications in pre-implantation embryos distribute and change in the genome and
how they regulate the expression of embryonic genes and the first differentiation of
the cell fate. By using the latest ultra-low- input micrococcal nuclease-based native
chromatin immunoprecipitation (ULI- NChIP) method, the study has detected the
changes of histone H3K4me3 and H3K27me3, which are associated with gene activa-
tion and repression respectively, during the stages of mouse pre-implantation embryo
development. This has been the first systematic genome-wide map of histone modi-
fications in mouse pre-implantation embryos. It has revealed the establishment of
3 Major Research Achievements 41

H3K4me3 and HK27me3 modifications during the pre-implantation embryo devel-

opment and found that the broad H3K4me3 domain plays an important role in gene
expression regulation during the pre-implantation development (Liu et al. 2016).
It is worth mentioning that another study funded by this MRP has been published
in the same issue of Nature. This “back-to-back” paper has reported a noncanon-
ical form of H3K4me3 in the untranscripted oocytes, one-cell and early two-cell
embryos before the genome activation, which exists as broad peaks in nonpromoter
regions, including the intergenic regions. Through downregulation of H3K4me3 in
oocytes by overexpression of the demethylase KDM5B, the researchers have unex-
pectedly found that noncanonical H3K4me3 is necessary for the genome silencing
(but not activation) during oogenesis (Zhang et al. 2016). The comment released
in the same issue of Nature, titled “Developmental Biology: Panoramic Views of
the Early Epigenome,” has pointed out that these studies detail changes in histone
modifications after the fertilization and during the early embryonic development,
the chromatin openness on the regulation of gene expression, and how epigenetic
information is inherited from parents to progeny. These findings are of great signif-
icance for the research of abnormal embryonic development and the improvement
of the success rate of assisted reproductive technology, which will benefit patients
with recurrent abortion, embryo damage, and infertility. They symbolize that China
has achieved research results with world influence in related fields. The study has
been selected as one of China’s top 10 research advances in life sciences in 2016 by
China Union of Life Science Societies.

3.4.2 Establishment of Single-Cell Transcriptome

Sequencing and Analysis Tools

The single-cell RNA-sequencing (SCRS) is a powerful technique to analyze genomic

expression in a single-cell or micro RNAs. Compared with the microarray tech-
nology, SCRS can detect more transcriptomes in a more sensitive manner. It can
analyze multiple transcripts of the same gene and their corresponding protein types
and also detect new splice sites in known genes. It features high accuracy and low
noise. SCRS helps to accurately display the detailed changes during cell program-
ming and reprogramming differentiation. In recent years, researchers have begun to
use this technique to overcome the bottleneck of small initial sample size. Through
the self-developed single-cell transcriptome analysis technique and weighted gene
co-expression network analysis (WGCNA), our scientists have uncovered molec-
ular properties of CD133+/GFAP-ependymal (E) cells in the adult mouse forebrain
neurogenic zone. Prominent hub genes of the gene network unique to ependymal
CD133+/GFAP-quiescent cells were enriched for immune-responsive genes, as well
as genes encoding receptors for angiogenic factors. Administration of vascular
endothelial growth factor (VEGF) activated CD133+ ependymal neural stem cells
(NSCs), lining both the lateral and the fourth ventricles and, together with basic
42 G. Pei et al.

fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation

and migration. This study has revealed the existence of dormant ependymal NSCs
throughout the ventricular surface of the central nervous system (CNS), as well as
the abundant signals after CNS injury for their activation (Luo et al. 2015). With
both patch-clamp recording and the single-neuron transcriptome analyses, another
study has identified the molecular markers of neuronal maturation and their rela-
tions with energy metabolism (Chen et al. 2016c). The hepatic stem cell lineage
and the activation mechanisms have been revealed with the single-cell transcriptome
analysis.

3.4.3 Establishment of Genome-Wide Methylation Profiles

to Reveal the Inheritance and Evolution Rules
of Epigenetic Modifications

With the support of this MRP, our scientists, using MethylC-Seq, have surveyed
the silkworm Bombyx mori that have a low level of methylation and established the
single-base resolution methylome of its silk gland (Xiang et al. 2010). The study has
generated the first insect epigenome, confirmed the existence of epigenetic mecha-
nism in insects and its important functional significance, clarified the long-standing
fuzzy understanding of insect epigenetic system, and inspired researchers to rethink
the research of insect DNA methylation and its functions. Since its publication, it has
been cited for over 170 times. It has been reported in Nature China as a highlight, and
in Nature Asia–Pacific as a research highlight titled “Threading Together a Map of
Silkworm DNA Modifications.” It has also received a review in Nature China titled
“Epigenetics, Few and Far Between”, writing that “this work shows that the evolution
of insects can be understood from the perspective of epigenetics, and the research
results provide valuable data for exploring the effect of epigenetics on silkworm
domestication.” Since the publication, the study has been cited in three English
monographs—Epigenetic Genetic Regulation and Epigenomics, Insect Molecular
Biology and Biochemistry, and Honeybee Neurobiology and Behavior: A Tribute to
Randolf Menzel.
DNA methylation, as important epigenetic mechanisms regulating gene expres-
sion, influences a series of biological processes, such as cell fate determina-
tion, development, and homeostasis maintenance of tissues and organs. Its modi-
fications, including 5-methylcytosine (5mC), N6-methyladenine (6 mA), and N4-
methylcytosine (4mC), have been found in genomic DNA from bacteria and eukary-
otes. 5mC and its derivatives during demethylation, 5hmC, are thought to be the
type of methylated base in mammalian genomic DNA. Unlike 5mC, 6 mA is present
in prokaryotes and some lower eukaryotes, especially bacteria, of high abundance.
It plays important roles in controlling a number of biological functions, such as
DNA replication and repair, gene expression, and host–pathogen interactions. Our
scientists have shown that 6 mA is present in Drosophila genome (Zhang et al.
3 Major Research Achievements 43

2015), and that the 6 mA modification is precisely regulated by the Drosophila

TET homolog, DNA 6 mA demethylase (DMAD), during early embryogenesis. The
study has uncovered a new DNA modification in eukaryotes, making it an original
breakthrough in epigenetic research.
Using MethylC-Seq, our scientists have measured genome-wide DNA methy-
lomes at single-base resolution in zebrafish gametes and early embryos (Fig. 3.4)
and studied the rule of their inheritance from parents to offspring (Jiang et al. 2013).
The study has found that paternal DNA stably maintains the sperm methylome; for
maternal DNA, the oocyte methylome is gradually discarded and reprogrammed to
a pattern similar to that of the sperm methylome; the sperm methylome facilitates
the epigenetic regulation of embryogenesis. This discovery has challenged the tradi-
tional view that “the majority of information for the early embryonic development
regulation is stored in egg, and sperm carries just one set of DNA.” This study on the
zebrafish methylation inheritance has illustrated that besides DNA that can be inher-
ited, the epigenetic information (DNA methylome) can also be completely inher-
ited into the offspring. Upon its publication, Cell released a preview titled “Beyond
DNA: Programming and Inheritance of Parental Methylomes.” The inheritance of
the epigenetic information into the offspring means that the variation of epigenetic
information may play an important role in regulating animal development, pheno-
types, and even diseases, just as that of genetic information does. After the study was
published, 1/4 of its citations come from the field of evolution, and it has triggered
the new reflection whether epigenetic information plays a driving role in evolu-
tion. Meanwhile, the study on the DNA methylation reprogramming in mammals
has reported that the oxidation products of the methylation exist in both maternal

Erasing the whole Setting sperm-oocyte-

Protection of genomic imprinting methylome in PGCs

Active depletion of 5mC de novo methylation Paternal

Maternal
Paternal Maternal

5hmC+5fC

2-cell 4-cell blastocyst/ICM E6.5 E7.5 E13.5

PGCs

Fig. 3.4 Active demethylation of maternal and paternal genomes in mammals (Wang et al. 2014a)
44 G. Pei et al.

and paternal genomes during their early development, so both DNA methylomes go
through active demethylation. This discovery has changed the long-standing under-
standing that the maternal genome is demethylated through passive dilution during
the early embryonic development in mammals.
Chapter 4
Outlook

Gang Pei, Yongfeng Shang, Fanglin Sun, Zuoyan Zhu, Runsheng Chen,
Xiaofeng Cao, and Zhen Xi

Epigenetics has emerged since the late 1980s. In the twenty-first century, with
advances in technological means, this new discipline is developing in an unprece-
dentedly rapid speed. Increasingly more biophysicists, developmental biologists,
chemists, bioinformaticians, and geneticists are participating in the exploration of
epigenetics. Focusing on the international frontier and development trends of this
discipline, they promptly apply the latest research ideas and technical means of
related disciplines to epigenetic research and have greatly improved our compre-
hensive understanding of the epigenetic system. With the promotion of this MRP,
Chinese scholars have made great progress in the areas of molecular basis of epige-
netics and theoretical innovation of cell reprogramming and contributed significantly
to the comprehensive leapfrog development of the epigenetic research in China.

G. Pei (B)
Tongji University, Shanghai, 200000, China
e-mail: [email protected]
Y. Shang
Hangzhou Normal University, Hangzhou, Zhejiang, China
F. Sun
Tongji University, Shanghai, China
Z. Zhu
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China
R. Chen
Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
X. Cao
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
Z. Xi
Nankai University, Tianjin, China

© Zhejiang University Press 2023 45

G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming,
Reports of China’s Basic Research,
https://doi.org/10.1007/978-981-19-7419-9_4
46 G. Pei et al.

At present when the cell reprogramming research sees rapid development, there
are major breakthroughs in both principle and technology in many directions, with
emerging hotspots and fast changes. In addition to the Yamanaka factors OSKM,
scientists have found more pluripotency reprogramming factors, such as NANOG,
PRDM14, SALL4, ESRRB, UTFL1, TET2, and GLIS1. To prevent the exogenous
genes from integrating into the genome of target cells and improve the safety of
the system, our scientists have achieved considerable progress in somatic cell repro-
gramming and cell transdifferentiation with non-viral vectors, RNAs, transmem-
brane proteins, and small chemical molecules. Meanwhile, the epigenetic research
in cell reprogramming witnesses rapid development. The successful primate somatic
cell nuclear transfer depends on the use of epigenetic factors. However, current
research focuses on the discovery and functional verification of key factors, and
the understanding on the chromosome dynamics in reprogrammed cells, changes
in cell epigenomes and the regulated transcriptomes, and mechanisms for deter-
mining cell fate transitions remains elusive. In this context, our scientists should
seize the opportunities, continue to give full play to the interdisciplinary advantages,
and apply the technologies, strategies, and experience accumulated in the research
of biomacromolecular structures, single-cell omics, and gene editing to the research
of cell reprogramming and epigenetics. Centering on the frontier questions in life
sciences, we should develop new technologies and methods and promote the focuses
in the epigenetic research to progress from a single molecule to multiple molecules,
from a single type to multi-type modifications, from qualitative to quantitative, from
simple to complex systems. We should improve the efficiency and quality of the
cell reprogramming processes, make qualitative leaps in the theory development and
technological innovation in life sciences, open up new directions and bring a bright
future for the treatment of human diseases.

4.1 Directions to Be Strengthened in China’s Epigenetic

Research

We need to strengthen the following weak research directions in epigenetics in China.

(1) We should enhance the research on epigenetic regulation of cell differentia-
tion, tissue homeostasis, and mechanisms of organ development and regenera-
tion; and focus on elucidating the molecular mechanisms that epigenetic factors
integrate changes in intracellular and extracellular environment and signals to
regulate cell fate.
(2) We should boost the research on the pathogenic mechanisms of epigenetic regu-
lation in relevant major diseases, establish disease-related epigenome maps, and
enhance interdisciplinary cooperation with clinical medicine.
(3) We should utilize the advantages of species diversity in China to develop new
model organisms. We should study the mechanisms of epigenetic factors on
learning, memory, social animal behaviors, and intergenerational inheritance of
4 Outlook 47

acquired traits, discover new epigenetic phenomena, and explore the regulation
mechanisms of epigenetic factors on the genome stability and biological traits
from the perspective of species evolution.
(4) We should strengthen the research on the composition and dynamic changes
of higher-order chromatin structures in nucleus, advance further development
of the single cell technologies, promote the interdisciplinary cooperation with
physics, materials chemistry, and computational biology, and elucidate the
general principles of the assembly of higher-order chromatin structures and
the regulation mechanisms of their dynamic changes.

4.2 Strategic Needs of the Epigenetic Research in China

Epigenetic regulation plays a decisive role in the development of complex conditions

such as cancer, diabetes, mental illness, nervous system diseases, and reproductive
system diseases. The orderly responses of individual life to environmental factors
(including those of nutrition, physical chemistry, and psychology) largely depend on
the effective operation of epigenetic regulation networks. Epigenetic regulation also
plays an important role in plant development, resistance, and formation of heterosis.
The basic biology research in epigenetics helps to better understand the pathogenesis
of relevant complex diseases, means and intensity of individual responses to envi-
ronmental changes, and regulation mechanisms of plant development and breeding,
and lays a foundation for us to develop new treatment methods and original drugs
for specific diseases, improve individual survival status, and enhance the quality of
agricultural production and food security.
Through the implementation of this MRP, China has cultivated and established
an internationally leading research team covering all major directions of epigenetic
research, whose various achievements prove their strength in the field of epigenetic
research. In future, it is necessary for China to continue supporting these researchers
and keep the epigenetics team stable. This is not only crucial for China to maintain
its international status in this field, but also to support the development of national
economy and people’s livelihood.

4.3 Conceptions and Suggestions for Further Research

Based on the achievements and summaries of this MRP, the research group puts
forward the following research conceptions.
The convergence between chromatin biology and physics, materials chemistry,
and computational biology needs to be facilitated to make breakthroughs in method-
ology, so as to realize in situ real-time measurement and tracking of chromatin
morphology and higher-order chromatin structures at the single cell level, and carry
out the epigenome research at the single cell level. This is of significance to the studies
48 G. Pei et al.

on the establishment of epigenetic information, cell fate regulation, and responses to

environmental changes.
The convergence between epigenetics and chemistry, pharmacy, and computa-
tional biology needs to be deepened to screen, identify, and synthesize a series of
small molecule drugs targeting epigenetic regulators, and promote the clinical treat-
ment of specific diseases. These small molecule drugs are important for both basic
research and clinical application.
The convergence between epigenetics and mathematics and computational
biology needs to be deepened to perform systematic analyses and simulations of
epigenomes and epigenetic regulation networks, and, combined with the interactions
with other regulation networks in cells, to elucidate the significance of epigenetic
regulation on individual development and responses to environmental changes from
the perspective of system biology.
The convergence between epigenetics and neuroscience, physiology, and bioin-
formatics needs to be deepened to discover new epigenetic phenomena, explore new
epigenetic regulation mechanisms, and expand the current research field.
On the basis of the important research directions supported by this MRP, the
research group advises to enhance attention to and support for the following directions
to continuously increase China’s international position in the epigenetic research.
(1) Research on cellular higher-order chromatin structures, especially the analysis
of their composition and dynamic changes in the cells of distinct differentia-
tion states and different types. China’s overall research level of higher-order
chromatin structures, especially the higher-order structure of 30-nm chromatin
consisting of nucleosomes, has been in the forefront of the world. The 30-nm
chromatin structure has been reported to be a twist of repeating tetranucleosomal
structural units. The gap between such units provides a window for epigenetic
regulation, which makes it possible to study epigenetic regulation mechanisms
and explain the basic epigenetic questions. Strengthening the research in this
direction can maintain our advantages in this field.
(2) Expansion of studies on the mechanisms and functions of newly discovered
epigenetic modifications. Our scientists have made major progress in discov-
ering new epigenetic regulators, for example, the RNA m6A modification. The
next step is to strengthen the investment in this direction. Meanwhile, we should
promote the studies on the mechanisms and functions of newly discovered epige-
netic modifications, possible interactions between DNA and RNA epigenetic
modifications, and the epigenetic regulation mechanisms of cell differentia-
tion, tissue homeostasis, organ development and regeneration. We should also
develop new model organisms and explore the regulation mechanisms of epige-
netic factors on the genome stability and biological traits from the perspective
of species evolution.
(3) Development of new technologies, theories, and methods. It is suggested to
develop and optimize epigenome editing technologies and single-cell epigenetic
information measurement technology of epigenetic transcriptomes, expand the
4 Outlook 49

bioinformatic algorithms of epigenetic networks, and study the network inter-

actions between epigenomics and transcriptomics at the single-cell level. We
should strengthen the research on the pathogenic mechanisms of epigenetic
regulation in relevant major diseases and enhance interdisciplinary cooperation
with clinical medicine.
(4) Research on the dynamic influences of OSKM and other pluripotency factors on
cell epigenomics and transcriptomics at the single-cell level during the human
cell reprogramming. We should improve the efficiency of cell reprogramming,
the quality of reprogrammed cells, and the stability and homogeneity of repro-
gramming system, to lay the foundation for regenerative medicine based on
cell reprogramming and the clinical application of iPSC and transdifferentiated
cells.
(5) Research on the epigenetic responses and memory of environment signals to
explore the mechanisms. The epigenetic systems in organisms can make cells
with the same genome present different epigenomes and transcriptomes in
different environmental conditions, to differentiate into different morphology
with divergent functions. It has been found that histone modifications are inher-
itable and subject to the feedback regulation by epigenetic modifying enzymes,
which indicates the existence of epigenetic response and memory systems in
organisms. However, we do not know much about them.
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Index

C G
Cell programming and reprogramming, 14 Gene expression, 38
Cell signaling pathways, 35

M
Modifications modifying enzymes, 13
D
DNA methylation, 43 R
Reader proteins, 13

E Y
Epigenetic modifications, 40 Yamanaka factors, 31

© Zhejiang University Press 2023 55

G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming,
Reports of China’s Basic Research,
https://doi.org/10.1007/978-981-19-7419-9