DNA

methylation
BS-seq, RRBS-seq, methylC-seq

Sabrina Cuomo & Martina D’Aniello

 Summary
Epigenetics
DNA Methylation
Genome-wide DNA methylation
analysis techniques
 Introduction

DNA methylation is a process involving the

addition of methyl groups to DNA,
influencing gene activity without altering
the genetic sequence. Primarily found in
gene promoters, it tends to suppress gene
transcription. DNA methylation plays
crucial roles in genomic imprinting, the
repression of transposable elements,
aging, and the development of cancer.
 DNA METHYLATION ANALYSIS

MethylC-seq, BS-seq, RRBS-seq

These methodologies all use bisulfite treatment to
distinguish methylated and unmethylated DNA
regions. Unmethylated cytosines become uracil,
detected as thymine in sequencing, while methylated
cytosines remain unchanged, aligning with the
reference sequence.
What is Epigenetics?

Epigenetics explores heritable

modifications in gene
expression and phenotypes
without altering the DNA
sequence. Altered chromatin
structure is a key player in this
process, influencing gene
expression and leading to
observable changes in cell or
organism characteristics.
Based on DNA presence and
chromosomal modifications,
genes can be activated or
deactivated.
In fact, epigenetic
modifications are like
punctuation marks on the
DNA sequence.
Epigenetic variation, often
termed "epimutations", involves
heritable changes without
altering the nucleotide
sequence.
Reversible and associated with
DNA and histone chemical
modifications, epigenetic
effects are influenced by factors
like RNA interference, leading to
RNA-dependent DNA
methylation and histone
modification.
 STEPS OF GENE REGULATION

1. Signals reach a cell, and proteins

relay information to the DNA.
2. Gene regulatory proteins attach to
specific DNA sequences, acting as
switches to activate or inhibit genes.
3. Enzymes associated with regulatory
proteins add or remove epigenetic
tags to the DNA or histones.
4. Epigenetic tags, like methyl groups,
provide a means for the cell to
"remember" and maintain gene
activity over the long term.
 DNA Methylation
DNA Methylation is the addition of methyl tags
to cytosines in the sequence CG. The addition of
a methyl group at the 5 position of cytosine
reduces gene expression. Active genes are less
methylated than inactive ones.

In somatic tissues, methylation typically

occurs in CG-rich regions called CpG
islands, usually upstream of the
promoter region.
CPG ISLANDS

CpG islands are regions in the

genome with a high frequency of
CpG sites, usually 300–3,000
base pairs long. CpG islands are
mainly located in promoter
regions and found in almost half
of all human genes. In embryonic
stem cells, non-CpG methylation,
involving CpA and CpT, is
prevalent.
DNA METHYLATION: KEY ROLE
IN CANCER DEVELOPMENT

Hypermethylation
repressing tumor suppressor
genes.

Hypomethylation refers to
the loss of DNA methylation.
It is a common feature in
various cancers, especially
in metastatic tumors.
 EPIGENETIC CONTROL

DNA Methylation and Histone

Deacetylation are crucial
epigenetic mechanisms that
influence gene expression.

An example of DNA
Methylation involves the
agouti gene in mice: it
influences phenotypic
traits.
 Genome-wide DNA
methylation analysis

MethylC-Seq

Bisulfite sequencing

Reduced Representation BS sequencing

 NGS-Based Genome-Wide
DNA Methylation Analysis

When DNA is bisulfite treated,

unmethylated cytosine is converted to
uracil. Methylcytosine is not affected.
After bisulfite treatment, unmethylated
Cs are read as T and so differ in the
treated and untreated samples. By
contrast, methyl-C is read as C and is
the same as the reference sequence.
Three techniques are utilized to generate
bisulfite sequencing (BS) libraries
compatible with next-generation
sequencing:
 1. MethylC-Seq

Sequencing of methylated regions of the genome.

1. Genomic DNA is fragmented into smaller pieces.
2. Double-stranded universal adapter sequences with methylated
cytosines are ligated to the fragmented DNA.
3. The ligated DNA is size-selected to isolate fragments of a specific size
range.
4. Sodium bisulfite treatment is applied to convert unmethylated cytosines
to thymine, while leaving methylated cytosines unchanged.
5. PCR amplification with primers complementary to the adapter
sequences enriches the library.
6. The final library, representative of the original methylation patterns, is
sequenced to analyze DNA methylation across the genome.
 Case study

The study employed an analysis

called "Genome-wide Methyl-Seq"
to investigate the effects of
glucocorticoid (GC) influence DNA
methylation in various tissues, with
a focus on comparisons between
the brain and blood.
 2. BS-seq

This method involves bisulfite treatment to distinguish methylated and

unmethylated cytosines.
1. Genomic DNA is fragmented.
2. Ligation of a first set of double-stranded adaptors containing methylated
adenine bases within DpnI restriction sites near the site of ligation with
genomic DNA.
3. After bisulfite (BS) conversion, PCR is conducted using primers
complementary to the converted adapter sequences, producing double-
stranded DNA. DpnI digestion removes only the first adapter set.
4. Sequencing adapters are then ligated to the double-stranded BS-converted
genomic DNA fragments.
5. PCR with primers complementary to the adapters is performed to generate
a sequencing library.
 Case study

The study focuses on switchgrass, a

promising bioenergy crop with traits
suitable for biofuel production. Two
switchgrass genotypes, AP13 and VS16,
derived from different ecotypes (lowland
and upland, respectively), were analyzed for
DNA methylation patterns using methylated
DNA immunoprecipitation-sequencing
(MeDIP-seq) and bisulfite-sequencing (BS-
seq).
Results indicate that both genotypes exhibit Dworkin, M., Xie, S., Saha, M. et al. Analyses of
methylomes of upland and lowland switchgrass
similar DNA methylation patterns. (Panicum virgatum) ecotypes using MeDIP-seq
and BS-seq. BMC Genomics 18, 851 (2017).
https://doi.org/10.1186/s12864-017-4218-0
 3. RRBS-seq

RRBS-Seq or scRRBS is a sequencing protocol that uses

restriction enzymes on genomic DNA to create sequence-
specific fragments.
1. Genomic DNA is digested with the methylation-insensitive
MspI restriction enzyme.
2. Digested DNA is separated by gel electrophoresis, and
specific size fractions are chosen.
3. The selected DNA undergoes end repair, ligation to double-
stranded methylated sequencing adapters, BS conversion,
and PCR amplification using adapter-specific primers.
 Case study

The study explores the correlation

between paternal exposure to childhood
maltreatment and alterations in sperm
small non-coding RNA (sncRNA) and DNA
methylation profiles. The aim is to
investigate the potential epigenetic
consequences of paternal early life stress
on the health and development of the
next generation. DNA methylation
analyzed using reduced-representation
bisulfite sequencing (RRBS-seq).
 IMPORTANCE OF
METHYLATION ANALYSIS

After bisulfite treatment, the analysis of these

regions provides insights into the methylation
levels within a biological context.
Understanding the methylation status is
essential for comparing normal and
pathological conditions.
Thank you for the attention!