21/10/2021

Epigenetic regulation of the immune system

1. Introduction to epigenetics

All cells in the body have the same DNA, but have different morphology and function because
the DNA is used in a different way. Gene regulation is part of what is epigenetics. Identical twins
have the same DNA, but there may be some differences such as one can be healthy and the
other can develop cancer, one of them can look much older than the other… Therefore, there is
something else regulating the physiology of our genes.

Epigenetics was coined (acuñado) by 1942:

- Epi: from Ancient Greek επί/epi = 'upon', 'over', 'above’, (additional)

- Genetics: γενετικός genetikos meaning "genitive"/"generative", which in turn derives
from γένεσις genesis meaning "origin”.

Conrad Waddington said that epigenetic are “the causal interactions between genes and their
products, which brings the phenotype into being”.

What is the basis of phenotypic variation? Phenotype arises from Genotype through
programmed change and interaction with the environment.

It was for describing heritable changes in the cellular phenotype and gene expression that are
independent of alterations in the DNA sequence during embryonic development. This man
studied the development of flies embryology, he made some changes in the environment of the
flies (temperature, etc.) and he found that there was an impact on the phenotypes. So the
ambient influences the genotype and the phenotype arises from this genotype through a
program changed by the interaction of the environment.

He uses Waddington's Epigenetic Landscape to explain it, in which a cell, once it differentiates,
there is no turning back. You should imagine a STEM cell on the top of the mountain and it
starts to roll downstream, at some point this cell has to make a decision (differentiation) which is
influenced by the environment.

→ How extracellular stimuli are received in the nucleus and interpreted?

External stimuli → intracellular signaling → transcriptional control → gene expression → cell
fate and behaviour.

The external stimuli are translated into intracellular signaling, this is translated into a
transcriptional control that regulates gene expression and this different types of gene expression
is what makes the cell what it is.

2. The Chromatin

In order to understand how these signals are interpreted in the nucleus of the cell, we need to
study the chromatin. Chromatin is a structure of DNA and proteins present in Eukaryotes, It is
the mix of the DNA molecule with different proteins that are organized in nucleosomes, each
nucleosome is composed by 8 histones. There are two H2A, two H2B, two H3 and two H4, and
they are bound together with the DNA by the histone H1 which acts as a link. This structure is

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important to package the long DNA molecules in a very tiny space, as for each cell the DNA is
about 2m.

When you look at the nucleus through the microscope there are some areas which are darker
and others which are clearer, the ones more compact are the heterochromatin which are
supposed to be silent whereas the ones that are less packed are the euchromatin which are
active.

2. 1 Chromatin domains are defined by epigenetic marks and factors

Signals that are on top of the DNA but modulate how DNA is expressed

Epigenetic marks:

● DNA/RNA methylation → one of the most studied one.

● Covalent histone modifications
● ncRNA (micro, lnc) → they can bind to certain areas of the genome

Epigenetic mechanisms: write or introduce modification on the DNA or on the histone tails

● Writers and erasers → enzymes which remove this modifications

● Readers (proteins, scaffolds, enzymes) → just bind there and don’t do anything. They
can be transcription factors.
● Nucleosome remodeling (multiprotein complexes with helicase or ATP-dependent
activity, such as SWI/SNF) → they slide the nucleosome exposing certain areas of DNA
for binding factors.

3. DNA methylation
DNA methylation mainly occurs in the cytosines. DNA methylation was discovered in Bacillus
tuberculosis in 1925, but its research slowed down for years. Its importance and structure was
discovered much later with the KO of mice. In 1953 they discovered the double helix, this
knowledge was very useful to study this. The function was not known until 1992, they use mice

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embryonically lethal. The lapse of time was due to the study in worms and drosophila that don’t
have DNA methylation, and for years these were the animals used in the labs. These were the
animal models used for research so if it was not present there, it meant it was not important.
This became a dogma until Li, Bestor and Jaenisch described that DMTase1 KO Mice was
embryonically lethal. DNA methylation role in mammals took more than 60 years to be identified.

DNA methylation is a stable gene silencing mechanism. Methylation occurs mainly in CpG
islands where the C and G are very concentrated, these CpG islands are present in promoters
and enhancers of genes and they are associated with transcriptional repression.

In this process the DNA methyltransferase deposits a methyl group in the cytosines. but also
there are other methylations such as 5-hydroxymethylcytosine, 5-formylcytosine or
5-carboxylcytosine but we are not sure about what they did. Also there is a methylation on the
adenosine by the N6-methyladenosine which increases upon stress as they repress LINE1
transposon expression.

If the CpG islands are not methylated, the transcription factors can bind to the consensus
sequence, there is a recruit of DNA polymerase and active transcription. But when CpG islands
are methylated there is the binding of other proteins and all together maintain the area in
repress state, it means in a non-transcriptional stage where the transcriptional factors cannot
bind. What it looks like is that high methylation levels means low expression, whereas low
methylation levels means high expression. DNA methylation in 5mC of CpGs is an anchor for
repressor factors.

In the CpG islands not methylated the RNAPII can go and transcribe the different TF that are
bounded in DNA. However if the DNA is methylated (MeCP) that are associated with HDACs
do not allow the binding of TF and therefore the activity of RNAPII enzyme. In conclusion, high
methylation levels mean low expression and vice-versa.

Do you think that the DNA methylation can be important for hematopoiesis?
Global DNA Methylation is different at cell types and genomic regions. They isolate the different
cell type of the hematopoiesis with different markers, for example hematopoietic STEM cells
were isolated by selecting CD90, CD34 and CD49f. They compared the cell types and they saw
that there are differences in the levels of DNA methylation in the different types of cells. They
saw it through looking at the regulatory regions, such as promoter, enhancer…; and there were
different levels of methylation. Also it is associated with areas where the transcriptional start
sites and the transcription factor binding sites are.

Myeloid-Lymphoid lineage choice is marked by DNA methylation depletion at key transcription

factor binding sites. The differences in methylation between lymphoid and myeloid progenitor
cells is maintain in mature cells, being myeloid cells always less methylated. They found that
the quantity of methylation in the myeloid progenitors in areas that are important for determining
one lineage or the other were very different. For instance, lower methylation in myeloid
progenitors in these areas is associated with higher transcription of the important factor that
determines the myeloid lineage. While there are some sites in the lymphoid progenitors which
also follow this rule, but there are some sites which don’t follow this rule. This is because not

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only DNA methylation is important, as histone modification gives this additional layer of
regulation.

DNA methylation differences are linked to cell-type-specific transcription levels in some genes.
In general, low methylation is expected to correlate with higher expression, but for some
particular genes this doesn’t occur: in GATA1, GATA2 and TAL1 this correlation exists; but other
genes has no correlation or opposite correlation. That is because there are other signaling that
coexists with methylation of the DNA.

4. Histone code
The HISTONE CODE : combination of post transcriptional modifications in histone tails. We
don't know everything about histone code.
Each of these tails has a bound of aminoacids which can be suitable and modified. For instance,
lysines can be acetilates or methylated, not at the same time, Thr/ser/Tyr can be
phosphorylated. There are many other modifications.
To give an idea of this complexity, Histone H3 contains nineteen lysines known to be
methylated—each can be un-, mono-, di- or tri-methylated. If modifications are independent, this
allows a potential 419 or 280 billion different lysine methylation patterns!! Far more than the
maximum number of histones in a human genome… (6.4 Gb / ~150 bp = ~44 million histones if
they are very tightly packed)
More than 50 modifications have been described. The main modifications of nucleosomes are:

- Acetylation
- Methylation
- Phosphorylation (Thr, Ser, Tyr)
- Ubiquitination

They are placed in the different histones that conform the nucleosome and in the histone 1,
which is out of the nucleosomes. As always, there are modifications to activate and inhibit the
transcription.

- Active transcription → acetylation and methylation in some histones.

- Repressive marks → methylation in the DNA, methylation and ubiquitination in histones.

4. 1 Histone modifications

Histone modifications are present in patterns or ”preferred” profiles. Moreover, they associate
with “chromatin states” (transcriptional activity, DNA repair…)

For sure we know that for instance, the tri-methylation of H3K4me3 is the association of
activation of promoters, MIRAR IMATGE. Acetylation means activation (generally). Also there
are repressor modifications: trimethyl K27 in H3 and trimethylation in K9 of H3. Sometimes we
found that these active and repressive marks come together in the same promoters, in those
cases the transcription is off and these promoter are called poised.

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4. 2 Histone - modifying enzymes

There are many other sites which have multiple enzymes that can make the same modification.
This is a way to regulate in a high-tune manner as depending on the signal which arrives to this
cell it can activate one or other complex.

Histone modifications act as anchor sites for proteins through different domains. In other words,
certain protein domains can read different histone modifications. For instance, PHD fingers bind
to this trimethylation of lysine 4, but then this same modification can bind enzymes which arise
this same modification. Sometimes understanding the dynamics is very difficult with the tools
that we have right now.

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5. How epigenetics contribute to control innate immune response?

LPS activates macrophages, and this induces the expression of IL12b genes (IL12 and IL23)
which activate T helper lymphocytes. While in the fibroblast LPS induces the expression of
collagen that helps to form fibrosis. Both cells have the same receptor for LPS, which is the
TLR4/MD2 receptor.

What makes the difference between these 2 cells is the way the chromative structure is in one
gene or the other. In the myeloid progenitor, the regulatory region for IL12b is partially
demethylated, this allows that when there is the lineage specification, there can bind
transcription factors which define the macrophage lineage, like PU1, but still this gene is
repressed. However, when the stimulation of LPS arrives, PU1 allows to engage with other
proteins like p300 which acetylate the histones and allows transcription to be activated. So there
is a step-way modification of this locus.

If a locus is repress how it gets activated? because something should be able to bind there, so
there are some factors which can bind to these areas in which most of them are methylated but
some areas area a little bit demethylated, these help the binding. But it is not so clear and it is
not always like this.

Fibroblasts help this configuration on the promoter areas and enhancers, so when stimuli of LPS
arrives this locus is never gonna be responsive.

5. 1 Rapid vs. slow responses depend on chromatin status at enhancers

The different conformation of these enhancers allow them to respond in a fast or slower way. if
you are half activated, when the signal arrives you can respond very fast. There are other sites
that are completely closed, and somehow the factor can bind the region and this allows the
binding of other signals that will activate the locus and start transcription.

The ones that are half activated are called POISED ENHANCERS and the ones which are
closed are called LATENT ENHANCERS.

The important thing is that the latent enhancers when receive the signal, they remodel the locus
and activate the transcription slower but when the signal disappears the transcriptional factors
go away. But something remains like a memory in these areas. In this case, there is
monomethylation in IL4. So this mark means that the locus remembers that it was activated
onces by Interferon gamma or IL4.

6. Chromatin structure contributes to innate immune response at several levels

Epigenetics controls the development and it is also important to define areas of the genome
which respond to the specific signals. But also they are important in the memory phase, such as
in macrophages. Macrophages are part of the innate immune system.

- Does epigenetic regulation in lymphocytes impact vaccine efficacy? Yes

Gene expression is precisely controlled in a time- and space-dependent manner by epigenetic

regulation to fulfil the function of memory T cells. Without memory there are NO vaccines.

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 - Is epigenetic regulation important for the lymphoid lineage?

This is important in T cells. Memory T cells when they are resting they already have a different
gene expression than naïve T cells, but they are still not activated. The difference between a
naive and a memory cell is that the memory T cells are fastly activated because they already
have genes that are poised (in a half activated state). Different genes have different
modifications.

In human CD8+ memory T cells include genes involved in migration, homeostasis and
readiness for activation such as:

- Cell lectin-like receptor genes (KLRC1, KLRF1 and KLRG1)

- Granzyme genes (GZMA, GZMB, GZMH and GZMK)

Activation-induced poised genes in memory T cells include

- IL3, IL5, IL10 and IL21 and these genes provide a fast and robust effector function for
memory T cells following activation

7. Epigenetic alterations can contribute to immune system diseases?

In cancer this has been well studied for many years. In recent years they have been associated
with autoimmune diseases, lupus, diabetes, etc. In cancer, in many cases, genes that are tumor
suppressors, acquire hypermethylation of promoters and this promotes accumulation of
mutations in certain gene regions. DNA methylation is strongly altered in virtually all cancers

But there are other areas of the genome which are highly methylated and somehow they lose
this methylation and contribute to chromosomal instability. We don’t know very well how this
occurs.

In many cases there has been shown that mutations exist, but nevertheless sometimes it is not
found, there is a tumour with high levels of hypermethylation in promoters, not always the same
promoter,, but the cause is not found.

7.1 Contribution of epigenetics to clonal evolution in leukemia

The order of the epigenetic alteration can influence the type of cancer. For instance TET2 and
JAK2 alteration are found in leukemia, but can occur before the other. This dynamics is
important for the treatment, but it is not easy. The tumor process is long and sometimes when
we look at the alteration we only see one stage of the disease. In leukemia we have more
access as we can get the blood, but in solid tumors it’s more complicated.

7. 2 Aberrant epigenetic modifications in autoimmune diseases triggering a cascade of

events

They alter the gene expression which affects the activity of the cells and induce the
inflammatory process. The good thing is that this modification can be reversed with drugs, we
have some tools to inhibit the enzymes that deposit methylation or acetylation. Combinating
these types of enzymes we can treat some of these cancers.

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8. Epigenetic changes contribute to cell fate decisions

→ It is a very complex field. Epigenetics also influences other processes like ageing. Now we
know that with these drugs impacting on epigenetic modifications we can alter cell
differentiation, so a differentiated cell can become multipotent.

9. Modern definitions of EPIGENETICS

- “The study of mitotically and/or meiotically heritable changes in gene function that
cannot be explained by changes in DNA sequence” (Riggs and Potter, 1996).
- “The inheritance of patterns of DNA and RNA activity that do not depend on the naked
nucleotide sequence. By “inheritance,” we mean a memory of such activity transmitted
from one cell generation to the next (through mitosis)” (Manel Esteller, 2005).
- “A change in the state of expression of a gene that does not involve a mutation, but that
is nevertheless inherited in the absence of the signal or event that initiated the change.”
(Mark Ptashne, 2007).
- “The structural adaptation of chromosomal regions so as to register, signal or
perpetuate altered activity states” (Adrian Bird, 2007).
- “The study of heritable changes in gene expression that occur independent of changes
in the primary DNA sequence” (Peter Jones, 2009).

All of them are correct but the negrita is the best one.