Gene repression and chromatin compaction

The concept of cellular memory. Schematic illustration of the involvement of PcG and TrxG complexes in the
determination of active and repressed states of gene expression and, thereby, cellular differentiation, which is
maintained over many cell divisions. TA, transcriptional activator; TR, transcriptional repressor
doi: 10.1101/cshperspect.a019331.
Developmentally regulated gene repression
is associated with H3K27me3
• H3K27me3 is found in facultative heterochromatin
• EZH2 trimethylates H3K27
• EZH2 is an HMT that is part of Polycomb Repressive Complex 2 (PRC2)
• Polycomb Repressive Complex 1 (PRC1) contains CBX which binds specifically to
H3K27me3 via a chromodomain
• PRC1 complex facilitates chromatin compaction to repress expression of
developmental genes, ensure lineage specific regulation
 Stability of Epigenetic Modifications
H3K27me3/H3K4me3

H3K27me3

H3K9me3

Robust silencing with

HP1α/DNA methylation

In mammals - DNA methylation may provide the cellular memory

Blomen & Boonstra (2011) Cel Mol Life Sci 68 27-44
Heterochromatin is associated with H3K9me2/3 especially constitutive domains

Weaver 5th Edition

Figure 13.32

Reading and writing

• SUV39H methylates histone H3 on lysine 9 and is in a complex with HP1α protein
• Chromo domain of HP1α binds to H3K9me2/3
• Suggests a mechanism for spreading of a silent compact heterochromatic state
• Facilitates formation of compact heterochromatin through oligomerization of chromo
shadow domain
 Histone Code Model for
Formation of
Heterochromatin

Lodish 8th Fig 8-29

Lecture 15
The role of DNA methylation

• One of the most extensively studied epigenetic marks

• In mammalian genomes, decorates the majority of cytosines involved
in transcriptional gene silencing and plays important roles during
mammalian development
• It is found at the promoters and enhancers of inactive genes, at
repetitive elements, and within transcribed gene bodies
• Its perturbation is often associated with human diseases ie. cancer
 DNA methylation

Occurs predominantly at CpG dinucleotides in

mammalian cells CpG
GpC

Usually both CpGs in a complementary pair are

methylated

Dnmt1 methylates CpG at hemimethylated sites-

"maintenance methylase"- patterns of CpG
methylation are inherited after replication.

Allis et al 2nd Edition Chapter 15 Figure 1

 DNA methyl transferases (DNMTs)

Ambrosi et al 2017
De novo and maintenance DNA methylation

self-complementary 5ʹ-
CG-3ʹ pairs marked as
vertical strokes

Bird 1999 DOI: 10.1126/science.286.5448.2287

DNA methylation Key early findings
• CpG is a rare dinucleotide in mammalian genomes
• 70% of CpG methylated in somatic cells
• CpG methylation associated with gene silencing
Evidence
1) no expression from methylated DNA template transfected into mammalian
cells
2) treatment of cells with 5-azacytidine, which inhibits CpG methylation, led to
expression from silent loci (eg on inactive X chromosome)
• Several proteins specifically bind to methylated CpG e.g. MeCP2
CpG Dinucleotide Repeats
• Regions of DNA where a cytosine nucleotide is followed by a guanine
nucleotide in the linear sequence of bases along its 5' → 3' direction
• Occur in high frequency in genomic regions called CpG islands

Wang et al 2018
 CpG Islands (CGIs)

CpG islands are regions of high CpG density (>50%),

which usually extend for 200–2000 bps

Shores are regions that reside up to 2kb from CGIs,

Shelves 2-4k b away

Lack CpG methylation in which the adjacent gene is

active.

Found at promoters of most human genes

Methylation ensures long term silencing

Allis et al 2nd Edition Chapter 15 Figure 5

Recruitment of corepressors
by methyl-CpG binding
proteins

Methyl DNA binding proteins silence

gene expression through recruitment
of chromatin modifying complexes

Allis et al 2nd Edition Chapter 15 Figure 9

Effects of DNA methylation on transcription

Active transcription Repression of transcription

Random transcription Repression of random transcription

Correct transcription initiation Incorrect transcription initiation

Miller & Grant 2019

Detection of CpG methylation
• Digest genomic DNA with restriction enzymes that cut CpG and are
methylation sensitive
e.g. HpaII and MspI both cut CCGG if C not methylated but only MspI cuts if C
methylated

• Bisulphite modification of genomic DNA. Methylated C protected

from deamination
 Bisulphite modification

• Bisulphite deaminates cytosine in a nucleophilic attack whilst the methyl group on 5-methylcytosine
protects the amino group from the deamination
• Perform Sanger sequencing
any C that was not methylated is read as a "T”
compare to reference DNA sequence all methylated and non-methylated CpG can be identified
• Method of choice but technically difficult and time consuming
Example: bisulfite genomic sequencing

Bisulfite genomic DNA sequencing of MLH1 CpG island

Weisenberger et al (2008) NAR, 36, 4689

Imprinting in Mammals
• Normally both maternal and paternal alleles of each gene are
transcribed
• Genomic imprinting is an epigenetic* mechanism that restricts the
expression of the gene to one of the two parental chromosomes
• Affects a few hundred genes (less than 1% of total genes)
• For some genes, it is the maternal allele that is active for others it is the
paternal

*epigenetics can be defined as a change in phenotype (gene expression) that is

heritable (meiotic or mitotic) but does not involve DNA mutations
Key Features of Imprinting
• Imprinted genes are clustered
3-10 genes over 100 to 3000kb
• At least one of the genes in the cluster does not code for protein
Usually, a very long non-coding RNA
• Each imprinted cluster contains a Differentially DNA Methylated
Region (DMR)
DMR is methylated on one chromosome but not the other
• Methylation status of DMR established in ovary or testis and
maintained on only ONE parental chromosome in diploid embryo
 Allele with methylated DMR results in no expression of non-coding RNA gene

The imprinted control element (ICE), a DMR, is methylated on the maternal but not paternal
chromosome

The IG-NC gene is transcribed on the paternal chromosome

The non-coding RNA represses some of the protein coding genes (IG) on the same
chromosome i.e. a cis-acting silencing mechanism

Allis et al 2nd Edition Chapter 26 Figure 5

 Imprinted expression is regulated by DMR

DMR is a positive regulator of IG-NC gene expression

Function of DMR must be blocked by DNA methylation

Allis et al 2nd Edition Chapter 26 Figure 6

 Cis-acting gene silencing at imprinted gene clusters Two models

Maternal chromosome
• CTCF binds to nonmethylated ICE
• CTCF is associated with insulators
• Repression of Igf2 is because
CTCF blocks enhancer access

Paternal chromosome
• Methylation of ICE, also spreads
to H19-NC.
• Enhancer activates in cis genes

Allis et al 2nd Edition Chapter 26 Figure 8

 Cis-acting gene silencing at imprinted gene clusters Two models

Maternal chromosome
• Methylated ICE silences Air-NC,
but Igf2r gene expressed along
with others

Paternal chromosome
Air-NC expressed, other genes in the
cluster silenced.
Air is a 108 kb ncRNA

Allis et al 2nd Edition Chapter 26 Figure 8

What is the function of genomic imprinting in mammals?

• At least half of imprinted genes are required for early development

• Other imprinted genes are important for neurological functions
But some imprinted genes are not essential
• Embryo cannot develop with only paternal or only maternal chromosomes

Two hypotheses
Parental conflict
Paternal genome should increase competitiveness by increasing growth, maternal
genome should fairly distribute resources by limiting growth.
Trophoblast defense
Imprinting could block spontaneous oocyte development by silencing maternal genes
required for placental development
Parental conflict hypothesis
• In some species, more than one male can father offspring from the same litter. A house cat, for
example, can mate more than once during a heat and have a litter of kittens with two or more
fathers. If one father's kittens grow larger than the rest, his offspring will be more likely to survive to
adulthood and pass along their genes. So it's in the interest of the father's genes to produce larger
offspring. The larger kittens will be able to compete for maternal resources at the expense of the
other father's kittens.
• On the other hand, a better outcome for the mother's genes would be for all of her kittens to survive
to adulthood and reproduce. The mother alone will provide nutrients and protection for her kittens
throughout pregnancy and after birth. She needs to be able to divide her resources among several
kittens, without compromising her own needs.
• It turns out that many imprinted genes are involved in growth and metabolism. Paternal imprinting
favours the production of larger offspring, and maternal imprinting favours smaller offspring. Often
maternally and paternally imprinted genes work in the very same growth pathways. This conflict of
interest sets up an epigenetic battle between the parents -- a sort of parental tug-of-war.