Transcript DNA methylation

Course Title:
Epigenetics
Lecture Titles:
Lecture I: General Overview and History of Epigenetics
Lecture II: DNA methylation
Lecture III: Alteration in DNA methylation and its transgenerational inheritance
Lecture IV: DNA methylation and genome stability
Lecture V: Epigenetic variation in genome evolution and crop improvement
Lecture VI: Histone modifications
Lecture VII: RNA interference
Lecture VIII: Epigenetics and gene expression
Lecture : TBD
Lecture : TBD
Lecture : Summary
Course Title:
Epigenetics
Lecture Titles:
Lecture I: General Overview and History of Epigenetics
Lecture II: DNA methylation
Lecture III: Alteration in DNA methylation and its transgenerational inheritance
Lecture IV: DNA methylation and genome stability
Lecture V: Epigenetic variation in genome evolution and crop improvement
Lecture VI: Histone modifications
Lecture VII: Non-coding small RNA and RNA interference
Lecture VIII: Epigenetics and gene expression
Lecture : TBD
Lecture : TBD
Lecture : Summary
How are epigenetic variations accomplished？
Epigenetic effects can be accomplished by several selfreinforcing and inter-related covalent modifications on
DNA and/or chromosomal proteins, such as DNA
methylation and histone modifications, and by chromatin
remodeling, such as repositioning of nucleosomes. These
heritable modifications are collectively termed
“epigenetic codes” (reviewed in Richards and Elgin, 2002).
表观遗传学机制 Epigenetic Mechanisms
(1) 染色体重塑
Chromatin remodeling
(2) DNA共价修饰
Covalent modifications in DNA
• Cytosine DNA methylation
(3) 染色体蛋白质共价修饰
Covalent modifications in
chromosomal proteins:
• 组蛋白乙酰化/去乙酰化
Histone acetylation/deacetylation
去
• 组蛋白H3-Lys9甲基化
Histone H3-Lys9 methylation
• Others
Histone code
epigenetic code
表观遗传学密码
Relationships between the four best understood
epigenetic markers:
Chromatin remodeling
Histone deacetylation
Histone methylation
Small RNAs
DNA methylation
This vicious cycles of DNA methylation and histone modifications
ensure that the silenced genes remain silent.
The Four bases of DNA
Genomes 3 (© Garland Science 2007)
Cytosine methylation
Cytocine methylation occurs predominantly in CpG dinucleotides in mammalian species. It is
interesting to be noted that the CpG dinucleotide is self-complementary.
Cytosine
Guanine
Difference between DNA base change and modification
Base changes
Methylation modifications
Cytosine
5-Methylcytosine
Hypothesis of DNA Methylation Memory
The idea that DNA methylation in animals could represent a mechanism of cell memory
arose independently in two laboratories in 1975 (Holliday and Pugh 1975, Science 186:226-232; Riggs
1975, Cytogenet. Cell Genet. 14: 9-25). Both groups proposed that patterns of methylated and nonmethylated CpG could be copies when cells divide.
To ensure copying of parental pattern onto the progeny strand, they postulated a
“maintenance methytransferase” that would exclusively methylate CpGs base-paired with
a methylated parental CpG.
METHYLATED
DNA
replication
Maintenance
methylation
HEMIMETHYLATED
Methylation patterns are heritable
The fact that methylation patterns are heritable was initially established using DNAmethylation-sensitive restriction enzymes (Bird and Southern 1978). The early studies also
showed that either both CpGs in a complementary pair were methylated, or neither was
methylated, which fitted well with the predictions of the maintenance model.
The mammalian maintenance DNA methytransferase
DNA methyltransferase was first purified in mammalian species in 1983 (Bestor & Ingram,
1983 PNAS 80: 5559-63). The preferred DNA substrate of this enzyme, Dnmt1, is DNA
methylated at CpG on one strand only (hemimetylated DNA). Thus, this enzyme seemed
to be a maintenance DNA methytransferase.
Dnmt1
Dnmt1
Discovery of de novo DNA methyltransferases
All known prokaryotic cytosine methyltransferases share a set of diagnostic protein motifs.
These features are also found in Dnmt1. These features eventually led to the discoveries of
the mammalian de novo methyltransferases (Okano et al. 1998).
Regulatory domain
PCNA
RFT
CXXC
Catalytic domain
BAH
I
Dnmt1
NLS
Dnmt2
(weak activity)
PWWP
Dnmt3a (de novo methylation of CpG)
Dnmt3b
(de novo methylation of CpG)
ATRX
IV VI
IX X
Nature Review Genetics (2010)
What sequences are methylated in our genome?
DNA from mammalian somatic tissues is methylated at 70% of all CpG sites. Highly
methylated sequences include satellite DNAs, repetitive elements including transposons,
nonrepetitive intergeneic DNA, and exons of genes. Key exceptions of this global
methylation of the mammalian genomes are the CpG islands (regions with high CpG density).
Most CpG islands marks the promoters and 5’ domains of genes. Approximately 60% of
human genes have CpG island promoters.
CpG island
ACTIVE
SILENCED
What protects CpG islands from DNA methylation?
(1) CpG islands are unmethylatable by the existing de novo methytransferases. However,
this is unlikely because they become densely methylated on the inactive X chromosome and
in cancer cells.
(2) CpG islands are protected from methylation by the binding of factors which exclude
Dnmts.
(3) CpG islands are maintained in a methylation-free state with the aid of DNA demethylase
that actively remove methyl-CpGs.
(4) The atypical base composition and lack of methylation reflect abnormal DNA metabolism
at these CpG islands. For example, recombination and/or repair may be concentrated at
these sites, which may result in high level of DNA turnover.
(5) Early embryonic transcription from a CpG island promoter is required to ensure that
DNA methylation is excluded. However, there is no evidence that transcription excludes
CpG methylation.
(6) A complex relationship between DNA methylation and chromatin structures in some
eukaryotes, including plants.
Regulation of gene expression by DNA methylation
(1) Several studies in early 1980s showed that genes can be silenced by artificial methylation
of CpG sites and silenced genes can be activated by treatment with 5-azacytidine, which
inhibits DNA methylation in living cells.
(2) Interference with transcription factor binding: Transcription factors that recognize GCrich sequence motifs can be interfered by the presence of the methyl groups in the
methylated CpGs.
(3) Attraction of methyl-CpG-binding proteins: methyl-CpG-binding proteins (MeCP1 and
MeCP2), methyl-CpG-binding domain (MBD) proteins (MBD1, MBD2, MBD3, MBD4), another
unrelated protein, Kaiso. These proteins recruit repressory protein complexes that in turn
interact with histone deacetylases (HDAC).
(4) Complex interrelationship between DNA methylation and histone modification, which
result in heterochromatin formation and gene silencing.
What are important future topics in the field?
1. Understand the evolutionary significance of DNA methylation.
2. Understand the complex relationship of DNA methylation with other
epigenetic marks.
3. What are the intrinsic and environmental factors that induce changes in
DNA methylation patterns?
4. Disorder of DNA methylation patterns has been found in many genetic
diseases, including cancers. We need to understand the exact role of DNA
methylation in these complex diseases.
Science 14 May, 2010:Vol. 3288. p. 872
Author Summary
The queen honey bee and her worker sisters do not seem to have much in common. Workers
are active and intelligent, skillfully navigating the outside world in search of food for the
colony. They never reproduce; that task is left entirely to the much larger and longer-lived
queen, who is permanently ensconced within the colony and uses a powerful chemical
influence to exert control. Remarkably, these two female castes are generated from identical
genomes. The key to each female’s developmental destiny is her diet as a larva: future queens
are raised on royal jelly.
This specialized diet is thought to affect a particular chemical modification, methylation,
of the bee’s DNA, causing the same genome to be deployed differently. To document
differences in this epigenomic setting and hypothesize about its effects on behavior, we
performed high-resolution bisulphite sequencing of whole genomes from the brains of
queen and worker honey bees. In contrast to the heavily methylated human genome, we
found that only a small and specific fraction of the honey bee genome is methylated.
Most methylation occurred within conserved genes that provide critical cellular functions.
Over 550 genes showed significant methylation differences between the queen and the
worker, which may contribute to the profound divergence in behavior. How DNA
methylation works on these genes remains unclear, but it may change their accessibility
to the cellular machinery that controls their expression. We found a tantalizing clue to a
mechanism in the clustering of methylation within parts of genes where splicing occurs,
suggesting that methylation could control which of several versions of a gene is
expressed. Our study provides the first documentation of extensive molecular differences
that may allow honey bees to generate different phenotypes from the same genome.
Genome reprogramming and small interfering RNA in the Arabidopsis germline
In the pollen grain, the two haploid sperm cells (orange circles) are supported by the larger haploid vegetative cell
(green circle); in the ovule, the haploid egg cell (red) is supported by the endosperm (yellow). In pollen, the 21nt
easiRNAs are synthesized in the vegetative cell and move to the sperm. In the ovule, the 24nt siRNA were found in the
seed and are proposed to be produced in the endosperm (yellow).
Chromatin states in pluripotent, differentiated, and reprogrammed cells
Cynthia L Fisher, Amanda G Fisher
Pluripotent embryonic stem cells (ESCs) possess the ability to self-renew indefinitely in culture and differentiate into all embryoderived lineages. As they do so, they pass through progenitor states, their differentiation potential decreases (blue triangle), and
they become further specialised in cellular function and morphology (purple triangle). The ultimate conclusion of any
differentiation process, and of development in vivo, results in terminally differentiated cell types with limited options to change
their characteristics under normal conditions. ESCs are characterised by a transcriptional network centred on the core
pluripotency transcription factors Oct4, Sox2, and Nanog, and by chromatin-based characteristics: transcriptional permissivity,
hyperdynamic accessiblility, and DNA methylation in a non-CpG context. As ESCs differentiate, those chromatin-based trends
decrease (in blue box), while other characteristics increase (in purple box), including levels of CpG DNA methylation,
heterochromatin, euchromatic H3K9me2 regions (called ‘LOCKS’), and the expression of lineage-specific factors. Under certain
artificial ‘reprogramming’ conditions, differentiated cells can be induced to revert back towards a pluripotent state.
Chromatin states in pluripotent, differentiated, and reprogrammed cells
Cynthia L Fisher, Amanda G Fisher
In embryonic stem cells (ESCs), developmental regulators necessary for lineage-specific gene expression programs are repressed (or expressed
at very low levels), yet are ‘primed’ for rapid induction of expression upon receiving differentiation cues. These primed genes are characterised
by ‘bivalent’ chromatin domains, containing H3K4me3 (associated with active transcription; deposited by a trithorax group Mll containing
histone methyltransferase (HMT) complex shown in green) and H3K27me3 (associated with gene repression; deposited by Ezh2 HMT within a
Polycomb group PRC2 complex containing Jarid2 shown in red), at promoters. Additionally, bivalent genes have non-methylated CpG DNA
regions (non-mCpG), and possess repressive H2AK119Ub1 marks (deposited by Ring1a/b ubiquitin E3 ligases within a PRC1 complex shown in
dark blue), at the promoter region and throughout the coding region, which is thought to restrain RNA Polymerase II (RNAP II, shown in light
blue) from productive elongation. This poised form of RNAP II is characterised by a unique combination of abundant Ser5 phosphorylation
(shown in yellow) but low levels of Ser2 phosphorylation (as indicated by Ser non-P, in white). Upon differentiation to progenitors, bivalently
marked ESC genes can resolve into active or inactive forms, or remain poised and bivalent; in addition, new bivalent domains can form at
different genes. Resolution of bivalency is thought to involve activity of lysine demethylases (KDMs), histone deubiquitylases (DUBs), and
DNA methyltransferases (DNMTs). Active genes show loss of repressive chromatin marks, an increase in H3K4me3, gain of H3K36me3 within
coding regions, and contain RNAP II with Ser5P and non-P Ser2 near promoter regions and Ser5P and Ser2P within coding regions. Inactive
genes lose active chromatin marks, retain repressive chromatin marks, and may gain CpG methylation (mCpG). Refer to the key for explanation
of symbols used.
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