2. Content
• Introduction
• Demethylation during early development
• De novo methylation at the time of implantaton
• Post-implantation methylation changes
• Tissue-specific methylation patterns
• Conclusion
• Future prospects
3. Introduction
• DNA methylation is
a biochemical process
involving the addition of
a methyl group to
the cytosine or adenine
DNA nucleotides.
• DNA methylation alters
the expression of genes
in cells as cells divide and
differentiate from ESCs
into specific tissues.
The methylation reaction
(Leonhardt and Cremer, 1995)
4. Some basics
• DNA methylation causes transcriptional repression
• H3K9 and H3K27 methylation causes transcriptional
repression
• H3K4 methylation causes transcriptional activation
• Acetylation of histones generally leads to transcriptional
activation
5. In DNA where methylations are happening?
• CpG islands
How to know which region of DNA undergoing
methylation?
• HpaII (CCGG) and HhaI (GCGC)
8. Demethylation during early development
• Methylation pattern of parental gametes are largely
erased in the preimplantation stage(morula and
blastula):
A. Active demethylation: begins in the zygote.
B. Passive demethylation: occurs during first few early
replication cycles. Dnmt1 relocate from nucleus to
cytoplasm.
9. De novo methylation at the time of implantaton
• Mediated by Dnmt3a and Dnmt3b.
• Generate bimodal pattern of methylation.
• CpG islands remain protected.
10. Mechanism of CpG island protection
Cedar and Bergman, 2009
H3K4me3 may be involved in CpG island protection
11. Post-implantation methylation changes
• Post-implantation methylation changes are of tissue
specific or gene specific nature.
• Example: silencing of pluripotency genes like Oct-3/4
and Nanog.
13. Cntd…
• Another major event occurring after implantation
throughout all cells of the embryo is the inactivation of
one X-chromosome in female animals.
• This is also achieved by changes in chromatin structure
followed by de novo methylation of CpG island
promoters.
• Probably it also mediated by histone methylases capable
of generating heterochromatin and then recruiting Dnmts
that carry out targeted local methylation many days after
the initial inactivation event (Cedar and Bergman, 2009).
14. Tissue-specific methylation patterns
• Tissue specific genes: non CpG island promoter
(methylated at implantation).
• These promoters are demethylated during tissue
development for which cell-type-specific factors
recognize them and then recruit demethylases.
• This demethylation occurs in an active manner(not
require DNA replication) and is mediated by cis acting
sequences and trans acting factors (Kirillov et al., 1996).
15. Cntd…
• CpG island methylation sometimes leads to up
regulation of gene.
• How cell target de novo methylations to specific sites on
the DNA?
16. Polycomb complex
• A complex of protein bound at specific gene.
• Almost all the sites that undergo targetted de novo
methylation are known polycomb targets (Straussman et
al., 2009).
• Mammals have 2 main polycomb complexes: PRC1 and
PRC2.
• Polycomb complex has an ability to recruit Dnmt3a and
Dnmt3b.
17. Targeted de novo methylation by polycomb
complex
Cedar and Bergman, 2012
18. DNA methylation and polycomb complexes:
linking development to cancer
Gopalakrishnan et al., 2008
PRC 1 PRC 2
19. Conclusion
• DNA methylation patterns are erased during pre-
implantation and then re-established throughout
development via sequence information in the DNA.
• Once established, DNA methylation patterns can be
maintained autonomously through many cell divisions.
• DNA methylation inhibits gene expression by affecting
chromatin structure.
• Changes in methylation during post-implantation
development are usually secondary to factor-mediated gene
activation or repression, but this subsequent methylation
pattern provides long-term stability.
20. Future Prospects
• What is the mechanism involved in setting up methylation
patterns? How is local sequence information translated in
epigenetic information?
• How does methylation play a role in lineage determination
during development?
• What are the roles of environment and aging on DNA
methylation? What are methylation’s effects on disease
susceptibility?
• How histone and DNA methylation are coordinated at
molecular level.
21. References:
1. Cedar, H. and Bergman, Y. 2009. Linking DNA methylation and histone
modification: patterns and paradigms. Nature Reviews Genetics. 10: 295-
304.
2. Cedar, H. and Bergman, Y. 2012. Programming of DNA methylation
patterns. Annu. Rev. Biochem. 81: 97–117.
3. Gopalakrishnan, S., Van Emburgh, B.O., Robertson,K.D. 2008. DNA
methylation in development and human disease. Mutation Research. 647:
30–38.
4. Kirillov, A., Kistler, B., Mostoslavsky, R., Cedar, H., Wirth, T. and Bergman,
Y. 1996. A role for nuclear NF-κB in B-cell-specific demethylation of the
Igκlocus. Nat. Genet. 13: 435–41.
5. Leonhardt, H. and Cremer, T. 1995. Functional Analysis of DNA
Methylation in Development and Disease. an der Fakultät für Biologie. 1-
117
6. Singal, R. and Ginder G.D. 1999. DNA Methylation. Blood. 93: 4059-4070.
7. Straussman, R., Nejman, D., Roberts, D., Steinfeld ,I. and Blum, B. 2009.
Developmental programming of CpG island methylation profiles in the
human genome. Nat. Struct. Mol. Biol. 16: 564–71.
22.
23.
24. - inhibition of DNMT1, -
- replication dependent
DNA DEMETHYLATION
PASSIVE
- replication independent
- clearly demonstrated in some cases
- unknown mechanism, unknown demethylase. DNA repair/glycosylase?
ACTIVE
- DNA methylation is stable but reversible
Editor's Notes
CpG island: a region that contains a high density of CpGs and is located at the promoters of many genes.
HpaII- from Haemophilus parainfluenza bacteria.
HhaI- from Haemophilus haemolyticus
DNMT3A and DNMT3B are responsible for establishing new DNA methylation patterns.
DNMT1 copies existing methylation patterns following DNA replication and hence is predominantly considered the maintenance methyltransferase.
DNMT3L is homologous to DNMT3A and DNMT3B within the N-terminal regulatory region and is highly expressed in germ cells. It stimulate the activity of DNMT3A and DNMT3B and it is required for establishing genomic imprints.
Key functional domains and protein–protein interaction domains indicated. All of the active DNA methyltransferases contain the active site motif IV in the C-terminal region. DNMT1 contains a region required for its interaction with PCNA, which is adjacent to the nuclear localization signal (NLS). The N-terminal region of DNMT1 also contains a cysteine-rich HRX-like region and a lysine-glycine repeat (KG(5)) region. DNMT3A and DNMT3B contain plant homeodomain (PHD) and PWWP domains. These two domains are required for targeting DNMT3A and DNMT3B to pericentromeric heterochromatin and contribute to protein–protein interactions by recognition of histone modifications.
Transcriptional repression by DNA methylation can be done by 3 mechanisms:
By direct interference with the binding of TF: some TF (eg, Sp1 and CTF) are not sensitive to methylation of their binding sites and many factors have no CpG dinucleotide residues in their binding sites.
By binding of specific transcriptional repressors to methylated DNA: Two such factors, MeCP-1 and MeCP-2 (methyl cytosine binding proteins 1 and 2) bind to methylated CpG residues. A component of the MeCP-1 complex, PCM1, has a methyl- CpG binding domain (MBD) through which MeCP-1 binds to methylated DNA.
MeCP-2 is more abundant than MeCP-1 in the cell and is able to bind to DNA containing a single methylated CpG pair. MeCP-2 has two domains: a MBD and a TRD(transcriptional repressor domain). It can inhibit transcription from a promoter at a distance. MeCP-2 associate with a corepressor complex containing mSin3A and histone deacetylases. Trichostatin A —Deacetylase inhibitor—Relieve transcriptional repression. DNA methylation and histone deacetylation, can be linked by MeCP-2.
C. By making inactive chromatin structure:
AP-2: Activating Protein-2 is a family of closely related transcriptional factors which plays a critical role in regulating gene expression during early development.
NFkB: nuclear transcription factor-kappaB
At the time of implantation the entire genome gets remodified through a wave of de novo methylation which is mediated by
This de novo methylation generate a bimodal pattern of methylation in which most sequences becoming methylated to high levels (>80%), while CpG island like windows remain protected.
Before implantation, most CpGs in the embryonic genome are unmethylated (light purple circles), but some regions are packaged with nucleosomes containing methylated (Me) lysine 4 of histone H3 (H3K4), perhaps as a result of RNA polymerase binding. At the time of implantation, the methyltransferases Dnmt3a and Dnmt3b are expressed. DNA methylation (dark purple circles) is facilitated by the Dnmt3 binding partner, DNMT3L, which binds to chromatin by recognizing the K4 residue on histone H3. If this histone moiety is methylated, however, the complex cannot bind and the underlying DNA region is thus protected from de novo methylation. This may be one of the mechanisms used to generate a bimodal methylation pattern characterized by methylation over most of the genome, but not at CpG islands.
During tissue development demethylation and methylation of specific genes happens.
Generally tissue specific genes have non CpG island promoter. So it is automatically methylated at the time of implantation as at that time there is a wave of de novo methylation. During tissue development these promoters are demethylated to make it accessible to transcriptional machinery. Cell-type-specific factors recognize these tissue specific genes and then recruit demethylases to the promoter. This demethylation occurs in an active manner (not require DNA replication) and is mediated by specific cis acting sequences and trans acting factors.
CpG island methylation sometimes leads to gene activation. This may happen when the CpG island are present in the promoters region of antisense transcripts. When methylated these antisense transcripts will not form and this will lead to activation of those genes which are silenced by these antisense transcripts.
To target de novo methylation at specific site on DNA polycomb complexes are required.
Targeted de novo methylation. Genes targeted by the polycomb complex, PRC2, normally have unmethylated CpG island promoters but are repressed by virtue of the histone methylase EZH2-mediated methylation of H3K27, which then recruits the chromodomain-containing complex, PRC1, generating a form of heterochromatin. Targeted de novo methylation can occur at specific sites during normal development or abnormally in cancer, and this probably occurs because EZH2 itself recruits Dnmt3a and Dnmt3b.
DNA methyltransferase-polycomb complex interactions in development and cancer. (A) In normal pluripotent cells (ES cells in this case, although tissue specific stem cells may be similar), genes involved in differentiation are expressed at low levels or are repressed. These genes tend to be marked by both activating (trimethylated H3K4, 3XMe-H3K4) and repressive (trimethylated H3K27, 3XMe-H3K27) marks. A subset of these genes may also be marked by DNA methylation. The regulation of the DNMTs in this state remains unknown. The DNMTs may constitutively associate with polycomb complexes but be rendered inactive or in a low activity state (bottom panel). Alternatively, other chromatin marks or differences in the activity/composition of polycomb complexes prevent DNMT recruitment (top panel). For example, the 3XMe-H3K4 may inhibit DNMT3L-mediated recruitment of de novo DNA methyltransferases. In this bivalent state, genes are able to respond to external developmental cues. (B) In tumor cells or tumor initiating cells (TIC), we propose that normal regulation of the DNMTs is disrupted. This may be due to aberrant recruitment of DNMTs to genes normally repressed by polycomb complexes and/or a change in the activity of bound DNMTs, possibly by post-translational modifications like sumoylation (indicated by the red stars) or a change in the chromatin environment such as acquisition of other repressive histone marks (e.g. trimethylated H3K9). Pro-differentiation genes are then locked in an ‘off’ state due to dense DNA methylation and cells no longer properly respond to differentiation cues and/or they acquire enhanced self-renewal capacity. Once dense DNA methylation is present, the DNMTs may have a role in maintaining PRC1/PRC2 binding.
