DNA Methylation Patterns During Development

Speaker: Bhupendra Singh Rawat

Ph.D scholar 1st year

Animal biochemistry

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

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)

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

In DNA where methylations are happening?

• CpG islands

How to know which region of DNA undergoing methylation?

• HpaII (CCGG) and HhaI (GCGC)

Mammalian DNA methylation machinery

Gopalakrishnan et al., 2008

DNA methylation and transcriptional repression

Singal and Ginder, 1999

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.

De novo methylation at the time of implantaton

• Mediated by Dnmt3a and Dnmt3b.

• Generate bimodal pattern of methylation.

• CpG islands remain protected.

Mechanism of CpG island protection

Cedar and Bergman, 2009

H3K4me3 may be involved in CpG island protection

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.

Inactivation of pluripotency genes.

Cedar and Bergman, 2012

Cntd…

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).

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).

Cntd…

• CpG island methylation sometimes leads to up regulation of gene.

• How cell target de novo methylations to specific sites on the DNA?

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.

Targeted de novo methylation by polycomb complex

Cedar and Bergman, 2012

DNA methylation and polycomb complexes: linking development to cancer

Gopalakrishnan et al., 2008

PRC 1 PRC 2

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.

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.

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.

- 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