Epigenetics:DNA methylation I

Requirements for epigenetic materials

• Need to be transmitted faithfully during mitosis and meiosis (possibly along with DNA)

-> cell need to have maintenance mechanisms similar to semi-conservative mechanism of DNA replication

• Need to store information for cells (cellular memory) – such as ‘Histone Code’

-> cell need to have system for writing and reading this store information.

-> Writer (DNA and Histone methylases) vs Reader (MBDs and Bromo and Chromo-domain proteins)

DNA and Histone modificatins during DNA replication

What is DNA methylation?

- Cytosine methylation only in 5’-CpG-3’- mCpG initially hypothesized as a mechanism for cellular memory during DNA replication (semi-conservative mechanism) - self-complementary (palindromic)- methylation-sensitive enzyme HpaII used for analyzing and proving the initial prediction two types of DNA (methylated and un-methy lated DNA but always symmetrical -> proving maintenance of mCpG)- transfected CpG DNA never methylated, transfected mCpG maintained

identification DNMT1 (Bestor and Ingram, 1983)

How DNA becomes methylated?

-predict two types of enzymes (un-methylated –de novo methylase hemi-methylated –maintenance methylase)

- late 1990s identification of DNMT3A, DNMT3B, DNMT3L, DNMT2

- hemi-methylated CpG good substrate for DNMT1

What enzymes are involved?

- DNMT1-KO: genome-wide loss of DNA methylation, embryonic lethal (e9.5)

- DNMT2 tRNA methylase, DNMT2-KO: no obvious phenotypic consequence

- DNMT3A work together with DNMT3L for de novo methylation, postnatal lethal

-DNMT3B-KO embryonic lathal (e14.5), demethylation on satellite DNA human ICF syndrome loss of methyl on repetitive DNA

Which portion of genes or genome get methylated?

- most CpG sites in vertebrate genomes methylated (~80%) deriving mutational decay of CpG site to CpA or TpG (CpG density under- represented in vertebrates’ genomes)

- promoter regions unmethylated CpG islands

- UCSC genome browser demo for CpG islands and DNA methylation data

When genomes become methylated during development?

Two stages during gametogenesis after implantation

demethylation-remethylation in these stages (active demethylaiton)

de novo methylases are very active in these

stages!!!

DNA methylation during Development

blastocyst

fertilization

meiosisPGC

Retrotransposons

Met

hyla

tion

(%)

P1 promoter

Promoter (developmental genes)

Promoter (CpG island)

100

50

0

Met

hyla

tion

(%)

100

50

0

What triggers DNA methylation or protection from methylation?

Triggers1) Histone modifications plants and fungus -> H3K9me and RNAi mammals -> HKMTs for H3K9 and H3K272) antisense transcriptionProtectors1) Transcription factor-binding2) Transcription during early embryogenesis

What are the consequences of DNA methylation?

Earlier experiments with methylated reporter no expression -> repression!5-azacytidine treatment -> de-repress genes in X -> repression!

This repression activity could be titrated out by adding extra non-specific methylated DNA

-> presence of factors binding to mCpG!

How methylated DNA becomes transcriptionally repressed?

-methyl-CpG-binding protein MeCP2 MeCP2-KO neurological defects Rett syndrome in humans

MBD1 no obvious phenotypeMBD2 high affinity to mCpG NuRD (mi-2) co-repressorMBD3 no affinity to mCpG NuRD co-repressor, emb lethalMBD4 DNA repair T:G mismatch glycosylase MBD-KO high CpG mutationKaiso zinc-finger protein

MBD in action

Why genomes need to get methylated?

• DNMTs-KO, ICF patients -> chromosomal aberrations (fusion, breakage, aneuploid)

-> accurate chromosome segregation -> genomic stability by repression retroposons

Evolution of DNA methylation as a host defense mechanism!

DNA methylation as an epigenetic materials

• Need to be transmitted faithfully during mitosis and meiosis (possibly along with DNA)

-> CpG methylation is transmitted in a semi-conservative manner due to its palindromic nature through DNMT maintenance enzyme.

• Need to store information for cells (cellular memory)

-> cell need to have system for writing and reading this store information.

-> Writer (DNA methylases) vs Reader (MBDs)