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 meiosis PGC Retrotransposons Methylation (%) P1 promoter Promoter (developmental genes) Promoter (CpG island) Methylation (%)

What triggers DNA methylation or protection from methylation? Triggers 1) Histone modifications plants and fungus -> H3K9me and RNAi mammals -> HKMTs for H3K9 and H3K27 2) antisense transcription Protectors 1)Transcription factor-binding 2)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 phenotype MBD2 high affinity to mCpG NuRD (mi-2) co-repressor MBD3 no affinity to mCpG NuRD co-repressor, emb lethal MBD4 DNA repair T:G mismatch glycosylase MBD-KO high CpG mutation Kaiso 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)