DNA methylation and epigenetics

DNA methylation in Eukaryots Non-methylated DNA : Saccharomyces cerevisiae, Drosophila, Caenorhabditis, …. mammals: 3-8% cytosine residues, CpG sequences (u embryonal stem cell also non-CG, in brain alsohydroxymethylC) plants : 25-30% cytosine residues (~ genome size) - mainly in repetitive heterochromatinic regions (non-methylated islands of gene-rich DNA in large genomes) - metC (CG) even in coding sequences of transcribed genes

Epigenetics Mitoticaly and/or meioticaly herritable changes in gene function, which are not based on changes in primary DNA sequence Mechanism:DNA methylation histon modifications (acetylation, methylation,..) - Regulation of gene expression in development, parental imprinting - Transgene and TE silencing, - Paramutations, - Conformational changes in chromatin (keeping heterochromatin structure)

Mechanism of DNA methylation - in Eukaryots metylation of cytosin on C5(also adenin in bacteria) N N N H C H O CH 3 5-methyl cytosine N N O H T O CH 3 thymine + H 2 O - NH 3 N N N H C H O cytosine + SAM „hot spot of mutagenesis“

Feature Value DNA molecule Chr.1Chr.2Chr.3Chr.4Chr.5SUM Length (bp) Top arm (bp) Bottom arm (bp) Base composition (%GC) Overall Coding Non-coding Number of genes Gene density (kb per gene ) Average gene Length (bp) Average peptide Length (bp) Exons Number Total length (bp) Average per gene Average size (bp) Number of genes With ESTs (%) Number of ESTs 29,105,111 14,449,213 14,655, , , ,482 8,772, ,522 19,646,945 3,607,091 16,039, , , ,631 5,100, ,989 23,172,617 13,590,268 9,582, , , ,570 6,654, ,732 17,549,867 3,052,108 14,497, , , ,073 5,150, ,605 25,53,409 11,132,192 14,803, , , ,226 7,571, , ,409,949 25, ,982 33,249, ,773 Genom Arabidopsis - statistics

DNA methylation in plants – target sequences - both symmetric (palindromatic) sekvence and asymmetric sequences: 5’ CG CNG C---C------G--C---- 3’ 3’ GC GNC G---G------C--G---- 5’ Symmetric – methylation pattern easily kept after replication hemimethylated state = signal for maintainance methylation -----C * G C * NG C * C * G G C G NC G G C * CG CNG C GC * GNC * G Asymmetric – after replication de novo methylation (by another signal – histon modification or presence of complementary sRNA)

Methylation analysis - hydrogensulphite sequencing (NaHSO 3 ) 1. denaturation CC C C CC---C------C C-- 2. modification of Cyt to Ura (5metC remains unchanged) CC C U UU---C------U U-- 3. shotgun sequencing, specific PCR + sequencing original sequence CC C C CC---C------C C G G GG---G------G G-- modified sequence CC C U UU---C------U U G A AA---G------A A--

Analysis of methylation in Arabidopsis „Chromosome epigenetic landscape“

Analysis of DNA methylation in Arabidopsis Strong methylation in heterochromatin near centromeres MetC even in coding sequences of expressed genes! (more than 30 % genes, middle expression) Metylation in promotors of developmentally regulated genes (less than 5 % genes)

Plant cytosine 5-methyltransferases MET1 (metyltransferase) CMT3 a CMT2 (chromomethyltransferases) DRM2 (a DRM1) (domain rearranged methyltransferase)

MET1 (metyltransferase1) - related to mammalian Dnmt1 - maintenance methylation of symmetric CG - asociated with replication - involvement of nucleosom remodeling complex (DDM1) (in heterochromatin) Signal – hemimethylated CG (CpG) ----C * G C * HG C * C * G G C G DC G G C * CG CHG C GC * GDC * G

CMT3 (chromomethyltransferase3) - unique for plants and fungi - related to mammalian Dnmt1 + chromodomain (meK) - mainly constitutive heterochromatin (repeats, retroTEs) - „maintenance“ methylation of CHG Activity associated with methylation of histon H3K9 ----C * G C * HG C * C * G G C G DC G G C * CG CHG C GC * GDC * G

CMT2 (chromomethyltransferase2) - related to CMT3 (chromodomain - meK) - mainly constitutive heterochromatin (repeats, retroTEs) - „maintenance“ methylation of CHH Activity associated with methylation of histon H3K9 ----C * G C * HG C * C * G G C G DC G G C * CG CHG C GC * GDC * G

DRM2 a 1 (domain rearranged methyltransferase) - related to mammalian Dnmt3, but with rearranged domains, - de novo methylation of all sequences (CG, CNG, asymmetric) - DRM1 – early seed development, DRM3 inactive (but necessary) - maintenance methylation of asymetric and CNG - regulation of gene expression, silencing Targed sequences determined by the presence of complementary siRNA C * G C * HG C * C * G G C G HC G G C * CG CHG C GC * GHC * G

RNA directed DNA methylation AGO4 – ARGONAUTE (carrying ss small RNA) DRM2 – de novo metyltransferase DRD1 – chromatin remodelling protein PolV – special RNA polymerase

Maintenance methylation of DNA Heterochromatine with histone H1 - middle part of longer TE (coding region) - chromatine remodelling factor DDM1 required - methylation driven with: H3K9me2 (CHH, CHG) – CMT2, CMT3 CG hemimethylation – MET1 Chromatine without histone H1 - TE borders, short TE, genes - chromatine remodelling factor DRD1 required - methylation driven with: siRNA (RdDM) – DRM2 (RdDM – H3K9me2, demet. H3K4, see later ) CG hemimethylation – MET1

Zemach et al Maintenance methylation of TE

DNA demethylation 1) by inactivation of maintenance enzymes after replication (MET1, CMT2, CMT3, histon methylatransferases) 2) DNA glycosylases ( cleavage of methylcytosine, cytosine incorporated by reparation ) ROS1 (REPRESSOR OF SILENCING) – somatic cells (RNA binding ROS3 required – unknown function) DML2, DML3 (DEMETER-LIKE) – somatic cells DME1 (DEMETER) – parental imprinting (endosperm)

DNA demethylation ROS1, DML2, DML3) - mutation in „demethylation“ genes → increased methylation in 3’ and 5’ UTR - protection of genes (promoters) against methylation (alt. modulation of transcription) In methyltransferase mutants – demethylation inhibited (feed back regulation) ros1 dml2 dml3 WT methylation increase in methylation

Modulation of DNA methylation - inactivation of methyltransferase genes MET1, DDM1, CMT3 - application of 5-azacytidine – inhibits MET1 - dihydroxypropyladenine (DHPA) – modulates [SAM] Fenotypic changes connected with demethylation: - variable: from negligible to strong (caused by activated TE) - sex reversion reversion, - cycloidea phenotype (hypermethylation)

Reactivation of silenced (trans)genes by 5-azacytidine - inhibitor of MET1 (5-azacytidine, AzaC) hemiMet? deMet Nocarová, Fischer, unpubl. + AzaC Demethylated - reactivation often only transient!

Interpretation of DNA methylation steric consequences of cytosine methylation - changes on the histone level - posttranslational modifications of histones - SRA domains: metylace (SUVH family), - Metyl CpG-binding d.: deacetylation (HDAC) - presence of histone forms (H2A.Z) changes in binding of interacting proteins - regulators of transcription - proteins involved in structural modifications of chromatin

example: MBD proteins (metyl-CpG-binding domain) - plant MBD proteins lack TRD (transcription repression domain) - specificity of binding affected with other interacting proteins - AtMBD5 interacts also with C*HH - MBD probably interact with histone modifying proteins (HDAC = histon deacethylases → inactivation) Model of chromatin compression by dimerazing AtMBD7 protein

- similar decrease in H3K9me2 in kyp a cmt3 Autocatalytic loop CHG methylation (CMT3) and H3K9 methylation(KYP, KRYPTONITE) - transcription induces demethylation H3K9me2 by IBM1) ROS1/ ROS3 ??? x DRM2

DNA methylation and chromatin - overview - correlation between DNA hypermethylation and hypoacethylation of histons and chromatin condensation (later replicated DNA) - inhibition of DNA methylation (MET1) and histone deacethylation (AtHD1) cause similar changes - presence of histone H2A.Z (promoters of active genes) mutually exclusive with DNA methylation !

- H3K9 di(tri-)methylation (KYP) signal for CHG methylation - CHG methylation signal for H3K9 methylation - H3K9 demethylation (IBM1) likely induced by transcription - H3K4 trimethylation typical for euchromatin, induced by transcription DNA methylation and chromatin - overview

Maintenance of epigenetic state - overview inactive chromatin - MET 1 – maintenance methylation of C*G - KYP (SUVH5,6) – H3K9me2  CMT3 - C*HG (CMT2 – C*HH) - DRM2, (DRM1) – maintenance met. CHH  complementary siRNA Aktivní chromatin - IBM1 – demethylation of H3K9  transcription - ROS1/ROS3, DML2, DML3 – demetC  transcription, sRNA?

Interconnections among DNA methylation and chromatin structure metC = d sDNA modification histone post- translational modifications and forms binding of interacting proteins TF, RNA pol., MBD, …

Roles of DNA methylation - herritable (mitotic and partially even meiotic modulation of transcription – gene expression - defence against invazive DNA (TE) - regulation of gene expression (ontogenesis, differenciation, cell memmory, stress reaction) - parental imprinting - modulation of chromatin structure (both - cause and result) - timing of DNA replication - regulation of homologous recombination (meiosis) - tool of evolution - genomes of polyploids (methylation changes) - new genes (mutagenesis of pseudogenes)