Egger 2023 with verified questions and answers

primary epigenetic marks = sequence specific - intrinsic DNA sequence - sequence-specific TFs - noncoding RNA secondary epigenetic marks = sequence independent - readers of DNA methylation - noncoding RNA methylation in bacteria - methylation A or C to identify & degrade invading DNA & track mismatch methylation in bacteria functions - timing of DNA replication - partitioning nascent chromosomes to daughter cells - repair of DNA - timing of transposition & conjugal transfer of plasmids + DNA methylation protects host genome from destruction Methylation in bacteria enzymes Dam: adenine methylation Dcm: cytosine methylation SeqA: neg. regulator of replication initiation, binds hemimethylated OriC methylation in different species - except in yeast - in vertebrates throughout the genome - in plants & invertebrates: mosaic methylation, on repetitive DNA & actively transcribed sequences - at CpG in mammals signals that direct DNA methylation - H3K9 methylation de novo in plants - interspersed repeats - RNAs in plants and pRNAs - Chromatin modifiers and remodellers role of DNA methylation in mammals - defense against foreign DNA - X-chromosome inactivation - imprinting - immune response (self vs bacteria) - gene regulation during differentiation highly methylated sequences in mammals satellite DNA repetitive sequences (transposons, non-repetitive intergenic DNA, exons) CpG islands - at least 500bp - 55% G+C - in 60% of human genes promotors - methylated in x-chromosome and imprinted genes shores = regions of genome that reside up to 2kb from CGIs shelves 2-4 kb away from CGIs mutagenicity of methylated cytosines - methylated cytosines may be bound by methyl DNA-binding proteins (MBDs) - unbound 5-methylcytosine is prone to spontanous mutation through deamination - Thymine DNA methylation reaction deoxycytidine + S-adenosyl-L-methionine (SAM) DNMT 5-methyl-cytidine + S-adenosyl homocysteine SAH DNMT1 - preferance for hemimethylated DNA - maintainance of DNA methylation - located at replication fork - KO: genomic loss of DNA methylation, abnormal expression of imprinted genes, activation of retrotransposons DNA methyltransferases domain CXXC domain binds unmethylated CpGs ATRX domain binds H3K4me0 UHRF1 definition binds to hemi-methylated DNA during S-phase and recruits the main DNA methyltransferase gene, DNMT1, to regulate chromatin structure and gene expression - catalyzes multiple mono-ubiquitylation of H3 at K14/K18/K23 UHRF1 domains 1. UBL - ubiquitination activity 2. Tandem Tudor Domain TTD - allows to bind to di-/trimethylated H3K9 3. Plant homeodomiain PHD - recognizes H3R2 & determines binding specifity of UHRF1 to H3K9me2/3 4. Set and RING associated (SRA) domain - facilitates DNA methylation and histone modifications by recruiting DNMT1 and HDAC1 5. Really Interesting new gene RING - intrinsic E3 ligase activity toward histones and nonhistone proteins UHRF1 function - multi-domain protein - recruits DNMT1 to replication foci - stimulates catalytic activity of DNMT1 - binds hemimethylated DNA - binds H3K9me3 - RING domain ubiquitinates H3 at K14, K18 and K23 retroviral DNA & methylation noninfectious in ES cells due to silencing of viral genes by DNA methylation in somatic cells - lack of DNA methylation - infections de novo methylatransferases DNMT3a/3B - no preference for hemimethylated DNA - KO - imprinting defects (3A), demethylation of majo satellite repeats (3B) ICF syndrome = immunodeficiency centromeric instability & facial abnormalities - loss of methylation in repetetive elements - pericentromeric heterochromatin - autosomal recessive DNMT3 & chromatin interaction - binding of H3 tail - stimulates DNMT3A activity) - PWWP domain binds H3K36me3 - intragenic DNA methylation deposited by DNMT3B - inhibits spurious transcription initiation DNMT3L lacks catalytic activity, but stimulates DNMT3A and DNMT3B DNMT3C duplication of Dnmt3b gene in mice, with specific role in the silencing of retrotransposons in the development of male germ cells DNMT2 methylation of tRNAs DNMT3 enyzmes capable of non-CpG methylation (CAG in ESCs, CAC in neurons) CG-specific readers = methyl-CpG binding proteins - MeCP2 - MBD1-4 Rett Syndrome - x-chromosomal neurodevelopmental - cause for mental retardation in woman- impaired motor skills, abnormal breathing, hand wringing - mostly de novo heterozygous mutation in MeCP2 gene MeCP2 mode of action binds mCG and mCAC in the brain to restrain transcription of genes critical for neuronal function MeCP2 function recognizes cytosine methylated trinucleotide and di-nucleotide sequences to tune transcription in the mammalian brain trans effect recruitment of chromatin-modifying enzymes (HDACs & DNMTs) via methyl-DNA binding protein - local chromatin condensation & transcriptional repression CG-specific readers - non-methyl-CpG binding proteins CXXC recognize non-methylated CpGs & target proteins containing this domain CFP1, KDM2A, KDM2B - help keep unmethylated state of CpG islands CFP1 binding correlates with H3K4me3 - active mark associated with transcriptional activation in CpG islands present in absence of transcription demethylation by oxidation - 5hmCG, - 5fCG - 5aCG de novo methylation enzymes DNMT3A DNMT3B DNMT3L 5-hydroxymethylcytosine by TET family, intermediate for DNA demethylation DNA demethylation ways 1. hydroxylation by TET family to form 5hmC oder 5fC 2. 5mC or 5hmC deamination ny AID/APOBEC - 5-mU or 5-hmU 3 differen erasers 1. TET family 2. AID/APOBEC 3. BER DNA glycosylase (base excision repair) TET family hydroxylation & subsequent oxidation of mC ten-eleven translocation AID/APOBEC family - deaminates 5mC or 5hmC - important function for AB class switching & hypermutation in B-cells (AB diversity) BER DNA glycosylases (base excision repair) - mediate DNA repair active DNA demethylation ways 1. signal response 2. in early mammalian development 3. in tissue specific differentiation active demethylation/ signal response 1. BDNF, FGF1 demethylation in postmitotic neurons 2. IL-2 stimulation of T-cells 3. Estrogen stimulation of breast cancer cells early mammalian development active demethylation - paternal pronuclei after fertilization (TET3 dependent) - mouse primordial germ cells (PGCs) tissue specific differentiation Skeletal muscle cells have the capacity to demethylate the MyoD promoter in fibroblasts, when fused to heterokaryons methods for DNA methylation mapping 1. methylation sensitive restriction enzymes (MSRE) 2. affinity purification based approaches 3. bisulfide modification MSRE digestions with HpaII (blocked by CG methylation) or Msp1 (not blocked by CG methylation) affinity purification MeDIP (AB against methylated cytosine) and MBD affinity capture denaturation of genomic DNA fragments, then affinity purification to get methylated DNA bisulfide modification deamination of unmethylated cytosines to uracil - protection of methylated cytosines - sequence polymorphism detected by sequencin, PCR or restriction bisulfide conversion steps 1. sulphonation 2. hydrolytic deamination 3. alkali-desulphonation Ilumina Infinitum 450K 1. U bead type matches unmethylated CpG site 2. M bead type matches methylate site RRBS digestion with CpG specific enzyme (MspI) - enrich for CpG sites (promotors, islands, enhancers) - needs lower sequencing coverage polycomb & tritorax group proteins cell cycle control + proliferation stress response + checkpoint activation cellular memory X-chr inactivation cell fate determination stem cell self-renewal tumorigenesis PcG proteins - have a key role in cell identity and the maintenance of a proper differentiation program during development - complexes dynamically regulate gene hierarchy during cell fate choice - repress TFs in ESCs - repress Hox genes stem cells & chromatin regions they have bivalent chromatin regions carrying H3K4me and H3K27 me - regulated by PCR2 & MLL2 complexes - poised promotors lncRNAs = long noncoding RNAs - recruit chromatin modifiers Beispiele für lncRNAs - hotair (Hox transcript antisense RNA) - repressor of HoxD, recruits PRC2 to genomic loci, overexpressed in cancer, promotes metastasis - xist - x-chromosome inactivation, recruits PRC2, H3K27me3 - coldair - plants, cold-induced flowering via PRC2-recruitment - ANRIL - recruits PRC1/PRC2 to silence INK4a via H3K27me, important for p53 regulation - Air, Kcnq1ot1 rcruit G9a (H3K9me) in cis, regulate imprinting - pRNA recruits DNMT3b to rDNA genes to repress transcription of ribosomal RNA Genomic DNA methylation - 70% of DNA in somatic tissue is methylated at CpG dinucleotides - exception: CpG islands highly methylated sequences satellite DNA repetitive sequences like transposons non-repetitive intergenic DNA exons of genes CpG Island definition - at least 500bp length - 55% GC (0.65) - 60% of genes have CpG island promoters DNMT1 again = maintenance DNA methyltransferase - preference for hemimethylated DNA - KO - lethal at E.9.5, abnormal expression of imprinted genes, activation of retrotransposons bisulfide conversion RRBS = reduced representation bisulfite sequencing - digestion with CpG specific enzyme (MSpl) - enrich CpG sites (CpG islands, promotors, enhancers) - advantage: lower sequencing coverage needed TrxG functions - cell cycle control + proliferation - tumorigenesis - general transcriptional ACTIVATION - stem cell self-renewal - cell fate det. - X chromosome inactivation - cellular memory PcG cassical: targeting of PRC2 leads to methylation of H3K27 - recognized by Cbx subunit of PRC1 - catalyzes monoubiquitination of H2A at Lys 119 Trithorax complexes mono- & di- & trimethylation on H3K4 at active promotors PcG in early development - PRC1/ 2 KO are early embryonic lethal - have key role in cell identity & maintainance of proper differentiation program during development