表观遗传学研究方法.pptVIP

Methods for studying epigenetic modifications Methods for studying epigenetic modifications DNA methylation– bisulfite sequencing Histone modification chromatin immunoprecipitation (ChIP) DNA adenosine methylation identification (DamID) siRNA production – deep sequencing TTCGCCGACTAA TTCGCCGAuTAA Bisulfite treatment differentiates cytosine and methylcytosine Bisulphite treatment TTCGCCGACTAA No treatment TTCGCCGACTAA TTCGCCGAuTAA Methyl-cytosine When DNA is bisulfite treated, unmethylated cytosine is converted to uracil. Methylcytosine is not affected. O N NH2 N ~ O N NH2 N ~ CH3 cytosine 5-methylcytosine O N NH2 N ~ CH3 O N O N ~ uracil 5-methylcytosine Bisulfite treatment Bisulfite treatment differentiates cytosine and methylcytosine Bisulphite treatment TTCGCCGACTAA No treatment TTCGCCGACTAA TTCGCCGAuTAA TTCGCCGACTAA TTCGCCGATTAA Methyl-cytosine After bisulfite treatment, unmethylated Cs are read as T and so differ in the treated and untreated samples. By contrast, methyl-C is read as C and is the same as the reference sequence. Chromatin Immunoprecipitation (ChiP) Modified histone (e.g. H3K4me) Cross-link DNA to histone Cross-link DNA to histone Shear DNA to smaller pieces; add specific antibody to modified histone; affinity purify antibody and bound histone/DNA complex. ` Chromatin Immunoprecipitation (ChiP) Cross-link DNA to histone Remove crosslink, purify DNA, examine by PCR, sequencing or microarray Chromatin Immunoprecipitation (ChiP) Shear DNA to smaller pieces; add specific antibody to modified histone; affinity purify antibody and bound histone/DNA complex. ` DNA adenine methylation ID (DamID) GATC Dam methylase from E. coli Protein of interest (e.g. LHP1) A fusion protein is made of Dam and the protein of interest The fusion protein binds to selected regions of chromatin (e.g. H3K27me3) and methylates adenines at nearby GATC sites Dam is an adenine methyltransferase with specificity for the sequence GATC Methylation can be detected by methyl