Fig. 1

Optimization of mononucleosome isolation and sequential ChIP. a Mononucleosomes were isolated from the indicated cell lines by sucrose gradient (S.G.), and assessed for purity by native PAGE analysis. Dinucleosomal fragments from NCCIT (NCCIT-Di) were included for size reference. b A quick protocol (Q.P.) was developed (see Experimental Procedures), and resulting mononucleosomes from NCCIT cells were assessed for purity by overloading of DNA onto an agarose gel. c Top shows a schematic representation of the TSS region of MLH1 with placement of primer pairs. Below, PCR analysis of mononucleosomes (mono, from Q.P.), dinucleosomes (di, from S.G.) and genomic DNA (gDNA) of NCCIT cells. d Mononucleosomes isolated from RKO and SW480 (using Q.P.) were subjected to ChIP, MLH1 promoter analysis (primer set described above, part C). e Mononucleosomes isolated from RKO and SW480 (using Q.P.) were subjected to sequential ChIP using indicated combinations of anti- H3, H3K4me3 and H3K27me3 immunoprecipitation (IP), and the MLH1 promoter was analyzed by primer set-B described above. f Amplicons depicted in E was quantitated by ImageJ (all data expressed as percentage of input and normalized to IgG background). g Mononucleosomes from NCCIT cells (using Q.P.) were subjected to single or sequential ChIP (precipitation of H3K27me3 followed by H3K4me3). Chromatin patterns of CDO1, SFRP1 and SOX17 were assessed between treatment groups and compared to MYC as a control. h The schematic depicts isolation of mononucleosomes and individual histone peptides, the latter as depicted by peaks in the HPLC analysis below. i Sequential IP of mononucleosomes (using Q.P.) or purified histone peptide substrates from NCCIT cells were analyzed by dot blot using anti-H3K4me3 antibody (left panel shows sequence of IP)