Figure 4
Application of PAT-ChIP to mouse LMD samples. Forty tissue slides were prepared from the lung of K-rasv12 transgenic mouse and subjected to LMD to isolate both normal and tumor cells. Chromatin was immunoprecipitated with the reported antibodies and the DNA analyzed by real-time qPCR for enrichment at specific loci (each sample amplified in triplicate). Enrichments of the amplified sequences are expressed as the ratio between bound and input (percentage). (A) Representative hematoxylin and eosin staining of the lung of one mice in which the expression of the oncogene was induced with 4-hydroxytamoxifen (4-OHT, right panel). (B) Evaluation of chromatin fragmentation by 1.3% AGE and SYBR® Gold staining of DNA purified from unbound fractions after ChIP with the H3K4me3 antibody. (C) Amplification of transcriptionally active (Actb and Gapdh) and inactive (Hapln1 and Col2a1) promoter regions after H3K4me3 immunselection. Mock, no antibody. (D) Amplification of transcriptionally active (Actb and Gapdh) and inactive (Hapln1 and Col2a1) promoter regions, and major satellite, after H3K9me3 immunoselection Mock, no antibody. (E) Amplification of regions located upstream and downstream from the transcription start site (TSS; at the indicated distance from TSS, see also Table 6) of the beta-actin and Gapdh genes, after H3K4me3 immunoselection. (F) Amplification of transcriptionally active (Actb and Gapdh) and inactive (Hapln1 and Col2a1) gene promoter regions, and major satellite sequence, after H3K4me3, H3K27me3, H3K27Ac, and Pol II immunoselections. Mock, no antibody; n.r. Ab, non-related antibody. (G) Amplification of two CTCF binding sites (CTCF-BS of DNA-methyltransferase 3a (Dnmt3a) and DNA-methyltransferase 1 (Dnmt1) genes) and two CTCF unrelated genomic regions as controls (CTCF neg. sequences 1 and 2), after CTCF immunoselection. Mock, no antibody; n.r. Ab, non-related antibody).