MLL3 ChIP sequencing in murine and human HCC cells

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3/KMT2C) histone methyltransferase occur in a range of solid tumors and heterozygous deletions encompassing MLL3 occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Mll3 cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduc...,

Histone ChIP was performed as previously described (Lee et al., 2006). Briefly, cell samples were cross-linked in 1% formaldehyde for 10 minutes, and the reaction was stopped by addition of glycine to 125 mM final concentration. Fixed cells were lysed in SDS lysis buffer, and the chromatin was fragmented by sonication (Covaris). Sheared chromatin was incubated with a final 10 μg/mL concentration of antibodies against either H3K4me3 (Abcam, ab8580, Lot:GR164706-1), H3K27ac (Abcam, ab4729, Lot:GR200563-1), H3K4me1 (Abcam; ab8895, Lot:GR114265-2) or normal rabbit IgG (Abcam, ab46540) at 4 °C for overnight. Antibodies were recovered by binding to protein A/G agarose (Millipore), and the eluted DNA fragments were used directly for qPCR or subjected to high-throughput sequencing (ChIP-Seq) using a HiSeq 2000 platform (Illumina). High-throughput reads were aligned to mouse genome assembly NCBI37/mm9 as previously described (Barradas et al., 2009). Reads that aligned to multiple loci in the m...,