Project description:We report the application of chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) for high-throughput profiling of H3K27me3 modifications in sk-hep-1(sk), sk-hep-1-shEZH2 (sks) and LO2 cells. Following standard ChIP procedure, 10 ng of each DNA sample was used for Illumina sequencing. Statistically significant ChIP-enriched regions (peaks) were identified by sk vs sks (p-value threshold of 10-2) or sk vs LO2 (p-value threshold of 10-3). Enriched peaks were annotated by the nearest gene using the newest UCSC RefSeq database. We identify genes modified by EZH2-mediated H3K27me3. This study provides insights into these down stream genes modified by EZH2 regulated H3K27me3 in HCC cell lines.
Project description:SK-UT-1 uterine leiomyosarcomas (Ut-LMS) cells were transduced with a fatty acid synthase (FASN)-containing retroviral vector to recapitulate the “lipogenic phenotype of cancer.” Consistent with this model, forced expression of FASN enhanced SK-UT-1 proliferation, migration, and cellular motion. Further investigation showed FASN promotes trimethylation of H3K9 (H3K9me3) and acetylation of H3K27 (H3K27ac) in SK-UT-1 cells. In contrast, siRNA targeting of FASN in high endogenous FASN expressing SK-LMS-1 Ut-LMS cells inhibits trimethylation of H3K9 and acetylation of H3K27. Palmitate, the predominant fatty acid product of FASN, increased H3K9me3, H3K27ac and H3K27me3 detection in SK-UT-1 cells. FASN promoted histone 3 methylation and acetylation through alteration of histone 3-modifying enzymatic activities (HDAC, HDM, HMT and HAT).ChIP-seq in SK-UT-1-FASN cells with anti-H3K9me3 antibody identified regions of enriched binding compared to vector-only cells. One differentially-enriched gene, CRISP1, was investigated further by ChIP-PCR. The transcriptionally repressive function of H3K9me3 was confirmed in CRISP1. Our results provide mechanistic insight into the pathobiology of the “lipogenic phenotype of cancer.” Here, FASN reprograms the Ut-LMS epigenome through chromatin remodeling to promote the “malignant phenotype.”
