EP400 Deposits H3.3 into Promoters and Enhancers During Gene Activation

Purpose: We wanted to know how histone variants H3.3 and H2AZ are deposited into genes and enhancers during gene activation Methods: U2OS cells were harvested at 70% confluency with formaldehyde crosslinking for ChIP-seq without crosslinking for RNA-seq. ChIP DNA were purified through standard chromatin immunoprecipitation using an Pol II, MED26, EP400, H3.3, H2AZ, H3k4me1 and K3K18ac antibodies. Libraries were prepared with a KAPA LTP kit and sequenced using the Illumina HiSeq 2000 platform. Total RNA was extracted with Trizol, digested with DNaseI and further purified by acid phenol. Libraries were prepared with Illumina TruSeq RNA Sample Prep Kits v2 and were sequenced on Illumina HiSeq 2000. Conclusion: Our biochemical and genomics (ChIP-Seq and mRNA-Seq) data show that EP400 contributes to H3.3 deposition in significantly with less of an effect on H2AZ in both genes and enhancers Overall design: Enrichment of EP400, Pol II, MED26, Histone varinat H3.3 and H2AZ, and enhancers marks H3K4me1 and H3K18ac in either Mock and/or EP400 knockdown conditions on chromatin were generated by ChIP-Seq. mRNA profiles under Mock siRNA or EP400siRNA were generated by deep sequencing, using Illumina HiSeq 2000.