Roles of the Histone Chaperone FACT in Drosophila Gene Regulation and Chromatin Architecture

The highly conserved histone chaperone FACT (Facilitates Chromatin Transcription) is thought to contribute to the disassembly and reassembly of nucleosomes in the wake of RNA polymerase II passage through chromatin. In yeast, mutation of FACT subunits leads to decreased nucleosome occupancy at the promoters and transcribed regions of genes. In addition, genetic experiments suggest that yeast FACT plays a critical role in restricting initiation of transcription to promoters, ensuring that only appropriate mRNAs are synthesized. However, FACT’s roles in chromatin biology and transcriptional regulation in higher eukaryotes are not well understood. Using Drosophila S2 cells as a model, I observe that depleting levels of the FACT complex results in aberrant transcriptional regulation of approximately 20% of expressed genes. We hypothesized that FACT-dependent alterations in transcription occur because nucleosome disassembly and reassembly are defective in FACT deficient S2 cells. To address that hypothesis, I monitored the nucleosome positioning using genome-wide micrococcal nuclease protection assays, and the distribution of bulk histones, histone variants, and histone modifications using ChIP-seq. I observed that FACT depletion has little effect on total nucleosome occupancy or positioning or on the distribution of either the histone variant H2Av or the histone 3 lysine 56 acetylation (H3K56ac) mark. On the other hand, the loss of FACT alters the distribution of other histone modifications, including histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 36 trimethylation (H3K36me3), in the promoters and transcribed regions of genes. In particular, H3K4me3, a mark of active promoters, was reduced at promoters and increased in gene bodies of the FACT depleted cells. Furthermore, levels of H3K36me3, a mark of active transcription over transcribed regions, was reduced in gene bodies but increased beyond the polyadenylation site. The levels of total (Rpb3) and transcriptionally engaged Pol II (PRO-seq) were reduced at promoters upon the loss of FACT. Taken together, our results are consistent with the model that FACT contributes to the interplay between chromatin architecture and control of promoter-proximal pausing