Chromatin and epigenetic memory

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

1. Brd4 promotes cell proliferation by preventing R-loop mediated DNA damage BRD4 binds to acetylated histones and promotes transcription of many genes. Recent studies show that BRD4 drives proliferation of many types of cancer cells. BRD4 inhibitors that block acetyl-histone binding or degrade BRD4 are reported to arrest cancer growth. Thus, some BRD4 inhibitor are used as anti-cancer drugs. However, the role of BRD4 in normal cell growth has remained unclear. Here, we investigated the role of BRD4 for proliferation of normal mouse embryonic fibroblasts, using conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells: they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. Our results indicate that BRD4 epigenetically marks genes controlling DNA damage and cell cycle genes and serves as a master regulator of normal cell growth. 2. Dynamics and Decay kinetics of the histone variant H3.3 as analyzed by live cell imaging. Incorporation of the variant histone H3.3 into the genome occurs in conjunction with gene expression throughout cell cycle. However, its precise regulatory mechanisms remain elusive. Traditional methods such as Chromatin Immunoprecipitation provide static views of H3.3 distribution, lacking dynamic insights. To gain insight into behavior of H3.3 in live cells, we conducted fluorescence recovery after photobleaching (FRAP) and examined mobility of H3.3 in mouse embryonic fibroblasts. The use of SNAP tag system allowed us to study mobility of all H3.3 pool and newly synthesized H3.3 pool. 3.3 was much more mobile as compared with the core histone H3.1 throughout 8 hours of FRAP assay. Importantly, H3.3 mobility was abolished when transcription was globally inhibited. Furthermore, deletion of histone chaperons HIRA and NSD2 substantially inhibited the mobility. . We also investigated the turnover i.e., decay dynamics, of H3.3 in live cell imaging over two days. Similar to the mobility, H3.3 decay was markedly delayed upon transcriptional inhibition and deletion of HIRA and NSD2. Our results revealed H3.3 dynamics and turn over are driven by ongoing transcription and dependent on chaperon mediated H3.3 loading on chromatin.