How genes are switched on or off in a controlled manner remains a fundamental question in the field of molecular biology. In eukaryotes, gene regulation involves physical restructuring of chromatin, which consists of DNA wound around octamers of histone proteins. Several core mechanisms have already been identified that partially explain how chromatin can be restructured to alter gene expression. Such mechanisms include covalent modifications to histone proteins and ATP-dependent remodelling of chromatin itself. The BET (Bromodomain Extra-Terminal domain) proteins bind acetylated histone peptides and localise to acetylated chromatin in vivo. They represent major therapeutic targets due to their role in aberrant gene regulation. We have identified the specific acetylation marks necessary to recruit BRD4 to nucleosomes in vitro by conducting pulldowns with endogenous nucleosomes and assembling modified recombinant nucleosomes. We have also characterised how BRD4 binds the nucleosome using a combination of binding assays and cryo-electron microscopy.
The BET proteins seem to recruit transcription factors to regions of acetylated chromatin to mediate some effect on gene transcription. One such transcriptional regulator which has been identified as an interaction partner of the BET proteins is CHD4 (Chromodomain Helicase DNA-binding protein 4), a ubiquitous, but poorly understood ATP-dependent chromatin remodeler found at the promoters of all active genes. The interaction between CHD4 and the BET proteins presents an opportunity to better understand how chromatin modifications can crosstalk with chromatin remodelling. We have used a range of tools including fluorescence and gel-based remodelling assays, binding assays, and cryo-electron microscopy to probe the activities of CHD4 and the BET proteins both together and in isolation of each other. This work serves to deepen our understanding of gene regulation and to shed light on the roles and mechanisms of two poorly understood groups of transcription factors.