The chromodomain helicase DNA binding protein 4 (CHD4) is an ATP-dependent DNA translocase that regulates chromatin structure in most or all tissues in complex animals. Despite its prominence, the mechanisms that regulate CHD4 remain unclear. It is known, however, that CHD4 can interact with acetylation sites on histones, as well as with a family of highly conserved regulators of gene expression – the bromodomain and extra-terminal domain (BET) proteins.
This project aimed to determine the effect of nucleosome acetylation and BET protein binding on CHD4 binding affinity and activity. This required the ability to produce nucleosomes with select acetyl-lysine (AcK) residues in order to ascertain which acetylation profiles are important for CHD4 activity. To achieve this selectivity, we used amber stop codon suppression to incorporate an AcK mimic, trifluoroacetyl-lysine (TFAK) directly into histone subunits expressed recombinantly in bacteria. We were able to use those purified histones to assemble nucleosomes with chosen acetylation profiles for remodelling assays with CHD4.
We present the results of two types of assays – gel-based electrophoretic mobility assays (EMSA) and fluorescence resonance energy transfer (FRET) assays – to show that the three ubiquitous BET proteins play functionally distinct roles in regulation of CHD4 activity. These data reveal substantial differences in protein interactions in lieu of strong homology and make headway into understanding the mechanisms that govern transcriptional regulation.