Structure-Function Analysis Of Bet Proteins In Transcription

The widely expressed bromodomain and extraterminal motif (BET) proteins bromodomain-containing protein 2 (BRD2), BRD3, and BRD4 are multifunctional transcriptional regulators that bind acetylated chromatin via their conserved tandem bromodomains. Small molecules that target BET bromodomains are being tested for various diseases, but typically do not discern between BET family members. Genomic distributions and protein partners of BET proteins have been described, but the basis for differences in BET protein function within a given lineage remains unclear. By establishing a gene knockout–rescue system in a Brd2-null erythroblast cell line, we compared a series of mutant and chimeric BET proteins for their ability to modulate cell growth, differentiation and gene expression. We found that the BET N-terminal halves bearing the bromodomains convey marked differences in protein stability, but do not account for specificity in BET protein function. Instead, when BET proteins were expressed at comparable levels, their specificity was largely determined by the C-terminal half. Remarkably, a chimeric BET protein comprised of the N-terminal half of the structurally similar short BRD4 isoform (BRD4S) and the C-terminal half of BRD2 functioned similarly to intact BRD2. We traced part of the BRD2-specific activity to a previously uncharacterized short segment predicted to harbor a coiled coil (CC) domain. Deleting the CC segment impaired BRD2’s ability to restore growth and differentiation, and the CC region functioned in conjunction with the adjacent ET domain (ETCC) to impart BRD2-like activity onto BRD4S. We found that despite having different functions, BET proteins share similar genome-wide chromatin binding profiles. Neither exchanging the bromodomain-containing N-terminal nor the ETCC-bearing C-terminal regions of BET proteins alters their distribution. Rather, BRD2-specific function may in part be mediated by ETCC interactions with the RNA polymerase II associated factor and casein kinase II complexes. In summary, our results identify distinct BET protein domains that regulate protein turnover and biological activities