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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, here 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 comprising 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 to impart BRD2-like activity onto BRD4S. In summary, our results identify distinct BET protein domains that regulate protein turnover and biological activities.

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, here 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 comprising 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 to impart BRD2-like activity onto BRD4S. In summary, our results identify distinct BET protein domains that regulate protein turnover and biological activities.

In Brief Chromatin reader protein BRD4 is thought to bookmark mitotic chromatin to propagate transcriptional states across mitosis. Behera et al. profiled and perturbed mitotic BRD4 chromatin occupancy to show that BRD4 is dispensable for this process. Instead, BRD4 mitotic chromatin association is likely a mere reflection of mitotically stable histone marks., SUMMARY Global changes in chromatin organization and the cessation of transcription during mitosis are thought to challenge the resumption of appropriate transcription patterns after mitosis. The acetyl-lysine binding protein BRD4 has been previously suggested to function as a transcriptional “bookmark” on mitotic chromatin. Here, genome-wide location analysis of BRD4 in erythroid cells, combined with data normalization and peak characterization approaches, reveals that BRD4 widely occupies mitotic chromatin. However, removal of BRD4 from mitotic chromatin does not impair post-mitotic activation of transcription. Additionally, histone mass spectrometry reveals global preservation of most posttranslational modifications (PTMs) during mitosis. In particular, H3K14ac, H3K27ac, H3K122ac, and H4K16ac widely mark mitotic chromatin, especially at lineagespecific genes, and predict BRD4 mitotic binding genome wide. Therefore, BRD4 is likely not a mitotic bookmark but only a “passenger.” Instead, mitotic histone acetylation patterns may constitute the actual bookmarks that restore lineage-specific transcription patterns after mitosis., Graphical Abstract

Erythroid transcription factors (TFs) control gene expression programs, lineage decisions, and disease outcomes. How transcription factors contact DNA has been studied extensively in vitro, but in vivo binding characteristics are less well understood as they are influenced in a reciprocal manner by chromatin accessibility and neighboring transcription factors. Here, we present a comparative analysis approach that takes advantage of non-coding sequence variation between functionally equivalent erythroid cell lines to conduct an in-depth analysis of erythroid TF binding profiles and chromatin features. Specifically, we analyzed ChIP-seq datasets to identify millions of genetic non-coding variants between the mouse erythroleukemia cell line (MEL), a GATA1-inducible erythroid progenitor cell line (G1E-ER4), and primary murine erythroblast cells. We found that while these cell lines are highly positively correlated in chromatin features, larger differences in TF binding intensity are correlated with higher degrees of genetic variation between cell lines. We next examined discriminatory genetic variants between the cell lines that are located in ChIP-seq peaks of the erythroid transcription factor GATA1. Hundreds of such variants fall within GATA1 motifs. Differential GATA1 binding intensities associated with the variants revealed nucleotide positions that contribute most to in vivo GATA1 chromatin occupancy and identified which alternative nucleotides are most likely to disrupt binding. Notably, this additional information about GATA1's in vivo nucleotide binding preferences improved prediction of GATA1 binding sites genome-wide. We applied similar approaches to determine the bp-resolution in vivo binding preferences of TAL1/SCL and CTCF. We additionally identified thousands of discriminatory genetic variants within GATA1 sites that fall outside canonical GATA elements but within binding sites of other known TFs. Association of these variants with differential GATA1 binding intensities revealed that the hematopoietic transcription factors TAL1/SCL and KLF1 positively regulate GATA1 chromatin occupancy. Strikingly, we identified a number of motifs not previously implicated in cooperating with GATA1 that positively impact GATA1 chromatin binding. Notably, we also defined motifs associated with negative regulation of GATA1 chromatin occupancy. Applying a similar analysis to TAL1/SCL and CTCF revealed additional motifs involved in regulating the chromatin occupancy of these TFs. Finally, we associated discriminatory genetic variation between erythroid cell lines with large changes in sub-kb-scale DNase hypersensitivity. We found that single base pair substitutions within or near a number of erythroid TF motifs, including that for the RUNX family of nuclear factors, are strongly associated with changes in chromatin accessibility. Our findings use novel methods in comparative ChIP-seq and DNase-seq analysis to reveal new insights about the genetic basis for erythroid TF chromatin occupancy and chromatin accessibility. Disclosures No relevant conflicts of interest to declare.