Stonestrom Aaron J, Hsu Sarah C, Jahn Kristen S, Huang Peng, Keller Cheryl A, Giardine Belinda M, Kadauke Stephan, Campbell Amy E, Evans Perry, Hardison Ross C, Blobel Gerd A
Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA; Perelman School of Medicine, University of Pennsylvania, Philadelphia PA; and.
Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA;
Blood. 2015 Apr 30;125(18):2825-34. doi: 10.1182/blood-2014-10-607309. Epub 2015 Feb 18.
Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.
含溴结构域和额外末端基序蛋白（BETs）的抑制剂正在被评估用于癌症和其他疾病的治疗，但关于BET蛋白在正常生理过程中的功能仍有许多有待了解之处。我们使用基因组学和遗传学方法，在由GATA1驱动的造血成熟系统中研究BET的功能，GATA1是一种先前已显示与BETs相互作用的乙酰化转录因子。我们发现BRD2、BRD3和BRD4被不同程度地招募到GATA1调控的基因上，其中BRD3结合的GATA1占据位点数量最多。全基因组范围内，药理学上的BET抑制损害了GATA1介导的转录激活，但不影响转录抑制。从机制上讲，BETs促进了GATA1在染色质上的占据，随后支持转录激活。通过结合CRISPR-Cas9介导的基因组工程和shRNA方法，我们观察到单独敲除BRD2或BRD4会减弱红系基因的激活。令人惊讶的是，仅在BRD2缺陷的情况下，BRD3的敲除才会影响红系转录。与BET蛋白之间的功能重叠一致，强制表达BRD3可显著挽救由BRD2缺陷导致的缺陷。这些结果表明，药理学上的BET抑制应在转录激活的不同步骤以及BET家族成员之间重叠功能的背景下进行解读。
