Functions of BET proteins in GATA1-mediated transcription [ChIP-seq]. Mus musculus

Transcription factor GATA1 binding in erythroblasts in the presence and absence of BET inhibitor JQ1, and BET protein BRD3 and BRD4 binding in erythroblasts in the presence and absence of GATA1. Inhibitors of Bromodomain and Extra-Terminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases yet their physiologic mechanisms remain largely unknown. 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 while BRD3 occupied the majority of GATA1 binding sites, BRD2 and BRD4 were also recruited to a subset of GATA1-occupied sites. Functionally, BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Co-activation by BETs was accomplished both by facilitating genomic occupancy of GATA1 and subsequently supporting transcription 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, while depletion of BRD3 only affected erythroid transcription in the setting of BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and partially overlapping functions among BET family members. Overall design: GATA1 null erythroblasts (G1E) conditionally expressing GATA1 as a GATA1-ER fusion protein were induced to express GATA1 by addition of 100nM estradiol for 24 hours. For GATA1 binding experiments this occurred in the absence or presence of 250nM JQ1. For BRD3 and BRD4 occupancy experiments G1E cells were compared to G1E cells with activated GATA1-ER fusion protein.

本数据集聚焦两类结合检测：一是在BET抑制剂JQ1存在与缺失条件下，成红细胞中的转录因子GATA1结合情况；二是在GATA1存在与缺失条件下，成红细胞中的BET家族蛋白BRD3与BRD4结合情况。溴结构域与额外末端基序（Bromodomain and Extra-Terminal motif, BET）蛋白抑制剂目前正被评估用于癌症及其他疾病的临床治疗，但其生理作用机制在很大程度上仍未明确。本研究借助基因组学与遗传学技术，在由GATA1（一种此前被证实可与BET蛋白相互作用的乙酰化转录因子）驱动的造血成熟系统中探究BET蛋白的功能。研究结果显示，尽管BRD3占据了绝大多数GATA1结合位点，但BRD2与BRD4同样会被招募至部分被GATA1占据的染色质结合位点。功能层面的实验结果表明，BET抑制剂会在全基因组范围内削弱GATA1介导的转录激活过程，但不会影响其转录抑制功能。BET蛋白的协同激活作用通过两种途径实现：一是促进GATA1在基因组上的结合占据，二是后续辅助转录激活过程的进行。本研究结合CRISPR/Cas9介导的基因组编辑与短发夹RNA（short hairpin RNA, shRNA）技术，观察到单独敲低BRD2或BRD4均可削弱红细胞系基因的激活过程；而仅在BRD2缺失的背景下，敲低BRD3才会对红细胞系转录产生影响。上述研究结果提示，在阐释药理BET抑制剂的作用效果时，应当结合转录激活过程中的不同步骤，以及BET家族成员间部分重叠的功能特性。实验设计概况：将GATA1缺失的成红细胞（G1E）改造为条件性表达GATA1-ER融合蛋白的细胞系，通过添加100nM雌二醇诱导24小时以激活GATA1的表达。在GATA1结合实验中，分别设置250nM JQ1存在与缺失两种条件开展对照实验。针对BRD3与BRD4结合位点的实验，则将未激活GATA1-ER融合蛋白的G1E细胞与激活后的G1E细胞进行对比分析。