Role of BRD4 in Normal Hematopoiesis and Hematopoietic Stem Cell Biology

含溴结构域蛋白4（Bromodomain-containing protein 4，BRD 4）是BET（bromodomain and extra terminal domain，溴结构域和额外末端结构域）的成员。 家族蛋白质，还包括BRD 2、BRD 3和BRDT。BRD 4促进了细胞的启动和延长， 通过与组蛋白尾部的乙酰化赖氨酸残基结合以促进RNA的募集来转录 聚合酶II复合物的活性转录位点。由于BRD 4是MYC癌基因表达所必需的， BRD 4抑制代表了通过小分子抑制剂靶向MYC依赖性癌症的有吸引力的策略。 BRD 4在实体瘤和骨髓恶性肿瘤（包括急性髓性白血病（AML））中均过表达。 BET抑制剂（BETi）已被证明对各种类型的肿瘤具有功效，尤其是MYC驱动的肿瘤。 癌的尽管对BRD 4在实体瘤中的作用进行了强有力的研究，但BRD 4在正常造血中的作用和BRD 4在肿瘤中的作用仍然是未知的。 BRD 4过表达对血液恶性肿瘤发病机制的影响在很大程度上仍是未知的。 填补这一关键的知识空白是这个为期3年的SHINE应用程序的主要目标。在目前的项目中， 我们的目的是确定BRD 4在造血干/祖细胞（HSC/HPC）功能中的作用， 探索Brd 4过表达是否影响HSC/HPC细胞命运和白血病转化。使用 在条件性Brd 4基因敲除（Mx 1Cre; Brd 4f/f）小鼠模型中，我们发现Brd 4基因杂合缺失 在小鼠的造血中没有引起明显的变化，造血中Brd 4的纯合缺失， 系统由于诱导凋亡而迅速减少HSC/HPCs和泛系细胞。因此 单独的条件性Brd 4敲除小鼠模型不适合于研究BRD 4在 HSC/HPC功能。因此，我们产生了几种具有不同BRD 4水平的Brd 4转基因（Tg）小鼠系 转基因表达（范围从25%至200%）。我们的初步数据显示BRD 4的过表达 在造血细胞中，Brd 4200%Tg改变了体内HSC/HPC池，并增加了HSC/HPC再铺板潜力 体外有趣的是，在Brd 4 Δ/Δ BMMNC（Brd 4 Δ/Δ;Brd425%Tg）中重新表达较低水平的BRD 4， 显著增加细胞存活率和CFU-Cs频率。我们假设一个亚型体 BRD 4小鼠模型（Mx 1Cre; Brd 4f/f;Brd425%Tg），通过在造血中表达可保护水平的BRD 4， 这使得HSC/HPC存活，将更适合于评估BRD 4在HSC/HPC中的隐藏作用 功能协调发展的我们还将检查BETi是否影响小鼠中的正常造血。此外，我们将 通过评估全基因组BRD 4，P-TEFb，Pol-II， H3 K27 ac和H3 K122 ac在HSC/HPC中存在，并与基因表达产物相关。这些 研究是及时的，对于填补知识的基本和关键差距至关重要， 揭示BRD 4在正常和恶性造血中的隐藏作用，从而填补了 关于造血中的Brd 4和用于治疗造血系统恶性肿瘤的BETi的知识。

Bromodomain-containing protein 4 (BRD4) is a member of the BET (bromodomain and extra terminal domain) family proteins that also include BRD2, BRD3, and BRDT. BRD4 facilitates the initiation and elongation of transcription by binding to acetylated lysine residues of histone tails to promote the recruitment of the RNA polymerase II complex to sites of active transcription. Since BRD4 is required for MYC oncogene expression, BRD4 inhibition represents an attractive strategy to target MYC-dependent cancers via small-molecule inhibitors. BRD4 is over-expression in both solid tumors and myeloid malignancies, including acute myeloid leukemia (AML). BET inhibitors (BETi) have been shown to have efficacy against various types of tumors, especially MYC-driven cancers. Despite robust studies of BRD4 in solid tumors, the role of BRD4 in normal hematopoiesis and the impact of BRD4 overexpression on the pathogenesis of hematological malignancies remain largely unknown. Filling this critical gap of knowledge is the primary goal of this 3-year SHINE application. In the current project, we aim to determine the roles of BRD4 in hematopoietic stem/progenitor cells (HSC/HPCs) function and explore whether Brd4 overexpression affects HSC/HPC cell fate and leukemic transformation. Using a conditional Brd4 knock- out (Mx1Cre;Brd4f/f) mouse model, we found that while heterozygous deletion of Brd4 in mice did not cause noticeable changes in hematopoiesis, homozygous deletion of Brd4 in the hematopoietic system quickly diminished HSC/HPCs and pan lineage cells due to the induction of apoptosis. Therefore, the conditional Brd4 knock-out mouse model alone is not suitable for studying the hidden role of BRD4 in HSC/HPC functions. We thus generated several Brd4 transgenic (Tg) mouse lines with different levels of BRD4 transgene expression (ranging from 25% to 200%). Our preliminary data showed that overexpression of BRD4 (Brd4200%Tg) in hematopoietic cells altered HSC/HPC pools in vivo and increased HSC/HPC replating potential in vitro. Interestingly, re-expression of a lower level of BRD4 in Brd4Δ/Δ BMMNCs (Brd4Δ/Δ;Brd425%Tg) significantly increased the cell survival and the frequencies of CFU-Cs. We hypothesize that a hypomorph BRD4 mouse model (Mx1Cre;Brd4f/f;Brd425%Tg), by expressing a protectable level of BRD4 in hematopoiesis which allow for HSC/HPC survival, would suit better for evaluating the hidden role of BRD4 in HSC/HPC functions. We will also examine whether BETi affect normal hematopoiesis in mice. Furthermore, we will decipher how BRD4 regulates the HSC/HPCs functions by assessing genome-wide BRD4, P-TEFb, Pol-II, H3K27ac, and H3K122ac occupancies in HSC/HPCs and correlating with the gene expression outputs. These studies are timely and fundamentally crucial for filling an essential and critical gap of knowledge towards uncovering the hidden roles of BRD4 in normal and malignant hematopoiesis, thus fill a critical gap in knowledge on Brd4 in hematopoiesis and BETi for the treatment of hematopoietic malignancies.