Role of BRD4 in Normal Hematopoiesis and Hematopoietic Stem Cell Biology_

家长助学金的简要摘要。这项补充基金申请的家长资助额为 1R01HL158081-01A1，题为“BRD4在正常造血和造血干细胞中的作用” 《细胞生物学》，2021年12月1日至2024年11月30日获得批准资助。 包含溴域的蛋白4(BRD4)是BET(溴域和Extra)的成员 终端域)家族蛋白，还包括BRD2、BRD3和BRDT。BRD4促进了 通过与组蛋白尾部的乙酰化赖氨酸残基结合来启动和延长转录 促进RNA聚合酶II复合体向活跃转录位点的募集。自.以来 BRD4是MYC癌基因表达所必需的，抑制BRD4是一种有吸引力的策略 通过小分子抑制剂靶向MYC依赖型癌症。BRD4在这两个组织中都过度表达 实体瘤和髓系恶性肿瘤，包括急性髓系白血病(AML)。Bit抑制剂 (Beti)已被证明对各种类型的肿瘤有疗效，特别是由MYC驱动的 癌症。尽管BRD4在实体肿瘤中的研究很活跃，但BRD4在正常 造血及BRD4过表达在血液病发病机制中的作用 恶性肿瘤在很大程度上仍不为人所知。填补这一关键的知识缺口是 这是一款为期3年的闪亮应用程序。在当前的项目中，我们的目标是确定BRD4在 造血干/祖细胞(HSC/HPC)的功能及Brd4 过表达影响HSC/HPC细胞命运和白血病转化。使用有条件的Brd4 在基因敲除(Mx1Cre；Brd4f/f)小鼠模型中，我们发现Brd4杂合性缺失在 小鼠未引起明显的造血改变，Brd4纯合子缺失 由于诱导，造血系统迅速减少了HSC/HPC和PAN系细胞 对细胞凋亡的影响。因此，单纯的条件性Brd4基因敲除小鼠模型不适用于 研究BRD4在HSC/HPC功能中的隐藏作用。因此，我们生成了几个Brd4 具有不同水平BRD4转基因表达的转基因(TG)小鼠(从25%到25%不等) 到200%)。我们的初步数据显示，BRD4(Brd4200%TG)在 造血细胞改变体内HSC/HPC池并增加HSC/HPC复制能力 在试管中。有趣的是，BRD4在BMMNC中的重新表达水平较低 (BRD4；Brd425%Tg)显著增加细胞存活率和CFU-Cs的频率。我们 假设一个低晶型BRD4小鼠模型(Mx1Cre；Brd4f/f；Brd425%Tg)，通过表达a 在允许HSC/HPC存活的造血保护水平下，BRD4将更适合 用于评估BRD4在HSC/HPC功能中的隐藏作用。我们还将检查Beti是否 影响小鼠的正常造血。此外，我们将破译BRD4是如何规范 HSC/HPC通过评估全基因组BRD4、P-TEFb、POL-II、H3K27ac和 H3K122ac在HSC/HPC中的占有率及其与基因表达输出的相关性。这些 研究对于填补基本的和关键的知识缺口是及时和根本的关键 为了揭示BRD4在正常和恶性造血中的隐藏作用，从而填补了 关于Brd4在造血中的关键知识差距和Beti用于造血治疗 恶性肿瘤。

A brief summary of the parent grant. The parent grant of this supplemental fund application is 1R01HL158081-01A1, entitled “Role of BRD4 in Normal Hematopoiesis and Hematopoietic Stem Cell Biology”, which was approved for funding from 12/01/2021 to 11/30/2024. 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 Brd4Ä/Ä BMMNCs (Brd4Ä/Ä;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.