Role of BRD4 in Normal Hematopoiesis and Hematopoietic Stem Cell Biology.

BRD4 在正常造血和造血干细胞生物学中的作用。

• 批准号: 10610129
• 负责人: Feng-Chun Yang
• 金额: $ 2.95万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-11 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10610129
• 关键词: Active Sites; Acute Myelocytic Leukemia; Admission activity; Affect; BRD2 gene; Behavior; Binding; Biological Assay; Breeding; Bromodomain; Cancer Biology; Cell Lineage; Cell Survival; Cells; Charge; Complex; DNA; DNA Polymerase II; Data; Doctor of Medicine; Doctor of Philosophy; Enrollment; Foundations; Frequencies; Funding; Gene Expression; Genetic Transcription; Genotype; Goals; Grant; Hematologic Neoplasms; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic System; Hematopoietic stem cells; Histones; In Vitro; Induction of Apoptosis; Knock-out; Knockout Mice; Knowledge; Laboratories; Lysine; MYC gene; Malignant - descriptor; Malignant Neoplasms; Master' s Degree; Molecular; Mus; Myeloproliferative disease; Output; Pathogenesis; Physicians; Positive Transcriptional Elongation Factor B; Protein Family; Proteins; RNA Polymerase II; Research; Research Assistant; Research Project Grants; Role; Scientist; Series; Solid; Solid Neoplasm; Tail; Techniques; Time; Tissues; Training; Transcription Elongation; Transgenic Mice; Transgenic Organisms; Translational Research; Work; Yang; base; clinically significant; epigenetic regulation; experience; genome-wide; in vivo; inhibitor; leukemic transformation; member; mouse model; overexpression; parent grant; programs; recruit; small molecule inhibitor; stem cell biology; stem cell function; transgene expression; tumor

1. A brief project 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.

1. 家长资助项目简介 该补充基金申请的母基金为 1R01HL158081-01A1，题为“BRD4 在正常情况下的作用” 造血与造血干细胞生物学”，于2021年12月1日至2021年12月1日获得资助 2024 年 11 月 30 日。 含布罗莫结构域蛋白 4 (BRD4) 是 BET（布罗莫结构域和额外末端结构域）的成员 家族蛋白还包括 BRD2、BRD3 和 BRDT。 BRD4 促进起始和延伸 通过与组蛋白尾部的乙酰化赖氨酸残基结合来促进 RNA 的募集来进行转录 聚合酶 II 与活性转录位点形成复合体。由于 MYC 癌基因表达需要 BRD4， BRD4 抑制代表了一种通过小分子抑制剂靶向 MYC 依赖性癌症的有吸引力的策略。 BRD4 在实体瘤和髓系恶性肿瘤中过度表达，包括急性髓系白血病 (AML)。 BET 抑制剂 (BETi) 已被证明对多种类型的肿瘤有效，尤其是 MYC 驱动的肿瘤 癌症。尽管对 BRD4 在实体瘤中的作用进行了深入的研究，但 BRD4 在正常造血中的作用和 BRD4 过度表达对血液恶性肿瘤发病机制的影响仍不清楚。 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。在当前的项目中， 我们的目标是确定 BRD4 在造血干/祖细胞 (HSC/HPC) 功能中的作用并探索 Brd4 过表达是否影响 HSC/HPC 细胞命运和白血病转化。使用有条件的 Brd4 在敲除 (Mx1Cre;Brd4f/f) 小鼠模型中，我们发现虽然小鼠中 Brd4 杂合缺失并没有 引起明显的造血变化，造血系统中Brd4的纯合缺失很快 由于诱导细胞凋亡，HSC/HPC 和泛谱系细胞减少。因此，有条件的Brd4 单独的敲除小鼠模型并不适合研究 BRD4 在 HSC/HPC 功能中的隐藏作用。我们 由此产生了多个具有不同水平的 BRD4 转基因表达的 Brd4 转基因 (Tg) 小鼠系 （范围从 25% 到 200%）。我们的初步数据表明，BRD4 (Brd4200%Tg) 在 造血细胞在体内改变了 HSC/HPC 池，并在体外增加了 HSC/HPC 移植潜力。 有趣的是，在 Brd4ä/ä BMMNC (Brd4ä/ä;Brd425%Tg) 中重新表达较低水平的 BRD4 显着 增加细胞存活率和 CFU-C 频率。我们假设亚型 BRD4 小鼠 模型（Mx1Cre；Brd4f/f；Brd425%Tg），通过在造血过程中表达可保护水平的 BRD4，从而允许 HSC/HPC 存活率，更适合评估 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功能。此外，我们将破译BRD4如何调节 HSC/HPC 通过评估全基因组 BRD4、P-TEFb、Pol-II、H3K27ac 和 H3K122ac 发挥功能 HSC/HPC 中的占据并与基因表达输出相关。这些研究是及时且有效的 对于填补基本和关键的知识空白以揭示隐藏的角色至关重要 BRD4 在正常和恶性造血中的作用，从而填补了 Brd4 在造血和恶性造血中的知识空白。 BETi 用于治疗造血系统恶性肿瘤。