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.家长拨款的项目摘要 这项补充基金申请的父母拨款是1R01HL158081-01A1，标题为“BRD4在正常情况下的作用” 《造血及造血干细胞生物学》，于2021年12月1日获批准拨款至 2024年11月30日。 含溴结构域的蛋白4(BRD4)是BET(溴结构域和额外末端结构域)的成员 还包括BRD2、BRD3和BRDT的家族蛋白。BRD4促进了基因的启动和延伸 通过与组蛋白尾部的乙酰化赖氨酸残基结合转录促进RNA的募集 聚合酶II复合体到活性转录位点。由于BRD4是MYC癌基因表达所必需的， BRD4抑制是通过小分子抑制剂靶向MYC依赖型癌症的一种有吸引力的策略。 BRD4在实体瘤和包括急性髓系白血病(AML)在内的髓系恶性肿瘤中均有过度表达。 BET抑制剂(Beti)已被证明对各种类型的肿瘤有效，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在正常造血和 BRD4过表达对血液系统恶性肿瘤发病机制的影响仍不清楚。 填补这一关键的知识缺口是这项为期3年的SHARE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 在基因敲除(Mx1Cre；Brd4f/f)小鼠模型中，我们发现Brd4杂合缺失在小鼠中没有 在造血系统中引起明显的变化，在造血系统中迅速纯合缺失Brd4 HSC/HPC和PAN系细胞因诱导凋亡而减少。因此，有条件的Brd4 单独的基因敲除小鼠模型不适合研究BRD4在HSC/HPC功能中的隐藏作用。我们 从而获得了几个具有不同水平BRD4转基因表达的转基因(TG)小鼠 (从25%到200%不等)。我们的初步数据显示，BRD4(Brd4200%TG)在 造血细胞在体内改变了HSC/HPC池，在体外增加了HSC/HPC的复制能力。 有趣的是，BRD4在BMMNC(Brd4；Brd425%Tg)中显著地重新表达较低水平的BRD4 增加细胞存活率和CFU-Cs的频率。我们假设一只低晶型的BRD4小鼠 模型(Mx1Cre；Brd4f/f；Brd425%TG)，通过在造血中表达可保护水平的BRD4，允许 HSC/HPC存活率更适合于评价BRD4在HSC/HPC功能中的潜在作用。我们还将 检测Beti是否影响小鼠的正常造血。此外，我们将破译BRD4是如何监管的 HSC/HPC通过评估全基因组BRD4、P-TEFb、POL-II、H3K27ac和H3K122ac的功能 HSC/HPC的占有率与基因表达输出相关。这些研究是及时的，而且 对于填补基本的和关键的知识缺口，揭示 BRD4在正常和恶性造血中的作用，从而填补了对Brd4在造血和恶性肿瘤中的重要认识空白。 贝替尼用于治疗血液系统恶性肿瘤。