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.

1.一个简短的项目摘要的父母补助金 本补充基金申请的母基金是1 R 01 HL 158081 - 01 A1，题为“BRD 4在正常 造血和造血干细胞生物学”，该项目于2021年1月12日获批资助， 11/30/2024. 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，并探索 Brd 4过表达是否影响HSC/HPC细胞命运和白血病转化。使用条件Brd 4 在Mx 1Cre; Brd 4f/f基因敲除小鼠模型中，我们发现Brd 4杂合缺失小鼠中 引起造血的显著变化，造血系统中Brd 4的纯合缺失迅速 减少HSC/HPC和泛系细胞由于诱导凋亡。因此，条件Brd 4 单独的基因敲除小鼠模型不适合研究BRD 4在HSC/HPC功能中的隐藏作用。我们 由此产生了几种具有不同水平的BRD 4转基因表达的Brd 4转基因（Tg）小鼠系 （范围从25%到200%）。我们的初步数据显示，BRD 4（Brd4200%Tg）的过表达在人乳腺癌细胞中是显著的。 造血细胞在体内改变了HSC/HPC池，并在体外增加了HSC/HPC再铺板潜力。 有趣的是，在Brd 4 <$/<$BMMNC（Brd 4 <$/<$;Brd425%Tg）中较低水平的BRD 4的再表达显著增加了BMMNC中BRD 4的表达。 增加细胞存活率和CFU-Cs频率。我们假设BRD 4亚型小鼠 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， HSC/HPC存活率，将更适合于评估BRD 4在HSC/HPC功能中的隐藏作用。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BRD 4如何调节 通过评估全基因组BRD 4、P-TEFb、Pol-II、H3 K27 ac和H3 K122 ac来评估HSC/HPC的功能 存在于HSC/HPC中，并与基因表达输出相关。这些研究是及时的， 从根本上说，这对于填补知识的一个重要和关键的空白，以揭示隐藏的作用， 因此，填补了关于Brd 4在造血中的知识的关键空白， 用于治疗造血系统恶性肿瘤的BETi。