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: 10610534
• 负责人: Feng-Chun Yang
• 金额: $ 2.46万
• 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/10610534
• 关键词: Acetylation; Active Sites; Acute Myelocytic Leukemia; Affect; BRD2 gene; Behavior; Binding; Biology; Bromodomain; C-terminal; Cancer Biology; Cell Cycle Progression; Cell Lineage; Cell Survival; Cells; Chromatin; Complex; DNA Polymerase II; Data; Data Set; Disease; Doctor of Philosophy; Fibrosis; Foundations; Frequencies; Funding; Future; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genomics; Goals; Hematologic Neoplasms; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic System; Hematopoietic stem cells; Histone Acetylation; Histones; In Vitro; Induction of Apoptosis; Inflammatory; Injections; Knock-out; Knockout Mice; Knowledge; Learning; Lysine; MYC gene; Malignant - descriptor; Malignant Neoplasms; Mediating; Mitotic Cell Cycle; Molecular; Mus; Myeloproliferative disease; Output; Pathogenesis; Positive Transcriptional Elongation Factor B; Protein Family; Proteins; RNA Polymerase II; Regulation; Research Personnel; Research Project Grants; Role; Solid; Solid Neoplasm; Stem Cell Research; System; Tail; Technical Expertise; Time; Training; Transcription Elongation; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; Work; Yang; clinically significant; cyclin T1; cytopenia; genome-wide; graduate student; histone acetyltransferase; histone modification; in vivo; inhibitor; leukemic transformation; member; mouse model; nuclease; overexpression; parent grant; recruit; single-cell RNA sequencing; small molecule; small molecule inhibitor; stem cell biology; stem cell function; transcriptome; transcriptome sequencing; transgene expression; tumor

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.

父母补助金的简要摘要。此补充基金申请的家长补助金为 1 R 01 HL 158081 - 01 A1，题为“BRD 4在正常造血和造血干细胞中的作用 细胞生物学”，获批准于2021年1月12日至2024年11月30日资助。 含溴结构域蛋白4（BRD 4）是BET（溴结构域和额外溴结构域）的成员。 末端结构域）家族蛋白，还包括BRD 2、BRD 3和BRDT。BRD 4促进了 通过与组蛋白尾部的乙酰化赖氨酸残基结合， 促进RNA聚合酶II复合物募集到活性转录位点。以来 BRD 4是MYC癌基因表达所必需的，BRD 4抑制代表了一种有吸引力的策略 通过小分子抑制剂靶向MYC依赖性癌症。BRD 4在两种细胞中都过表达， 实体瘤和骨髓恶性肿瘤，包括急性骨髓性白血病（AML）。BET抑制剂 （BETi）已被证明对各种类型的肿瘤有效，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在正常肿瘤中的作用仍然存在。 BRD 4过表达对造血系统的影响 恶性肿瘤仍然是未知的。填补这一关键的知识空白是 这三年的闪耀申请在目前的项目中，我们的目标是确定BRD 4在以下方面的作用： 造血干/祖细胞（HSC/HPC）功能，并探索Brd 4 过表达影响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， 小鼠的造血没有引起明显的变化，Brd 4的纯合缺失， 造血系统迅速减少HSC/HPCs和泛系细胞由于诱导 细胞凋亡。因此，单独的条件性Brd 4敲除小鼠模型不适用于 研究BRD 4在HSC/HPC功能中的隐藏作用。因此，我们生成了几个Brd 4 具有不同BRD 4转基因表达水平的转基因（Tg）小鼠系（范围从25%至25%）， 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 <$/<$BMMNC中重新表达较低水平的BRD 4， (Brd4<$/<$Brd425%Tg）可显著提高细胞存活率和CFU-Cs频率。我们 假设通过表达一种低型BRD 4小鼠模型（Mx 1Cre; Brd 4f/f;Brd425%Tg）， 造血中允许HSC/HPC存活的BRD 4的保护水平将更适合 用于评估BRD 4在HSC/HPC功能中的隐藏作用。我们还将研究是否贝蒂 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主要存在于HSC/HPC中，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和用于治疗造血疾病的BETi的知识的关键差距 恶性肿瘤