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: 10384169
• 负责人: Feng-Chun Yang
• 金额: $ 31万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384169
• 关键词: Acetylation; Active Sites; Acute Myelocytic Leukemia; Affect; Apoptosis; BRD2 gene; Behavior; Binding; Biology; Bromodomain; C-terminal; CD34 gene; Cell Cycle Progression; Cell Lineage; Cell Survival; Cell physiology; Cells; Chromatin; Chromatin Structure; Clinical Trials; Complex; DNA Polymerase II; Data; Development; Disease; Exhibits; Fibrosis; Frequencies; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Genomics; Goals; Hematologic Neoplasms; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic System; Hematopoietic stem cells; Histone Acetylation; Histones; Human; Impairment; In Vitro; Induction of Apoptosis; Inflammatory; Injections; Knock-out; Knockout Mice; Knowledge; Lysine; MYC gene; Malignant - descriptor; Malignant Neoplasms; Mediating; Mitotic Cell Cycle; Molecular; Mus; Myeloproliferative disease; Output; Pathogenesis; Population; Positive Transcriptional Elongation Factor B; Protein Family; Proteins; RNA Polymerase II; Regulation; Role; Solid; Solid Neoplasm; Tail; Testing; Thrombocytopenia; Time; Toxic effect; Transcription Elongation; Transgenic Mice; Transgenic Organisms; clinically significant; cyclin T1; genome-wide; histone acetyltransferase; histone modification; in vivo; inhibitor; knock-down; leukemic transformation; member; mouse model; nuclease; overexpression; recruit; single-cell RNA sequencing; small molecule; small molecule inhibitor; stem cell biology; stem cell function; transcriptome; transgene expression; tumor

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 Brd4 BMMNCs (Brd4;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.

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物。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 在造血干细胞/祖细胞 (HSC/HPC) 功能中的作用， 探讨 Brd4 过表达是否影响 HSC/HPC 细胞命运和白血病转化。使用 条件 Brd4 敲除 (Mx1Cre;Brd4f/f) 小鼠模型中，我们发现，虽然 Brd4 杂合缺失 在小鼠中并没有引起造血功能的明显变化，造血功能中 Brd4 的纯合缺失 由于诱导细胞凋亡，系统迅速减少 HSC/HPC 和泛谱系细胞。因此， 单独条件性 Brd4 敲除小鼠模型并不适合研究 BRD4 的隐藏作用 HSC/HPC 功能。因此，我们生成了多个具有不同 BRD4 水平的 Brd4 转基因 (Tg) 小鼠品系 转基因表达（范围从 25% 到 200%）。我们的初步数据表明 BRD4 的过度表达 造血细胞中的 (Brd4200%Tg) 改变了体内 HSC/HPC 池并增加了 HSC/HPC 重铺潜力 体外。有趣的是，在 Brd4 BMMNC 中重新表达较低水平的 BRD4 (Brd4;Brd425%Tg) 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 允许 HSC/HPC 存活，更适合评估 BRD4 在 HSC/HPC 中的隐藏作用 功能。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 通过评估全基因组 BRD4、P-TEFb、Pol-II、破译 BRD4 如何调节 HSC/HPC 功能 H3K27ac 和 H3K122ac 在 HSC/HPC 中的占据并与基因表达输出相关。这些 研究对于填补基本和关键的知识空白至关重要 揭示BRD4在正常和恶性造血中的隐藏作用，从而填补了一项关键空白 Brd4 在造血作用和 BETi 治疗造血系统恶性肿瘤方面的知识。