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.

含溴构域的蛋白4（BRD4）是BET的成员（溴结构域和额外终端结构域） 还包括BRD2，BRD3和BRDT的家庭蛋白质。 BRD4促进了主动性和延长 通过与组蛋白尾巴的乙酰化赖氨酸残基结合来转录，以促进RNA的募集 聚合酶II复合物与活性转录部位。由于Myc癌基因表达需要BRD4，因此 BRD4抑制是通过小分子抑制剂靶向MYC依赖性癌症的一种有吸引力的策略。 BRD4在实体瘤和髓样恶性肿瘤中均过表达，包括急性髓样白血病（AML）。 BET抑制剂（BETI）已被证明对各种类型的肿瘤具有效率，尤其是MYC驱动的 癌症。尽管对实体瘤的BRD4进行了强有力的研究，但BRD4在正常造血和 BRD4过表达对血液学恶性肿瘤发病机理的影响仍然很大未知。 填补这一重要的知识差距是这项为期三年的Shine应用程序的主要目标。在当前项目中 我们旨在确定BRD4在造血干/祖细胞（HSC/HPC）功能和 探索BRD4的过表达是否影响HSC/HPC细胞命运和白血病转化。使用 有条件的BRD4敲除（MX1CRE; BRD4F/F）鼠标模型，我们发现虽然BRD4的杂合缺失 在小鼠中没有引起造血的明显变化，造血中Brd4的纯合缺失 由于凋亡的诱导，系统迅速减少了HSC/HPC和Pan Lineage细胞。因此， 有条件的BRD4敲除鼠标模型不适合研究BRD4在 HSC/HPC功能。因此，我们生成了几个具有不同级别BRD4的BRD4转基因（TG）小鼠系 转基因表达（从25％到200％）。我们的初步数据表明BRD4的过表达 （BRD4200％TG）在造血细胞中改变了体内HSC/HPC池，并增加了HSC/HPC 体外。有趣的是，在BRD4BMMNC中重新表达了BRD4较低水平（BRD4; BRD425％TG） 显着增加了细胞存活和CFU-CS的频率。我们假设是hypomorph BRD4小鼠模型（MX1CRE; BRD4F/F; BRD425％TG），通过在造血中表达受保护的BRD4水平 允许HSC/HPC存活，将更适合评估BRD4在HSC/HPC中的隐藏作用 功能。我们还将检查BETI是否会影响小鼠的正常造血。此外，我们会的 通过评估全基因组BRD4，P-TEFB，POL-II，DETER BRD4如何调节HSC/HPC的功能 HSC/HPC中的H3K27AC和H3K122AC占用率与基因表达输出相关。这些 研究对于填补对知识的基本和关键差距至关重要 揭示BRD4在正常和恶性造血中的隐藏作用，从而填补了一个关键的空白 关于造血和BETI的BRD4的知识，用于治疗造血性恶性肿瘤。