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.

父母赠款的简要摘要。该补充基金申请的父母授予是 1R01HL158081-01A1，标题为“ Brd4在正常造血和造血茎中的作用 细胞生物学”，从12/01/2021至11/30/2024获得批准。 含溴构域的蛋白质4（BRD4）是BET的成员（溴结构域和额外 末端域）系列蛋白，还包括BRD2，BRD3和BRDT。 BRD4促进 通过与组蛋白尾巴的乙酰化赖氨酸残基的结合与转录的伸长和伸长 促进RNA聚合酶II复合物募集到活性转录部位。自从 MYC癌基因表达需要BRD4，BRD4抑制代表了一个有吸引力的策略 通过小分子抑制剂靶向MYC依赖性癌症。 BRD4在两者中都过表达 实体瘤和髓样恶性肿瘤，包括急性髓样白血病（AML）。下注抑制剂 （BETI）已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在正常中的作用 造血和BRD4过表达对血液学发病机理的影响 恶性肿瘤基本上仍然未知。填补这一重要的知识差距是 这个为期3年的Shine应用程序。在当前项目中，我们旨在确定BRD4在 造血干/祖细胞（HSC/HPC）的功能，并探索BRD4是否是否 过表达会影响HSC/HPC细胞命运和白血病转化。使用有条件的BRD4 敲除（mx1cre; brd4f/f）小鼠模型，我们发现虽然杂合删除了brd4 小鼠没有引起造血的明显变化，BRD4的纯合缺失 造血系统由于诱导而迅速减少HSC/HPC和Pan Lineage细胞 凋亡。因此，仅有条件的BRD4敲除鼠标模型不适合 研究BRD4在HSC/HPC函数中的隐藏作用。因此，我们产生了几个BRD4 具有不同级别BRD4转化表达的转基因（TG）小鼠系（从25％范围 至200％）。我们的初步数据表明，BRD4（BRD4200％TG）的过表达 造血细胞在体内改变了HSC/HPC池，并增加了HSC/HPC的潜力 体外。有趣的是，在Brd4ä/äbmmncs中重新表达了较低的BRD4级 （BRD4ä/ä; Brd425％TG）显着提高了CFU-CS的细胞存活和频率。我们 假设一个假设是通过表达A hypomorph BRD4小鼠模型（MX1CRE; BRD4F/F; BRD425％TG） 可保护HSC/HPC生存的造血中的BRD4水平可更好地适合 用于评估BRD4在HSC/HPC函数中的隐藏作用。我们还将检查beti是否 影响小鼠正常造血。此外，我们将破译BRD4如何调节 HSC/HPC通过评估全基因组BRD4，P-TEFB，POL-II，H3K27AC和 HSC/HPC中的H3K122AC占用物，与基因表达输出相关。这些 研究对于填补知识的基本和关键差距至关重要 旨在发现正常和恶性造血中Brd4的隐藏作用，从而填充 在造血的BRD4和BETI的BRD4知识中的临界差距用于造血的差距 恶性肿瘤。