Chromatin and epigenetic memory

染色质和表观遗传记忆

• 批准号: 9550387
• 负责人: Keiko Ozato
• 金额: $ 97.72万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9550387
• 关键词: Adult; Affect; Antibodies; Binding; Binding Proteins; Bone Marrow; Bromodomain; Cell division; Cells; ChIP-seq; Chromatin; Chromosomes; Complementary DNA; Coupled; DNA biosynthesis; Deposition; Development; Embryo; Employee Strikes; Enhancers; Environment; Erythrocytes; Family; Fibroblasts; Gene Expression; Generations; Genes; Genetic Transcription; Goals; Growth; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic stem cells; Histone H3; Histones; Immune; Inflammatory; Inflammatory Response; Interferon Type II; Interferon-beta; Knock-out; Knockout Mice; Lymphocyte; Memory; Mitosis; Mouse Strains; Mus; Myeloid Cells; Nuclear Protein; Nucleosomes; Physiological; Process; Production; Property; Proteins; Reactive Oxygen Species; Role; Solid Neoplasm; Tissues; Transcriptional Activation; Variant; c-myc Genes; cell growth regulation; cell type; chemokine; cytokine; daughter cell; epigenetic memory; epigenetic regulation; inhibitor/antagonist; macrophage; progenitor; response; small molecule inhibitor; stem

Development of small molecule inhibitors such as JQ1 and I-BET which specifically block binding of BET proteins to acetylated histone showed that BRD4 has a major role in cells growth regulation, affecting transcription of c-Myc and Fos. Thus these inhibitors can restrict growth of various blood cancers and solid tumors. These inhibitors are also shown to effectively inhibit certain types of inflammatory responses in immune cells. Although these inhibitors facilitated our understanding of BRD4 action, it has remained unclear what BRD4 does and how it does it during development and in adult cells. Our aim for the project has been to elucidate the role of BRD4 and its mode of action by studying Brd4 conditional knockout (cKO) mice. We found that BRD4 is critically required for the development of hematopoietic cells, in that deletion of Brd4 in embryonic hematopoietic stem cells (HSC) led to almost complete blockade of the subsequent progenitor differentiation required for generation of erythrocytes, lymphocytes and myeloid cells. As a results, Brd4 cKO were found embryonic lethal. We found that the requirement of BRD4 for inflammatory responses in macrophages is less striking, in that Brd4 deletion caused only a partial reduction in the production of proinflammatory factors (cytokines, chemokines and reactive oxygen species). Nevertheless, BRD4 was broadly present in super-enhancers in both quiescent and inflammatory macrophages. Our long-standing effort has produced new mouse strains in which two of the H3,3 loci (H3f3a, H3.3A, H3f3b, H3.3B) are replaced by a HA-tagged H3.3 cDNA and free GFP. H3f3a and H3f3b heterozygous mice are found viable and fertile. The advantage of these mice has been verified when we found that the H3.3 expression and the chromatin deposition can be studied simply by anti-HA antibody. These mice overcome the long standing difficulty in studying H3.3 that stems from the fact that high quality antibodies specific for H3.3 are not easily available. We investigated H3.3-HA expression in adult tissues by immunoblot, and in different immune cells by flowcytometry and showed that both H3.3A and H3.3B are expressed on most of adult cells. Likewise, H3.3 chromatin distribution can be clarified by anti-HA antibody. By ChIP -seq analysis of bone marrow derived macrophages and fibroblasts, we have shown that H3.3 (both A and B) are rapidly incorporated into transcriptionally activated genes in both cell types in response to interferon gamma or interferon beta.

小分子抑制剂（例如JQ1和I-BET）的发展，这些抑制剂特异性地阻断了BET蛋白与乙酰化组蛋白的结合表明，BRD4在细胞生长调节中具有重要作用，影响C-Myc和FOS的转录。因此，这些抑制剂可以限制各种血液癌和实体瘤的生长。这些抑制剂也被证明可以有效抑制免疫细胞中某些类型的炎症反应。尽管这些抑制剂促进了我们对BRD4作用的理解，但尚不清楚BRD4在发育和成人细胞中的作用以及它的作用。 我们对该项目的目的是通过研究BRD4条件敲除（CKO）小鼠来阐明BRD4及其作用方式。我们发现，在胚胎造血干细胞（HSC）中删除BRD4的造血细胞的发展至关重要，导致几乎完全阻断了随后产生红细胞，淋巴细胞和髓样细胞所需的祖细胞分化。结果，发现BRD4 CKO胚胎致死。我们发现，BRD4对巨噬细胞中炎症反应的需求较小，因为BRD4缺失仅导致促炎性因子（细胞因子，趋化因子和活性氧）的产生仅部分降低。然而，在静止和炎症性巨噬细胞中，BRD4在超级增强剂中广泛存在。 我们长期的努力产生了新的小鼠菌株，其中H3,3基因座（H3F3A，H3.3A，H3F3B，H3.3B）中有两个被HA标记的H3.3 cDNA和自由GFP取代。发现H3F3A和H3F3B杂合小鼠可行且肥沃。当我们发现可以简单地通过抗HA抗体研究H3.3表达和染色质沉积时，这些小鼠的优势已得到证实。这些小鼠克服了研究H3.3的长期困难，这是由于对H3.3所特有的高质量抗体不容易获得的事实。我们通过免疫印迹研究了成人组织中的H3.3-HA表达，并通过流动性测定术在不同的免疫细胞中进行了研究，并表明H3.3a和H3.3b均在大多数成年细胞上表达。同样，H3.3染色质分布可以通过抗HA抗体阐明。通过对骨髓衍生的巨噬细胞和成纤维细胞的芯片序列分析，我们表明，H3.3（A和B）在两种细胞类型中迅速掺入转录活化的基因中，以响应干扰素伽马或干扰素β。