Bromodomain protein Brd4 and its role in epigenetic memory

溴结构域蛋白 Brd4 及其在表观遗传记忆中的作用

• 批准号: 7594265
• 负责人: Keiko Ozato
• 金额: $ 80.21万
• 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/7594265
• 关键词: Address; Adverse effects; Alleles; Back; Binding; Biological Assay; Bromodomain; Cell Cycle Progression; Cell Proliferation; Cell division; Cells; Chromatin; Chromosomes; Data; Disruption; Embryo; Epigenetic Process; Excision; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Green Fluorescent Proteins; Growth; Interphase; Knockout Mice; Life; Malignant - descriptor; Malignant Neoplasms; Memory; Metaphase; Mitosis; Mitotic; Mitotic Chromosome; Modification; Nocodazole; Nuclear Protein; Nuclear Proteins; Numbers; Nuts; Paclitaxel; Pattern; Pharmaceutical Preparations; Positive Transcriptional Elongation Factor B; Proteins; Recruitment Activity; Role; Series; Tubulin; Work; base; cell fixing; cell growth; cell type; chromatin immunoprecipitation; daughter cell; knock-down; outer space; promoter; telophase

During this period, we wished to address the role of Brd4 in mitotic progression and the regulation of gene expression relevant to cell growth. The role for Brd4 in cell division has been proposed based on early embryonic lethality observed in Brd4 knock out mice and growth inhibition in Brd4 knock down cells. In line with this, it has been shown that the BRD4 gene is truncated and fused to the NUT gene in some malignant cancer. Our work began with the finding that a series of anti-tublin drugs that cause mitotic arrest perturb Brd4-chromosome interactions. In the presence of anti-tubulin drugs such as nocodazole, taxol and colchicines, the bulk of Brd4 moved from condensed chromosomes to the outer space within dividing cells. This was seen both in live cells with exogenous Brd4 fused to GFP as well as in fixed cells with endogenous Brd4 from metaphase to telophase. Interestingly, this effect was reversed when nocodazole was washed away from culture. Although Brd4 remained outside of chromosomes as long as nocodazole was present in the media, within 30 min after drug removal, Brd4 was reloaded onto chromosomes. Furthermore, after nocodazole wash-out, wild type cells expressing normal levels of Brd4 resumed mitosis and produced new daughter cells. However, cells expressing less Brd4 (due to disruption of one Brd4 allele (Brd4 +/-) were incompetent in reloading Brd4 onto chromosomes. Importantly those cells that filed to bring Brd4 back onto chromosomes were defective in the ensuing cell division. Thus, a large fraction of Brd4+/- cells did not complete mitosis, resulting in a significantly fewer number of daughter cells. These data indicate that the Brd4-chromosome interaction has an important role in normal progression of mitosis and that This interaction is required for protecting cells from adverse effects of anti-tublin drugs. Our data with chromatin immunoprecipitation assays indicate that Brd4 is recruited to the promoters of genes that regulate cell cycle progression, presumably by binding to the chromatin. Some agents including anti-tubulin drugs appear to interfere with Brd4 recruitment. It would be of importance to study genome-wide Brd4 recruitment and to identify agents that alter its pattern.

在此期间，我们希望解决BRD4在有丝分裂进程中的作用以及与细胞生长相关的基因表达的调节。 BRD4在细胞分裂中的作用是基于在BRD4中观察到的早期胚胎致死性的，在BRD4敲除小鼠和BRD4敲低细胞中的生长抑制作用。与此相一致，已经表明，在某些恶性癌中，BRD4基因被截短并融合到了坚果基因。我们的工作始于这样的发现，该发现是一系列引起有丝分裂停滞扰动BRD4-染色体相互作用的抗呼吸毒药。在存在抗微管蛋白药物（例如诺科唑，紫杉醇和秋水仙碱）的情况下，大部分BRD4从凝结的染色体转移到分裂细胞内的外太空。在具有与GFP融合的外源性BRD4的活细胞以及内源性BRD4从中期到末期的固定细胞中都可以看到这一点。有趣的是，当诺科唑被洗掉培养物时，这种效果被逆转。尽管只要在培养基中出现诺科唑，但BRD4仍留在染色体外，但在药物去除后的30分钟内，BRD4被重新加载到染色体上。 此外，在诺科唑洗涤后，表达正常水平BRD4水平的野生型细胞恢复有丝分裂并产生新的子细胞。但是，表达较少BRD4的细胞（由于一个BRD4等位基因（BRD4 +/-）的破坏，将BRD4重新加载到染色体上是无能的。重要的是，重要的是，将BRD4恢复到染色体上的细胞在随后的细胞分裂中有缺陷。 BRD4-染色体的相互作用在有丝分裂的正常进展中具有重要作用，并且这种相互作用是保护细胞免受抗抗蛋白药物的不良反应，而我们的数据具有染色质免疫沉淀测定法，这表明BRD4表明与细胞循环的促进者募集了与某些相关性倾斜的基因相关的基因。 BRD4招聘。