Regulation of Chromatin Structure byPhosphorylation

通过磷酸化调节染色质结构

• 批准号: 6776371
• 负责人: Kristen M Johansen
• 金额: $ 23.99万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6776371
• 关键词: Drosophilidae; biological signal transduction; cell cycle; cell nucleus; chromatin; chromosome aberrations; developmental genetics; gender difference; gene dosage; gene expression; gene targeting; genetic regulation; genetically modified animals; growth /development; histones; immunocytochemistry; invertebrate embryology; microscopy; molecular site; northern blottings; nucleic acid structure; phosphorylation; phosphotransferases; protein structure function; western blottings; yeast two hybrid system

APPLICANT?S DESCRIPTION: The long term objective of this study is to determine how signal transduction pathways in the nucleus regulate chromosomal architecture and gene expression. Towards this end, we have identified a novel tandem kinase in Drosophila, JIL-1, that associates with the chromosomes throughout the cell cycle, localizes specifically to the gene-active interband regions of the larval polytene chromosomes, is capable of phosphorylating histone H3 in vitro, and is enriched almost two-fold on the transcriptionally hyperactive male larval polytene X chromosome due to its association with the MSL dosage compensation complex. Histone H3 serine 10 phosphorylation levels are severely reduced in embryos from a hypomorphic JIL-1 mutant, thus placing JIL-l in the pathway mediating histone H3 phosphorylation. In addition, with high penetrance these mutants (which contain low levels of JIL-l kinase) show a number of chromosomal abnormalities, including lack of condensation at mitosis, disorganization in alignment at the mitotic spindle, separation from centrosomes, and ultimately chromosomal breakdown and fragmentation. Only 5 percent of the embryos survive to hatching, and of the animals that survive to adulthood, females outnumber males by a 2:1 ratio, supporting the hypothesis that JIL-1 is essential in dosage compensation mechanisms. Thus, these findings suggest a model where JIL-l signaling plays a direct functional role in chromosomal regulation and maintenance throughout the cell cycle, including condensation and segregation of chromosomes at metaphase as well as modification of chromatin and transcriptional regulation at interphase. We propose to test this model by generating a range of JIL-l mutant alleles, including a complete null, which will allow us to genetically dissect the functional requirements for JIL-l in different pathways and at different stages. We will determine whether histone H3 is a direct or indirect target of JIL-1 using biochemical approaches, and we will identify other potential substrates and interaction partners using genetic and yeast two-hybrid approaches. Our expectation is that JIL-l function is necessary in more than one chromatin remodeling complex, since JIL-l is found in females and on autosomes in addition to its presence on the male X-specific chromatin remodeling complex. The powerful molecular, genetic, and cellular approaches available in Drosophila are uniquely suited to the analysis of such complex signaling pathways. Thus, these studies will provide valuable new insights into the role of phosphorylation in regulation of gene expression and chromatin structure of fundamental significance for understanding both normal and cancerous developmental processes.

申请人的描述：这项研究的长期目标是确定 核中信号转导途径如何调节染色体 建筑和基因表达。为此，我们确定了一本小说 果蝇中的串联激酶，JIL-1，与染色体相关联 在整个细胞周期中，专门定位于基因活跃的边界 幼虫聚染色体的区域能够磷酸化 体外组蛋白H3，在转录上富集了几乎两倍 多活跃的雄性幼虫聚tene x染色体，因为它与 MSL剂量补偿复合物。组蛋白H3丝氨酸10磷酸化水平 从肌肌jil-1突变体中严重降低了胚胎，从而放置 JIL-L在介导组蛋白H3磷酸化的途径中。另外，与 这些突变体（含有低水平的JIL-L激酶）显示出一个 染色体异常数量，包括缺乏有丝分裂的凝结， 在有丝分裂主轴上对齐的混乱，与 中心体，最终是染色体分解和碎片化。只有5 胚胎的百分比生存到孵化，以及生存的动物的百分比 成年，女性的人数超过2：1的比例，支持假设 JIL-1在剂量补偿机制中至关重要。因此，这些发现 建议一个模型，其中JIL-L信号在 在整个细胞周期中，包括 中期和染色体的凝结和分离 在相间的染色质和转录调节的修饰。我们 建议通过生成一系列JIL-L突变等位基因来测试此模型， 包括完整的零，这将使我们能够基因剖析 在不同途径和不同的途径中JIL-L的功能要求 阶段。我们将确定组蛋白H3是直接还是间接目标 JIL-1使用生化方法，我们将确定其他潜力 使用遗传和酵母双杂交的底物和相互作用伙伴 方法。我们的期望是，JIL-L功能在不仅仅 一种染色质重塑复合物，因为在女性中发现了JIL-L 常染色体除了在雄性X特异性染色质上的存在外 重塑复合物。强大的分子，遗传和细胞方法 果蝇中可用 信号通路。因此，这些研究将为您提供宝贵的新见解 磷酸化在基因表达和染色质调节中的作用 理解正常和 癌性发育过程。