Regulation of Chromatin Structure byPhosphorylation

申请人-S描述：这项研究的长期目标是确定 核内信号转导通路如何调控染色体 建筑和基因表达。为此，我们确定了一部小说 果蝇中与染色体相关的串联蛋白JIL-1 在整个细胞周期中，特异性地定位于基因活跃的相互作用带 幼虫多线染色体的区域，能够磷酸化 组蛋白H3在体外，并在转录上富含近两倍 过度活跃的雄性幼虫多线X染色体与 MSL剂量补偿复合体。组蛋白H3丝氨酸10的磷酸化水平 在低形态的JIL-1突变体的胚胎中严重减少，因此将 JIL-L在介导组蛋白H3磷酸化中的作用。此外，使用 这些突变体(含有低水平的JIL-L激酶)表现出高外显性 染色体异常的数量，包括有丝分裂时缺乏凝集， 有丝分裂纺锤体排列紊乱，与 中心体，最终导致染色体破裂和碎裂。只有5个 胚胎存活到孵化的百分比，存活到孵化的动物 成年后，女性的数量超过男性的比例为2：1，支持了这一假设 IL-1在剂量补偿机制中起着至关重要的作用。因此，这些发现 建议一个JIL-L信号在其中发挥直接作用的模型 整个细胞周期的染色体调节和维持，包括 中期和中期染色体的凝聚和分离 染色质的修饰和间期的转录调节。我们 建议通过产生一系列吉林-L突变等位基因来测试这一模型， 包括一个完整的零，这将允许我们从基因上解剖 吉林-L在不同途径、不同阶段的功能需求 各阶段。我们将确定组蛋白H3是直接还是间接靶点 JIL-1使用生化方法，我们将确定其他潜在的 使用遗传和酵母双杂交的底物和相互作用伙伴 接近了。我们的期望是，吉利-L函数在超过 一个染色质重塑复合体，因为Jil-L在女性和其他人中发现 常染色体除了存在于男性X特异染色质外 重塑综合体。强大的分子、遗传和细胞方法 在果蝇中可用的是唯一适合于分析这种复合体的 信号通路。因此，这些研究将提供有价值的新见解 磷酸化在基因表达和染色质调控中的作用 具有基本意义的结构，对于理解正常和 癌症的发育过程。

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.