Chromosome Architecture: Cohesion of Transcriptionally Silenced Domains

染色体结构：转录沉默域的内聚

基本信息

批准号：
7989672
负责人：
Marc R. Gartenberg
金额：
$ 5.32万
依托单位：
UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-18 至 2011-05-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7989672
关键词：
Accounting Address Anaphase Appearance Architecture Binding Biological Assay Biological Models Bruck-de Lange syndrome Chromatids Chromatin Chromosomal Instability Chromosomes Complex DNA DNA biosynthesis Defect Development Disease Employee Strikes Eukaryota Fluorescence Microscopy Genes Genetic Recombination Genetic Transcription Genome Genome Stability Heterochromatin Histone Deacetylase Human Human Cell Line In Vitro Lead Malignant Neoplasms Maps Meiosis Membrane Proteins Mitotic Molecular Molecular Genetics Names Nature Pathway interactions Proteins Recruitment Activity Research Personnel Roberts-SC phocomelia syndrome Role Saccharomyces cerevisiae Sister Chromatid Site Structure Testing Time Transfer RNA Yeast Model System Yeasts chromatin immunoprecipitation cohesin cohesion gene repression histone modification histone-binding proteins human disease novel protein complex research study

项目摘要

DESCRIPTION (provided by applicant): DNA replication generates pairs of sister chromatids that are held together until anaphase onset by a protein complex named cohesin. The phenomenon, termed sister chromatid cohesion, is critical for the proper loading of chromatids onto mitotic and meiotic spindles. In human cell lines, defects in the pathway produce levels of chromosome instability that match those seen in cancers. In humans, other pathway defects are responsible for at least three developmental diseases: Cornelia de Lange Syndrome (CdLS), Roberts syndrome (RBS) and SC phocomelia (SC). At the cellular level, RBS and SC are characterized by a striking loss of cohesion in chromatid domains containing heterochromatin, a form of chromatin that was first noted for its constitutively condensed appearance. Heterochromatin is now known to contain specialized histone modifications and non-histone binding proteins that hinder a variety of DNA transactions, including transcription of most genes. Using a combined approach of fluorescence microscopy and site-specific recombination, my lab discovered that cohesion of heterochromatin-like domains in yeast Saccharomyces cerevisiae is distinct from cohesion elsewhere in the genome. More recently we found that cohesion of the transcriptionally silenced HMR locus relies specifically on Sir2, an integral heterochromatin component with NAD-dependent histone deacetylase activity, and a tRNA gene that serves as a boundary to one heterochromatin domain. Our yeast model system provides an ideal opportunity to investigate the mechanisms of heterochromatic cohesion. We have developed a novel set of cytological assays to study cohesion at specific sites that will be used in combination with chromatin immunoprecipitation to address four questions. First, we will identify the features of the tRNA gene that facilitate cohesion and determine the step in the cohesion pathway that they act. Second, we will determine whether Sir2 can recruit cohesin directly and identify the protein surfaces and binding partners necessary for recruitment. Third, we will test whether typical components of the cohesin pathway contribute to the specialized form of cohesion at heterochromatin. We will also isolate heterochromatin fragments to examine the binding mode of cohesin in vitro. Last, we will use molecular genetic assays to assess the role of cohesion in heterochromatic silencing and genome stability.

描述（由申请人提供）：DNA复制会产生成对的姐妹染色单体，这些姐妹染色单体被固定在一起，直到被称为粘蛋白的蛋白质络合物开始。该现象称为姐妹染色质剂，对于将染色单体适当地加载到有丝分裂和减数分裂纺锤体上至关重要。在人类细胞系中，途径中的缺陷会产生与癌症中看到的染色体不稳定性水平。在人类中，其他途径缺陷至少导致三种发育疾病：Cornelia de Lange综合征（CDLS），Roberts综合征（RBS）和SC Phocomelia（SC）。在细胞水平上，RB和SC的特征是在含有异染色质的染色单体结构域中的凝聚力丧失，这是一种染色质的形式，该形式以其组成性凝结的外观而最初引起。现在已知异染色质包含专门的组蛋白修饰和非固定结合蛋白，这些蛋白会阻碍各种DNA交易，包括大多数基因的转录。使用荧光显微镜和特异性重组的组合方法，我的实验室发现，酿酒酵母中异染色质样结构域的凝聚力与基因组其他地方的凝聚力不同。最近，我们发现转录沉默的HMR基因座的凝聚力特别依赖于SIR2，具有NAD依赖性组蛋白脱乙酰基酶活性的积分异染色质成分，以及一个tRNA基因，该基因充当一个异质素域的边界。我们的酵母模型系统提供了研究异质凝聚力机制的理想机会。我们已经开发了一组新型的细胞学测定，以研究特定部位的凝聚力，这些凝聚力将与染色质免疫沉淀一起使用，以解决四个问题。首先，我们将确定促进凝聚力的tRNA基因的特征，并确定它们作用的内聚力途径的步骤。其次，我们将确定SIR2是否可以直接募集粘蛋白并确定募集所需的蛋白质表面和结合伙伴。第三，我们将测试粘着素途径的典型成分是否有助于异染色质的特殊凝聚力形式。我们还将分离出异染色质片段，以检查体外粘蛋白的结合模式。最后，我们将使用分子遗传测定法来评估凝聚力在异质沉默和基因组稳定性中的作用。