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Biochemical and Genetic Analysis of yFACT, A Novel Nucleosome Reorganizing Factor

新型核小体重组因子 yFACT 的生化和遗传分析

基本信息

批准号：
8450938
负责人：
Timothy G Formosa
金额：
$ 29.98万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-03-01 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8450938
关键词：
Adopted Affect Attention Biochemical Genetics Biological Assay Cell physiology Cells Characteristics Chromatin Chromatin Modeling Chromatin Structure Complex Crosslinker Crystallography DNA DNA Packaging DNA biosynthesis DNA replication origin Defect Deposition Development Embryonic Development Energy-Generating Resources Enhancers Eukaryota Eukaryotic Cell Fluorescent Dyes Gene Expression Genetic Genetic Screening Genetic Transcription Genome Goals Grant Health Histone H2A Histones Human In Vitro Label Lead Learning Life Maps Measurement Methods Modeling Molecular Chaperones Mutation Nature Nucleosomes Organism Outcome Phase Physiological Physiology Play Polymerase Process Property Proteins RNA Reactive Oxygen Species Repression Research Role Stem cells Structure Systems Biology Techniques Technology Testing Transcript Work Yeast Model System Yeasts abstracting chromatin immunoprecipitation dimer genetic analysis genetic manipulation genome sequencing histone-binding proteins in vivo insight mutant novel pluripotency prevent promoter protein complex public health relevance research study tool

项目摘要

DESCRIPTION (provided by applicant): Project Summary/Abstract FACT is a complex of two proteins that are found in all eukaryotes, including humans, and is essential for viability in all organisms tested. Nucleosomes are the fundamental subunit of chromatin, which is the DNA packaging in eukaryotes. Their properties govern the flow of information from the genome. The goals of this project are to determine how FACT causes nucleosomes to adopt an alternative structure and to learn when and where the ability to form and resolve such structures is useful in living cells. FACT is unique among factors that regulate chromatin because it does not modify the histones that make up nucleosomes, it does not use an energy source to move the histone cores along the DNA, and it works through a mechanism that has not been observed with any other histone chaperone. FACT has been identified in a wide range of systems biology studies seeking factors that regulate cellular physiology, promote normal embryonic development, and allow stem cells to maintain pluripotency. Both the mechanism of FACT action and the circumstances in which it is used are therefore of high importance for understanding basic eukaryotic physiology. An integrated approach using a yeast model system is proposed for these studies to take advantage of the range of tools available in these organisms. The first aim of the proposal is to analyze the properties of nucleosomes that have been altered by FACT to produce what is called a "reorganized" form. This structure is significantly different from canonical nucleosomes, but its nature remains poorly characterized. Purified histones and DNA fragments labeled with fluorescent dyes will be used to probe the properties of reorganized nucleosomes, and mutant histone and FACT proteins will be used in these assays to allow the results to be compared with the properties of FACT in living cells. The second aim is to characterize the roles of FACT by identifying mutations that make it easier or harder to tolerate FACT defects using whole-genome sequencing, and by asking how distinct mutations in FACT and other histone chaperones affect the ability to maintain repression of cryptic promoters and promote stability of distinct chromatin states using quantitative PCR measurements of transcripts and chromatin immunoprecipitations (ChIPs). The third aim is to ask how FACT affects chromatin structure near origins of DNA replication, how this contributes to regulating origin firing, and to test a hypothetical role for FACT in depositing new chromatin after the genome is duplicated during replication. This aim uses some established ChIP technology but also requires development of some novel methods.

描述（由申请人提供）：