HISTONE/DNA INTERACTIONS DURING CHROMATIN BIOSYNTHESIS

染色质生物合成过程中的组蛋白/DNA 相互作用

• 批准号: 2178088
• 负责人: Anthony Annunziato
• 金额: $ 16.47万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178088
• 关键词: DNA replication; HeLa cells; acetylation; acyltransferase; chemical association; chromatin; enzyme inhibitors; histones; immunoprecipitation; nucleosomes; phosphorylation; protein biosynthesis

DESCRIPTION (Adapted from Applicant's Abstract): The long-term objective of this research program is to elucidate the mechanisms of somatic chromatin biosynthesis, and to understand the propagation of epigenetic information to progeny cells. To this end, the present proposal focuses on the function of histone acetylation and phosphorylation during chromatin replication, and on the analysis of nucleosome pre-assembly complexes that have been observed in HeLa, a transformed human cell line. Experiments will be performed to dissect the composition of soluble non- nucleosomal histone complexes that accumulate when DNA replication is inhibited, and to identify possible somatic histone escort proteins. This will be accomplished by 1) immunoprecipitating radiolabeled cell extracts with anti-histone antibodies, including antibodies specific for acetylated H4; and 2) probing the purified complexes with antibodies that recognize known somatic nucleosome assembly factors. The involvement of H4 acetylation in escort protein recognition will also be assessed, by challenging somatic cell extract with synthetic polypeptides that represent either the acetylated or the unacetylated N-terminus of histone H4. In parallel experiments, the ability of soluble histone complexes to participate in nucleosome assembly in vivo and in vitro will also be determined. As part of the above studies, the properties of a cytosolic histone acetyltransferase, which preliminary experiments strongly suggest is involved in acetylation of newly synthesized histone H4, will be examined. The activity, specificity, and cell cycle regulation of this acetyltransferase (which is specific for H4, and appears to be a member of the "acetyltransferase B" family) will be determined through a straightforward in vitro assay, using either purified histones or acetylated and unacetylated H4 N-terminal peptides as substrates. The ability of this acetyltransferase to facilitate histone complex formation will also be examined. Finally, antibodies that recognize the phosphorylated, but not the unphosphorylated forms of histone H1 will be used to study the role of H1 phosphorylation in chromatin replication. Immunoprecipitations of cross- linked (radiolabeled) chromatin fragments will be performed to determine the distribution of phosphorylated H1 with respect to newly replicated DNA, and to newly assembled nucleosomes. By immunoprecipitating chromatin DNA labeled during replication in cycloheximide, the relative degree to which phosphorylated H1 is presented on segregated parental nucleosomes will also be analyzed.

描述（改编自申请人摘要）：长期目标