HISTONE/DNA INTERACTIONS DURING CHROMATIN BIOSYNTHESIS

描述（改编自申请人的摘要）：长期目标 该研究计划的目的是阐明体细胞的机制 染色质生物合成，并了解表观遗传的传播 传递给子代细胞的信息。 为此，本提案重点 组蛋白乙酰化和磷酸化的功能 染色质复制以及核小体预组装的分析 在 HeLa（一种转化的人类细胞）中观察到的复合物 线。 将进行实验来剖析可溶性非-的成分 DNA 复制时积累的核小体组蛋白复合物 抑制，并鉴定可能的体细胞组蛋白护航蛋白。 这将通过 1) 免疫沉淀放射性标记细胞来完成 提取物含有抗组蛋白抗体，包括特异性抗体 乙酰化H4； 2) 用抗体探测纯化的复合物 识别已知的体细胞核小体组装因子。 的 H4 乙酰化参与护送蛋白识别也将 通过用合成物挑战体细胞提取物来进行评估 代表乙酰化或非乙酰化的多肽 组蛋白 H4 的 N 末端。 在平行实验中，能力 可溶性组蛋白复合物参与体内核小体组装 体外实验也将被确定。 作为上述研究的一部分，胞质组蛋白的特性 乙酰转移酶，初步实验强烈表明 参与新合成的组蛋白 H4 的乙酰化，将 检查了。 该物质的活性、特异性和细胞周期调节 乙酰转移酶（H4 特异，似乎是 “乙酰转移酶 B”家族的成员）将通过 直接的体外测定，使用纯化的组蛋白或 乙酰化和未乙酰化的 H4 N 端肽作为底物。 的 这种乙酰转移酶促进组蛋白复合物的能力 还将检查其形成。 最后，抗体识别磷酸化，但不识别磷酸化 组蛋白 H1 的非磷酸化形式将用于研究 染色质复制中的 H1 磷酸化。 免疫沉淀 将进行交联（放射性标记）染色质片段 确定磷酸化 H1 相对于新的分布 复制的DNA，以及新组装的核小体。 由 复制过程中标记的免疫沉淀染色质 DNA 放线菌酮，H1 磷酸化的相对程度 还将分析分离的亲本核小体的呈现。

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.