Parental nucleosomes segregated to newly replicated chromatin are underacetylated relative to those assembled de novo.
Parental nucleosomes segregated to newly replicated chromatin are underacetylated relative to those assembled de novo.
复制标题
与新复制的染色质分离的亲本核小体相对于从头组装的亲本核小体乙酰化程度较低。
DOI：
10.1021/bi00212a029
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Annunziato,AT
中科院分区：
文献类型：
--
作者：
Perry,CA;Allis,CD;Annunziato,AT
摘要：乙酰化组蛋白H4的特异性抗体被用来检测分离到新复制DNA的亲本组蛋白的乙酰化状态。为了产生仅含有分离的亲本核小体的新复制的染色质，在体外用[3 H] TTP标记分离的细胞核;或者，在放线菌酮存在下用[3 H]胸苷标记整个细胞。通过微球菌核酸酶消化制备可溶性染色质，并用“penta”抗体进行免疫沉淀（Lin et al.，1989年）。与此形成鲜明对比的是，含有新合成的二乙酰化H4的核小体（佩里等人，1993），体外复制的染色质仅对免疫沉淀轻微敏感。对照实验确定，真正的乙酰化染色质选择性免疫沉淀通过相同的技术和分离的核小体不解体与“五”抗体治疗期间。当复制与体外组蛋白乙酰化系统偶联时，免疫颗粒中分离的核小体的富集增加了约3倍，表明分离的组蛋白乙酰化状态的变化可以通过免疫学检测到，并且新DNA上的亲本组蛋白在DNA复制期间或之后立即可被乙酰转移酶接近。在放线菌酮存在下进行的体内脉冲追踪实验证实了这些结果。摄取实验进一步证实，同时组蛋白乙酰化并不改变体外DNA合成的速率。我们的研究结果提供了证据表明，复制能力的染色质是不强制乙酰化，并表明，乙酰化状态的分离组蛋白可能会保持在染色质复制。在DNA复制过程中，染色质高级结构的调节，以及转录活性与非活性染色质结构的传播方面，这可能具有的意义进行了讨论.有两种途径参与包装新的DNA进入核小体：从头核小体组装（需要新合成的H3和H4），和回收（或分离）的预先存在的组蛋白到新生的DNA.尽管核小体分离的方式，保守的还是分散的，一直是一个争论的问题[Annunziato（1990）]，但是亲本组蛋白分离成新DNA已经通过在没有同时组蛋白合成的情况下复制的染色质的分析得到证实，使用（1）翻译的替代物（Weintraub，1974，1976;西尔和Simpson，1975;西尔，1976年; Cremisi等人，1978;杰克逊和Chalkley，1981; Cusick等人，1984），（2）ts突变体（Roufa，1978），或（3）体外分离的细胞核（西尔，1978）。在这些条件下合成的DNA对核酸酶消化的敏感性大约是对照染色质的两倍，并且核酸酶抗性级分在含有组蛋白H1和HMG蛋白的典型核小体中（Annunziato&
Revised Manuscript Received August 31, 19939 abstract: Antibodies specific for acetylated histone H4 were used toexamine the acetylation state of parental histones that segregate to newlyreplicated DNA. To generate newly replicated chromatin containing only segregated parental nucleosomes, isolated nuclei were labeled with [3H] TTP in vitro; alternatively, whole cells were labeled with [3H] thymidine in the presence of cycloheximide. Soluble chromatin was prepared by micrococcal nuclease digestion, and subjected to immunoprecipitation with “penta” antibodies (Lin et al., 1989). In sharp contrast tonucleosomes containing newly synthesized, diacetylated H4 (Perry et al., 1993), chromatin replicated in vitro was only marginally susceptible to immunoprecipitation. Control experiments established that bona fide acetylated chromatin was selectively immunoprecipitated by the same techniques and that segregated nucleosomes were not disassembled during treatment with “penta” antibodies. When replication was coupled to an in vitro histone acetylation system, the enrichmentfor segregated nucleosomes in the immunopellet increased approximately 3-fold, demonstrating that changes in the acetylation state of segregated histones can be detected immunologically and that parental histones on new DNA are accessible to acetyltransferases during, or immediately after, DNA replication. In vivo pulse-chase experiments, performed in the presence of cycloheximide, confirmed these results. Uptake experiments further established that concurrent histone acetylation did not alter the rate of DNA synthesis in vitro. Our results provideevidence that replication-competent chromatin is not obligatorily acetylated, and indicate that the acetylation status of segregated histones may be maintained during chromatin replication. The possible significance of this, with respect to the regulation of chromatin higher order structures during DNA replication, and the propagation of transcriptionally active vs inactive chromatin structures, is discussed.There are two pathways involvedin packaging new DNA into nucleosomes: de novo nucleosome assembly (requiring newly synthesized H3 and H4), and the recycling (or segregation) of preexisting histones onto nascent DNA. Although the mode of nucleosome segregation, conservative or dispersive, has been a matter of debate [reviewedin Annunziato (1990)], the segregation of parental histones to new DNA has been confirmed by analyses of chromatin replicated in the absence of concurrent histone synthesis, using either (1) inhibitorsof translation (Weintraub, 1974, 1976; Seale & Simpson, 1975; Seale, 1976; Cremisi et al., 1978; Jackson & Chalkley, 1981; Cusick et al., 1984),(2) a ts mutant (Roufa, 1978), or (3) isolated nuclei in vitro (Seale, 1978). DNA synthesized under these conditions is approximately twice as sensitive to nuclease digestion than control chromatin, and the nuclease-resistant fraction is in typical nucleosomes that contain histone H1 and HMG proteins (Annunziato&