Xenopus Egg Extracts Increase Dynamics of Histone H1 on Sperm Chromatin
Xenopus Egg Extracts Increase Dynamics of Histone H1 on Sperm Chromatin
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DOI：
10.1371/journal.pone.0013111
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发表时间：
2010-09-29
期刊：
影响因子：
3.7
通讯作者：
Heald, Rebecca
中科院分区：
文献类型：
--
作者：
Freedman, Benjamin S.;Miller, Kelly E.;Heald, Rebecca
工作背景：已经在体内和使用重建的染色质研究了连接体组蛋白H1，但是很少有系统的研究细胞环境对其功能的影响。由于存在许多其他染色质因子和特定的伴侣，如RanBP 7/输入蛋白β，调节组蛋白H1，接头组蛋白可能在体内的功能不同，比在纯化systems.Methodology/主要发现：我们直接比较了H1结合到精子细胞核的缓冲液与非洲爪蟾卵提取物的细胞质，并监测加入核输入伴侣的影响。在缓冲液中，RanBP 7使精子核去致密化，而H1与染色质紧密结合并挽救RanBP 7介导的去致密化。H1结合在细胞质中减少，H1在细胞质中表现出快速的FRAP动力学，但在缓冲液中则不然。RanBP 7减少H1结合染色质在缓冲液和提取物，但不显着影响H1动力学在任何条件下。Importin β对精子染色质去凝聚和H1结合的影响小于RanBP 7，而RanBP 7/importin β的组合并不比单独的RanBP 7更有效。在补充RanBP 7的提取物中，H1定位于染色体病灶，其在DNA损伤后增加。与体细胞H1不同，与缓冲液相比，胚胎接头组蛋白H1 M同样良好地结合细胞质中的染色质。H1 M的氨基球状和羧基末端结构域在缓冲液中将染色质结合到全长蛋白质上，但在细胞质中被抑制10倍。高水平的H1或其截断扭曲有丝分裂染色体，并阻止他们的分离在后期。结论/意义：RanBP 7可以deconcense精子核和减少H1结合，但H1对染色质的快速动力学依赖于其他细胞质因子。细胞质极大地削弱了单个H1结构域的活性，只有全长蛋白质才能正确地浓缩染色质。我们的发现开始弥合纯化和体内染色质系统之间的差距。
Background: Linker histone H1 has been studied in vivo and using reconstituted chromatin, but there have been few systematic studies of the effects of the cellular environment on its function. Due to the presence of many other chromatin factors and specific chaperones such as RanBP7/importin beta that regulate histone H1, linker histones likely function differently in vivo than in purified systems.Methodology/Principal Findings: We have directly compared H1 binding to sperm nuclei in buffer versus Xenopus egg extract cytoplasm, and monitored the effects of adding nuclear import chaperones. In buffer, RanBP7 decondenses sperm nuclei, while H1 binds tightly to the chromatin and rescues RanBP7-mediated decondensation. H1 binding is reduced in cytoplasm, and H1 exhibits rapid FRAP dynamics in cytoplasm but not in buffer. RanBP7 decreases H1 binding to chromatin in both buffer and extract but does not significantly affect H1 dynamics in either condition. Importin beta has a lesser effect than RanBP7 on sperm chromatin decondensation and H1 binding, while a combination of RanBP7/importin beta is no more effective than RanBP7 alone. In extracts supplemented with RanBP7, H1 localizes to chromosomal foci, which increase after DNA damage. Unlike somatic H1, the embryonic linker histone H1M binds equally well to chromatin in cytoplasm compared to buffer. Amino-globular and carboxyl terminal domains of H1M bind chromatin comparably to the full-length protein in buffer, but are inhibited similar to 10-fold in cytoplasm. High levels of H1 or its truncations distort mitotic chromosomes and block their segregation during anaphase.Conclusion/Significance: RanBP7 can decondense sperm nuclei and decrease H1 binding, but the rapid dynamics of H1 on chromatin depend on other cytoplasmic factors. Cytoplasm greatly impairs the activity of individual H1 domains, and only the full-length protein can condense chromatin properly. Our findings begin to bridge the gap between purified and in vivo chromatin systems.