Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.
Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.
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DNA 复制相关染色质解缩分析：了解所选蛋白质染色质重塑机制的体内测定。
DOI：
10.1007/978-1-4939-2474-5_16
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Alexandrow,MarkG
中科院分区：
文献类型：
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作者：
Borysov,Sergiy;Bryant,VictoriaL;Alexandrow,MarkG
对基因表达的转录控制的许多事件至关重要的是对核心组蛋白和连接组蛋白的修饰，其调节染色质背景下反式作用因子对DNA底物的可及性。同样地，已知对DNA复制起始的控制以及复制机制在延伸期间通过可能遇到的多个水平的染色质浓缩进行的能力涉及染色质可接近性的产生。在后一种情况下，染色质接近可能需要是短暂的事件，以防止对细胞有害的染色质的总基因组解开。虽然有许多分子和生物化学方法用于研究组蛋白变化及其与转录和染色质可及性的关系，但很少有技术允许对DNA复制控制的基础事件进行分子解剖，因为它涉及染色质变化和可及性。在这里，我们概述了一种新的实验策略，用于解决特定蛋白质的能力，以诱导大规模的染色质展开（解凝聚）后，在体内位点特异性靶向工程基因座。我们的实验室已经以新的方式使用这个强大的系统来直接解决DNA复制蛋白质创建染色质可及性的能力，并对基本方法进行了修改，允许对引起感兴趣的蛋白质的染色质去凝聚的机制和相关因素进行分子遗传学分析。涉及其他蛋白质（竞争对手或刺激），同时药物治疗，共定位组蛋白修饰的分析共表达的替代方法也得到了解决，所有这些都说明了这个实验系统的实用性，用于扩展基本的发现生理相关的机制。虽然我们的小组使用的DNA复制相关的染色质可及性分析机制，这个独特的和强大的实验系统有可能是一个有价值的工具，了解染色质重塑机制编排的其他细胞过程，如DNA修复，重组，有丝分裂染色体凝聚，或其他染色体动力学涉及染色质改变和可及性。
Of critical importance to many of the events underlying transcriptional control of gene expression are modifications to core and linker histones that regulate the accessibility oftrans-acting factors to the DNA substrate within the context of chromatin. Likewise, control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is known to involve the creation of chromatin accessibility. In the latter case, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility. Here, we outline a novel experimental strategy for addressing the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. Our laboratory has used this powerful system in novel ways to directly address the ability of DNA replication proteins to create chromatin accessibility, and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the mechanisms and associated factors involved in causing chromatin decondensation by a protein of interest. Alternative approaches involving co-expression of other proteins (competitors or stimulators), concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, all of which are illustrative of the utility of this experimental system for extending basic findings to physiologically relevant mechanisms. Although used by our group to analyze mechanisms underlying DNA replication associated chromatin accessibility, this unique and powerful experimental system has the propensity to be a valuable tool for understanding chromatin remodeling mechanisms orchestrated by other cellular processes such as DNA repair, recombination, mitotic chromosome condensation, or other chromosome dynamics involving chromatin alterations and accessibility.