Multiple Overlapping Mammalian Chromatin Remodeling Systems Collaborate Genome-wide at Dynamic Chromatin Transitions [Affymetrix]

Expression data from 3134 mouse mammary epithelial cell line -/+ Tet-inducible chromatin remodeler mutant variants ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. We also examined effects of mutant remodeler variants on selective gene expression by global analysis of RNA expression patterns and found more than 800 genes are deregulated by these variants. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes. Flag tagged dominant negative variants of the chromatin remodelers Brg1, Chd4, and Snf2h were each stably integrated into a cell line containg the tetracycline transactivator regulatory system (3134Tet (7110)). The 3134Tet control cells and each of the dominant-negative remodeler cell lines were grown in media with or without tetracycline for 48 hrs to induce expression of the tetracycline transactivator alone (3134Tet cells) or tetracycline transactivator and dominant negative remodeler variant. Following treatment, cells were harvested for RNA extraction and hybridization to Affymetrix Mouse Gene 1.0 ST arrays.

本数据集源自3134小鼠乳腺上皮细胞系，实验分为添加与不添加四环素诱导型染色质重塑因子（chromatin remodeler）突变体变体两组。ATP依赖型染色质重塑是染色质结构动态组织所必需的核心过程。本研究报道了小鼠基因组中三种具备不同生理功能的重塑因子——Brg1、Chd4与Snf2h——的全基因组定位与活性特征。三种因子的定位模式彼此高度重合，且与开放染色质区域高度重叠。进一步通过诱导型突变体变体实验，本研究证实这些因子的催化活性可介导全基因组范围内的染色质重塑，且每种重塑因子均可独立调控特定位点的染色体重构。诸多染色质区域的可及性调控需要多种重塑因子协同发挥作用。本研究还通过全局RNA表达谱分析，检测了重塑因子突变体变体对选择性基因表达的影响，发现超过800个基因的表达受这些变体调控失调。上述研究结果为染色质组织模式提供了动态视角，并凸显了不同重塑因子在高等真核生物染色质维持中的差异化贡献。将带有Flag标签的染色质重塑因子Brg1、Chd4与Snf2h的显性负突变体，分别稳定整合至携带四环素反式激活因子调控系统的细胞系3134Tet (7110)中。将3134Tet对照细胞与各显性负重塑因子细胞系，分别置于添加或不添加四环素的培养基中培养48小时，以诱导仅表达四环素反式激活因子（3134Tet细胞），或同时表达四环素反式激活因子与显性负重塑因子变体。处理结束后，收集细胞用于RNA提取，并与Affymetrix小鼠基因1.0 ST芯片进行杂交。