Siddiqui Hasan, Fox Sejal R, Gunawardena Ranjaka W, Knudsen Erik S
Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521, USA.
J Cell Physiol. 2007 Apr;211(1):131-7. doi: 10.1002/jcp.20913.
Heterochromatin domains are important for gene silencing, centromere organization, and genomic stability. These genomic domains are marked with specific histone modifications, heterochromatin protein 1 (HP1) binding and DNA methylation. The retinoblastoma tumor suppressor, RB mediates transcriptional repression and functionally interacts with a number of factors that are involved in heterochromatin biology including HP1, Suv39h1, DNMT1, and components of the SWI/SNF chromatin remodeling complex. To analyze the specific influence of RB loss on chromatin modification, mouse adult fibroblasts (MAFs) derived from Rb(loxP/loxP) mice were utilized to acutely knockout RB. In this setting, target genes of RB are deregulated. Additionally, changes in histone modifications were observed. Specifically, histone H4 lysine 20 trimethylation was absent from heterochromatin domains following loss of RB and there were changes in the relative levels of histone modifications between RB-proficient and deficient cells. While RB loss significantly altered the modifications associated with heterochromatin domains, these domains were readily identified and efficiently mediated the recruitment of HP1alpha. Kinetic analyses of HP1alpha within the heterochromatin domains present in RB-deficient cells indicated that loss of RB retarded HP1alpha dynamics, indicating that HP1alpha is paradoxically more tightly associated with heterochromatin in the absence of RB function. Combined, these analyses demonstrate that loss of RB has global effects on chromatin modifications and dynamics.
异染色质结构域对于基因沉默、着丝粒组织和基因组稳定性至关重要。这些基因组结构域通过特定的组蛋白修饰、异染色质蛋白1（HP1）结合和DNA甲基化进行标记。视网膜母细胞瘤肿瘤抑制因子RB介导转录抑制，并与许多参与异染色质生物学的因子发生功能相互作用，包括HP1、Suv39h1、DNMT1以及SWI/SNF染色质重塑复合体的成分。为了分析RB缺失对染色质修饰的具体影响，利用源自Rb(loxP/loxP)小鼠的小鼠成年成纤维细胞（MAF）急性敲除RB。在这种情况下，RB的靶基因失调。此外，还观察到组蛋白修饰的变化。具体而言，RB缺失后，异染色质结构域中不存在组蛋白H4赖氨酸20三甲基化，并且RB功能正常和缺陷细胞之间组蛋白修饰的相对水平发生了变化。虽然RB缺失显著改变了与异染色质结构域相关的修饰，但这些结构域很容易被识别，并有效地介导了HP1α的募集。对RB缺陷细胞中存在的异染色质结构域内的HP1α进行动力学分析表明，RB缺失阻碍了HP1α的动态变化，这表明在缺乏RB功能的情况下，HP1α反常地与异染色质结合更紧密。综合这些分析表明，RB缺失对染色质修饰和动态变化具有全局性影响。
