Loss of RB compromises specific heterochromatin modifications and modulates HP1alpha dynamics.

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. (c) 2007 Wiley-Liss, Inc.