RBP1 Recruits the mSIN3-Histone Deacetylase Complex to the Pocket of Retinoblastoma Tumor Suppressor Family Proteins Found in Limited Discrete Regions of the Nucleus at Growth Arrest

Abstract

Retinoblastoma (RB) tumor suppressor family pocket proteins induce cell cycle arrest by repressing transcription of E2F-regulated genes through both histone deacetylase (HDAC)-dependent and -independent mechanisms. In this study we have identified a stable complex that accounts for the recruitment of both repression activities to the pocket. One component of this complex is RBP1, a known pocket-binding protein that exhibits both HDAC-dependent and -independent repression functions. RB family proteins were shown to associate via the pocket with previously identified mSIN3-SAP30-HDAC complexes containing exclusively class I HDACs. Such enzymes do not interact directly with RB family proteins but rather utilize RBP1 to target the pocket. This mechanism was shown to account for the majority of RB-associated HDAC activity. We also show that in quiescent normal human cells this entire RBP1-mSIN3-SAP30-HDAC complex colocalizes with both RB family members and E2F4 in a limited number of discrete regions of the nucleus that in other studies have been shown to represent the initial origins of DNA replication following growth stimulation. These results suggest that RB family members, at least in part, drive exit from the cell cycle by recruitment of this HDAC complex via RBP1 to repress transcription from E2F-dependent promoters and possibly to alter chromatin structure at DNA origins.

ACKNOWLEDGMENTS

We thank Bill Kaelin and Jim DeCaprio for providing monoclonal antibodies against RBP1. We also thank Eva Lee, Betty Moran, and Bob Eisenman for providing additional reagents. Wei-Ming Yang and Ed Seto provided class I HDAC-related reagents; those for class II HDAC were from Xiang Jiao Yang and Nick Bertos. Deconvolution microscopy was performed at the Whitehead Institute Microscopy Facility.

This work was supported through grants to P.E.B. from the National Cancer Institute of Canada and the Canadian Institutes for Health Research and to D.R. from NIH (GM485180) and the HHMI. A. L. is the recipient of Terry Fox Biomedical Studentship supported by National Cancer Institute of Canada. M.-C.T is supported by a scholarship from the Fonds pour la Formation de Chercheurs et l' Aide à la Recherche (FRSQ-FCAR-Santé). B.K.K. is supported by a Leukemia Society of America Fellowship, D.A.B. is supported by a Karen Grunebaum Cancer Research Fellowship, and Y.Z. is supported by an NIH Fellowship.