Abstract 266: Differential activation of the alkaline phosphatase promoter by pRB and p107

Abstract

Abstract The retinoblastoma susceptibility gene, Rb1, is closely linked with osteosarcoma as well as retinoblastoma. Functional analysis of pRB and the closely related family members, p107 and p130, has tended to focus on repression of proliferation, but genetic evidence from our lab and others indicates an active requirement for pRB in osteoblast differentiation that correlates more directly with osteosarcoma susceptibility. Our prior studies linked the pRB family with activation of the early osteoblast marker, alkaline phosphatase (the product of the Alpl gene). We have shown by Chromatin immunoprecipitation (ChIP) analysis the Alpl promoter is targeted directly by pRB and p107 when active. We addressed the issue of the specific contribution of pRB with the use of shRNA to create stable knockdown of pRB in an osteoblast precursor line. These studies showed pRB is required for several activation events. pRB displaces the repressor histone demethylase, KDM5A, from the inactive promoter. pRB is also necessary for recruiting a BRG1containing SWI/SNF complex to the promoter, which in turn is required for the recruitment of RNA polymerase II. The presence of p107 on the active Alpl promoter suggests it also contributes to activation, even though p107 is not implicated in suppression of osteosarcoma. p107 is not sufficient for the pRB-dependent functions discussed above, but it is unknown whether p107 is necessary. We addressed this issue with the use of shRNA to create stable knockdown of p107 in the osteoblast precursors. Depletion of p107 resulted in impaired induction of alkaline phosphatase, revealing that p107 is also essential for activation. However, the p107 depleted line remained able to displace KDM5A, showing that the functions of pRB and p107 are distinct. We considered the possibility that p107 is required to recruit specific transactivators to the promoter. While transcription factors that target Alpl directly have not previously been identified, SP1 and RUNX2 are the most strongly implicated. ChIP assays show both RUNX2 and SP1 bound to the active promoter in parental cells. Depletion of pRB resulted in the failure of both factors to associate with the promoter, but depletion of p107 had no effect on their promoter occupation. However, in contrast to the dispensability of p107 in displacement of KDM5A and recruitment of RUNX2 and Sp1, depletion of p107 resulted in failure of recruitment of BRG1-SWI/SNF, and in SWI/SNF-dependent recruitment of RNA polymerase II. Thus, pRB and p107 are both required for activation of Alpl expression, but their roles are not entirely overlapping. Further study of osteogenic genes targeted by pRB and/or p107 during differentiation, and the contribution of each, will help to understand the specific requirement for pRB in suppression of osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 266. doi:10.1158/1538-7445.AM2011-266