Regulation of interlocking gene regulatory network subcircuits by a small molecule inhibitor of retinoblastoma protein (RB) phosphorylation: Cancer cell expression of HLA-DR

Abstract

The induction of the major histocompatibility (MHC), antigen-presenting class II molecules by interferon-gamma, in solid tumor cells, requires the retinoblastoma tumor suppressor protein (Rb). In the absence of Rb, a repressosome blocks the access of positive-acting, promoter binding proteins to the MHC class II promoter. However, a complete molecular linkage between Rb expression and the disassembly of the MHC class II repressosome has been lacking. By treating A549 lung carcinoma cells with a novel small molecule that prevents phosphorylation-mediated, Rb inactivation, we demonstrate that Rb represses the synthesis of an MHC class II repressosome component, YY1. The reduction in YY1 synthesis correlates with the advent of MHC class II inducibility; with loss of YY1 binding to the promoter of the HLA–DRA gene, the canonical human MHC class II gene; and with increased Rb binding to the YY1 promoter. These results support the concept that the Rb gene regulatory network (GRN) subcircuit that regulates cell proliferation is linked to a GRN subcircuit regulating a tumor cell immune function.

Introduction

The discovery of the requirement of Rb for the IFN-γ induction of the MHC class II genes in nonprofessional antigen presenting cells represented one of the earliest linkages between cell cycle dysregulation and defects in tumor cell immune functions (Blanck, 2004, Lu et al., 1994). In Rb defective cells, the promoter of the prototypical MHC class II gene, HLA–DRA, is occupied by an Oct-1/YY1 containing repressosome that prevents the occupancy of the promoter by a series of positive-acting, promoter binding proteins (Osborne et al., 1997, Osborne et al., 2001, Osborne et al., 2004) (Fig. 1). In Rb positive cells, the promoter binding proteins, RFX, CREB, and NF-Y occupy the promoter to a certain degree in the absence of HLA–DRA induction (Kara and Glimcher, 1991, Osborne et al., 1997). Following exposure to IFN-γ, cells synthesize CIITA, due to STAT1α and IRF-1 co-occupancy of the CIITA promoter (Muhlthaler-Mottet et al., 1998). CIITA complexes with the above, HLA–DRA positive-acting promoter binding proteins, increasing the stability and frequency of the promoter interaction with the promoter binding proteins (Steimle et al., 1993). Also, CIITA recruits a HAT to the promoter, leading to promoter occupancy by the pre-initiation complex and to HLA–DRA transcription (Beresford and Boss, 2001).

Rb expression leads to disassembly of the repressosome through mechanisms that are not fully understood. Here we report that active Rb represses YY1 promoter activity and thereby YY1 synthess, thus leading to reduced YY1 binding at the HLA–DRA gene, which in turn would lead to a disassembly of the Oct-1/YY1 repressosome. These results suggest a complete mechanistic link between Rb expression and HLA–DRA de-repression. Furthermore, the small molecule, RRD-251, previously shown to prevent Rb inactivation by blocking Raf1 kinase phosphorylation of Rb, also facilitates HLA–DRA induction in the cells studied here. (Dasgupta et al., 2004, Kinkade et al., 2008). This type of induction is apparently indirect, thereby indicating the value of assembling GRN subcircuits in predicting the indirect effects of small molecule treatments and other regulatory manipulations.