Retinoid Regulated Association of Transcriptional Co-regulators and the Polycomb Group Protein SUZ12 with the Retinoic Acid Response Elements of Hoxa1, RARβ2, and Cyp26A1 in F9 Embryonal Carcinoma Cells

Abstract

Hox gene expression is activated by all- trans retinoic acid (RA), through binding to retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) of target genes. The RARs and RXRs each have three isotypes (α, β, and γ), which are encoded by distinct genes. Hox genes are also repressed by polycomb group proteins (PcG), though how these proteins are targeted is unclear. We used chromatin immunoprecipitation assays to investigate the association of RXRα, RARγ, cofactors, and the PcG protein SUZ12 with the Hoxa1, RARβ2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells (teratocarcinoma stem cells) during RA treatment. We demonstrate that RARγ and RXRα are associated with RAREs prior to and during RA treatment. pCIP, p300, and RNA polymerase II levels increased at target RAREs upon exposure to RA. Conversely, SUZ12 was found associated with all RAREs studied and these associations were attenuated by treatment with RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2, and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalent domain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreased at target RAREs whereas H3ac and H3K4me2 levels remained constant. These studies provide insight into the dynamics of association of co-regulators with RAREs and demonstrate a novel link between RA signaling and PcG repression.