Epigenetic Regulatory Mechanisms Distinguish Retinoic Acid-mediated Transcriptional Responses in Stem Cells and Fibroblasts

Vasundhra Kashyap,Lorraine J Gudas

doi:10.1074/jbc.m110.115345

Abstract

Retinoic acid (RA), a vitamin A metabolite, regulates transcription by binding to RA receptor (RAR) and retinoid X receptor (RXR) heterodimers. This transcriptional response is determined by receptor interactions with transcriptional regulators and chromatin modifying proteins. We compared transcriptional responses of three RA target genes (Hoxa1, Cyp26a1, RARbeta(2)) in primary embryo fibroblasts (mouse embryonic fibroblasts), immortalized fibroblasts (Balb/c3T3), and F9 teratocarcinoma stem cells. Hoxa1 and Cyp26a1 transcripts are not expressed, but RARbeta(2) transcripts are induced by RA in mouse embryonic fibroblasts and Balb/c3T3 cells. Retinoid receptors (RARgamma, RXRalpha), coactivators (pCIP (NCOA3, SRC3)), and p300 and RNA polymerase II are recruited only to the RARbeta(2) RA response element (RARE) in Balb/c3T3, whereas these proteins are recruited to RAREs of all three genes by RA in F9 cells. In F9, RA reduces polycomb (PcG) protein Suz12 and the associated H3K27me3 repressive epigenetic modification at the RAREs of all three genes. In contrast, in Balb/c3T3 cells cultured in the +/-RA, Suz12 is not associated with the Hoxa1, RARbeta(2), and Cyp26a1 RAREs, whereas slow levels of the H3K27me3 mark are seen at these RAREs. Thus, Suz12 is not required for gene repression in the absence of RA. Even though the Hoxa1 RARE and proximal promoter show high levels of H3K9,K14 acetylation in Balb/c3T3, the Hoxa1 gene is not transcriptionally activated by RA. In Balb/c3T3, CpG islands are methylated in the Cyp26a1 promoter region but not in the Hoxa1 promoter or in these promoters in F9 cells. We have delineated the complex mechanisms that control RA-mediated transcription in fibroblasts versus stem cells.

Highlights

Because the transcriptional state of a gene is determined by the overall chromatin structure, we examined the chromatin states of the RA response element (RARE) [8, 9, 40, 41] that regulate the expression of these genes by chromatin immunoprecipitation (ChIP) assays
We show that the lack of transcriptional activity of Hoxa1 and Cyp26a1 in Balb/c3T3 cells is an outcome of the differential chromatin signatures at these genes as we observed different epigenetic modifications associated with the Hoxa1 and Cyp26a1 RAREs in F9 stem cells
The Coactivators, pCIP and p300, Are Recruited to the RA receptor (RAR)␤2 RARE in Balb/c3T3 Cells—Because we found that the RAR/ retinoid X receptor (RXR) heterodimers associated with the RAR␤2 RARE in Balb/ c3T3 cells, we assessed the recruitment of coactivators pCIP and p300 using the conventional ChIP assay. pCIP was associated with the RAR␤2 RARE before Retinoic acid (RA) treatment, as shown by the ϳ4-fold enrichment at the RAR␤2 RARE relative to the Ϫ18-kb Hoxb1 negative control intergenic region in Balb/c3T3 cells (Fig. 3c)

Summary

Introduction

According to the current model of retinoid signaling, in the absence of the ligand RAR/RXR, heterodimers are bound to RAREs, and the receptors interact directly with nuclear co-repressor proteins such as nuclear receptor corepressor (NCoR) [16] and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) [17] These co-repressors can recruit histone deacetylase complexes I/II, which deacetylate the lysine residues of histone tails. This study is aimed at determining if the dynamic association of PcG proteins at retinoid-responsive genes is a regulatory mechanism specific to stem cells or if it is a mechanism important in other differentiated cellular lineages. We show that the lack of transcriptional activity of Hoxa and Cyp26a1 in Balb/c3T3 cells is an outcome of the differential chromatin signatures at these genes as we observed different epigenetic modifications associated with the Hoxa and Cyp26a1 RAREs in F9 stem cells

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