Cell-Specific Interaction of Retinoic Acid Receptors with Target Genes in Mouse Embryonic Fibroblasts and Embryonic Stem Cells

Abstract

All-trans retinoic acid (RA) induces transforming growth factor beta (TGF-␤)-dependent autocrine growth of mouse embryonic fibroblasts (MEFs). We have used chromatin immunoprecipitation to map 354 RA receptor (RAR) binding loci in MEFs, most of which were similarly occupied by the RAR␣ and RAR␥ receptors. Only a subset of the genes associated with these loci are regulated by RA, among which are several critical components of the TGF-␤ pathway. We also show RAR binding to a novel series of target genes involved in cell cycle regulation, transformation, and metastasis, suggesting new pathways by which RA may regulate proliferation and cancer. Few of the RAR binding loci contained consensus direct-repeat (DR)-type elements. The majority comprised either degenerate DRs or no identifiable DRs but anomalously spaced half sites. Furthermore, we identify 462 RAR target loci in embryonic stem (ES) cells and show that their occupancy is cell type specific. Our results also show that differences in the chromatin landscape regulate the accessibility of a subset of more than 700 identified loci to RARs, thus modulating the repertoire of target genes that can be regulated and the biological effects of RA.

Retinoic Acid and the Development of the Endoderm

“…Indeed, the cell specificity of the response to RA signaling is probably due to interactions with different regulatory proteins [73,74]. For example, fibroblasts and embryonic stem cells show clear differences in binding sites for RARs [73] as do embryonic stem cells and differentiated neurons [70].…”

Transcription factor TEAD4 regulates expression of Myogenin and the unfolded protein response genes during C2C12 cell differentiation

“…For ChIP-chip, the total input chromatin and ChIPed material were hybridised to the extended promoter array from Agilent (Santa Clara, CA, USA) covering À5 kb to þ 2 kb regions of around 17 000 cellular promoters, as previously described. 38 Data were analysed with ChIP Analytics from Agilent, further details are described in the Supplementary Material. Flag ChIP was performed with AntiFlag M2 Affinity Gel (Sigma-Aldrich, A2220).…”

“…C2C12 cell lines expressing Flag-HAtagged TEAD4 and the Flag-tagged TEAD4-DBD were generated by infection with the corresponding pBABE retroviruses, and infected cell populations were selected with continuous presence of puromycin, as described. 38 Cell populations expressing the shRNAs were generated by infection with the appropriate pLKO.1 lentiviral vectors and selection with continuous presence of puromycin. The TEAD4 shRNA sequences are, shA (5 0 -CCGGCCGCCAAATCTATGACAAGTTCTCGAGAACTTGTCATAGATTT GGCGGTTTTTG-3 0 ), and shB (5 0 -CCGGGCTGAAACACTTACCCGAGAACTC GAGTTCTCGGGTAAGTGTTTCAGCTTTTTG-3 0 ) and were ordered from SigmaAldrich (St. Louis, MO, USA).…”

Common fibroid-associated genes are differentially expressed in phenotypically dissimilar cell populations isolated from within human fibroids and myometrium

“…Activation of the retinoic acid (RA) pathway significantly reduces growth and extracellular matrix composition of uterine fibroid cells via regulation of the TGFB pathway (Malik et al 2008). In fibroblasts, differential regulation can occur via the RA receptors (RAR), which are cell-type specific with distinctive receptor binding properties between different cell types (Delacroix et al 2010), providing a mechanism for paracrine interaction in the tumour microenvironment. Uterine fibroids are hormonally regulated, as are cancerous tumours of the breast.…”