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. 2010 Jan;30(1):231-44.
doi: 10.1128/MCB.00756-09.

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

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Free PMC article

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

Laurence Delacroix et al. Mol Cell Biol. .
Free PMC article

Abstract

All-trans retinoic acid (RA) induces transforming growth factor beta (TGF-beta)-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 alpha and RAR gamma receptors. Only a subset of the genes associated with these loci are regulated by RA, among which are several critical components of the TGF-beta 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.

Figures

FIG. 1.
FIG. 1.
Expression of tagged RARs in MEFs and ES cells. (A) Schematic representation of RAR with a C-terminal 3×Flag-HA tag and Western blot assay showing expression of tagged RARs in total extracts from cells expressing tagged RARα or RARγ or control cells (Tα, Tγ, and C, respectively). In the upper panel, tagged RARs were detected with anti-Flag antibody, and in the lower panels, endogenous and tagged RARs were detected with isotype-specific antibodies and endogenous RXR was detected with a pan-RXR antibody. (B) Phase-contrast microscopy after 5 days of culture in 10% or 0% serum showing that RA treatment of Taf4lox/− MEFs expressing tagged RARγ leads to serum-independent growth. (C) ChIP-qPCR on the Crabp2 RARE and the Prm1 promoter in cells expressing tagged RARα and control cells treated for 2 h with RA. The antibodies used are shown below the graph. The value obtained with each antibody and the control cells was assigned a value of 1, and fold enrichment relative to this value is indicated. GFP, green fluorescent protein.
FIG. 2.
FIG. 2.
Identification of RAR binding sites in MEFs. (A) Graphic representation of tandem Flag ChIP-chip results on cells expressing tagged RARα and RARγ in the UCSC web browser (http://genome.ucsc.edu/) at the indicated loci. The values on the y axis show the normalized immunoprecipitate/input ratio. (B) Pie chart representation of the locations of RAR binding sites relative to the TSS. (C) Summary of the presence of classical DR-type RAR binding motifs at ChIPed loci. The total number of potential sites with one mismatch is shown along with the number of consensus sites. (D) Summary of gene ontology analysis of target genes. The number of genes in each category is shown along with representative examples.
FIG. 3.
FIG. 3.
Binding of RARs to target loci. (A) qPCR on the indicated loci after tandem Flag ChIP on cells expressing tagged RARα or RARγ in the absence of RA or after 2 h of RA treatment. The value obtained for each amplicon in the control cells was set to 1, and fold enrichment relative to this value is indicated. Oligonucleotide primers are designed at the center of the ChIP peak at each locus. (B) ChIP-qPCR at the indicated loci using pan-RAR and pan-RXR antibodies. The value obtained for each amplicon after control ChIP with anti-GFP antibody was set to 1, and fold enrichment relative to this value is indicated.
FIG. 4.
FIG. 4.
Characterization of DR-type RAR binding sites. (A to E) Competition EMSAs. EMSAs were performed using Rarbwt DR5 as a radioactively labeled probe and a 100-fold excess of the unlabeled competitor (Comp) oligonucleotides shown above each lane. The sequences of the DRs within the oligonucleotides are indicated at the right of each panel. Mismatches with the consensus sequence are shown in red. For the chimeric DR, a color code for each half site is used. The results of competition EMSAs and ChIP assays are summarized on the right. (F and G) ChIP-qPCR on the indicated loci using amplicons centered on the DR element.
FIG. 5.
FIG. 5.
Expression of RAR target genes. (A) Venn diagram of RAR-bound versus RAR-regulated genes. The numbers refer only to the ChIPed genes that are represented on the Affymetrix array as described in the text. (B) Expression of the indicated target genes in MEFs in the absence or presence of RA over a 24-h period as measured by qPCR and normalized to the ribosomal Rplp0 transcript. (C) Normalized expression in ES cells in the absence or presence of RA for the indicated times as measured by qPCR. In panels B and C, the relative expression at each time point is shown in arbitrary units.
FIG. 6.
FIG. 6.
ChIP at RA-regulated genes. (A and B) Schematic representation of the Rarb promoter with DR5 located close to the TSS. The localization of each amplicon is shown. ChIP was performed with the antibodies indicated below the graph, GFP, RAR, RXR, Pol II, H3K4me3, and H3K9ac. Results are expressed as percentages of the input. (C and D) Schematic representation of the Inmt promoter. The nonconsensus DR5 RAR binding site is located 4.2 kb upstream of the TSS. ChIP was performed as described above. (E and F) Schematic representation of the Gas2 promoter with the DR2 located 300 bp downstream of the TSS.
FIG. 7.
FIG. 7.
Identification of RAR binding sites in ES cells. (A) Schematic representation of RARs with a C-terminal 3×Flag-SBP or 3×Flag-HA tag and Western blot assay showing the expression of tagged and endogenous RARs in extracts of recombined or control ES cells. (B) Summary of classical DR-type RAR binding motifs at ChIPed ES cell loci as described in the legend to Fig. 1C. (C) Summary of gene ontology analysis of target genes. (D to F) Graphic representation of tandem Flag ChIP-chip results on ES cells and MEFs expressing tagged RARγ in the UCSC web browser at the indicated loci. The y axis shows the normalized immunoprecipitate/input ratio. (G and H) Flag ChIP-qPCR at the indicated loci in tagged and control MEFs and ES cells.
FIG. 8.
FIG. 8.
Chromatin at RAR-bound loci. (A to H) ChIP-qPCRs using the indicated antibodies at loci in MEFs (A, C, E, and G) and ES cells (B, D, F, and H). Amplicons are centered on the TSS. (I and J) Venn diagrams comparing RAR-bound loci and their H34Kme3 status in MEFs (I) and ES cells (J). The total number of loci with H3K4me3 in MEFs and H3K4me3 in addition to the bivalent loci is shown as an intersection with the RAR loci bound in MEFs or ES cells.

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