Identification of RAR target genes in MEFs.
To identify RAR target genes in Taf4lox/− MEFs, we used retroviral vectors to generate cell lines that stably express RARα and RARγ carrying a 3×Flag-HA tandem affinity purification (TAP) tag at the C terminus. The presence of the TAP tag does not affect RAR function in transient expression assays or activation of RAR target genes in the MEFs (data not shown). Western blotting on extracts of the infected cells showed that the exogenous TAP-tagged RARs made up only around 10 to 20% of the total RARα or RARγ (Fig. ). Cells expressing exogenous RARs also displayed RA-induced morphological changes and serum-independent growth (Fig. ), consistent with the idea that their presence does not affect the proper RA response of the cells. ChIP assay using either Flag or HA antibodies showed that tagged RARs and endogenous RXR were efficiently recruited to the DR2-type RARE in the cellular RA binding protein 2 gene (Crabp2) promoter, while no enrichment was seen at the control protamine 1 gene (Prm1) promoter (Fig. and data not shown).
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 (more ...)
We used these cells to perform ChIP-chip experiments with the Agilent extended promoter array comprising the kb −5 to +2 regions of around 17,000 cellular promoters. Duplicate tandem Flag ChIP-chip and HA ChIP-chip assays were performed on cells expressing RARγ that had been treated with RA for 2 h and compared with a tandem Flag ChIP-chip on untagged cells as a control. These experiments identified 354 loci bound by RARγ after Flag and HA ChIP in tagged cells but not in control cells (see Table S1 and Fig. S1A in the supplemental material). For example, occupancy of the characterized DR5 element in the Rarb
promoter was observed along with occupancy of several other known targets such as Crabp2
]), and the ubiquitin-conjugating enzyme E2C gene (Ube2c
) (Fig. ; data not shown). Among the identified loci are previously characterized target genes known to be RA regulated, but not as direct targets, such as the ceruloplasmin (Cp
), matrix metallopeptidase 2 (Mmp
), cathepsin D (Ctsd
), or type 1 alpha collagen (Col1a1
) gene (for a review, see reference 4
). Most of the occupied regions are, however, in novel target genes such as the topoisomerase II beta gene (Top2b
), which is transcribed convergently to Rarb
, or the interleukin-1 receptor antagonist (Il1rn
), pyroglutamyl-peptidase I (Pgpep1
), and tensin like C1 domain-containing phosphatase (Tenc1
) gene promoters (Fig. ). Analysis of 200 of the most sharply defined RAR binding sites showed that 49% were located upstream of the transcription start site (TSS), while 42% were located downstream, principally in the first intron (Fig. ).
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 (more ...)
Gene ontology analysis (http://david.abcc.ncifcrf.gov/
) showed that the largest class of target genes are involved in transcription regulation, for example, transcription factors such as Hoxa10
and the RAR coactivators Hmga1
), and Top2b
). One of the main functions of RARs is therefore to regulate other transcriptional networks (Fig. ). RARs also bind a significant number of genes involved in signal transduction, protein modification, and metabolism, as well as developmental regulatory genes.
Very similar results were observed in ChIP-chip experiments with cells expressing tagged RARα (Fig. ), as the vast majority of sites were similarly bound by RARα and RARγ (Fig. ; see Table S1 and Fig. S1A in the supplemental material). ChIP-qPCR confirmed specific binding of RARα and RARγ to promoters in the absence of RA (Fig. ). RA substantially increased RARα binding on the Rarb, Pgpep1, Cebpb, and F11 receptor (F11r) promoters, while the strongest increase in RARγ binding was seen at the Wnt10b locus, but little or no effect was observed on the other sites. RAR and RXR occupancy was also verified using pan-RAR and pan-RXR antibodies against the endogenous proteins in MEFs that do not express the tagged RARs (Fig. ; see also below). Together, the above results show that most, if not all, of the sites were occupied in the absence of RA by both RARα and RARγ but that the influence of RA is promoter specific.
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 (more ...) Characterization of RAR binding motifs.
To identify the RAR binding motifs, the 1,200 nucleotides flanking the highest-scoring oligonucleotide in each peak were analyzed for the presence of DR1, -2, and -5 elements that had either a perfect match to the known consensus [5′-RGKTCA-(n)1,2,5-RGKTCA-3′] or a single mismatch (see Table S2 in the supplemental material). While 149 potential DRs were identified, only 23 were fully consensus sites (Fig. ). This was confirmed by a more restricted analysis of the 300 nucleotides around the top 100 most sharply defined peaks that identified only 7 consensus elements (see Table S2 in the supplemental material). This analysis also revealed that 71 of these sites contained at least one consensus half site with a majority containing several anomalously spaced half sites but no obvious DR1, -2, or -5.
We verified RAR binding to several of the novel DRs by competition EMSAs in vitro or ChIP-qPCRs using primers centered on the DR element. In the EMSAs, the retarded RAR/RXR complex formed using an oligonucleotide comprising Rarb DR5 was efficiently competed by consensus DR5 elements from the Atxn2, F11r, Top2b, and STIP1 homology and U-box-containing protein 1 (Stub1) genes but not by mutated Rarb or Atxn2 oligonucleotides (Fig. , lanes 1 to 7). Efficient competition was also seen with DR5 elements with a single mismatch in the 5′ or 3′ half sites from the dehydrogenase/reductase (SDR family) member 3 (Dhrs3), Toll-interleukin-1 receptor domain-containing adaptor protein (Tirap), Rous sarcoma oncogene (Src), RB1-inducible coiled-coil 1 (Rb1cc1), and procollagen C-endopeptidase enhancer protein (Pcolce) genes (Fig. , lanes 9, 10, and 12 to 14). In contrast, little or no competition was observed with the potential DR5 element from the fibroblast growth factor 18 (Fgf18) or sedoheptulokinase (Shpk) gene (lanes 8 and 11), both of which contain a single mismatch with respect to the consensus.
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 (more ...)
These results were confirmed by ChIP-qPCR using RAR and RXR antibodies, where occupancy of the Rarb, Atxn2, Dhrs3, Tirap, and F11r loci was observed (Fig. ), suggesting that these elements bind the RAR/RXR both in vitro and in MEFs. In contrast, although the Fgf18 and Shpk loci are clearly occupied in MEFs (see Fig. S1A in the supplemental material), no occupancy was observed using the amplicon centered on the potential DRs (Fig. ). The potential DRs that were identified do not coincide well with the peak detected by ChIP-chip, indicating that the RAR binds not to these DRs but rather another sequence at these loci.
Consensus or single-mismatch DR2 elements from the homeobox a10 (Hoxa10), periplakin (Ppl), Crabp2, growth arrest-specific 2 (Gas2), Ctgf, neuropilin 1 (Nrp1), and nudix (nucleoside diphosphate-linked moiety X)-type motif 6 (Nudt6) genes efficiently competed the formation of the retarded complex in vitro (see appropriate lanes in Fig. ) and were occupied in MEFs (Fig. and not shown). In contrast, competition with the DR2 elements from the protein phosphatase 1-like (Ppm1l) and FCH domain only 2 (Fcho2) genes were much less efficient and no competition was seen with the cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22) element.
For DR1 elements, competition was observed using those of the chromatin-modifying protein 1A (Chmp1a), calcitonin receptor activity-modifying protein 2 (Ramp2), NR subfamily 1, group D, member 1 (Nr1d1), and death effector domain-containing (Dedd) genes (Fig. and data not shown). Surprisingly, however, no significant competition was observed with the consensus DR1 motif from the glycerophosphodiester phosphodiesterase 1 (Gde1) element. We noted that the spacer nucleotide in this element was C, while it was A in the Ramp2 and Dedd elements that compete. Exchange of the C in Gde1 for an A resulted in efficient competition (lane 5). Thus, half-site sequences and spacer nucleotides can play important roles in the formation of a complex with RAR/RXR. As observed in vitro, the Nr1d1 locus, but not Gde1, was occupied in cells (Fig. ). Similar to Fgf18, this indicates that binding to the Gde1 locus must be mediated by another element.
The above results indicate that many single mismatches do not abrogate RAR/RXR binding even when located at highly conserved positions such as the G in position 2 or 5′-TCA-3′. In contrast, in other elements, the equivalent mutations lead to a loss of binding. For example, in Cyp2d22 and Rb1cc1, 5′-TCA-3′ is mutated to 5′-TCG-3′ but only Rb1cc1 binds RAR/RXR. In Cyp2d22, replacing the G with an A to create a consensus leads to a strong increase in competition (Cyp2d22C, Fig. , lanes 2 and 3). We exchanged the 3′ half site of Cyp2d22 with that of Rb1cc1 (i.e., changing T to G at the third position). The resulting Cyp2d22R was a significantly better competitor than native Cyp2d22 although less efficient than the full consensus (lanes 2 to 4). Hence, the exact sequence of one half site plays a critical role in compensating for mismatches in the other half site.
A more striking example of this is found upon analysis of the indolethylamine N-methyltransferase (Inmt) gene binding site. At this locus, we did not find any consensus or single-mismatch DR element. However, visual inspection identified a DR5 with two mismatches that efficiently competes in an EMSA (Fig. ). Inverting the 5′ and 3′ half sites (Inmt-RV) led to a decrease in competition, showing that the mismatches are less well tolerated in the 5′ position, where RXR normally binds (lane 4). Similarly, transforming the DR5 to a DR2 also decreases competition (lane 5). Strikingly, however, replacing 5′-GGG-3′ in the 5′ half site with 5′-AGG-3′ or 5′-AGT-3′ leads to a strong reduction in competition (lanes 6 to 7), while 5′-GGT-3′ competes, but less well than native 5′-GGG-3′ (lanes 3 and 8). These results show that in nonconsensus elements, mutations in one half site can be compensated for by the exact sequence of the other half site. In the case of Inmt, a G in the first position seems critical to compensate for mutations in the second half site.
Association of RAR binding sites with other transcription factors.
Many RAR-bound loci did not contain identifiable DR1, -2, and -5 elements but contained consensus half sites. Multiple alignments of the regions adjacent to these half sites did not reveal the presence of an additional consensus sequence for another transcription factor that would bind together with RAR/RXR (see Fig. S2A in the supplemental material). Analysis of a larger 300 nucleotides around the peaks identified numerous consensus sites for SP1 and AP2. However, they were also commonly found in control promoter sets and were not enriched in the RAR-bound set.
It has recently been shown in MCF7 cells that almost 60% of the RAR-bound loci were also bound by estrogen receptor (ER) (21
). We therefore examined the 354 RAR-bound loci for ER binding sites by using the consensus 5′-RGGTCWnnnWGACMY-3′ (61
) but identified only 3 estrogen response elements (EREs) in the MEF data set. Similarly, Hua et al. reported a significant overlap between RAR and FOXA1 binding sites. Using the consensus 5′-TRTTTRYWYW-3′, we observed 46 potential FOXA1 sites. Thus, assuming that they are all bona fide
binding sites, only 12% of the RAR-bound loci show an associated FOXA1 site. Similar results were obtained upon the analysis of 211 of the ES RAR-bound loci (see below), where we found 44 potential FOXA1 sites (20%) but no EREs.
RA regulation of target gene expression.
We have identified 354 RAR-bound loci, but taking into account the divergent promoters and genes with close neighbors, there are 383 potential RA-regulated genes. Comparison with our previous transcriptome data shows that 267 of the 383 genes were present on the Affymetrix array, but only 48 genes were upregulated and 26 were repressed by RA (Fig. ; see Table S1 in the supplemental material). As the transcriptome analysis was performed after 24 h of RA treatment, we verified gene expression to determine whether we had missed many transiently regulated genes. Reverse transcription coupled to qPCR identified several categories of genes. Rarb and Inmt are not expressed in the absence of RA and are strongly induced over a 24-h period (Fig. ). Others, such as Ctgf, show significant expression in the absence of ligand but are transiently stimulated by RA. In contrast, genes such as Gas2 or Kit ligand (Kitl) are repressed by RA. However, the expression of wingless-related MMTV integration site 10b (Wnt10b), like that of many other genes (data not shown), is not affected by RA. These observations indicate that only around 27% of the RAR-bound genes are regulated by RA in these cells under the experimental conditions used.
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 (more ...)
We investigated several promoters more closely by using primer pairs designed to amplify the TSS and regions located at −2 kb and +2 kb relative to the TSS. RAR/RXR occupancy of Rarb
DR5 was observed in the absence of RA and was strongly increased after 2 to 24 h with RA (Fig. ). In contrast, Pol II was observed only in the presence of ligand. This result differs from that observed previously in P19 cells, where some Pol II was present at Rarb
in the absence of RA (31
). Interestingly, significant H3K4me3 and H3K9ac covalent histone modifications tightly associated with transcriptional activity (57
) were observed in the absence of RA and strongly increased after RA treatment (Fig. ). Lower levels of both of these markers were also observed downstream of the TSS.
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, (more ...)
At the Inmt promoter, RAR/RXR binding to the nonconsensus DR5 located 4.2 kb upstream of the TSS was observed in the absence of RA and increased by RA (Fig. ). No Pol II was observed at the TSS in the absence of RA, but it was strongly recruited in its presence. Interestingly, a minor but significant RA-dependent recruitment of Pol II at the RAR binding site was also observed. H3K4me3 was seen only in the presence of RA at the TSS and in the downstream region (Fig. ). Thus, unlike Rarb, where H3K4me3 was seen in the absence of RA, this marker is fully RA dependent at Inmt. H3K9ac was observed at the TSS, in the downstream region, and also at the RAR binding site and was stimulated in each region by RA.
We were unable to detect such striking changes at RA-repressed promoters. At the Gas2 promoter, RAR and RXR occupancy of the downstream DR2 element could be observed in the absence of RA and was not altered in its presence (Fig. ). Pol II occupancy of this promoter was observed in the absence of ligand, consistent with its constitutive expression; however, no significant changes in Pol II promoter occupancy were observed in the presence of RA. Similarly, high levels of the H3K4me3 and H3K9ac markers were observed in the absence of RA (Fig. ), but a transient reduction was seen in the presence of RA concomitant with the transient repression (Fig. ). Thus, while the strong de novo activation is reflected by Pol II recruitment and the appearance and/or increase of histone modifications associated with transcription, at repressed genes, only minor and transient changes were seen.
Cell-specific binding of RAR to its cognate elements.
We have previously noted that the repertoire of RA-regulated genes in MEFs is significantly different from that observed in other cell types such as F9 or ES cells (15
). To examine the basis for this specificity, we generated ES cells in which the genes encoding RARα and RARγ were modified by homologous recombination to introduce a TAP tag at the C terminus (Fig. ). Western blot analysis of extracts from the tagged ES cells showed somewhat lower expression of tagged than untagged RAR, probably due to the presence of the neomycin resistance cassette in the 3′ untranslated region.
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 (more ...)
We compared duplicate tandem Flag ChIP-chip on ES cells expressing tagged RARγ to that on untagged cells to identify 462 bound loci. Among these are many previously identified genes, such as the Rarb, caudal-type homeobox 1 (Cdx1), left-right determination factor 1 (Lefty1), and pluripotency factor Pou5f1 (Oct4) genes, as well as a series of novel genes (see Table S3 and Fig. S1B in the supplemental material). Analysis of the 211 genes with the sharpest peaks indicated that, as observed in MEFs, the majority of the binding sites contained not consensus DR elements but either degenerate DRs or no identifiable DRs but anomalously spaced consensus half sites (Fig. ; see Table S3 in the supplemental material). Gene ontology analysis indicated that the largest functional classes of target genes were related to transcription regulation and proteolysis (Fig. ).
Comparison with MEFs showed that only 58 loci were bound in both cell types (see Table S3 in the supplemental material). For example, the brain serine/threonine kinase 1 (Brsk1
) locus is occupied in ES cells but not in MEFs (Fig. ). At the Wnt10b
locus, RAR binds to a site upstream of the Wnt10b
gene in MEFs not occupied in ES cells, whereas in ES cells, RAR occupies a site within the Wnt1
gene (Fig. ). Similarly, the HoxA and HoxB loci are differentially occupied in MEFs and ES cells (see Fig. S1C in the supplemental material). In contrast, the retinitis pigmentosa isocitrate dehydrogenase 3 (NAD+
) beta (Idh3b
) locus (18
) was comparably occupied in both cell types (Fig. ).
These results were verified by ChIP-qPCR. In ES cells, RAR occupancy of the Lefty1, human immunodeficiency virus type I enhancer binding protein 3 (Hivep3; also know as Schnurri-3 [Shn3]), gastrulation brain homeobox 2 (Gbx2), Brsk1, and cell death-inducing DNA fragmentation factor alpha subunit-like effector A (Cidea) loci, but not of the Wnt10b and Inmt loci, was observed (7G). In contrast, in MEFs, occupancy of the Inmt and Wnt10b loci was observed, but not of the Lefty1, Gbx2, Hivep3, and Brsk1 loci, while the Rarb and Ctgf loci are occupied in both cell types (Fig. and [discussed above]). These results show clearly that binding of RAR to many of its cognate elements is cell type specific.
We assessed the expression and RA regulation of several genes in the different cell types. In ES cells, Rarb is not expressed in the absence of RA and is progressively and strongly induced by RA (Fig. ). In contrast, Inmt is neither expressed nor induced by RA in ES cells. Wnt10b and Gas2 were expressed at low levels in ES cells and were not regulated by RA. Hivep3 and Wnt1 are strongly upregulated by RA in ES cells, while they are neither expressed nor RA regulated in MEFs. Gbx2 is also not RA regulated in MEFs.
Chromatin modifications at target loci.
We compared the chromatin states of these loci in the two cell types. In MEFs, the highest level of H3K4me3 is observed at the constitutively active Gas2 promoter, while it is strongly increased by RA at Rarb and Inmt (Fig. ). In contrast, no significant H3K4me3 was observed at Hivep3 and Wnt1 in the presence or absence of RA. In ES cells, a high level of H3K4me3 was seen at Rarb that was only mildly increased by RA (Fig. ). H3K4me3 was also observed at Hivep3 and Wnt1 and significantly increased by RA. In contrast, there was no H3K4me3 at Inmt in the presence or absence of RA, while at Gas2, levels were much lower in ES cells than in MEFs. Similar results were seen for H3K9ac (Fig. ), although it is worth noting that low but significant levels (compared to major satellite as a negative control) were seen at Hivep3 in MEFs and at Inmt in ES cells, where the corresponding genes were not expressed.
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 (more ...)
In MEFs, a significantly higher level of the repressive H3K27me3 marker was observed at Hivep3 and Wnt1 than at the others (Fig. ). In ES cells, H3K27me3 at Rarb and Hivep3 was decreased by RA, while it was unaffected at the other loci (Fig. ). Unlike MEFs, however, the low or inactive Gas2 and Inmt promoters did not show H3K27me3 levels significantly higher than those of the expressed genes. In MEFs, the Hivep3 and Wnt1 promoters also showed higher levels of H3K9me3 than did the others, while in ES cells, none of the promoters showed a high level of this marker (Fig. ).
Together, these results show that the Hivep3 and Wnt1 promoters are in a repressed state in MEFs, suggesting that this chromatin conformation may restrict RAR/RXR binding to these loci. In ES cells, the Inmt locus shows no significant H3K4me3, in agreement with the lack of its expression, and neither this promoter nor the Gas2 promoter showed higher levels of the repressive H3K9 and H3K27me3 markers. Thus, these promoters are not in a repressed heterochromatin state that would account for the lack of RAR binding in ES cells.
These results are similar, but not identical, to those obtained in the global mapping of H3K4me3 in ES cells and MEFs in the absence of RA (36
). In this study, differential H3K4me3 at Rarb
was observed in MEFs, but Hivep3
had H3K4me3, while we clearly do not detect this marker at these loci. This perhaps reflects differences between individual MEF lines. In contrast, in ES cells, our results are in agreement with those of Mikkelsen et al.
These observations prompted us to make a global comparison of RAR binding and H3K4me3 in MEFs and ES cells. We performed H3K4me3 ChIP-seq on our MEFs and used these data and the Mikkelsen ES cell data for comparison (see Table S4 in the supplemental material). This analysis shows that the vast majority of genes bound by RAR in MEFs show significant H3K4me3 in the absence of RA, only a minority are in a repressed state (Fig. ; see Fig. S1D in the supplemental material). Similarly, although the Wnt1 and Hivep3 loci are in a repressed state in MEFs, the majority of the genes bound selectively in ES cells by RAR are in an active state in MEFs. A similar situation exists in ES cells, where most RAR-bound genes, whether in MEFs or in ES cells, show H3K4me3 and are in an active state or are bivalent promoters marked by both K4 and K27 trimethylation (Fig. ). Together, these results indicate that while the heterochromatinization in MEFs of some genes active in ES cells may explain the loss of RAR binding, this is not a general mechanism to account for the cell-specific association of RAR with its binding loci.