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Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology - PubMed

  • ️Sun Jan 01 2012

. 2012 Jul 27;287(31):26328-41.

doi: 10.1074/jbc.M112.361790. Epub 2012 Jun 1.

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Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology

Emmanuel Moutier et al. J Biol Chem. 2012.

Abstract

Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.

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Figures

FIGURE 1.
FIGURE 1.

DR and IR frequencies at RAR-occupied sites in embryoid bodies and F9 cells. A, frequencies of the indicated half-site spacings at the high, middle, and low occupied sites in the embryoid body RAR ChIP-seq data set are shown. B, DR/IR frequencies in the total data set of 13385 embryoid body sites are shown. C, DR/IR frequencies at promoter proximal sites are shown. D, DR/IR frequencies at the high, middle, and low occupied sites in the RAR F9 cell RAR ChIP-seq data set are shown. E, DR/IR frequencies at the top 13385 F9 sites are shown. F, DR/IR frequencies at VDR-occupied sites from the data set of (27) are shown.

FIGURE 2.
FIGURE 2.

RAR-RXR binding to DR0 elements. A, shown is a University of California at Santa Cruz web browser view of sequence tag density in .wig file format of the RAR-occupied site at the Socs3 gene comprising a DR0 in EBs. B, EMSA analysis shows the ability of the indicated DR0 elements to compete with the labeled Rarb DR5 element for RAR-RXR complex formation. The sequences of the DR0 elements within the competing oligonucleotides are shown with the repeated half-sites indicated by arrows. Variations from the consensus half-site sequence are indicated in red. All competitors were used a 100-fold excess. C, competition was performed with increasing quantities (10-, 25-, 50-, and 100-fold excess) of the oligonucleotides shown above each lane. Lane 1 is the oligonucleotide probe with no recombinant RAR-RXR, and lane 2 is the oligonucleotide probe with RAR-RXR but no competitor. D, EMSA competition analysis of the indicated IR0 elements is shown. The sequences of the IR0 motifs within the competing oligonucleotides are shown with the inverted half-sites indicated by arrows. Mutated nucleotides are indicated in red. Competition was performed with increasing quantities as above.

FIGURE 3.
FIGURE 3.

Quantification of the interaction between RAR-RXR and different RAR binding element oligonucleotides by ITC. A, representative ITC titrations of RAR/RXR to the indicated DR/IR elements are shown. RAR-RXR binds DR5, DR2, DR0, and IR0 with a stoichiometry of 1 DNA per heterodimer. B, thermodynamic parameters (dissociation constant (Kd), enthalpy (ΔH), and entropy (−TΔS)) of DNA binding to RAR-RXR was determined by ITC.

FIGURE 4.
FIGURE 4.

Composite DR8 elements. A, consensus sequence of the composite DR8 element and the DR0 elements derived from MEME analysis are shown. Numbers of DR0 elements in the 1000 highest occupied sites (a total of 431) in EBs with the indicated additional 5′ half-sites or without an additional half-site are calculated. B, EMSA competition was performed with increasing quantities (10-, 25-, 50-, and 100-fold excess) of the oligonucleotides shown above each lane. Lane 1 is the oligonucleotide probe with no recombinant RAR-RXR, and lane 2 with RAR-RXR but no competitor. The wild-type and mutated oligonucleotides are shown below the EMSA panel, and mutations are indicated in red. C, shown is are the location of the half-sites 1, 2, and 3 (see panel A) of the DR8 elements from the 1000 highest occupied sites within the 150 bp window around the peak summit. The location relative to the summit is indicated on the x axis, and the number of half-sites is indicated on the y axis. The red arrows indicate the major population where the DR2 spacing is closest to the peak center, and the black arrows the minor population more consistent with DR8/DR0 occupancy. D, location of the DR8, DR0, and DR2 elements within the 150-bp window around the peak summit in the 1000 high, medium, and low occupied classes of the EB data set is shown. x and y axes are as indicated in panel B. Red and black arrows are as described above.

FIGURE 5.
FIGURE 5.

DR8, IR0, but not DR0 act as RAREs. A, shown is a schematic representation of the reporter vector where three copies of the indicated motifs spaced by 12 nucleotides are inserted upstream of a TATA element and the CAT reporter gene. B, shown is relative CAT activity of the different reporter vectors 48 h after transfection. EV is empty vector, and the elements in the other vectors are shown below each lane. The DR5 is from the Rarb gene, DR0 is from the Socs3 gene, the IR0 is from the Trim16, gene, DR0 (P) is the pseudo-DR0 motif from the Hoxb13 locus, DR8 (C) is the composite DR8 element from the Mafa gene, and DR8 is the simple DR8 from the Dedd gene. C, relative CAT activity from a second series of transfections shows the activity of the mutated composite Mafa DR8 element. Wt, wild-type; Mt, mutant.

FIGURE 6.
FIGURE 6.

Identification of a pseudo-DR0 motif. A, shown is the sequence of the pseudo-DR0 motif identified from the MEME analysis. B, shown is the University of California at Santa Cruz web browser view of sequence tag density in .wig file format of the RAR-occupied sites at the Hoxb13 and Wsb2 genes comprising the pseudo-DR0 in embryoid bodies. C, EMSA competition analysis of the various indicated pseudo-DR0s to compete for RAR/RXR complex formation. The sequences of the pseudo-DR0 elements within the competing oligonucleotides are shown with the half-sites indicated by arrows. Mutated nucleotides are indicated in red, and the conserved G at position 2 of the half-sites are boxed in blue. All competitors were used a 100-fold excess.

FIGURE 7.
FIGURE 7.

Association of RAR-occupied sites with gene expression. A, shown is a Venn diagram representing the overlap between the total number of transcripts associated with RAR binding sites and those regulated by RA. B and C, shown are DR and IR frequencies at the RAR-occupied sites associated with RA-induced and repressed transcripts, respectively. D, DR5/DR0 ratios in the indicated classes of RAR-occupied sites.

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