Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis

Abstract

Background: Among its many roles in development, retinoic acid determines the anterior-posterior identity of differentiating motor neurons by activating retinoic acid receptor (RAR)-mediated transcription. RAR is thought to bind the genome constitutively, and only induce transcription in the presence of the retinoid ligand. However, little is known about where RAR binds to the genome or how it selects target sites.

Results: We tested the constitutive RAR binding model using the retinoic acid-driven differentiation of mouse embryonic stem cells into differentiated motor neurons. We find that retinoic acid treatment results in widespread changes in RAR genomic binding, including novel binding to genes directly responsible for anterior-posterior specification, as well as the subsequent recruitment of the basal polymerase machinery. Finally, we discovered that the binding of transcription factors at the embryonic stem cell stage can accurately predict where in the genome RAR binds after initial differentiation.

Conclusions: We have characterized a ligand-dependent shift in RAR genomic occupancy at the initiation of neurogenesis. Our data also suggest that enhancers active in pluripotent embryonic stem cells may be preselecting regions that will be activated by RAR during neuronal differentiation.

Figure 1

RAR binding shifts in response to RA exposure. (a) The plots in the two leftmost columns show enrichment over all 1,822 pre-RA and 1,924 post-RA RAR binding sites (± 1 kbp over the binding site), where the blue shading corresponds to the ChIP-seq read count in the region. (b) Examples of constitutive and ligand-specific RAR binding at four loci (Rqcd1, Cyp26a1, Hoxa1, Hoxb4/Hoxb5).

Figure 2

Direct binding of RAR mediates the initial response to RA during early neurogenesis. Genes with more than five-fold differential expression after 8 hours of RA exposure are listed. RAR binds to many of the up-regulated genes, with binding more likely for greater degrees of up-regulation. Red arrows indicate post-RA RAR binding within 20 kbp of the gene. Black dashed lines indicate pre-RA RAR binding within 20 kbp. Three functional groups of genes are indicated by coloring the gene names. Information for all more than two-fold differentially expressed genes is tabulated in Additional file 2.

Figure 3

Constitutive RAR binding without ES cell-poised Pol2 at Stra8 and Rarb. RAR is constitutively bound at these targets, but no enrichment of poised/initiating polymerase (Pol2-S5P) is observed in ES cells at these loci. Within 8 hours of retinoid exposure, the initiating and elongating forms of Pol2 are recruited to these genes.

Figure 4

RAR binding sites are coincident with ES cell transcription factor binding and H3K4 methylation. (a) Percentages of binding sites within 200 bp of ES cell binding events. Coincidence rates between 10,000 random genomic locations and ES cell binding events are shown for reference. In cases where the same protein was profiled by multiple labs, we denote the source using the following abbreviations: B, Bernstein lab [38-40]; N, Ng lab [24]; Y, Young lab [37]. (b) Rates of post-ES cell binding sites where at least one ES cell TF binding site (of 13 profiled TFs) is within 200 bp. HSC, hematopoietic stem cell.

Figure 5

Both constitutively bound and exclusively post-RA RAR binding sites are coincident with ES cell regulatory events. Line-plot clustergram of ChIP-seq enrichment in 1-kbp windows centered on 1,924 post-RA RAR binding sites. Color shading denotes scaled ChIP-seq read depth (see Materials and methods).

Figure 6

ChIP-seq data improves motif specificity. The true positive and additional prediction rates are shown when predicting post-RA RAR binding sites by ranking sites according to motif similarity or when combining motif information with various other data sources (see Materials and methods).