Figure 5. Bhlhb5 and Prdm8 bind to common genomic loci.

DNA sequences to which Bhlhb5 is bound in the dorsal telencephalon at E17.5 were identified by chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). A) Bhlhb5 consensus binding motif identified by the analysis of genomic Bhlhb5 binding sites. E-box is underlined. B – D) ChIP-seq data shows Bhlhb5 binding at its own proximal promoter (B), the proximal promoter of RP58 (C) and the first intron of Cdh11 (D). The height of the black bars indicates the number of input-normalized ChIP-Seq reads, representing the amount of Bhlhb5 binding. Consensus binding motifs for Bhlhb5 are found within the Bhlhb5 binding site at each gene, shown in red. Vertical gray bars indicate the genomic regions amplified in subsequent ChIP-qPCR experiments, either at the Bhlhb5 binding site (BS) or at a negative control region located −2 or −3 kb away, as indicated. The binding site for Bhlhb5 within the first intron of Cdh11 is indicated by the red bar. E – G) ChIP-qPCR using antibodies to Bhlhb5 confirms that Bhlhb5 is bound to its own proximal promoter (E), the proximal promoter of RP58 (F) and the first intron of Cdh11 (G). Experiments show significantly enriched Bhlhb5 binding at the identified Bhlhb5 binding site (BS) relative to the negative control region. H – J) ChIP-qPCR using antibodies to Prdm8 reveals that Prdm8 is also bound to the Bhlhb5 binding site at the proximal promoter of Bhlhb5 (H), the proximal promoter of RP58 (I) and the first intron of Cdh11 (J). Experiments show significantly enriched Prdm8 binding at the identified Bhlhb5 binding site (BS) relative to the negative control region. Also see Figure S7, in which 12 additional genomic loci are tested for the co-occupancy of Bhlhb5 and Prdm8. For E – J, chromatin immunoprecipitates were prepared from the dorsal telencephalon of P0 mice and y-axis (Binding) represents enrichment of gDNA over input (×10⁻³). For each ChIP experiment, antibody and/or knockout controls were performed in parallel, and in each case, these showed extremely low apparent binding, indicating that the ChIP experiments were specific (e.g., Figures S4C, S4D, 6A, 6B and S8). Data are representative of at least three independent experiments. * indicates significant (p < 0.05, t-test). K) Bhlhb5 and Prdm8 co-associate in neurons. Co-immunoprecipitation experiments (using ChIP conditions to preserve protein-DNA complexes) were performed to address whether Bhlhb5 and Prdm8 exist in a common complex. Bhlhb5 was immunoprecipited from the dorsal telencephalon of WT or Bhlhb5^−/− mice and subjected to western blotting using antibodies to Prdm8 or Bhlhb5, as indicated. Data are representative of four independent experiments. This interaction appears to be specific since the transcriptional activator CREB was not observed in Bhlhb5-associated complexes (data not shown). Note that we were unable to co-immunoprecipitate Bhlhb5 and Prdm8 under native conditions, possibly because the interaction between Bhlhb5 and Prdm8 is indirect, or because they interact selectively in the presence of DNA.