For these
new gene expression profiling experiments, we chose P0 as the time point, both for practical reasons MLN2238 chemical structure and with the hope of discovering new target genes. Interestingly, we found that just as Prdm8 mRNA is upregulated in Bhlhb5 mutant mice, so Bhlhb5 mRNA is upregulated in Prdm8 mutant mice ( Figures 4A and 4B). But what about other Bhlhb5 target genes—are they likewise upregulated in Prdm8 mutant mice? We found that loss of either Bhlhb5 or Prdm8 resulted in changes in gene expression in a small number of genes and, remarkably, all of the genes that were significantly upregulated in one mutant were also upregulated in the other. These genes include Antxr2, Connexin36, NMDA3A, and Paqr3 ( Figures 4C–4F) as well as Fgf5 and Netrin1 (data not shown). We therefore conclude that Bhlhb5 and Prdm8 inhibit the expression of a common set of genes, consistent with the possibility that they function together as part of the same repressor complex. We next investigated more Ponatinib directly the possibility that Bhlhb5 and Prdm8 form a
repressor complex by characterizing Bhlhb5 occupancy throughout the genome and testing whether Prdm8 is bound to the same genomic loci as Bhlhb5. The genomic binding sites of Bhlhb5 in the dorsal telencephalon were mapped by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). As a negative control, we also performed ChIP-seq using tissue from Bhlhb5−/− heptaminol mice, thereby confirming the specificity of the Bhlhb5 antibody. In these ChIP-seq experiments, we identified ∼2,300 specific Bhlhb5 binding sites, representing approximately one binding site per million bases (see ftp://ross-et-al-2012.hms.harvard.edu to visualize genomic data online). In addition to describing Bhlhb5 binding sites in the brain across the genome, the identification of these sequences allowed us to uncover an eight nucleotide consensus binding motif for Bhlhb5: CATATGNTNT ( Figure 5A). Thus, Bhlhb5 binds to a sequence element consisting of a canonical E-box (underlined),
a motif common to many members of the basic helix-loop-helix family, together with several other key nucleotides that likely confer additional sequence specificity. Having identified genomic Bhlhb5 binding sites in the brain, we were in a position to ask whether Prdm8 binds to the same DNA sequence elements. To address this question, we chose several genes from the ChIP-seq data to test, including two genes that showed Bhlhb5 binding in the proximal promoter, Bhlhb5 itself and Repressor Protein 58 (RP58) ( Figures 5B and 5C). In addition, we selected one of the putative Bhlhb5 target genes identified by expression profiling, Cdh11, which showed Bhlhb5 binding within its first intron ( Figure 5D).