Figure 6. A Bhlhb5 dimer recruits Prdm8 to form a repressor complex at specific DNA targets.

A) Bhlhb5 forms a homodimer. HEK293T cells were transfected with the indicated constructs, immunoprecipitated with antibodies to myc, and blotted using antibodies to both myc and HA, revealing that myc-tagged Bhlhb5 can co-immunoprecipitate HA-tagged Bhlhb5 but not HA-tagged E2-2. Top blot shows the immunoprecipitated protein (IP); bottom blot shows 2.5% of input. Note that myc-Bhlhb5 (with 6 myc tags) and the IgG heavy chain have the same apparent molecular weight (~ 50 kDa), whereas HA-Bhlhb5 (with 3 HA tags) is ~ 40 kDA. The isoform of E2-2 used in these experiments was the longer form, E2-2B. B) ChIP experiments performed using tissue from knockout mice reveal that Prdm8 cannot target to Bhlhb5 binding sites in the absence of Bhlhb5. In WT mice, Bhlhb5 (red) and Prdm8 (blue) bind to the proximal promoter of RP58, as revealed by ChIP-qPCR for Bhlhb5 and Prdm8 (i). In Prdm8^−/− mice, Bhlhb5 still binds to the proximal promoter RP58 (ii). In Bhlhb5^−/− mice, Prdm8 can no longer bind to Bhlhb5 binding sites, indicating that Bhlhb5 is required for Prdm8 targeting to this loci (iii). Similar results were seen for the Bhlhb5 binding sites at the Cdh11 and Bhlhb5 genes (see Figure S4). Data are representative of three independent experiments. The y-axis (Binding) represents enrichment of gDNA over input (×10⁻³).