This analysis revealed that four of the eight genes are significantly Dasatinib order reduced in the absence of NFIA or Sox9: Apcdd1, Mmd2, Zcchc24, and Hod-1 ( Figure 4M and S4). Next we performed in situ hybridization on E12.5 NFIA- or Sox9-deficient and heterozygote control embryos and confirmed the reduced levels of expression of Apcdd1, Mmd2,

and Zcchc24 ( Figures 4A–4L). These data provide genetic evidence that expression of these genes is dependent on both NFIA and Sox9. We next sought to determine whether Sox9 and NFIA are capable of interacting with their binding sites in the promoter regions of Apcdd1, Mmd2, and Zcchc24 by performing ChIP on E12.5 spinal cord. To determine whether endogenous Sox9 and NFIA interact with their binding sites, we designed primers flanking their sites and used PCR to detect ChIP of these regions ( Figures 4N, 4P, and 4R, black arrows; Figure S5). These ChIP assays demonstrate that NFIA and

Sox9 bind regions of the Apcdd1, Mmd2, and Zcchc24 promoters that contain their consensus binding sites ( Figures 4N, 4P, and 4R), indicating a direct regulatory relationship. Although the foregoing data indicate that Sox9 and NFIA can directly regulate the expression of Apcdd1, Mmd2, and Zcchc24, they do not distinguish between individual and collaborative regulation of these genes. To determine whether Sox9 and NFIA collaborate Ruxolitinib mouse to activate Apcdd1, Mmd2, and Zcchc24 expression, we cloned their promoter regions and examined the ability of Sox9 and NFIA to activate these regulatory elements. Our reporter assays indicate that NFIA and Sox9 alone are not sufficient to activate the Zcchc24 promoter, but combined expression resulted in a 3.5-fold induction in promoter activity ( Figure 4S). Similarly, analysis of the Apcdd1 and Mmd2 promoters indicated that combined expression of Sox9 and NFIA resulted in a 4-fold Dichloromethane dehalogenase increase in activity compared to individual expression ( Figures 4O and 4Q). These data

indicate that Sox9 and NFIA collaborate to drive activation of these regulatory elements. In parallel, we used two mutant versions of Sox9, one that is not capable of binding DNA (Sox9-F12L) and another that is deficient in protein dimerization (Sox9-A76E) ( Mertin et al., 1999 and Sock et al., 2003). We found that for all three promoters, combined induction is dependent upon both dimerization and DNA binding, as shown by the fact that synergistic activation with NFIA was significantly reduced with both Sox9 mutants ( Figures 4O, 4Q, and 4S). We next sought in vivo evidence for collaborative regulation of Apcdd1, Mmd2, and Zcchc24 by Sox9 and NFIA by assessing these regulatory relationships in the chick model.