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Roles for SOX9 have been extensively studied in development and particular emphasis has been placed on SOX9 roles in cell lineage determination in a number of discrete tissues. Aberrant expression of SOX9 in many cancers, including colorectal cancer, suggests roles in these diseases as well and recent studies have suggested tissue- and context-specific roles of SOX9. Our genome wide approach by chromatin immunoprecipitation sequencing (ChIP-seq) in human colorectal cancer cells identified a number of physiological targets of SOX9, including ubiquitously expressed cell cycle regulatory genes, such as CCNB1 and CCNB2, CDK1, and TOP2A. These novel high affinity-SOX9 binding peaks precisely overlapped with binding sites for histone-fold NF-Y transcription factor. Furthermore, our data showed that SOX9 is recruited by NF-Y to these promoters of cell cycle regulatory genes and that SOX9 is critical for the full function of NF-Y in activation of the cell cycle genes. Mutagenesis analysis and in vitro binding assays provided additional evidence to show that SOX9 affinity is through NF-Y and that SOX9 DNA binding domain is not necessary for SOX9 affinity to those target genes. Collectively, our results reveal possibly a context-dependent, non-classical regulatory role for SOX9.

To understand the role of the CCAAT-binding factor, CBF, in transcription, we developed a strategy to purify the heterotrimeric CBF complex from HeLa cell extracts using two successive immunoaffinity chromatography steps. Here we show that the p32 protein, previously identified as the ASF/SF2 splicing factor-associated protein, copurified with the CBF complex. Studies of protein–protein interaction demonstrated that p32 interacts specifically with CBF–B subunit and also associates with CBF–DNA complex. Cellular localization by immunofluorescence staining revealed that p32 is present in the cell throughout the cytosol and nucleus, whereas CBF is present primarily in the nucleus. A portion of the p32 colocalizes with CBF-B in the nucleus. Interestingly, reconstitution of p32 in an in vitro transcription reaction demonstrated that p32 specifically inhibits CBF-mediated transcription activation. Altogether, our study identified p32 as a novel and specific corepressor of CBF-mediated transcription activation in vitro.

Mutations in SOX9, a gene essential for chondrocyte differentiation cause the human disease campomelic dysplasia (CD). To understand how SOX9 activates transcription, we characterized the DNA binding and cell-free transcription ability of wild-type SOX9 and a dimerization domain SOX9 mutant. Whereas formation of monomeric mutant SOX9–DNA complex increased linearly with increasing SOX9 concentrations, formation of a wild-type SOX9–DNA dimeric complex increased more slowly suggesting a more sigmoidal-type progression. Stability of SOX9–DNA complexes, however, was unaffected by the dimerization mutation. Both wild-type and mutant SOX9 activated transcription of a naked Col2a1 DNA template. However, after nucleosomal assembly, only wild-type and not the mutant was able to remodel chromatin and activate transcription of this template. Using a cell line, in which the Col2a1 vector was stably integrated, no differences were seen in the interactions of wild-type and mutant SOX9 with the chromatin of the Col2a1 vector using ChIP. However, the mutant was unable to activate transcription in agreement with in vitro results. We hypothesize that the SOX9 dimerization domain is necessary to remodel the Col2a1 chromatin in order to allow transcription to take place. These results further clarify the mechanism that accounts for CD in patients harboring SOX9 dimerization domain mutations.

We performed in vivo dimethylsulfate footprinting of the 220 bp mouse proximal proalpha1(I) collagen promoter and the 350 bp mouse proximal proalpha2(I) collagen promoter in BALB/3T3 fibroblasts, primary mouse skin fibroblasts, S-194 B cells, NMuLi liver epithelial cells and RAG renal adenocarcinoma cells and in vitro DNase I footprinting of these promoters using nuclear extracts of these different cell types. Whereas proalpha1(I) and proalpha2(I) collagen RNAs were present in BALB/3T3 fibroblasts and primary fibroblasts, these RNAs could not be detected in the three other cell lines. Comparison of in vitro DNase I footprints for each of the two proximal collagen promoters indicated that the patterns of protection were very similar with the different nuclear extracts, suggesting that the DNA binding proteins binding to these promoters were present in all cell types tested. In contrast, in vivo footprints over these proximal promoters were cell-specific, occurring only in fibroblast cells and not in the other three cell types. The in vivo footprints were generally located within the in vitro footprinted regions. Our results suggest that although all cell types tested contained nuclear proteins that can bind to the proximal proalpha1(I) and proalpha2(I) collagen promoters in vitro , it is only in fibroblasts that these proteins bind to their cognate sites in vivo . We discuss possible regulatory mechanisms in type I collagen genes that can contribute to the cell-specific in vivo protein-DNA interactions at the proximal promoters.