SOX9cre1: a distal SOX9 regulator that interacts with Hh signaling factors
Chromosomal rearrangements, such as translocations, can sometimes alter a nearby gene’s expression resulting in disease, despite the rearrangement breakpoints mapping at great distances from the disease-causing gene. Such phenomena can be defined as position effects, and have led to speculation of the importance of chromatin interactions occurring upwards of a megabase upstream or downstream to certain genes. Position effects have been reported with SOX9SOX9 results in campomelic dysplasia (CD), several clinical cases of this phenotype have been described with translocation breakpoints as far as 932 upstream kb from the gene. Studying one such case (900 kb upstream to SOX9) in the context of an apparent clustering of breakpoints has led to the prediction of a possible cis-acting regulatory element 1.1 Mb upstream of SOX9, named SOX9cre1, which may be responsible for a subtle CD phenotype when displaced. We investigated the role of this putative regulator in SOX9 expression using reporter assays in a SOX9-expressing chondrosarcoma cell line, and evaluated the tissue-specific activation of the gene. SOX9cre1 increases the activity of a minimal SOX9 promoter in reporter constructs nearly three-fold, in a tissue-specific manner, consistent with an enhancer role. In silico studies identify a Gli1 binding site in the 2.1 Kb SOX9cre1, suggesting that SOX9 may be activated directly through the Sonic hedgehog (Shh) or related Hedgehog (Hh) cell signaling pathways known to be involved in SOX9 expression. Functional assays in which reporter constructs are co-transfected with Gli1 and Smad1 of the bone morphogenic protein (Bmp) signaling pathway suggest these transcription factors may directly interact with SOX9cre1. EMSA studies confirm physical interactions between Gli1 and SOX9cre1 at the putative binding site. These data support a direct link between the Hh signaling pathways and SOX9 regulation, and suggest a mechanism of regulation through distal chromatin interactions.