The SOX9 upstream region prone to chromosomal aberrations causing campomelic dysplasia contains multiple cartilage enhancers

Abstract

Two decades after the discovery that heterozygous mutations within and around SOX9 cause campomelic dysplasia, a generalized skeleton malformation syndrome, it is well established that SOX9 is a master transcription factor in chondrocytes. In contrast, the mechanisms whereby translocations in the –­350/–50-kb region 5′ of SOX9 cause severe disease and whereby SOX9 expression is specified in chondrocytes remain scarcely known. We here screen this upstream region and uncover multiple enhancers that activate Sox9-promoter transgenes in the SOX9 expression domain. Three of them are primarily active in chondrocytes. E250 (located at –250 kb) confines its activity to condensed prechondrocytes, E195 mainly targets proliferating chondrocytes, and E84 is potent in all differentiated chondrocytes. E84 and E195 synergize with E70, previously shown to be active in most Sox9-expressing somatic tissues, including cartilage. While SOX9 protein powerfully activates E70, it does not control E250. It requires its SOX5/SOX6 chondrogenic partners to robustly activate E195 and additional factors to activate E84. Altogether, these results indicate that SOX9 expression in chondrocytes relies on widely spread transcriptional modules whose synergistic and overlapping activities are driven by SOX9, SOX5/SOX6 and other factors. They help elucidate mechanisms underlying campomelic dysplasia and will likely help uncover other disease mechanisms.

Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone

“…The notion of redundant or “shadow” enhancers is now well established (for reviews, see [66, 67]). Indeed, multiple enhancers upstream of Sox9 have recently been shown to regulate its expression in cartilage [68]. Additional enhancers relevant to the gonad are likely to be outside the 120 kb BAC that was originally screened by transgene reporter assays [37].…”

SOX9 and the many facets of its regulation in the chondrocyte lineage

“…To date, several long-range enhancers of SOX9 have been proposed based on histone modification marks and reporter activities in transgenic embryos (Figure 3B) (28–33). The CD/ACD-prone region includes a −70kb enhancer active in multiple cell types expressing SOX9 ; −84 and −196kb enhancers primarily active in growth plate chondrocytes; and a −250kb enhancer specific to precartilaginous condensations (29,33).…”

An Emerging Regulatory Landscape for Skeletal Development

“…Global comparative analyses among different tissues have provided tissue-distinct putative active enhancer profiles [26, 53]. By using datasets obtained from mouse embryonic limbs, putative regulatory elements to control Sox9 transcription were identified [54, 55]. Some of these elements were further confirmed by in vivo reporter assays [54, 55] .…”

“…By using datasets obtained from mouse embryonic limbs, putative regulatory elements to control Sox9 transcription were identified [54, 55]. Some of these elements were further confirmed by in vivo reporter assays [54, 55] . Importantly, some of the confirmed regions are located in the genomic regions that have been associated with campomelic dysplasia and the Pierre Robin sequence in which genomic translocation, deletion or duplication is frequently observed, suggesting a link involved in the disease.…”