AP-1 family members act with Sox9 to promote chondrocyte hypertrophy

Abstract

An analysis of Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif. Here, we have explored the functional interplay between Sox9 and AP-1 in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun-and Sox9-bound regions throughout the chondrocyte genome, reflecting direct binding of each factor to the same enhancers and a potential for protein-protein interactions within AP-1-and Sox9-containing complexes. In vitro reporter analysis indicated that direct co-binding of Sox9 and AP-1 at target motifs promoted gene activity. By contrast, where only one factor can engage its DNA target, the presence of the other factor suppresses target activation consistent with protein-protein interactions attenuating transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 confirmed the requirement of Sox9 for hypertrophic chondrocyte development, and in vitro and ex vivo analyses showed that AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model in which AP-1 family members contribute to Sox9 action in the transition of chondrocytes to the hypertrophic program.

An Emerging Regulatory Landscape for Skeletal Development

“…These interactions have been confirmed and functionally analyzed in a recent report that showed that most Sox9 binding occurs in conjunction with Jun, in which Jun binds either directly at AP-1 target sites or indirectly in a complex with Sox9 [9]. …”

“…Analyses of Sox9 −/− ∷WT chimeras demonstrated that Sox9 −/− cells do not contribute to mesenchymal condensation [6]. The removal of Sox9 within both proliferating and prehypertrophic chondrocytes prevented hypertrophic progression in mice [7-9], indicating a broad requirement for Sox9 throughout the chondrogenic program. Thus, Sox9 is necessary for mesenchymal condensation and the survival, proliferation and differentiation of chondrocytes, and for the maintenance of the growth plate.…”

Transcriptional control of chondrocyte specification and differentiation

“…Many of these proteins are expressed in GPCs [152]. AP1 factors are activated in response to various growth factors and inflammatory cytokines, and regulate cell proliferation, differentiation and death.…”

“…JUN is thus necessary to repress the chondrogenic fate of JPs, but is dispensable during EC and GPC differentiation. Nevertheless, a recent study showed that a large majority of SOX9-bound genomic regions coincided with a small fraction of JUN-bound regions in non-hypertrophic GPCs [152]. In vitro assays have suggested that SOX9 and JUN are able to physically interact with one another and to additively activate a Col10a1 enhancer.…”

Dmrt2 promotes transition of endochondral bone formation by linking Sox9 and Runx2

“…These results t the idea that Sox9 plays essential role in chondrogenesis. However, it is now established that Sox9 is required for chondrocyte hypertrophy 15,16 and our ndings suggest that target genes of Sox5/6/9 positively regulate chondrocyte hypertrophy. In addition to Dmrt2, FoxA2 and AP-1 family members, which were identi ed as Sox5/6/9-inducible genes (Supplemental Data 1),…”