Synergistic co-regulation and competition by a SOX9-GLI-FOXA phasic transcriptional network coordinate chondrocyte differentiation transitions

Abstract

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.

Disruption of a Hedgehog-Foxf1-Rspo2 Signaling Axis Leads to Tracheomalacia and a Loss of Sox9+ Tracheal Chondrocytes

“…We also examined previously published ATAC-seq and H3K4me3 ChIP-seq performed in the E9.5 cardiopulmonary foregut progenitors to help identify active promoter and enhancer regions (Steimle et al, 2018). Examination of genome browsers showed that Gli3 was bound to Foxf1 and Sox9 promoter regions that overlapped with H3K4me3 peaks ( Figure 5D), consistent with previous reports that they are direct HH/Gli targets (Hoffmann et al, 2014;Tan et al, 2018;Vokes et al, 2008). Gli3 binding regions were also detected on putative regulatory elements of Notum and Wnt 11 but not on the Rspo2 or Wnt4 loci ( Figure 5D and Supplemental Figure 3F), suggesting that Rspo2 and Wnt4 might be indirectly regulated by Gli.…”

“…Previous studies have shown that both Foxf1 and Sox9 are direct transcriptional targets of HH/Gli in different cellular contexts (Bien-Willner et al, 2007;Hoffmann et al, 2014;Madison et al, 2009;Tan et al, 2018). In order to discover additional Gliregulated genes that might mediate tracheal chondrogenesis, we performed RNA sequencing on E10.5 foreguts and E11.5 tracheas dissected from control and Table 1).…”

Insights into Gene Regulatory Networks in Chondrocytes

“…The Ihh-Gli axis plays crucial roles in chondrocyte differentiation and proliferation through a negative feedback loop formed with parathyroid hormone-related peptide (PTHrP) and by directly acting on chondrocytes, respectively [21]. Integrative analysis of the ChIP-seq data of Sox9 binding in mouse rib chondrocytes and the data on Gli1 and Gli3 binding in E11.5 mouse limb bud cells revealed that Sox9 and Gli cooperatively regulate common target genes in proliferating chondrocytes, including Trps1, Sox9, Sox5, Sox6, Col2a1, and Ptch1 [40].…”

Regulation of terminal hypertrophic chondrocyte differentiation in Prmt5 mutant mice modeling infantile idiopathic scoliosis

“…We have not addressed how hypertrophic chondrocyte differentiation is able to proceed without these key regulators of terminal chondrocyte differentiation. It is possible that undetectable levels of RUNX2 and Mmp13 are sufficient to promote some endochondral ossification, albeit in a severely delayed manner, or that other RUNX family members or co-factors, such as RUNX3, MEF2C or FOXA2, are compensating (Arnold et al, 2007;Tan et al, 2018;Yoshida et al, 2004).…”