and 2 other(s)
INTRODUCTION: The gene Indian hedgehog (Ihh), expressed in prehypertrophic and hypertrophic chondrocytes, is indispensable for the specification of osteoblast lineages in endochondral ossification . Recent reports support the involvement of Ihh signaling in osteoarthritis (OA)-related cartilage degeneration [2,3]. However, the regulatory mechanisms underlying Ihh transcription in chondrocytes are not established. Here we aimed to identify tissue-specific regulatory elements for Ihh transcription in chondrocytes by a genome-wide approach and to validate in vivo roles of the regions. METHODS: First, we obtained chromatin immunoprecipitation sequencing (ChIP-seq) data for histone modification (H3K36me3 and H3K4me2), Sox9 (master transcriptional factor for chondrogenesis), and Sox6 in mouse P1 rib chondrocytes . Next, we checked enhancer activities of the H3K4me2+ regions by in vitro reporter assays and assessed the enhancer activity in vivo using lacZ reporter transgenic mice. We verified the mechanism of the enhancer activation using in vitro reporter assays and electrophoresis mobility shift assay (EMSA). To test the functional relevance of Sox9 to the Ihh expression, we examined the effect of dominant negative inhibition of Sox9 on Ihh expression in an insulin-mediated chondrogenic culture of ATDC5 cells. Lastly, we generated enhancer-deleted mouse lines using a CRISPR/Cas9 approach and checked their phenotypes in developmental stages by the histological analysis and RT-qPCR. To examine the contribution of the enhancer to OA progression, we assessed: (1) the enhancer activity in the lacZ reporter transgenic mice under a cartilage-degenerated condition and (2) OA severity in an OA model created in the enhancer deleted-mice using a histology-based OARSI scoring system compared to that in WT mice. All animal studies were approved by the Animal Care and Use Committee of The University of Tokyo. Significant differences between two quantitative data were evaluated by Student's t-test. P-values <0.05 were considered significant. RESULTS SECTION: H3K36me3 peaks (transcribed regions) were observed in the Ihh gene region. We identified five H3K4me2 peaks (putative enhancer regions) upstream of the Ihh gene region (referred to as A, B, C, D, and E in order from distal to proximal). We confirmed the strong enhancer activity of the region A, which is a ~400-bp region highly conserved among vertebrates, in in vitro reporter assays. Transgenic mice carrying the lacZ reporter driven by the region A and a mouse Ihh promoter fragment (−105 bp to +58 bp)  showed beta-galactosidase activity exclusively in skeletal tissues at E16.5; six out of nine transgene-positive pups (67%) had the tissue-specific beta-galactosidase activity in the G0 analysis. The histological analysis of the growth plate at E16.5 confirmed that the activity of the region A was consistent with endogenous Ihh expression in prehypertrophic chondrocytes. Considering that the region A was silent in organs like kidney, gut, stomach and liver, which are known to express Ihh at E16.5, the region A may work as a chondrocyte-specific Ihh enhancer in vivo. In addition, ChIP-seq for Sox9 and Sox6 and subsequent de novo motif analyses predicted their binding to the region A. The region A showed Sox trio (Sox5, Sox6, and Sox9)-responsive enhancer activity in in vitro reporter assays, and Sox dimeric motif, which is located in the center of the region A, was necessary for its enhancer activity. We also confirmed Sox9 and Sox6 binding to the Sox dimeric motif in the region A by EMSA. These results supported the requirement of cooperative binding of Sox9 and Sox6 (and Sox5) to the region A for its enhancer activity. The dominant-negative inhibition of Sox9 suppressed Ihh and Col2a1 expression as early as 2 days after the adenovirus infection. Thus, Sox9, together with Sox5/6, is likely to positively regulate Ihh transcription at least partially through distal enhancers including the region A. Lastly, we examined the function of the region A using a mutant mouse line, in which the region A is deleted by the CRISPR/Cas9 approach. The Region-A-/- mice showed subtle phenotypes in Ihh expression and skeletal tissues compared to Ihh-null mutants in embryos. In contrast, at 8 weeks after surgical OA induction, Region-A-/- mice showed a trend toward the suppression of OA progression compared to WT mice. We also confirmed the beta-galactosidase activity in articular cartilage in 7-month-old lacZ reporter transgenic mice, which naturally developed cartilage degeneration. DISCUSSION: We identified the chondrocyte-specific Ihh enhancer and clarified the activation mechanism through Sox trio. In addition, the result of in vitro loss of function of Sox9 suggests that Ihh is a direct target of Sox9. Although the trend toward the reduction of Ihh expression in Region-A-/- mice may partly support the requirement of the region A in the maintenance of the proper level of Ihh expression, an enhancer network in Ihh expression is likely to compensate for lack of the region A in the mutant embryos . Furthermore, the phenotypes of enhancer-deleted mice and reporter transgenic mice under cartilage degenerative conditions may partly support the involvement of the region A in cartilage degeneration probably through induction of Ihh expression. SIGNIFICANCE/CLINICAL RELEVANCE: This study is an important first step in identifying the regulatory landscape for Ihh expression in the developing skeleton. In addition, we propose a new concept that modulating the enhancer activity of the region A may be a new disease-modifying approach for OA associated with Ihh signaling. REFERENCES:  St-Jacques B et al. Genes Dev, 1999  Wei F et al. Osteoarthritis Cartilage, 2012  Zhou J et al. Arthritis Res Ther, 2014  Ohba S. et al. Cell Report, 2015  Yoshida CA et al. Genes Dev, 2004  Will AJ et al. Nat Genet, 2017
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