Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression

• Published in: Developmental cell 2018-09, Vol.46 (6), p.794-806.e6
• Main Authors: Mochizuki, Yusuke, Chiba, Tomoki, Kataoka, Kensuke, Yamashita, Satoshi, Sato, Tempei, Kato, Tomomi, Takahashi, Kenji, Miyamoto, Takeshi, Kitazawa, Masashi, Hatta, Tomohisa, Natsume, Tohru, Takai, Shinro, Asahara, Hiroshi
• Format: Article
• Language: English
• Subjects: acampomelic campomelic dysplasia; ACD; Animals; cartilage; Cartilage - cytology; Cartilage - metabolism; Cells, Cultured; Chondrocytes - cytology; Chondrocytes - metabolism; Chromatin - metabolism; clustered regularly interspaced short palindromic repeats; CRISPR; CRISPR-Cas Systems; enhancer; Enhancer Elements, Genetic; Female; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Organ Specificity; Sequence Deletion; signal transducer and activator of transcription 3; Sox9; SOX9 Transcription Factor - genetics; SOX9 Transcription Factor - metabolism; SRY-box 9; Stat3; STAT3 Transcription Factor - metabolism; transcription complex
• ISSN: 1534-5807; 1878-1551
• DOI: 10.1016/j.devcel.2018.07.024

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation. •CRISPR/Cas9-ChIP-seq approach revealed Sox9 enhancer candidate•SIN3A-dCas9-mediated epigenetic silencing and 3C confirmed far-upstream Sox9 enhancer•CRISPR/Cas9-mediated enhancer deletion mice showed an ACD-like phenotype•CRISPR/dCas9-ChIP-MS identified STAT3 as a trans-acting mediator of Sox9 enhancer Mochizuki et al. develop combinatorial and systematic CRISPR/Cas9-based approaches to identify tissue-specific enhancers. They apply this to exploring Sox9 regulation in chondrocytes and identify a cartilage-specific enhancer important for SOX9 expression and skeletal development. CRISPR/dCas9-ChIP-mass spectrometry analysis further implicated STAT3 in acting at the enhancer to regulate SOX9 expression.