Combinatorial CRISPR/Cas9 approach to elucidate a far-upstream enhancer complex for tissue-specific Sox9 expression

Combinatorial CRISPR/Cas9 approach to elucidate a far-upstream enhancer complex for tissue-specific Sox9 expression

Abstract

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.

Journal Article

JOUNAL:
Developmental Cell

TITLE:
Combinatorial CRISPR/Cas9 approach to elucidate a far-upstream enhancer complex for tissue-specific Sox9 expression

DOI:

https://doi.org/10.1016/j.devcel.2018.07.024

Correspondence to

Hiroshi ASAHARA, Professor
Department of Systems BioMedicine
Graduate School of Medical and Dental Sciences
Tokyo Medical and Dental University (TMDU)
E-mail: asahara.syst(at)tmd.ac.jp