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

Developmental Cell
Yusuke MochizukiHiroshi Asahara

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.

Citations

Feb 23, 2020·International Journal of Molecular Sciences·Riko NishimuraLerdluck Ruengsinpinya
Jul 18, 2020·Bone & Joint Research·Jamie Fitzgerald
Dec 19, 2019·International Journal of Molecular Sciences·Hironori Hojo, Shinsuke Ohba
May 13, 2020·Biology Methods and Protocols·Toshitsugu Fujita, Hodaka Fujii
Jul 3, 2020·Clinical & Experimental Metastasis·Yahui HuangQiongqiong He
Mar 12, 2020·Animal Cells and Systems·Hyunji LeeKyoungmi Kim
Nov 10, 2020·Frontiers in Endocrinology·Katerina Trajanoska, Fernando Rivadeneira
Nov 17, 2020·Nucleic Acids Research·Agustin Sgro, Pilar Blancafort
Feb 19, 2021·Laboratory Animals·Christophe Galichet, Robin Lovell-Badge
Feb 13, 2021·Genome Research·Núria Bosch-GuiterasRory Johnson
Apr 21, 2021·The CRISPR Journal·Hirotaka FujitaHodaka Fujii
Jun 27, 2021·Proceedings of the National Academy of Sciences of the United States of America·Prem Swaroop YadavYingzi Yang

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