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.
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DOI：
10.1016/j.devcel.2018.07.024
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发表时间：
2018-09-24
影响因子：
11.8
通讯作者：
Asahara H
中科院分区：
文献类型：
--
作者：
Mochizuki Y;Chiba T;Kataoka K;Yamashita S;Sato T;Kato T;Takahashi K;Miyamoto T;Kitazawa M;Hatta T;Natsume T;Takai S;Asahara H
SRY-box 9（SOX 9）是调节软骨发育的主要转录因子。据报道，在肢端畸形（acampomelic campomelic dysplasia，ACD）患者中，SOX 9上游约1 Mb区域的断裂点导致SOX 9单倍不足，这表明软骨发育所必需的SOX 9增强子区域位于该长的非编码序列中。然而，调控软骨特异性SOX 9表达的顺式作用增强子区域仍有待鉴定。为了鉴定远端软骨Sox 9增强子，我们利用多种CRISPR/Cas9技术的组合，包括启动子-增强子复合物的富集，随后进行下一代测序和质谱（MS），SIN 3A-dCas 9介导的表观遗传沉默和增强子缺失小鼠的产生。因此，我们可以确定一个关键的远上游顺式元件和Stat 3作为反式作用因子，调节软骨特异性Sox 9表达和随后的骨骼发育。我们的策略可以促进明确的ACD诊断，并应有助于揭示详细的染色质构象和调控。Mochizuki等人开发了基于CRISPR/Cas9的组合和系统方法来鉴定组织特异性增强子。他们将其应用于探索软骨细胞中的Sox 9调节，并确定了对Sox 9表达和骨骼发育重要的软骨特异性增强子。CRISPR/dCas 9-ChIP-质谱分析进一步表明STAT 3作用于增强子以调节SOX 9表达。
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 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. 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.
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影响因子：
4.6
作者：
Inui M;Miyado M;Igarashi M;Tamano M;Kubo A;Yamashita S;Asahara H;Fukami M;Takada S
通讯作者：
Takada S
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
DOI：
10.1016/0922-3371(90)90079-c
发表时间：
1990-05-01
期刊：
CELL DIFFERENTIATION AND DEVELOPMENT
影响因子：
--
作者：
ATSUMI, T;MIWA, Y;IKAWA, Y
通讯作者：
IKAWA, Y
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
--
作者：
Fonseca AC;Bonaldi A;Bertola DR;Kim CA;Otto PA;Vianna-Morgante AM
通讯作者：
Vianna-Morgante AM