Structural variants (SVs) can affect protein-coding sequences as well as gene regulatory elements. However, SVs disrupting protein-coding sequences that also function as cis-regulatory elements remain largely uncharacterized. Here, we show that craniosynostosis patients with SVs containing the histone deacetylase 9 (HDAC9) protein-coding sequence are associated with disruption of TWIST1 regulatory elements that reside within the HDAC9 sequence. Based on SVs within the HDAC9‐TWIST1 locus, we defined the 3′-HDAC9 sequence as a critical TWIST1 regulatory region, encompassing craniofacial TWIST1 enhancers and CTCF sites. Deletions of either Twist1 enhancers (eTw5-7^Δ/Δ) or CTCF site (CTCF-5^Δ/Δ) within the Hdac9 protein-coding sequence led to decreased Twist1 expression and altered anterior/posterior limb expression patterns of SHH pathway genes. This decreased Twist1 expression results in a smaller sized and asymmetric skull and polydactyly that resembles Twist1^+/− mouse phenotype. Chromatin conformation analysis revealed that the Twist1 promoter interacts with Hdac9 sequences that encompass Twist1 enhancers and a CTCF site, and that interactions depended on the presence of both regulatory regions. Finally, a large inversion of the entire Hdac9 sequence (Hdac9^INV/+) in mice that does not disrupt Hdac9 expression but repositions Twist1 regulatory elements showed decreased Twist1 expression and led to a craniosynostosis-like phenotype and polydactyly. Thus, our study elucidates essential components of TWIST1 transcriptional machinery that reside within the HDAC9 sequence. It suggests that SVs encompassing protein-coding sequences could lead to a phenotype that is not attributed to its protein function but rather to a disruption of the transcriptional regulation of a nearby gene.

中文翻译：

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破坏 TWIST1 转录调控的 HDAC9 结构变异导致颅面和肢体畸形

结构变体 (SV) 可以影响蛋白质编码序列以及基因调控元件。然而，破坏蛋白质编码序列的 SVs 也作为cis调节元件在很大程度上仍然没有被表征。在这里，我们显示具有包含组蛋白去乙酰化酶 9 (HDAC9 ) 蛋白编码序列的SV 的颅缝早闭患者与HDAC9序列内的TWIST1调节元件的破坏有关。基于HDAC9 - TWIST1基因座内的 SV，我们将 3' - HDAC9序列定义为关键的TWIST1调节区，包括颅面TWIST1增强子和 CTCF 位点。Hdac9蛋白编码序列中Twist1增强子 (eTw5-7 ^Δ/Δ ) 或 CTCF 位点 (CTCF-5 ^Δ/Δ ) 的缺失导致Twist1表达降低并改变了 SHH 通路基因的前肢/后肢表达模式。这种减少的Twist1表达导致更小的和不对称的头骨和多指畸形，类似于Twist1 ^+/-小鼠表型。染色质构象分析显示Twist1启动子与包含Twist1的Hdac9序列相互作用增强子和 CTCF 位点，这种相互作用取决于两个调节区的存在。最后，在不破坏Hdac9表达但重新定位Twist1调节元件的小鼠中，整个Hdac9序列 ( Hdac9 ^{INV/+ ) 的大量倒置显示}Twist1表达降低，并导致颅缝早闭样表型和多指畸形。因此，我们的研究阐明了位于HDAC9内的TWIST1转录机制的重要组成部分序列。这表明包含蛋白质编码序列的 SV 可能导致一种表型，该表型并非归因于其蛋白质功能，而是归因于附近基因转录调控的破坏。