Unraveling the transcriptional regulation of TWIST1 in limb development.
Unraveling the transcriptional regulation of TWIST1 in limb development.
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DOI：
10.1371/journal.pgen.1007738
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发表时间：
2018-10
期刊：
影响因子：
4.5
通讯作者：
Birnbaum RY
中科院分区：
文献类型：
--
作者：
Hirsch N;Eshel R;Bar Yaacov R;Shahar T;Shmulevich F;Dahan I;Levaot N;Kaplan T;Lupiáñez DG;Birnbaum RY
转录因子 TWIST1 在中胚层发育中发挥着至关重要的作用，特别是在肢体和颅面形成中。因此，TWIST1 的单倍体不足可导致肢体和颅面畸形，这是 Saethre-Chotzen 综合征的一部分。然而，TWIST1在发育过程中转录调控的分子基础尚未阐明。在这里，我们表征了 TWIST1-HDAC9 基因座中驱动发育中肢体和鳃弓转录的活性增强子。利用可用的 p300 和 H3K27ac ChIP-seq 数据，我们鉴定了 12 个候选增强子，它们位于邻近基因组蛋白脱乙酰酶 9 (HDAC9) 的编码序列内部和外部。使用斑马鱼和小鼠增强子检测，我们发现其中八个候选者具有类似于 Twist1 表达的肢/鳍和鳃弓增强子活性。使用 4C-seq，我们发现 Twist1 启动子区域在第 11.5 天时与小鼠胚胎的肢芽和鳃弓中的三个增强子（eTw-5、6、7）相互作用。此外，我们发现两种转录因子 LMX1B 和 TFAP2 结合这些增强子并调节它们的增强子活性。最后，使用 CRISPR/Cas9 基因组编辑，我们发现 eTw5-7 增强子的纯合删除会降低肢芽中 Twist1 的表达，并导致轴前多指畸形，这是在 Twist1+/- 小鼠中观察到的一种表型。总而言之，我们的研究结果表明，每个增强子都具有离散的活动模式，并且共同构成了发育中的四肢/鳍和鳃弓中 Twist1 转录的时空调节网络。我们的研究表明，TWIST1 增强子的突变可能导致 TWIST1 表达减少，从而导致 TWIST1 编码突变所见的表型结果。 TWIST1 单倍体不足可导致肢体和颅面畸形，例如 Saethre-Chotzen 综合征。然而，控制 TWIST1 表达和防止发育异常的 DNA 调控元件尚不清楚。在这里，我们鉴定并表征了几个 TWIST1 转录增强子。每个增强子激活肢体或鳃弓的离散亚组织中的表达，并共同对应于模拟 TWIST1 时空表达的一组元件。此外，还发现两个重要的转录因子 LMX1B 和 TFAP2 可以结合这些增强子并参与其活性。最后，三个 Twist1 增强子 (eTw5-7) 的纯合缺失降低了肢芽中 Twist1 的表达，并导致多指畸形，如杂合 Twist1-/+ 小鼠中所见。这项研究阐明了 TWIST1 调控元件及其转录机制的重要组成部分，表明这些增强子的改变可能导致功能丧失，从而可能导致与 TWIST1 编码突变相似的表型结果。
The transcription factor TWIST1 plays a vital role in mesoderm development, particularly in limb and craniofacial formation. Accordingly, haploinsufficiency of TWIST1 can cause limb and craniofacial malformations as part of Saethre-Chotzen syndrome. However, the molecular basis of TWIST1 transcriptional regulation during development has yet to be elucidated. Here, we characterized active enhancers in the TWIST1-HDAC9 locus that drive transcription in the developing limb and branchial arches. Using available p300 and H3K27ac ChIP-seq data, we identified 12 enhancer candidates, located both within and outside the coding sequences of the neighboring gene, Histone deacetyase 9 (HDAC9). Using zebrafish and mouse enhancer assays, we showed that eight of these candidates have limb/fin and branchial arch enhancer activity that resemble Twist1 expression. Using 4C-seq, we showed that the Twist1 promoter region interacts with three enhancers (eTw-5, 6, 7) in the limb bud and branchial arch of mouse embryos at day 11.5. Furthermore, we found that two transcription factors, LMX1B and TFAP2, bind these enhancers and modulate their enhancer activity. Finally, using CRISPR/Cas9 genome editing, we showed that homozygous deletion of eTw5-7 enhancers reduced Twist1 expression in the limb bud and caused pre-axial polydactyly, a phenotype observed in Twist1+/- mice. Taken together, our findings reveal that each enhancer has a discrete activity pattern, and together comprise a spatiotemporal regulatory network of Twist1 transcription in the developing limbs/fins and branchial arches. Our study suggests that mutations in TWIST1 enhancers could lead to reduced TWIST1 expression, resulting in phenotypic outcome as seen with TWIST1 coding mutations. TWIST1 haploinsufficiency can cause limb and craniofacial malformations, such as Saethre-Chotzen syndrome. However, the DNA regulatory elements that control TWIST1 expression and prevent developmental abnormalities are not known. Here, we identified and characterized several TWIST1 transcriptional enhancers. Each enhancer activates expression in a discrete sub-tissue of a limb or branchial arch and together correspond to a cluster of elements that mimic TWIST1 spatiotemporal expression. Furthermore, two important transcription factors, LMX1B and TFAP2, were found to bind these enhancers and participate in their activity. Finally, homozygous deletion of three Twist1 enhancers (eTw5-7) reduced Twist1 expression in the limb bud and led to polydactyly, as seen in heterozygous Twist1-/+ mice. This study elucidates TWIST1 regulatory elements and essential components of its transcriptional machinery, suggesting that alteration of these enhancers could lead to loss of function that may result in similar phenotypic outcomes as seen with TWIST1 coding mutations.