The Zic family homologue Odd-paired regulates Alk expression in Drosophila.
The Zic family homologue Odd-paired regulates Alk expression in Drosophila.
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DOI：
10.1371/journal.pgen.1006617
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发表时间：
2017-04
期刊：
影响因子：
4.5
通讯作者：
Palmer RH
中科院分区：
文献类型：
--
作者：
Mendoza-García P;Hugosson F;Fallah M;Higgins ML;Iwasaki Y;Pfeifer K;Wolfstetter G;Varshney G;Popichenko D;Gergen JP;Hens K;Deplancke B;Palmer RH
间变性淋巴瘤激酶 (Alk) 受体酪氨酸激酶 (RTK) 在果蝇内脏中胚层 (VM) 胚胎发生过程中的创始细胞 (FC) 规范中发挥着关键作用。报告基因和 CRISPR/Cas9 缺失分析揭示了 Alk 基因座及其上游的增强子区域影响羊膜浆膜 (AS)、VM 和表皮的组织特异性表达。通过使用果蝇转录因子 (TF) 文库进行高通量酵母单杂交筛选 (Y1H)，我们鉴定出脊椎动物 Zic 转录因子家族的果蝇同源物奇配对 (Opa) 作为胚胎 Alk 表达的新型调节因子。进一步的表征鉴定了 Alk 相关增强子元件之一中进化保守的 Opa 结合顺式调节基序。利用 Alk 报告系以及 CRISPR/Cas9 介导的 Alk 基因座调节元件去除，我们展示了 Opa 在胚胎 AS、表皮和 VM 中对 Alk 表达的调节。此外，我们还确定了整合来自其他 TF 的输入的增强子元件，例如 Binou (Bin) 和 Bagpipe (Bap)，以组合方式调节 Alk 的 VM 表达。综上所述，我们的数据表明 Opa 锌指 TF 是胚胎 Alk 表达的新型调节因子。 Alk 受体酪氨酸激酶在果蝇发育过程中被反复使用，以驱动多种组织中的信号传导事件。 Alk 基因的空间和时间表达模式受到严格调控。识别影响 Alk 表达的因素对于更好地了解 Alk 信号传导的控制方式非常重要。在本文中，我们描述了 Alk 基因座中的顺式调控序列以及结合它们以控制果蝇胚胎中 Alk 表达的转录因子。使用机器人蛋白质-DNA 相互作用测定，我们鉴定了 Zic 家族转录因子奇数配对为与 Alk 基因座中的调控元件结合的因子。奇数对与 Alk 顺式调控元件的结合在空间上有所不同，揭示了在 Alk 表达组织的子集中需要额外的转录因子，例如 NK3 和 FoxF 直向同源物 Bagpipe 和 Biniou。我们的研究结果为胚胎发生过程中 Alk 受体的时间和空间调节的动态提供了新的见解。
The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression. The Alk receptor tyrosine kinase is employed repeatedly during Drosophila development to drive signaling events in a variety of tissues. The spatial and temporal expression pattern of the Alk gene is tightly regulated. Identifying factors that influence the expression of Alk is important to better understand how Alk signaling is controlled. In this paper we characterize cis-regulatory sequences in the Alk locus and the transcription factors that bind them to govern Alk expression in the Drosophila embryo. Using a robotic protein-DNA interaction assay, we identified the Zic family transcription factor Odd-paired as a factor that binds to regulatory elements in the Alk locus. Binding of Odd-paired to Alk cis-regulatory elements varies spatially, revealing a requirement for additional transcription factors such as the NK3 and FoxF orthologues Bagpipe and Biniou in a subset of Alk-expressing tissues. Our findings provide new insight into the dynamics underlying temporal and spatial regulation of the Alk receptor during embryogenesis.