State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716, China.
Biological Science Research Center, Southwest University, Chongqing, 400716, China.
Insect Sci. 2022 Feb;29(1):111-127. doi: 10.1111/1744-7917.12916. Epub 2021 Apr 16.
Clip-domain serine proteases (CLIPs) play important roles in insect innate immunity and development. Our previous studies indicated that CLIP13, an epidermis-specific gene, was involved in cuticle remodeling during molting and metamorphosis in the silkworm, Bombyx mori. However, the transcriptional regulatory mechanism and regulatory pathways of CLIP13 remained unclear. In the present study, we investigated CLIP13 expression and the regulation pathway controlled by 20-hydroxyecdysone (20E) in the silkworm. At the transcriptional level, expression of CLIP13 exhibited pronounced spatial and temporal specificity in different regions of the epidermis; homeodomain transcription factors POU-M2, antennapedia (Antp), and abdominal-B (Abd-B) showed similar expression change trends as CLIP13 in the head capsule, thorax, and abdomen, respectively. Furthermore, results of cell transfection assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation demonstrated that POU-M2, Antp, and Abd-B were involved in the transcriptional regulation of CLIP13 by directly binding to their cis-response elements in CLIP13 promoter. RNA interference-mediated silencing of POU-M2, Antp, and Abd-B led to a decrease of CLIP13 expression in the head capsule, the epidermis of the 1st to 3rd thoracic segments and the 7th to 10th abdominal segments, respectively. Consistent with CLIP13, 20E treatment significantly upregulated expression of POU-M2, Antp, and Abd-B in the silkworm epidermis. Taken together, these data suggest that 20E positively regulates transcription of CLIP13 via homeodomain proteins POU-M2, Antp, and Abd-B in different regions of the silkworm epidermis during metamorphosis, thus affecting the molting process. Our findings provide new insight into the functions of homeodomain transcription factors in insect molting.
剪接域丝氨酸蛋白酶 (CLIPs) 在昆虫先天免疫和发育中发挥重要作用。我们之前的研究表明，表皮特异性基因 CLIP13 参与了家蚕蜕皮和变态过程中的表皮重塑。然而，CLIP13 的转录调控机制和调控途径尚不清楚。在本研究中，我们研究了 CLIP13 在蚕中的表达及其受 20-羟基蜕皮酮 (20E) 调控的途径。在转录水平上，CLIP13 的表达在表皮的不同区域具有明显的时空特异性；同源域转录因子 POU-M2、触角足（Antp）和 Abd-B 在头壳、胸部和腹部的表达变化趋势与 CLIP13 相似。此外，细胞转染实验、电泳迁移率变动分析和染色质免疫沉淀实验的结果表明，POU-M2、Antp 和 Abd-B 通过直接结合 CLIP13 启动子中的顺式反应元件参与 CLIP13 的转录调控。POU-M2、Antp 和 Abd-B 的 RNA 干扰介导的沉默导致头壳、第 1 至 3 胸段和第 7 至 10 腹段表皮中 CLIP13 的表达减少。与 CLIP13 一致，20E 处理显著上调了蚕表皮中 POU-M2、Antp 和 Abd-B 的表达。综上所述，这些数据表明，20E 通过不同区域表皮中的同源域蛋白 POU-M2、Antp 和 Abd-B 正向调节 CLIP13 的转录，从而影响蜕皮过程。我们的研究结果为同源域转录因子在家蚕蜕皮中的功能提供了新的见解。
