He Qianyu, Chen Jinxia, Chen Shanshan, Gao Xinyu
College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing 163319, China.
College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing 163319, China.
J Adv Res. 2025 Jul 22. doi: 10.1016/j.jare.2025.07.032.
Insects undergo intricate metamorphosis processes governed by hormonal regulatory mechanisms, exemplified by the antagonistic actions of 20-hydroxyecdysone (20E) and juvenile hormone (JH). Krüppel homolog 1 (Kr-h1) is a key transcription factor mediating JH's antagonistic effects on 20E. Previous studies have shown that Kr-h1 plays dual roles in regulating the 20E signaling gene Broad-Complex (Br-C), repressing it in larvae and activating it during pupation. However, the mechanisms underlying this dual functionality remain elusive.
This study aimed to determine whether SUMOylation, a post-translational modification, functions as part of a molecular switch mechanism for Kr-h1's stage-specific transcriptional roles.
Using Drosophila melanogaster, we analyzed Kr-h1 SUMOylation dynamics via Western blot, immunoprecipitation combined with Drosophila genetics. Immunostaining, RT-qPCR, and luciferase reporter assays were performed to investigate the impact of SUMOylation on the bidirectional transcriptional regulation of Kr-h1 on Br-C. SUMOylation sites were identified through mutagenesis and bioinformatics. Coimmunoprecipitation and mass spectrometry explored Kr-h1 interactors. Hormonal effects on regulation of Kr-h1 SUMOylation were tested using JH mimic (methoprene) and 20E treatments.
Kr-h1 undergoes stage-specific SUMOylation: it is highly SUMOylatedin early third-instar larvae, enabling interaction with the histone methyltransferase SmydA-8 to repress Br-C. Conversely, at the white prepupal stage, deSUMOylation disrupts the Kr-h1- SmydA-8 interaction, thereby activating Br-C expression. K113 was identified as the primary SUMOylation site in Kr-h1. JH stimulated Kr-h1 SUMOylation by enhancing its interaction with the SUMO-conjugating enzyme Ubc9. Conversely, 20E promotedKr-h1 deSUMOylation via increased association with the deSUMOylating enzyme ubiquitin-like protease 1 (Ulp1). Co-stimulation with JH and 20E synergistically amplified deSUMOylation.
SUMOylation of Kr-h1 serves as a molecular switch governing its dual regulatory functions in Br-C expression. JH and 20E antagonistically control this post-translational modification. Our study establishes SUMOylation as a critical regulator of Kr-h1's stage-specific activity, revealing how it orchestrates crosstalk between JH and 20E signaling during insect development. These findings provide a mechanistic framework for understanding how post-translational modifications confer functional plasticity to transcription factors at pivotal developmental transitions. Moreover, identifying SUMOylation as a central node in JH/20E crosstalk opens new avenues for targeting this pathway in insect growth regulators for pest management.
昆虫经历由激素调节机制控制的复杂变态过程，以20-羟基蜕皮激素（20E）和保幼激素（JH）的拮抗作用为例。Krüppel同源物1（Kr-h1）是介导JH对20E拮抗作用的关键转录因子。先前的研究表明，Kr-h1在调节20E信号基因Broad-Complex（Br-C）中起双重作用，在幼虫期抑制它，在化蛹期间激活它。然而，这种双重功能背后的机制仍然难以捉摸。
本研究旨在确定SUMO化这种翻译后修饰是否作为Kr-h1阶段特异性转录作用的分子开关机制的一部分发挥作用。
使用黑腹果蝇，我们通过蛋白质免疫印迹、免疫沉淀结合果蝇遗传学分析Kr-h1的SUMO化动力学。进行免疫染色、RT-qPCR和荧光素酶报告基因测定以研究SUMO化对Kr-h1对Br-C的双向转录调控的影响。通过诱变和生物信息学鉴定SUMO化位点。免疫共沉淀和质谱法探索Kr-h1相互作用蛋白。使用JH模拟物（烯虫酯）和20E处理测试激素对Kr-h1 SUMO化调节的影响。
Kr-h1经历阶段特异性SUMO化：它在三龄早期幼虫中高度SUMO化，能够与组蛋白甲基转移酶SmydA-8相互作用以抑制Br-C。相反，在白色蛹前期，去SUMO化破坏了Kr-h1与SmydA-8的相互作用，从而激活Br-C表达。K113被鉴定为Kr-h1中的主要SUMO化位点。JH通过增强其与SUMO缀合酶Ubc9的相互作用刺激Kr-h1 SUMO化。相反，20E通过增加与去SUMO化酶泛素样蛋白酶1（Ulp1）的结合促进Kr-h1去SUMO化。JH和20E共同刺激协同放大去SUMO化。
Kr-h1的SUMO化作为一种分子开关，控制其在Br-C表达中的双重调节功能。JH和20E拮抗地控制这种翻译后修饰。我们的研究确定SUMO化是Kr-h1阶段特异性活性的关键调节因子，揭示了它如何在昆虫发育过程中协调JH和20E信号之间的串扰。这些发现提供了一个机制框架，用于理解翻译后修饰如何在关键发育转变中赋予转录因子功能可塑性。此外，将SUMO化鉴定为JH/20E串扰中的中心节点，为在害虫管理的昆虫生长调节剂中靶向该途径开辟了新途径。
