Regulation of Drosophila Metamorphosis by Xenobiotic Response Regulators

Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional responses to xenobiotic compounds as well as native cellular and developmental processes. The relationships between the functions of these proteins in xenobiotic responses and in development were unknown. We investigated the genes regulated by CncC and dKeap1 during development and the signal transduction pathways that modulate their functions. CncC and dKeap1 were enriched within the nuclei in many tissues, in contrast to the reported cytoplasmic localization of Keap1 and Nrf2 in cultured mammalian cells. CncC and dKeap1 occupied ecdysone-regulated early puffs on polytene chromosomes. Depletion of either CncC or dKeap1 in salivary glands selectively reduced early puff gene transcription. CncC and dKeap1 depletion in the prothoracic gland as well as cncCK6/K6 and dKeap1EY5/EY5 loss of function mutations in embryos reduced ecdysone-biosynthetic gene transcription. In contrast, dKeap1 depletion and the dKeap1EY5/EY5 loss of function mutation enhanced xenobiotic response gene transcription in larvae and embryos, respectively. Depletion of CncC or dKeap1 in the prothoracic gland delayed pupation by decreasing larval ecdysteroid levels. CncC depletion suppressed the premature pupation and developmental arrest caused by constitutive Ras signaling in the prothoracic gland; conversely, constitutive Ras signaling altered the loci occupied by CncC on polytene chromosomes and activated transcription of genes at these loci. The effects of CncC and dKeap1 on both ecdysone-biosynthetic and ecdysone-regulated gene transcription, and the roles of CncC in Ras signaling in the prothoracic gland, establish the functions of these proteins in the neuroendocrine axis that coordinates insect metamorphosis.哺乳动物Nrf2-Keap1与同源的果蝇CncC-dKeap1蛋白复合物，可同时调控针对外源性化合物的转录应答以及天然细胞进程与发育过程。此前对于这两类蛋白在外源性化合物应答与发育过程中的功能关联尚不清楚。本研究针对发育过程中受CncC与dKeap1调控的基因，以及调控二者功能的信号转导通路展开了探究。CncC与dKeap1在多种组织的细胞核中富集，这与已报道的培养哺乳动物细胞中Keap1与Nrf2定位于细胞质的结果形成鲜明对比。CncC与dKeap1可结合多线染色体上受蜕皮激素调控的早期膨突区域。在唾液腺中敲低CncC或dKeap1，均可选择性降低早期膨突相关基因的转录水平。在前胸腺中敲低CncC或dKeap1，以及胚胎中cncCK6/K6与dKeap1EY5/EY5功能丧失突变，均可降低蜕皮激素合成相关基因的转录水平。与之相反，在幼虫体内敲低dKeap1，以及胚胎中dKeap1EY5/EY5功能丧失突变，可分别提升外源性化合物应答相关基因的转录水平。在前胸腺中敲低CncC或dKeap1，可通过降低幼虫蜕皮类固醇水平，延缓化蛹进程。在前胸腺中组成型激活Ras信号会引发过早化蛹与发育停滞，而敲低CncC可抑制这一现象；反之，组成型激活Ras信号会改变CncC在多线染色体上的结合位点，并激活这些位点上的基因转录。CncC与dKeap1对蜕皮激素合成相关基因及受蜕皮激素调控的基因转录的调控作用，以及CncC在前胸腺Ras信号通路中的功能，共同证实了这两类蛋白在协调昆虫变态的神经内分泌轴中发挥的功能。