RESUMO
The steroid hormone ecdysone, which controls insect molting and metamorphosis, is synthesized in the prothoracic gland (PG), and several steroidogenic enzymes that are expressed specifically in the PG are involved in ecdysteroidogenesis. In this study, we identified new regulators that are involved in the transcriptional control of the silkworm steroidogenic enzyme genes. In silico analysis predicted several potential cis-regulatory elements (CREs) for the homeodomain transcription factors Antennapedia (Antp) and POU-M2 in the proximal promoters of steroidogenic enzyme genes. Antp and POU-M2 are expressed dynamically in the PG during larval development, and their overexpression in silkworm embryo-derived (BmE) cells induced the expression of steroidogenic enzyme genes. Importantly, luciferase reporter analyses, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Antp and POU-M2 promote the transcription of the silkworm steroidogenic enzyme gene Phantom (Phm) by binding directly to specific motifs within overlapping CREs in the Phm promoter. Mutations of these CREs in the Phm promoter suppressed the transcriptional activities of both Antp and POU-M2 in BmE cells and decreased the activities of mutated Phm promoters in the silkworm PG. In addition, pulldown and co-immunoprecipitation assays demonstrated that Antp can interact with POU-M2. Moreover, RNA interference-mediated down-regulation of either Antp or POU-M2 during silkworm wandering not only decreased the ecdysone titer but also led to the failure of metamorphosis. In summary, our results suggest that Antp and POU-M2 coordinate the transcription of the silkworm Phm gene directly, indicating new roles for homeodomain proteins in regulating insect ecdysteroidogenesis.
Assuntos
Proteína do Homeodomínio de Antennapedia/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Insetos/metabolismo , Metamorfose Biológica , Oxigenases de Função Mista/metabolismo , Fatores do Domínio POU/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Proteína do Homeodomínio de Antennapedia/genética , Bombyx , Biologia Computacional , Ecdisona/química , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/genética , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , Oxigenases de Função Mista/genética , Muda , Mutação , Nucleopoliedrovírus/genética , Fatores do Domínio POU/genética , Regiões Promotoras Genéticas , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Interferência de RNA , Fatores de Transcrição/genéticaRESUMO
Transcription factor Broad Complex (BR-C) is an ecdysone primary response gene in insects and participates in the regulation of insect growth and development. In this study, we performed a genome-wide identification of BR-C target genes in silkworm (Bombyx mori) using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). As a result, a total of 1006 BR-C ChIP peaks were identified, and 15% of peaks were located in the promoter regions of 133 protein-coding genes. Functional annotation revealed that these ChIP peak-associated genes, as potential BR-C targets, were enriched in pathways related to biosynthetic process, metabolic process, and development. Transcriptome analysis and quantitative real-time polymerase chain reaction (PCR) examination revealed that developmental changes in expression patterns of a portion of potential BR-C targets, including HR96 and GC-α1, were similar to those of BR-C. ChIP-PCR examination confirmed that BR-C could directly bind to the promoters of potential targets. Further, dual luciferase assays demonstrated that HR96 promoter activity was significantly upregulated following BR-C overexpression, and this upregulation was abolished when the binding motif in the promoter was truncated. This study will be helpful for deciphering the regulatory roles of BR-C during insect growth and development.
Assuntos
Bombyx , Proteínas de Insetos , Fatores de Transcrição , Animais , Bombyx/genética , Bombyx/metabolismo , Ecdisona , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The fat body plays key roles in metabolism and sustenance of growth throughout the life of a silkworm, and thus represents a model tissue for studying development and metamorphosis. Analysis of 18,480 ESTs derived from larval and pupal fat body cDNA libraries allowed characterization of gene expression patterns in the fat body during metamorphosis. By Phrap assembling, 6,814 estimated transcription units (TUs) were generated, 2,673 of which contained 60% of the total ESTs and represented 1,366 distinctive known genes. These genes were classified into 14 categories based on their known or putative functions, and most were found to be involved in metabolism and cell defense. Further comparative analysis of the expression profiles revealed that the gene expression patterns in the larval fat body strikingly differed from those in the pupal fat body. From this, we identified a subset of genes potentially associated with metamorphic events such as the formation or destruction of specific tissues, and simultaneously confirmed the stage-specific expression patterns of several identified genes using RT-PCR examination. This work will provide a valuable resource for studying regulatory mechanisms associated with fat body developmental changes and silkworm metamorphosis.