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Synergistic action of the transcription factors Kruppel homolog 1 and Hairy in juvenile hormone/Methoprene-tolerant-mediated gene-repression in the mosquito Aedes aegypti.

ABSTRACT: Arthropod-specific juvenile hormones control numerous essential functions in development and reproduction. In the dengue-fever mosquito Aedes aegypti, in addition to its role in immature stages, juvenile hormone III (JH) governs post-eclosion (PE) development in adult females, a phase required for competence acquisition for blood feeding and subsequent egg maturation. During PE, JH through its receptor Methoprene-tolerant (Met) regulate the expression of many genes, causing either activation or repression. Met-mediated gene repression is indirect, requiring involvement of intermediate repressors. Hairy, which functions downstream of Met in the JH gene-repression hierarchy, is one such factor. Krüppel-homolog 1, a zinc-finger transcriptional factor, is directly regulated by Met and has been implicated in both activation and repression of JH-regulated genes. However, the interaction between Hairy and Kr-h1 in the JH-repression hierarchy is not well understood. Our RNAseq-based transcriptomic analysis of the Kr-h1-depleted mosquito fat body revealed that 92% of Kr-h1 repressed genes are also repressed by Met, supporting the existence of a hierarchy between Met and Kr-h1 as previously demonstrated in various insects. Notably, 130 genes are co-repressed by both Kr-h1 and Hairy, indicating regulatory complexity of the JH-mediated PE gene repression. A mosquito Kr-h1 binding site in genes co-regulated by this factor and Hairy was identified computationally. Moreover, this was validated using electrophoretic mobility shift assays. A complete phenocopy of the effect of Met RNAi depletion on target genes could only be observed after Kr-h1 and Hairy double RNAi knockdown, suggesting a synergistic action between these two factors in target gene repression. This was confirmed using a cell-culture-based luciferase reporter assay. Taken together, our results indicate that Hairy and Kr-h1 not only function as intermediate downstream factors, but also act together in a synergistic fashion in the JH/Met gene repression hierarchy.

SUBMITTER: Saha TT

PROVIDER: S-EPMC6818763 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Synergistic action of the transcription factors Krüppel homolog 1 and Hairy in juvenile hormone/Methoprene-tolerant-mediated gene-repression in the mosquito Aedes aegypti.

Saha Tusar T TT Roy Sourav S Pei Gaofeng G Dou Wei W Zou Zhen Z Raikhel Alexander S AS

PLoS genetics 20191029 10

Arthropod-specific juvenile hormones control numerous essential functions in development and reproduction. In the dengue-fever mosquito Aedes aegypti, in addition to its role in immature stages, juvenile hormone III (JH) governs post-eclosion (PE) development in adult females, a phase required for competence acquisition for blood feeding and subsequent egg maturation. During PE, JH through its receptor Methoprene-tolerant (Met) regulate the expression of many genes, causing either activation or ...[more]

PMID: 31661489

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Project description:Recent studies reported that JH-regulated phosphorylation status of the JH-receptor complex contributes to its transcription activity in Aedes aegypti. However, phosphorylation sites of these proteins have not yet been identified. In this study, we found that the fusion of an EGFP tag to Ae. aegypti Kr-h1 (AaKr-h1) and Met (AaMet) improved their stability in mosquito Aag-2 cells, which allowed their purification. The liquid chromatography and tandem mass spectrometry analysis of the purified AaKr-h1 showed that the phosphoserine residue at position 694, located in the evolutionarily conserved SVIQ motif, is dephosphorylated when the cells are exposed to JH. The AaKr-h1 dephosphorylation mutant (S694V) showed significantly higher activity in inducing the luciferase gene regulated by JH response elements. The phosphorylation profile of Met also changed after exposing Aag-2 cells to JH III. The Ser-77 and Ser-710 residues of Met were phosphorylated after JH III treatment. In contrast, the two phosphoserine residues at positions 73 and 747 were dephosphorylated after JH III treatment. JH exposure also induced transient and reversible phosphorylation of Thr-664 and Ser-723 residues. Overall, these data show that JH induces changes in post-translational modifications of AaMet and AaKr-h1. SIGNIFICANCE: Female Aedes aegypti mosquitoes are known to vector many disease agents, including Zika virus, dengue virus chikungunya virus, and Mayaro and yellow fever virus. In the present study, we developed an efficient method to prepare Ae. aegypti Met and Kr-h1, which are typically difficult to produce and purify, using a mosquito cell line expression system. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches were utilized to map the phosphorylation profiles of the isolated proteins. We then monitored the changes induced by JH activation in the phosphorylation profiles to check if the JH modulates post-translation modification of its key transcription factors. We found that the JH induced alterations in the phosphorylation profiles of the multiple residues of AaMet. In contrast, activation of the JH signaling pathway was accompanied by dephosphorylation of AaKr-h1 at phosphoserine-694, increasing its transcriptional activity. In addition, S694 of AaKr-h1 was located in the RMSSVIQYA motif highly conserved in orthologous proteins from other insect species. These results can help us further understand how JH modulates its key transcription factors and provide a basis for the development of novel insect control strategies.

Dataset Information

Synergistic action of the transcription factors Kruppel homolog 1 and Hairy in juvenile hormone/Methoprene-tolerant-mediated gene-repression in the mosquito Aedes aegypti.

Publications

Synergistic action of the transcription factors Krüppel homolog 1 and Hairy in juvenile hormone/Methoprene-tolerant-mediated gene-repression in the mosquito Aedes aegypti.

Similar Datasets

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