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

Tusar T Saha,Wei Dou,Alexander S Raikhel,Zhen Zou,Gaofeng Pei,Sourav Roy,Lynn M Riddiford

doi:10.1371/journal.pgen.1008443

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.

Highlights

Arthropod-specific juvenile hormones (JHs) are key regulators of a large array of physiological processes, including growth, metamorphosis and reproduction [1, 2]
JH acting through its receptor Methoprene-tolerant (Met) regulates the expression of large gene cohorts
JH mediated gene repression, unlike activation that is directly mediated by Met, is indirect and requires intermediate transcriptional repressors Hairy and Kruppel-homolog 1 (Krh1)

Summary

Introduction

Arthropod-specific juvenile hormones (JHs) are key regulators of a large array of physiological processes, including growth, metamorphosis and reproduction [1, 2]. Recent studies have indicated the existence of a not-yet fully characterized membrane JH receptor that is responsible for Met phosphorylation, a necessary step for the activity of the JH receptor complex [20, 21]. This mode of action involving JH has been confirmed for several genes, including early trypsin (ET), regulator of ribosomal synthesis 1 (RRS1), Hairy, and Krüppel-homolog 1 (Kr-h1) [2, 10, 12, 16, 17, 22, 23]. Some targets of the JH signaling pathway, such as Hairy and Kr-h1, are transcription factors (TFs) that in turn can regulate the expression of downstream genes [2, 22, 23]

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Conclusion