Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti.

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis.

The DNA chaperone HMGB1 potentiates the transcriptional activity of Rel1A in the mosquito Aedes aegypti.

High Mobility Group protein 1 (HMGB1) is a non-histone, chromatin-associated nuclear protein that functions in regulating eukaryotic gene expression. We investigated the influence and mechanism of action of Aedes aegypti HMGB1 (AaHMGB1) on mosquito Rel1A-mediated transcription from target gene promoters. The DNA-binding domain (RHD) of AaRel1A was bacterially expressed and purified, and AaHMGB1 dramatically enhanced RHD binding to consensus NF-kB/Rel DNA response elements. Luciferase reporter analyses using a cecropin gene promoter showed that AaHMGB1 potentiates the transcriptional activity of AaRel1A in Aag-2 cells. Moreover, overexpression of AaHMGB1 in Aag-2 cells led to an increase in mRNA levels of antimicrobial peptide genes. In vitro GST pull-down assays revealed that the presence of DNA is a pre-requisite for assembly of a possible ternary complex containing DNA, AaHMGB1 and AaRel1A. Notably, DNA bending by AaHMGB1 enhanced the binding of AaRel1A to a DNA fragment containing a putative NF-kB/Rel response element. Importantly, AaHMGB1 was identified as a potential immune modulator in A. aegypti through AaHMGB1 overexpression or RNAi silencing in Aag-2 cells followed by bacterial challenge or through AaHMGB1 RNAi knockdown in mosquitoes followed by Dengue virus (DENV) infection. We propose a model in which AaHMGB1 bends NF-kB/Rel target DNA to recruit and allow more efficient AaRel1A binding to activate transcription of effector genes, culminating in a stronger Toll pathway-mediated response against DENV infection.

Comprehensive DNA methylation analysis of the Aedes aegypti genome.

Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation.