Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division

Engineered <i>Aedes aegypti</i> JAK/STAT Pathway-Mediated Immunity to Dengue Virus

<div><p>We have developed genetically modified <i>Ae</i>. <i>aegypti</i> mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role.</p></div

Generation of transgenic <i>Ae</i>. <i>aegypti</i> over-expressing Dome and Hop under the control of the Vg promoter.

<p>(A) Schematic of the piggyBac (pBac) transformation plasmids used to generate the VgDome and VgHop lines. pBacL, pBacR: pBac left and right arms, 3xP3: eye-specific promoter with either EGFP or DsRed as markers, Vg promoter: vitellogenin promoter, Dome: Dome coding sequence, Hop: Hop coding sequence, TrypT: trypsin terminator sequence. (B) Transcript abundance of transgenes and effector genes in the fat body of VgDome and VgHop lines from before blood feeding (0 hr) up to 48 hpbm. Each bar represents the relative fold change of Dome, Hop or DVRF1 (DENV restriction factor 1), compared between transgenic lines and WT <i>Ae</i>. <i>aegypti</i>. The S7 ribosomal gene was used to normalize cDNA templates. Error bars indicate standard error of the mean.</p

Midgut transcriptomic profiles of transgenic mosquitoes compared to WT at 24 hpbm.

<p>Relative gene expression of (A) Fold change in Dome and Hop gene expression (fat body/midgut) (B) Fold change in DVRF1 gene expression in the midgut of the transgenic lines as compared to WT. (C) Number of differentially expressed transcripts in the midgut of the transgenic lines as compared to WT mosquitoes, classified according to functional groups as previously described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005187#pntd.0005187.ref008" target="_blank">8</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005187#pntd.0005187.ref015" target="_blank">15</a>]. Abbreviations: CS, cytoskeletal and structural; CSR, chemosensory reception; DIV, diverse functions; DIG, blood and sugar food digestive; IMM, immunity; MET, metabolism; PROT, proteolysis; RSM, redox, stress, and mitochondrion; RTT, replication, transcription, and translation; TRP, transport; UKN, unknown functions. (D) Percentage of genes enriched or depleted in each functional group in the midguts of the VgDome or VgHop mosquitoes as compared to WT.</p

Impact of transgenesis on mosquito fitness.

<p>(A) Lifespans of male and female mosquitoes maintained on 10% sucrose solution, or of female mosquitoes that were provided a bloodmeal to induce transgene expression. Statistical analyses of survival curves was performed using the log rank test with Prism software. ***: p<0.001. (B) Fecundity of WT and transgenic <i>Ae</i>. <i>aegypti</i>, as represented by the number of eggs produced by each female mosquito. Statistical analyses were performed using the Mann-Whitney test with Prism software **: p<0.01 as compared to WT. (C) Expression of vitellogenin at 24 hpbm in the transgenic lines as compared to WT. mRNA levels were measured by real-time PCR, with ribosomal gene S7 as the normalization control. Error bars indicate standard error of the mean.</p

Gene ontology terms over-represented among transcripts commonly regulated in the fat body of VgDome and VgHop compared to WT.

<p>The analyses were performed using the GOstats package in R with a list of 50 commonly enriched and 18 commonly depleted transcripts. Gene ontology (GO) terms with p-values ≤0.01 were considered statistically significant.</p

Effect of JAK/STAT pathway activation on DENV infection in transgenic <i>Ae</i>. <i>aegypti</i>.

<p>The JAK/STAT pathway was induced in the transgenic lines by providing them a naïve bloodmeal; 2 days later, JAK/STAT-activated mosquitoes were orally infected with DENV2 or DENV4. DENV2 titers of the VgDome and VgHop lines were determined for (A) midgut infection at 7 dpibm, (B) disseminated infection at 14 dpibm, and (C) salivary gland infection at 21 dpibm. (D) Midgut DENV2 infection without prior activation of the JAK/STAT pathway through a naïve blood meal at 7 dpibm. (E) Disseminated DENV2 infection of the JAK/STAT pathway-activated hybrid VgDomexVgHop line at 14 dpibm. (F) Disseminated DENV4 infection of the JAK/STAT-activated VgDome and VgHop lines at 14 dpibm. WT mosquitoes were used as a control in parallel in all experiments. Horizontal red lines indicate medians. (G) Prevalence of DENV infection represents data from graphs A-F. Data are pools of results from at least three replicates. Statistical analyses comparing median virus titers were performed using either the Mann-Whitney test or Kruskal-Wallis test with Dunn’s post-test, using Prism software. Statistical analyses comparing virus prevalence were determined by chi-square test. *: p<0.05, **: p<0.01, ***: p<0.001 compared to WT. Descriptive statistics is presented in supplementary <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005187#pntd.0005187.s012" target="_blank">S7 Table</a>.</p

Fat body transcriptomic profiles of transgenic mosquitoes compared to WT at 24 hpbm.

<p>(A) Number of differentially expressed transcripts between the fat body of transgenic and WT mosquitoes., classified according to functional groups as previously described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005187#pntd.0005187.ref008" target="_blank">8</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005187#pntd.0005187.ref015" target="_blank">15</a>]. Abbreviations: CS, cytoskeletal and structural; CSR, chemosensory reception; DIV, diverse functions; DIG, blood and sugar food digestive; IMM, immunity; MET, metabolism; PROT, proteolysis; RSM, redox, stress, and mitochondrion; RTT, replication, transcription, and translation; TRP, transport; UKN, unknown functions. (B) Percentage of genes enriched or depleted, in the fatbody of transgenic lines compared to the WT, for each functional group (C) Venn diagram showing genes significantly regulated in VgDome and VgHop mosquitoes. Green arrows and circle represent the VgDome strain, and red arrows and circle represent the VgHop strain. Upward arrows represent genes significantly enriched, downward arrows represent genes significantly depleted in each strain when compared to WT mosquitoes.</p