The proper balance of gene expression is essential for cellular health, organismal development, and maintaining homeostasis. In response to complex internal and external signals, the cell needs to modulate gene expression to maintain proteostasis and establish cellular identity within its niche. On a genome level, single-celled prokaryotic microbes display clustering of co-expressed genes that are regulated as a polycistronic RNA. This phenomenon is largely absent from eukaryotic microbes, although there is extensive clustering of co-expressed genes as functional pairs spread throughout the genome in Saccharomyces cerevisiae. While initial analysis demonstrated conservation of clustering in divergent fungal lineages, a comprehensive analysis has yet to be performed. Here we report on the prevalence, conservation, and significance of the functional clustering of co-regulated genes within the opportunistic human pathogen, Candida albicans. Our analysis reveals that there is extensive clustering within this organism—although the identity of the gene pairs is unique compared with those found in S. cerevisiae—indicating that this genomic arrangement evolved after these microbes diverged evolutionarily, rather than being the result of an ancestral arrangement. We report a clustered arrangement in gene families that participate in diverse molecular functions and are not the result of a divergent orientation with a shared promoter. This arrangement coordinates the transcription of the clustered genes to their neighboring genes, with the clusters congregating to genomic loci that are conducive to transcriptional regulation at a distance.
more »
« less
Reliance of Host-Encoded Regulators of Retromobility on Ty1 Promoter Activity or Architecture
The Ty1 retrotransposon family is maintained in a functional but dormant state by its host, Saccharomyces cerevisiae . Several hundred RHF and RTT genes encoding co-factors and restrictors of Ty1 retromobility, respectively, have been identified. Well-characterized examples include MED3 and MED15 , encoding subunits of the Mediator transcriptional co-activator complex; control of retromobility by Med3 and Med15 requires the Ty1 promoter in the U3 region of the long terminal repeat. To characterize the U3-dependence of other Ty1 regulators, we screened a library of 188 known rhf and rtt mutants for altered retromobility of Ty1 his3AI expressed from the strong, TATA-less TEF1 promoter or the weak, TATA-containing U3 promoter. Two classes of genes, each including both RHF s and RTT s, were identified. The first class comprising 82 genes that regulated Ty1 his3AI retromobility independently of U3 is enriched for RHF genes that restrict the G1 phase of the cell cycle and those involved in transcriptional elongation and mRNA catabolism. The second class of 51 genes regulated retromobility of Ty1 his3AI driven only from the U3 promoter. Nineteen U3-dependent regulators (U3DRs) also controlled retromobility of Ty1 his3AI driven by the weak, TATA-less PSP2 promoter, suggesting reliance on the low activity of U3. Thirty-one U3DRs failed to modulate P PSP2 -Ty1 his3AI retromobility, suggesting dependence on the architecture of U3. To further investigate the U3-dependency of Ty1 regulators, we developed a novel fluorescence-based assay to monitor expression of p22-Gag, a restriction factor expressed from the internal Ty1i promoter. Many U3DRs had minimal effects on levels of Ty1 RNA, Ty1i RNA or p22-Gag. These findings uncover a role for the Ty1 promoter in integrating signals from diverse host factors to modulate Ty1 RNA biogenesis or fate.
more »
« less
- Award ID(s):
- 1818197
- NSF-PAR ID:
- 10348986
- Date Published:
- Journal Name:
- Frontiers in Molecular Biosciences
- Volume:
- 9
- ISSN:
- 2296-889X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Background Frog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae . Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions. Results Using a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis -regulatory elements ( CREs ) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, but also viral mimics of CREs interacting with several transcription factors including CEBPs, CREBs, IRFs, NF-κB, and STATs, which are critical for regulation of cellular immunity and cytokine responses. Our study suggests that intergenic regions immediately upstream of highly expressed FV3 genes have evolved to bind IRFs, NF-κB, and STATs more efficiently. Moreover, we found an enrichment of putative microRNA (miRNA) sequences in more than five intergenic regions of the FV3 genome. Our sequence analysis indicates that a fraction of these viral miRNAs is targeting the 3’-UTR regions of Xenopus genes involved in interferon (IFN)-dependent responses, including particularly those encoding IFN receptor subunits and IFN-regulatory factors (IRFs). Conclusions Using the FV3 model, this study provides a first genome-wide analysis of non-coding regulatory mechanisms adopted by ranaviruses to epigenetically regulate both viral and host gene expressions, which have co-evolved to interact especially with the host IFN response.more » « less
-
Babitzke, Paul (Ed.)ABSTRACT Oxidative stress causes cellular damage, including DNA mutations, protein dysfunction, and loss of membrane integrity. Here, we discovered that a TrmB (transcription regulator of mal operon) family protein (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulator of oxidative stress response. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons, including genes associated with thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel α helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea. IMPORTANCE TrmB-like proteins, while not yet associated with redox stress, are found in bacteria and widespread in archaea. Here, we expand annotation of a large group of TrmB-like single winged-helix DNA binding domain proteins from diverse archaea to function as thiol-based transcriptional regulators of oxidative stress response. Using Haloferax volcanii as a model, we reveal that the TrmB-like OxsR functions during hypochlorite stress as a transcriptional activator and repressor of an extensive gene coexpression network associated with thiol relay and other related activities. A conserved cysteine residue of OxsR serves as the thiol-based sensor for this function and likely forms an intersubunit disulfide bond during hypochlorite stress that stabilizes a homodimeric configuration with enhanced DNA binding properties. A CG-rich DNA motif in the promoter region of a subset of sites identified to be OxsR-bound is required for regulation; however, not all sites have this motif, suggesting added complexity to the regulatory network.more » « less
-
null (Ed.)Insect systemic immune responses to bacterial infections have been mainly studied using microinjections, whereby the microbe is directly injected into the hemocoel. While this methodology has been instrumental in defining immune signaling pathways and enzymatic cascades in the hemolymph, it remains unclear whether and to what extent the contribution of systemic immune defenses to host microbial resistance varies if bacteria invade the hemolymph after crossing the midgut epithelium subsequent to an oral infection. Here, we address this question using the pathogenic Serratia marcescens (Sm) DB11 strain to establish systemic infections of the malaria vector Anopheles gambiae, either by septic Sm injections or by midgut crossing after feeding on Sm. Using functional genetic studies by RNAi, we report that the two humoral immune factors, thioester-containing protein 1 and C-type lectin 4, which play key roles in defense against Gram-negative bacterial infections, are essential for defense against systemic Sm infections established through injection, but they become dispensable when Sm infects the hemolymph following oral infection. Similar results were observed for the mosquito Rel2 pathway. Surprisingly, blocking phagocytosis by cytochalasin D treatment did not affect mosquito susceptibility to Sm infections established through either route. Transcriptomic analysis of mosquito midguts and abdomens by RNA-seq revealed that the transcriptional response in these tissues is more pronounced in response to feeding on Sm. Functional classification of differentially expressed transcripts identified metabolic genes as the most represented class in response to both routes of infection, while immune genes were poorly regulated in both routes. We also report that Sm oral infections are associated with significant downregulation of several immune genes belonging to different families, specifically the clip-domain serine protease family. In sum, our findings reveal that the route of infection not only alters the contribution of key immunity genes to host antimicrobial defense but is also associated with different transcriptional responses in midguts and abdomens, possibly reflecting different adaptive strategies of the host.more » « less
-
more » « less
Abstract Glutaredoxins (GRXs) are small oxidoreductase enzymes that can reduce disulfide bonds in target proteins. The class III GRX gene family is unique to land plants, and
Arabidopsis thaliana has 21 class III GRXs, which remain largely uncharacterized. About 80% ofA. thaliana class III GRXs are transcriptionally regulated by nitrate, and several recent studies have suggested roles for these GRXs in nitrogen signaling. Our objective was to functionally characterize two nitrate‐induced GRX genes,AtGRXS5 andAtGRXS8 , defining their roles in signaling and development in theA. thaliana root. We demonstrated thatAtGRXS5 andAtGRXS8 are primarily expressed in root and shoot vasculature (phloem), and that the corresponding GRX proteins display nucleo‐cytosolic subcellular localization. Ectopic expression ofAtGRXS8 in transgenic plants caused major alterations in root system architecture: Normal primary root development, but a near absence of lateral roots. RNA sequencing demonstrated that the roots ofAtGRXS8 ‐overexpressing plants show strongly reduced transcript abundance for many primary nitrate response genes, including the major high‐affinity nitrate transporters. Correspondingly, high‐affinity nitrate uptake and the transport of nitrate from roots to shoots are compromised inAtGRXS8 ‐overexpressing plants. Finally, we demonstrated that the AtGRXS8 protein can physically interact with the TGA1 and TGA4 transcription factors, which are central regulators of early transcriptional responses to nitrate inA. thaliana roots. Overall, these results suggest thatAtGRXS8 acts to quench both transcriptional and developmental aspects of primary nitrate response, potentially by interfering with the activity of the TGA1 and TGA4 transcription factors.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmolb.2022.896215
