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1. Pyruvate decarboxylase and thiamine biosynthetic genes are regulated differently by Pdc2 in S. cerevisiae and C. glabrata

   https://doi.org/10.1371/journal.pone.0286744  

   Iosue, Christine L.; Ugras, Julia M.; Bajgain, Yakendra; Dottor, Cory A.; Stauffer, Peyton L.; Hopkins, Rachael A.; Lang, Emma C.; Wykoff, Dennis D. (June 2023, PLOS ONE)

   Misra, Hari S. (Ed.)

   Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially defective in C . glabrata , but the transcription factor Cg Pdc2 upregulates some thiamine biosynthetic and transport genes. One of these genes encodes a recently evolved thiamine pyrophosphatase ( CgPMU3 ) that is critical for accessing external thiamine. Here, we demonstrate that Cg Pdc2 primarily regulates THI genes. In Saccharomyces cerevisiae , Pdc2 regulates both THI and pyruvate decarboxylase (PDC) genes, with PDC proteins being a major thiamine sink. Deletion of PDC2 is lethal in S . cerevisiae in standard growth conditions, but not in C . glabrata . We uncover cryptic cis elements in C . glabrata PDC promoters that still allow for regulation by Sc Pdc2, even when that regulation is not apparent in C . glabrata . C . glabrata lacks Thi2, and it is likely that inclusion of Thi2 into transcriptional regulation in S . cerevisiae allows for a more complex regulation pattern and regulation of THI and PDC genes. We present evidence that Pdc2 functions independent of Thi2 and Thi3 in both species. The C-terminal activation domain of Pdc2 is intrinsically disordered and critical for species differences. Truncation of the disordered domains leads to a gradual loss of activity. Through a series of cross species complementation assays of transcription, we suggest that there are multiple Pdc2-containing complexes, and C . glabrata appears to have the simplest requirement set for THI genes, except for CgPMU3 . CgPMU3 has different cis requirements, but still requires Pdc2 and Thi3 to be upregulated by thiamine starvation. We identify the minimal region sufficient for thiamine regulation in CgTHI20 , CgPMU3 , and ScPDC5 promoters. Defining the cis and trans requirements for THI promoters should lead to an understanding of how to interrupt their upregulation and provide targets in metabolism for antifungals. 

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2. Evolution of a cis -Acting SNP That Controls Type VI Secretion in Vibrio cholerae

   https://doi.org/10.1128/mbio.00422-22  

   Ng, Siu Lung; Kammann, Sophia; Steinbach, Gabi; Hoffmann, Tobias; Yunker, Peter J.; Hammer, Brian K. (May 2022, mBio)

   Storz, Gisela (Ed.)

   ABSTRACT Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes and other organisms to new niches. Comparative genomics can be used to infer rewiring of regulatory architecture based on large effect mutations like loss or acquisition of transcription factors but may be insufficient to identify small changes in noncoding, intergenic DNA sequence of regulatory elements that drive phenotypic divergence. In human-derived Vibrio cholerae , the response to distinct chemical cues triggers production of multiple transcription factors that can regulate the type VI secretion system (T6), a broadly distributed weapon for interbacterial competition. However, to date, the signaling network remains poorly understood because no regulatory element has been identified for the major T6 locus. Here we identify a conserved cis -acting single nucleotide polymorphism (SNP) controlling T6 transcription and activity. Sequence alignment of the T6 regulatory region from diverse V. cholerae strains revealed conservation of the SNP that we rewired to interconvert V. cholerae T6 activity between chitin-inducible and constitutive states. This study supports a model of pathogen evolution through a noncoding cis -regulatory mutation and preexisting, active transcription factors that confers a different fitness advantage to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches. IMPORTANCE Organisms sense external cues with regulatory circuits that trigger the production of transcription factors, which bind specific DNA sequences at promoters (“ cis ” regulatory elements) to activate target genes. Mutations of transcription factors or their regulatory elements create phenotypic diversity, allowing exploitation of new niches. Waterborne pathogen Vibrio cholerae encodes the type VI secretion system “nanoweapon” to kill competitor cells when activated. Despite identification of several transcription factors, no regulatory element has been identified in the promoter of the major type VI locus, to date. Combining phenotypic, genetic, and genomic analysis of diverse V. cholerae strains, we discovered a single nucleotide polymorphism in the type VI promoter that switches its killing activity between a constitutive state beneficial outside hosts and an inducible state for constraint in a host. Our results support a role for noncoding DNA in adaptation of this pathogen. 

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3. Prediction and experimental validation of a new salinity-responsive cis-regulatory element (CRE) in tilapia

   https://doi.org/10.6069/0n4f-6v50  

   Kim, Chanhee; Kültz, Dietmar (January 2022, Panorama Public)

   Quantitative DIA proteomics in combination with the transcription inhibitor actinomycin D was performed on the tilapia OmB cell line to identify proteins that are upregulated by transcriptional regulation during hyperosmotic stress. This analysis revealed proteins that are transcriptionally up-regulated by hyperosmolality in these cells. The promoter regions of these proteins were compared and a novel hyperosmolality-induced cis-regulatory element (CRE) and corresponding transcription factor candidate were identified. The CRE was experimentlly validated by site-directed mutagenesis in combination with reporter assays. 

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4. Antagonizing cis- regulatory elements of a conserved flowering gene mediate developmental robustness

   https://doi.org/10.1073/pnas.2421990122  

   Lanctot, Amy; Hendelman, Anat; Udilovich, Peter; Robitaille, Gina M; Lippman, Zachary B (February 2025, Proceedings of the National Academy of Sciences)

   Developmental transitions require precise temporal and spatial control of gene expression. In plants, such regulation is critical for flower formation, which involves the progressive maturation of stem cell populations within shoot meristems to floral meristems, followed by rapid sequential differentiation into floral organs. Across plant taxa, these transitions are orchestrated by the F-box transcriptional cofactor geneUNUSUAL FLORAL ORGANS(UFO). The conserved and pleiotropic functions ofUFOoffer a useful framework for investigating how evolutionary processes have shaped the intricatecis-regulation of key developmental genes. By pinpointing a conserved promoter sequence in an accessible chromatin region of the tomato ortholog ofUFO, we engineered in vivo a series ofcis-regulatory alleles that caused both loss- and gain-of-function floral defects. These mutant phenotypes were linked to disruptions in predicted transcription factor binding sites for known transcriptional activators and repressors. Allelic combinations revealed dosage-dependent interactions between opposing alleles, influencing the penetrance and expressivity of gain-of-function phenotypes. These phenotypic differences support that robustness in tomato flower development requires precise temporal control ofUFOexpression dosage. Bridging our analysis toArabidopsis, we found that although homologous sequences to the tomato regulatory region are dispersed within theUFOpromoter, they maintain similar control over floral development. However, phenotypes from disrupting these sequences differ due to the differing expression patterns ofUFO. Our study underscores the complexcis-regulatory control of dynamic developmental genes and demonstrates that critical short stretches of regulatory sequences that recruit both activating and repressing machinery are conserved to maintain developmental robustness. 

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5. Genetic dissection of cis -regulatory control of ZmWUSCHEL1 expression by type B RESPONSE REGULATORS

   https://doi.org/10.1093/plphys/kiad652  

   Chen, Zongliang; Cortes, Liz; Gallavotti, Andrea (December 2023, Plant Physiology)

   Abstract Mutations in cis-regulatory regions play an important role in the domestication and improvement of crops by altering gene expression. However, assessing the in vivo impact of cis-regulatory elements (CREs) on transcriptional regulation and phenotypic outcomes remains challenging. Previously, we showed that the dominant Barren inflorescence3 (Bif3) mutant of maize (Zea mays) contains a duplicated copy of the homeobox transcription factor gene ZmWUSCHEL1 (ZmWUS1), named ZmWUS1-B. ZmWUS1-B is controlled by a spontaneously generated novel promoter region that dramatically increases its expression and alters patterning and development of young ears. Overexpression of ZmWUS1-B is caused by a unique enhancer region containing multimerized binding sites for type B RESPONSE REGULATORs (RRs), key transcription factors in cytokinin signaling. To better understand how the enhancer increases the expression of ZmWUS1 in vivo, we specifically targeted the ZmWUS1-B enhancer region by CRISPR-Cas9-mediated editing. A series of deletion events with different numbers of type B RR DNA binding motifs (AGATAT) enabled us to determine how the number of AGATAT motifs impacts in vivo expression of ZmWUS1-B and consequently ear development. In combination with dual-luciferase assays in maize protoplasts, our analysis reveals that AGATAT motifs have an additive effect on ZmWUS1-B expression, while the distance separating AGATAT motifs does not appear to have a meaningful impact, indicating that the enhancer activity derives from the sum of individual CREs. These results also suggest that in maize inflorescence development, there is a threshold of buffering capacity for ZmWUS1 overexpression. 

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