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The article discusses the use of CRISPR-mediated gene editing to generate transgene-free, gain-of-function alleles in plants by targeting repressive cis-regulatory elements (CRE) within native promoters. Wang et al. demonstrate the potential to achieve endogenous gene overexpression by deleting specific repressive motifs from rice and soybean promoters, resulting in increased protein content. The study emphasizes a systematic and rational approach to promoter engineering, highlighting the importance of targeting conserved repressive motifs across plant species for achieving desired phenotypes. The research provides insights into tissue-specific gene expression enhancement and offers a roadmap for implementing targeted promoter engineering strategies in plant science. [Extracted from the article]

Since a transcriptional regulator has yet to be identified within the tunicamycin biosynthetic gene cluster in Streptomyces clavuligerus, we conducted a comprehensive investigation by focusing on the possible function of the pleiotropic regulator AdpA on tunicamycin. The genes encoding early steps of tunicamycin biosynthesis were significantly upregulated in S. clavuligerus ΔadpA. At the same time, they were downregulated in adpA overexpressed strain as shown by RNA-sequencing (RNA-seq) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis. The tunicamycin gene cluster's co-transcription pattern was understood by reverse transcriptase polymerase chain reaction (RT-PCR). Our Electrophoretic Mobility Shift Assay (EMSA) data clearly showed AdpA's binding to the upstream sequence of the tunA gene, asserting its regulatory control. In addition to its direct negative regulation of tunicamycin biosynthesis, AdpA operates at a global level by orchestrating various regulatory genes in S. clavuligerus, such as wblA, whiB, bldM, arpA, brp, and adsA involved in morphological differentiation and secondary metabolite biosynthesis as depicted in RNA-seq data. This study represents a significant milestone by unveiling the AdpA regulator's pathway-specific and global regulatory effect in S. clavuligerus. [ABSTRACT FROM AUTHOR]

B cell lymphoma 6 (BCL6) is a conserved multi-domain protein that functions principally as a transcriptional repressor. This protein regulates many pivotal aspects of immune cell development and function. BCL6 is critical for germinal center (GC) formation and the development of high-affinity antibodies, with key roles in the generation and function of GC B cells, follicular helper T (Tfh) cells, follicular regulatory T (Tfr) cells, and various immune memory cells. BCL6 also controls macrophage production and function as well as performing a myriad of additional roles outside of the immune system. Many of these regulatory functions are conserved throughout evolution. The BCL6 gene is also important in human oncology, particularly in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), but also extending to many in other cancers, including a unique role in resistance to a variety of therapies, which collectively make BCL6 inhibitors highly sought-after. [ABSTRACT FROM AUTHOR]

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E coli (EAEC)–specific virulence factors. Here, we identified an E coli O104:H4 isolate that carried a single-nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS , encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E coli O104:H4 Δ stx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and Δ rpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR , encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E coli O104:H4 virulence, primarily through an AggR-dependent mechanism. [ABSTRACT FROM AUTHOR]

Peach varieties that differ in red coloration due to varied anthocyanin accumulation result from transcriptional regulation by PpMYB10s, a group of specific R2R3 MYBs. Here we investigated the mechanisms driving a lack of anthocyanin in yellow‐skinned 'Jinxiu' peach peel, as well as accumulation induced by UV irradiance. It was found that PpMYB10.1, PpMYB10.2 and PpMYB10.3 were positive regulators of anthocyanin accumulation, but the stimulation by PpMYB10.2 was weak. Low expression of PpMYB10.1 causes natural anthocyanin deficiency in 'Jinxiu' peel. However, the promoter sequences of PpMYB10.1 were identical in 'Jinxiu' and a naturally red‐coloured peach 'Hujingmilu'. Therefore, potential negative regulator(s) upstream of PpMYB10.1 were explored. A novel R2R3‐MYB repressor termed PpMYB80 was identified through comparative transcriptomic analysis and then functionally confirmed via transiently overexpressing and silencing in peach fruit, as well as transformation in tobacco. PpMYB80 directly binds to the promoter of PpMYB10.1 and inhibits its expression, but does not affect PpMYB10.3. In UV‐exposed 'Jinxiu' fruit, expression of PpMYB10.3 was upregulated, while PpMYB10.1 remained low and PpMYB80 enhanced, which results in accumulation of anthocyanin in peel. This study revealed a transcriptional cascade involving PpMYB activators and repressors in regulating basal and UV‐induced anthocyanin accumulation in peach peel. Summary Statement: To explore the mechanisms for natural deficient and UV‐induced anthocyanin accumulation in 'Jinxiu' peach peel, the study was conducted and it was revealed that anthocyanin accumulation is regulated by a transcriptional cascade involving activators PpMYB10.1/10.3 and repressor PpMYB80. [ABSTRACT FROM AUTHOR]

Abstract The color of red-skinned pear (Pyrus spp.) is primarily attributed to accumulation of anthocyanins, which provide nutritional benefits for human health and are closely associated with the commercial value of fruits. Here, we reported the functional characterization of a R2R3-MYB repressor PyMYB107, which forms an ‘activator-repressor’ loop to control anthocyanin accumulation in the red-skinned pear. PyMYB107 overexpression inhibited anthocyanin biosynthesis in both pear calli and fruits, while virus-induced gene silencing of PyMYB107 increased anthocyanin accumulation in pear fruits. Furthermore, ectopic expression of PyMYB107 decreased anthocyanin accumulation in tomato, strawberry and tobacco. PyMYB107 can competitively bind to PybHLH3 with PyMYB10/MYB114, thereby suppressing the transcriptional activation of key anthocyanin biosynthesis genes, PyANS and PyUFGT. Site-directed mutagenesis showed that mutations within the R3 domain and EAR motif of PyMYB107 eliminated its repressive activity. Additionally, PyMYB107 exhibited a comparable expression pattern to PyMYB10/MYB114 and was transcriptionally activated by them. Our finding advanced comprehension of the repression mechanism underlying anthocyanin accumulation, providing valuable molecular insights into improving quality of pear fruits.

Summary: In the early 1900s, Erwin Baur established Antirrhinum majus as a model system, identifying and characterising numerous flower colour variants. This included Picturatum/Eluta, which restricts the accumulation of magenta anthocyanin pigments, forming bullseye markings on the flower face.We identified the gene underlying the Eluta locus by transposon‐tagging, using an Antirrhinum line that spontaneously lost the nonsuppressive el phenotype. A candidate MYB repressor gene at this locus contained a CACTA transposable element. We subsequently identified plants where this element excised, reverting to a suppressive Eluta phenotype. El alleles inhibit expression of anthocyanin biosynthetic genes, confirming it to be a regulatory locus. The modes of action of Eluta were investigated by generating stable transgenic tobacco lines, biolistic transformation of Antirrhinum petals and promoter activation/repression assays.Eluta competes with MYB activators for promoter cis‐elements, and also by titrating essential cofactors (bHLH proteins) to reduce transcription of target genes. Eluta restricts the pigmentation established by the R2R3‐MYB factors, Rosea and Venosa, with the greatest repression on those parts of the petals where Eluta is most highly expressed.Baur questioned the origin of heredity units determining flower colour variation in cultivated A. majus. Our findings support introgression from wild species into cultivated varieties. [ABSTRACT FROM AUTHOR]

The color of red-skinned pear (Pyrus spp.) is primarily attributed to accumulation of anthocyanins, which provide nutritional benefits for human health and are closely associated with the commercial value of fruits. Here, we reported the functional characterization of a R2R3-MYB repressor PyMYB107, which forms an 'activator-repressor' loop to control anthocyanin accumulation in the red-skinned pear. PyMYB107 overexpression inhibited anthocyanin biosynthesis in both pear calli and fruits, while virus-induced gene silencing of PyMYB107 increased anthocyanin accumulation in pear fruits. Furthermore, ectopic expression of PyMYB107 decreased anthocyanin accumulation in tomato, strawberry and tobacco. PyMYB107 can competitively bind to PybHLH3 with PyMYB10/MYB114, thereby suppressing the transcriptional activation of key anthocyanin biosynthesis genes, PyANS and PyUFGT. Site-directed mutagenesis showed that mutations within the R3 domain and EAR motif of PyMYB107 eliminated its repressive activity. Additionally, PyMYB107 exhibited a comparable expression pattern to PyMYB10/MYB114 and was transcriptionally activated by them. Our finding advanced comprehension of the repression mechanism underlying anthocyanin accumulation, providing valuable molecular insights into improving quality of pear fruits. [ABSTRACT FROM AUTHOR]

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. Key points: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea [ABSTRACT FROM AUTHOR]

Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here, we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore, gene repression in ZF-R-expressing T cells was highly efficient in vitro and in vivo during the entire monitoring period (up to 10 weeks), and it was accompanied by epigenetic remodeling events. Finally, we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion, we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing.

Anthocyanins play a paramount role in color variation and significantly contribute to the economic value of ornamental plants. The conserved activation complex MYB-bHLH-WD40 (MBW; MYB: v-myb avian myeloblastosis viral oncogene homolog; bHLH: basic helix–loop–helix protein; WD40:WD-repeat protein) involved in anthocyanin biosynthesis has been thoroughly researched, but there have been limited investigations into the function of repressor factors. In this study, we characterized TgMYB4, an R2R3-MYB transcriptional repressor which is highly expressed during petal coloration in red petal cultivars. TgMYB4-overexpressing tobaccos exhibited white or light pink petals with less anthocyanin accumulation compared to control plants. TgMYB4 was found to inhibit the transcription of ANTHOCYANIDIN SYNTHASE (TfANS1) and DIHYDRO-FLAVONOL-4-REDUCTASE (AtDFR), although it did not bind to their promoters. Moreover, the TgMYB4 protein was able to compete with the MYB activator to bind to the :bHLHprotein, thereby suppressing the function of the activator MBW complex. These findings demonstrate that TgMYB4 plays a suppressive role in the regulation of anthocyanin synthesis during flower pigmentation. [ABSTRACT FROM AUTHOR]

Serotonin 1A (5-HT1A) autoreceptors located on serotonin neurons inhibit their activity, and their upregulation has been implicated in depression, suicide and resistance to antidepressant treatment. Conversely, post-synaptic 5-HT1A heteroreceptors are important for antidepressant response. The transcription factor deformed epidermal autoregulatory factor 1 (Deaf1) acts as a presynaptic repressor and postsynaptic enhancer of 5-HT1A transcription, but the mechanism is unclear. Because Deaf1 interacts with and is phosphorylated by glycogen synthase kinase 3β (GSK3β)—a constitutively active protein kinase that is inhibited by the mood stabilizer lithium at therapeutic concentrations—we investigated the role of GSK3β in Deaf1 regulation of human 5-HT1A transcription. In 5-HT1A promoter-reporter assays, human HEK293 kidney and 5-HT1A-expressing SKN-SH neuroblastoma cells, transfection of Deaf1 reduced 5-HT1A promoter activity by ~45%. To identify potential GSK3β site(s) on Deaf1, point mutations of known and predicted phosphorylation sites on Deaf1 were tested. Deaf1 repressor function was not affected by any of the mutants tested except the Y300F mutant, which augmented Deaf1 repression. Both lithium and the selective GSK3 inhibitors CHIR-99021 and AR-014418 attenuated and reversed Deaf1 repression compared to vector. This inhibition was at concentrations that maximally inhibit GSK3β activity as detected by the GSK3β-sensitive TCF/LEF reporter construct. Our results support the hypothesis that GSK3β regulates the activity of Deaf1 to repress 5-HT1A transcription and provide a potential mechanism for actions of GSK3 inhibitors on behavior. [ABSTRACT FROM AUTHOR]

Inhibitor of differentiation 1 (ID1) protein is highly expressed in undifferentiated cells and associated with tumor progression and metastasis. Restricted regulation of the gene expression is necessary for cell maintenance. We performed the characterization of the response elements involved in the regulation of ID1 promoter activity using deletions and mutations. In silico analysis was used to predict regulatory binding sites. Mutants of the 5' upstream region of the ID1 promoter were constructed with nucleotide deletion or mutation, and the promoter activity was systematically analyzed. In silico analysis revealed several transcription binding sites presented in upstream region of the ID1 gene. Cluster of four Sp1 binding sites at position -1296 to -1078 contributed to the maximal expression activity of ID1 gene. The deletion and mutation of these Sp1 binding sites resulted in reduction of the expression level. Moreover, it is suggested that mutation of the Sp1 binding site at position -1078 could permit repressor proteins to suppress the gene expression. This study yields a new insight to a better understanding of the ID1 gene regulation, which pave the way to obtain the normal expression level of ID1 for cancer therapy development. [ABSTRACT FROM AUTHOR]

Mutations leading to upregulation of efflux pumps can produce multiple drug resistance in the pathogen Pseudomonas aeruginosa. Changes in their DNA binding regions, i.e., palindromic operators, can compromise pump depression and subsequently enhance resistance against several antibacterials and biocides. Here, we have identified (pseudo)palindromic repeats close to promoters of genes encoding 13 core drug-efflux pumps of P. aeruginosa. This framework was applied to detect mutations in these repeats in 17,292 genomes. Eighty-nine percent of isolates carried at least one mutation. Eight binary genetic properties potentially related to expression were calculated for mutations. These included palindromicity reduction, mutation type, positioning within the repeat and DNA-bending shift. High-risk ST298, ST308 and ST357 clones commonly carried four conserved mutations while ST175 and the cystic fibrosis-linked ST649 clones showed none. Remarkably, a T-to-C transition in the fourth position of the upstream repeat for mexEF-oprN was nearly exclusive of the high-risk ST111 clone. Other mutations were associated with high-risk sublineages using sample geotemporal metadata. Moreover, 1.5% of isolates carried five or more mutations suggesting they undergo an alternative program for regulation of their effluxome. Overall, P. aeruginosa shows a wide range of operator mutations with a potential effect on efflux pump expression and antibiotic resistance. [ABSTRACT FROM AUTHOR]

LysR-type transcriptional regulators (LTTRs) form one of the largest families of bacterial regulators. They are widely distributed and contribute to all aspects of metabolism and physiology. Most are homotetramers, with each subunit composed of an N-terminal DNA-binding domain followed by a long helix connecting to an effector-binding domain. LTTRs typically bind DNA in the presence or absence of a small-molecule ligand (effector). In response to cellular signals, conformational changes alter DNA interactions, contact with RNA polymerase, and sometimes contact with other proteins. Many are dual-function repressor–activators, although different modes of regulation may occur at multiple promoters. This review presents an update on the molecular basis of regulation, the complexity of regulatory schemes, and applications in biotechnology and medicine. The abundance of LTTRs reflects their versatility and importance. While a single regulatory model cannot describe all family members, a comparison of similarities and differences provides a framework for future study. [ABSTRACT FROM AUTHOR]

In organisms that use reduced sulfur compounds as alternative or additional electron donors to organic compounds, transcriptional regulation of genes for enzymes involved in sulfur oxidation is needed to adjust metabolic flux to environmental conditions. However, little is known about the sensing and response to inorganic sulfur compounds such as thiosulfate in sulfur-oxidizing bacteria. In the Alphaproteobacterium Hyphomicrobium denitrificans, one strategy is the use of the ArsR–SmtB-type transcriptional regulator SoxR. We show that this homodimeric repressor senses sulfane sulfur and that it is crucial for the expression not only of sox genes encoding the components of a truncated periplasmic thiosulfate-oxidizing enzyme system but also of several other sets of genes for enzymes of sulfur oxidation. DNA binding and transcriptional regulatory activity of SoxR are controlled by polysulfide-dependent cysteine modification. The repressor uses the formation of a sulfur bridge between two conserved cysteines as a trigger to bind and release DNA and can also form a vicinal disulfide bond to orchestrate a response to oxidizing conditions. The importance of the sulfur bridge forming cysteines was confirmed by site-directed mutagenesis, mass spectrometry, and gel shift assays. In vivo, SoxR interacts directly or indirectly with a second closely related repressor, sHdrR. [ABSTRACT FROM AUTHOR]

Simple Summary: Lipid metabolism has a substantial impact on the quality of meat in swine farming. PPARγ has a close association with lipid metabolism and displays high expression in adipose tissues. It is of paramount importance to control lipid metabolism to regulate PPARγ expression and activity in pigs. In this study, the association between the expression of the PPARγ gene and the backfat thickness of pigs was investigated. In recent years, retrotransposon insertion polymorphisms (RIPs), a new type of molecular marker, have emerged as a potentially valuable tool for genetic breeding in livestock and poultry. In this study, a combined retrotransposon insertion in the PPARγ gene of pigs was discovered that could be used as an efficient marker in selecting pigs for growth rate and lean percentage. PPARs are essential regulators of mammalian fatty acid and lipid metabolism. Although the effects of genetic variations, including single nucleotide polymorphisms (SNPs) in PPARs genes on the phenotype of domestic animals have been investigated, there is limited information on the impact of retrotransposon insertion polymorphisms (RIPs). In this study, a combined comparative genome and polymerase chain reaction (PCR) was used to excavate the RIPs in porcine PPARs. We also investigated the potential effects of retrotransposon insertion on phenotype and expression patterns. This study identified the two RIPs in PPARs genes, namely an ERV in intron 1 of PPARα and a combined retrotransposon in intron 2 of PPARγ, designated as PPARα-ERV-RIP and PPARγ-COM-RIP, respectively. These RIPs exhibited different distribution patterns among Chinese indigenous breeds and Western commercial breeds. Individuals with the PPARα-ERV-RIP+/+ genotype (+/+ indicated homozygous with insertion) among Large White pigs had significantly higher (p < 0.05) corrected backfat thickness compared to those with the other two genotypes. Similarly, those with the PPARγ-COM-RIP−/− genotype had significantly higher (p < 0.05) corrected backfat thickness than those with the other two genotypes in Large White pigs. Moreover, in 30-day-old Sujiang piglets, the PPARγ gene expression in the backfat of those with the PPARγ-COM-RIP−/− genotype (−/− indicated homozygous without insertion) was significantly greater (p < 0.01) than those with other genotypes. The dual luciferase reporter gene assay demonstrated that the combined retrotransposon insertion significantly reduced the activity of the MYC promoter in both C2C12 and 3T3-L1 cells (p < 0.01). Therefore, the combined retrotransposon insertion could function as a repressor to decrease the expression of PPARγ, making PPARγ-COM-RIP a valuable molecular marker for assisted selection of backfat thickness in pig breeding. [ABSTRACT FROM AUTHOR]

In our previous study, the phenazine-1-carboxylic acid (PCA) 1,2-dioxygenase gene cluster (pcaA1A2A3A4 cluster) in Sphingomonas histidinilytica DS-9 was identified to be responsible for the conversion of PCA to 1,2-dihydroxyphenazine (Ren Y, Zhang M, Gao S, Zhu Q, et al. 2022. Appl Environ Microbiol 88:e00543-22). However, the regulatory mechanism of the pcaA1A2A3A4 cluster has not been elucidated yet. In this study, the pcaA1A2A3A4 cluster was found to be transcribed as two divergent operons: pcaA3-ORF5205 (named A3-5205 operon) and pcaA1A2-ORF5208-pcaA4-ORF5210 (named A1-5210 operon). The promoter regions of the two operons were overlapped. PcaR acts as a transcriptional repressor of the pcaA1A2A3A4 cluster, and it belongs to GntR/FadR family transcriptional regulator. Gene disruption of pcaR can shorten the lag phase of PCA degradation. The results of electrophoretic mobility shift assay and DNase I footprinting showed that PcaR binds to a 25-bp motif in the ORF5205-pcaA1 intergenic promoter region to regulate the expression of two operons. The 25-bp motif covers the 210 region of the promoter of A3-5205 operon and the 235 region and 210 region of the promoter of A1-5210 operon. The TNGT/ANCNA box within the motif was essential for PcaR binding to the two promoters. PCA acted as an effector of PcaR, preventing it from binding to the promoter region and repressing the transcription of the pcaA1A2A3A4 cluster. In addition, PcaR represses its own transcription, and this repression can be relieved by PCA. This study reveals the regulatory mechanism of PCA degradation in strain DS-9, and the identification of PcaR increases the variety of regulatory model of the GntR/FadR-type regulator. IMPORTANCE Sphingomonas histidinilytica DS-9 is a phenazine-1-carboxylic acid (PCA)- degrading strain. The 1,2-dioxygenase gene cluster (pcaA1A2A3A4 cluster, encoding dioxygenase PcaA1A2, reductase PcaA3, and ferredoxin PcaA4) is responsible for the initial degradation step of PCA and widely distributed in Sphingomonads, but its regulatory mechanism has not been investigated yet. In this study, a GntR/FadR-type transcriptional regulator PcaR repressing the transcription of pcaA1A2A3A4 cluster and pcaR gene was identified and characterized. The binding site of PcaR in ORF5205-pcaA1 intergenic promoter region contains a TNGT/ANCNA box, which is important for the binding. These findings enhance our understanding of the molecular mechanism of PCA degradation. [ABSTRACT FROM AUTHOR]

A critical step in intracellular Chlamydia infection is the production of infectious progeny through the expression of late genes. This differentiation step involves conversion from a reticulate body (RB), which is the replicating form of the bacterium, into an elementary body (EB), which is the developmental form that spreads the infection to a new host cell. EUO is an important chlamydial transcription factor that controls the expression of late genes, but the mechanisms that regulate EUO are not known. We report that a plasmid-encoded protein, Pgp4, enhanced the repressor activity of EUO. Pgp4 did not function as a transcription factor because it did not bind or directly modulate transcription of its target promoters. Instead, Pgp4 increased the ability of EUO to bind and repress EUO-regulated promoters in vitro and physically interacted with EUO in pulldown assays with recombinant proteins. We detected earlier onset of EUO-dependent late gene expression by immunofluorescence microscopy in Pgp4-deficient C. trachomatis and C. muridarum strains. In addition, the absence of Pgp4 led to earlier onset of RB-to-EB conversion in C. muridarum These data support a role for Pgp4 as a negative regulator of chlamydial transcription that delays late gene expression. Our studies revealed that Pgp4 also has an EUO-independent function as a positive regulator of chlamydial transcription.IMPORTANCEChlamydia trachomatis is an important human pathogen that causes more than 150 million active cases of genital and eye infection in the world. This obligate intracellular bacterium produces infectious progeny within an infected human cell through the expression of late chlamydial genes. We showed that the ability of a key chlamydial transcription factor, EUO, to repress late genes was enhanced by a plasmid-encoded protein, Pgp4. In addition, studies with Chlamydia Pgp4-deficient strains provide evidence that Pgp4 delays late gene expression in infected cells. Thus, Pgp4 is a novel regulator of late gene expression in Chlamydia through its ability to enhance the repressor function of EUO.

Retrotransposons, a type of DNA fragment that can mobilize itself on genome, can generate genetic variations and develop for molecular markers based on the insertion polymorphism. Zinc finger proteins (ZNFs) are among the most abundant proteins in eukaryotic animals, and their functions are extraordinarily diverse and particularly important in gene regulation. In the current study, bioinformatic prediction was performed to screen for retrotransposon insertion polymorphisms (RIPs) in six ZNF genes (ZNF2, ZNF3, ZNF7, ZNF8, ZNF10 and ZNF12). Six RIPs in these ZNFs, including one short interspersed nuclear element (SINE) RIP in intron 1 and one long interspersed nuclear element 1 (L1) RIP in intron 3 of ZNF2, one SINE RIP in 5′ flanking region and one SINE RIP in intron 2 of ZNF3, one SINE RIP in 3′ UTR of ZNF7 and one L1 RIP in intron 2 of ZNF12, were discovered and their presence was confirmed by PCR. The impact of the SINE RIP in the first intron of ZNF2, which is close to the core promoter of ZNF2, on the gene activity was investigated by dual-luciferase assay in three cell lines. Our results showed that the SINE insertion in the intron 1 of ZNF2 repressed the core promoter activity extremely significantly (P

Abstract Background Anthocyanins are metabolites of phenylpropanoid pathway, and involves in diverse processes of plant development and adaptation, which are regulated by the MYB-bHLH-WD40 (MBW) protein complexes. Many R2R3-MYB activators have been well characterized, but the MYB repressors in anthocyanin biosynthesis were recognized recently, which are also important in modulating phenylpropanoid metabolism in plants. The regulatory mechanism of anthocyanin biosynthesis in oil crop Brassica napus remains to be revealed. Results In this study, we identified an anthocyanin repressor BnCPC in B. napus. BnCPC encoded a typical R3-MYB protein containing a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif for interaction with bHLH proteins. Overexpression of BnCPC in B. napus inhibited anthocyanin accumulation, especially under anthocyanin inducible conditions. Protein–protein interaction and dual-luciferase assays confirmed that BnCPC could compete with BnPAP1 to interact with bHLHs (BnTT8 and BnEGL3), and repress the expression of anthocyanin biosynthetic genes (e.g., BnDFR) that activated by MBW complexes. Moreover, we found BnCPC inhibited the MBW complex-induced BnCPC activity. Conclusions Overall, this research demonstrated that BnCPC repressed anthocyanin biosynthesis by affecting the formation of MBW complex, and formed a feedback loop to regulate anthocyanin accumulation in B. napus.

Transcriptional factors such as the TetR family of transcriptional regulators (TFTRs) are widely found amongst bacteria, including mycobacteria, and are accountable for their survival. Here, we characterized a novel TFTR, Ms6244, from Mycobacterium smegmatis that negatively autoregulates its expression and represses its neighbouring gene, Ms6243. We also report the binding of Ms6244 to the inverted repeats in the intergenic region of Ms6244 and Ms6243. Further, an Ms6244‐deleted strain shows various morpho‐physiological differences compared to the wild type. We further confirmed that the deletion of Ms6244 itself and not the resultant Ms6243 overexpression is the cause of the altered physiology. Our data thus suggest that Ms6244 is an essential regulator, having far‐reaching effects on M. smegmatis physiology. [ABSTRACT FROM AUTHOR]

Saro_0803 is a transcriptional factor modulating the transcription of the stilbene-degrading enzyme gene nov1 in Novosphingobium aromaticivorans DSM 12444. Reportedly, Saro_0803 undergoes resveratrol-mediated dissociation from the nov1 promotor and distinguishes resveratrol from its precursors, p-coumaric acid and trans-cinnamic acid, enabling the transcriptional factor to serve as a biosensor component for regulating resveratrol biosynthesis. However, little is known about the molecular mechanisms underlying the Saro_0803 interactions with either the nov1 promotor gene or resveratrol, which undermines the potential for Saro_0803 to be further modified for improved biosynthetic performance and other applications. Here, we report the discovery of the 22 bp A/T-rich Saro_0803 binding site near the −10 box of the nov1 promotor (named nov1p22bp). As validated by molecular docking-guided mutagenesis and binding affinity assays, the Saro_0803 binding of its target DNA sequence relies on charge-predominating interactions between several typical positively charged residues and nucleic acid. Furthermore, we semi-quantified the influence of resveratrol presence on Saro_0803–nov1p22bp interaction and identified a bilateral hydrophobic pocket within Saro_0803 comprising four aromatic residues that are crucial to maintaining the resveratrol binding capability of the transcriptional factor. Our data are beneficial to understanding saro_0803′s structural and functional properties, and could provide theoretical clues for future adaptations of this transcriptional factor. [ABSTRACT FROM AUTHOR]

A multi-step phosphorelay system is the main conduit of cytokinin signal transduction. However, several groups of additional factors that also play a role in this signaling pathway have been found—among them the Cytokinin Response Factors (CRFs). In a genetic screen, CRF9 was identified as a regulator of the transcriptional cytokinin response. It is mainly expressed in flowers. Mutational analysis indicates that CRF9 plays a role in the transition from vegetative to reproductive growth and silique development. The CRF9 protein is localized in the nucleus and functions as a transcriptional repressor of Arabidopsis Response Regulator 6 (ARR6)—a primary response gene for cytokinin signaling. The experimental data suggest that CRF9 functions as a repressor of cytokinin during reproductive development. [ABSTRACT FROM AUTHOR]

Clostridioides difficile may constitute a small part of normal gut microbiota in humans without causing any symptoms, but an uncontrolled growth common to hospitalized patients can cause Clostridioides difficile infection (CDI) leading to severe colonic symptoms. As the bacteria are attaining resistance to various antibiotics worldwide, CDI is becoming a serious public health problem. Although a family of transcription factors called MarR (M ultiple a ntibiotic r esistance R egulator) plays a key role in the bacterial response to various environmental stresses including antibiotics, most of the 14 MarRs predicted to exist in the C. difficile genome lack structural or functional studies. In this respect, X-ray crystal structure of a C. difficile MarR CD0473 with a yet unknown function has been determined using a Hg-soaked crystal. In the structure, two closely located flexible conformations of Hg-bound cysteines suggested a possibility of intra-subunit disulfide bridge formation. By searching the neighboring intergenic regions of CD0473, two pseudo-palindromic DNA sites were found and shown to bind the protein. MarR CD0473 binding stronger to the DNA in an oxidizing condition supported further that it may function as a redox regulated switch likely via its oxidized disulfide formation. [ABSTRACT FROM AUTHOR]

Hairy and Krüppel homolog 1 (Kr-h1) are transcriptional repressors that act synergistically to mediate the gene-repressive action of juvenile hormone (JH). However, whether a regulatory relationship exists between Hairy and Kr-h1 remains unclear. In this study, an inhibitory effect of Hairy on Kr-h1 expression was found. Genetic studies in Drosophila have shown that the simultaneous overexpression of Hairy and Kr-h1 can rescue the defective phenotypes caused by the overexpression of a single factor. Reduced expression of Kr-h1 was observed in Hairy-overexpressing flies and cells, whereas the expression levels of Hairy were unaffected in cells with ectopic expression of Kr-h1. The inhibitory effect of Hairy on Kr-h1 expression was found to occur at the transcriptional level, as Hairy bound directly to the B-box within the Kr-h1 promoter via the bHLH motif and recruited the corepressors C-terminal binding protein (CtBP) and Groucho (Gro) through the PLSLV and WRPW motifs, respectively. Our findings revealed a regulatory relationship between two JH response factors, which advances our understanding of the molecular mechanism of JH signaling. [Display omitted] • Hairy represses Kr-h1 expression. • BHLH motif of Hairy is necessary for its binding to the B-box within the Kr-h1 promoter. • PLSLV and WRPW motifs of Hairy are crucial for recruiting CtBP and Gro to facilitate the repression. [ABSTRACT FROM AUTHOR]

Mutations leading to upregulation of efflux pumps can produce multiple drug resistance in the pathogen Pseudomonas aeruginosa. Changes in their DNA binding regions, i.e., palindromic operators, can compromise pump depression and subsequently enhance resistance against several antibacterials and biocides. Here, we have identified (pseudo)palindromic repeats close to promoters of genes encoding 13 core drug-efflux pumps of P. aeruginosa. This framework was applied to detect mutations in these repeats in 17,292 genomes. Eighty-nine percent of isolates carried at least one mutation. Eight binary genetic properties potentially related to expression were calculated for mutations. These included palindromicity reduction, mutation type, positioning within the repeat and DNA-bending shift. High-risk ST298, ST308 and ST357 clones commonly carried four conserved mutations while ST175 and the cystic fibrosis-linked ST649 clones showed none. Remarkably, a T-to-C transition in the fourth position of the upstream repeat for mexEF-oprN was nearly exclusive of the high-risk ST111 clone. Other mutations were associated with high-risk sublineages using sample geotemporal metadata. Moreover, 1.5% of isolates carried five or more mutations suggesting they undergo an alternative program for regulation of their effluxome. Overall, P. aeruginosa shows a wide range of operator mutations with a potential effect on efflux pump expression and antibiotic resistance.

Background: Anthocyanins are metabolites of phenylpropanoid pathway, and involves in diverse processes of plant development and adaptation, which are regulated by the MYB-bHLH-WD40 (MBW) protein complexes. Many R2R3-MYB activators have been well characterized, but the MYB repressors in anthocyanin biosynthesis were recognized recently, which are also important in modulating phenylpropanoid metabolism in plants. The regulatory mechanism of anthocyanin biosynthesis in oil crop Brassica napus remains to be revealed. Results: In this study, we identified an anthocyanin repressor BnCPC in B. napus. BnCPC encoded a typical R3-MYB protein containing a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif for interaction with bHLH proteins. Overexpression of BnCPC in B. napus inhibited anthocyanin accumulation, especially under anthocyanin inducible conditions. Protein–protein interaction and dual-luciferase assays confirmed that BnCPC could compete with BnPAP1 to interact with bHLHs (BnTT8 and BnEGL3), and repress the expression of anthocyanin biosynthetic genes (e.g., BnDFR) that activated by MBW complexes. Moreover, we found BnCPC inhibited the MBW complex-induced BnCPC activity. Conclusions: Overall, this research demonstrated that BnCPC repressed anthocyanin biosynthesis by affecting the formation of MBW complex, and formed a feedback loop to regulate anthocyanin accumulation in B. napus. [ABSTRACT FROM AUTHOR]

Purpose: Deregulation of SNCA encoding α-synuclein (α-SYN) has been associated with both the familial and sporadic forms of Parkinson disease (PD). Epigenetic regulation plays a crucial role in PD. The intron1 of SNCA harbors a large unmethylated CpG island. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1), a CpG island binding protein, can repress gene expression by occupying hypomethylated CpG-rich promoters, and therefore SNCA could be a target for TET1. We investigated whether TET1 binds to SNCA-intron1 and regulates gene expression. Methods: The dopaminergic neuronal cell line, ReNcell VM, was used. Reverse transcription-polymerase chain reaction (RTPCR), real time-quantitative PCR, Western blot, dot-blot, and Chromatin immunoprecipitation were conducted. The substantia nigra tissues of postmortem PD samples were used to confirm the level of TET1 expression. Results: In the human dopaminergic cell line, ReNcell VM, overexpression of the DNA-binding domain of TET1 (TET1- CXXC) led to significant repression of α-SYN. On the contrary, knocking down of TET1 led to significantly higher expression of α-SYN. However, overexpression of the DNA-hydroxymethylating catalytic domain of TET1 failed to change the expression of α-SYN. Altogether, we showed that TET1 is a repressor for SNCA, and a CXXC domain of TET1 is the primary mediator for this repressive action independent of its hydroxymethylation activity. TET1 levels in PD patients are significantly lower than that in the controls. Conclusions: We identified that TET1 acts as a repressor for SNCA by binding the intron1 regions of the gene. As a high level of α-SYN is strongly implicated in the pathogenesis of PD, discovering a repressor for the gene encoding α-SYN is highly important for developing novel therapeutic strategies for the disease. [ABSTRACT FROM AUTHOR]

Landomycin A (LaA) is the largest member of the landomycin group of angucyclic polyketides. Its unusual structure and strong anticancer properties have attracted great interest from chemists and biologists alike. This, in particular, has led to a detailed picture of LaA biosynthesis in Streptomyces cyanogenus S136, the only known LaA producer. LanK is a TetR family repressor protein that limits the export of landomycins from S136 cells until significant amounts of the final product, LaA, have accumulated. Landomycins carrying three or more carbohydrate units in their glycosidic chain are effector molecules for LanK. Yet, the exact mechanism that LanK uses to distinguish the final product, LaA, from intermediate landomycins and sense accumulation of LaA was not known. Here, we report crystal structures of LanK, alone and in complex with LaA, and bioassays of LanK's interaction with synthetic carbohydrate chains of LaA (hexasaccharide) and LaE (trisaccharide). Our data collectively suggest that the carbohydrate moieties are the sole determinants of the interaction of the landomycins with LanK, triggering the latter's dissociation from the lanK–lanJ intergenic region via structure conversion of the helices in the C‐terminal ligand‐binding domain. Analysis of the available literature suggests that LanK represents an unprecedented type of TetR family repressor that recognises the carbohydrate portion of a natural product, and not an aglycon, as it is the case, for example, with the SimR repressor involved in simocyclinone biosynthesis. [ABSTRACT FROM AUTHOR]

It has been established that through binding to bone morphogenetic proteins (BMPs), bone morphogenetic protein receptor I B (BMPR1B) can mediate transforming growth factor β (TGF-β) signal transduction, and is involved in the regulation of several biological processes, such as bone and muscle formation and homeostasis, as well as folliculogenesis. Also known as FecB, BMPR1B has been reported as the major gene for sheep prolificacy. A number of previous studies have analyzed the relationship between single nucleotide polymorphisms (SNPs) in this gene and its related performance. In recent years, with the illustration of the effect of retrotransposon insertion on the expression of the proximal genes or phenotypic variation, retrotransposon insertion polymorphisms (RIPs) have been used as a novel type of molecular marker in the evaluation of evolution, population structure and breeding of plant and domestic animals. In this study, the RIPs in porcine BMPR1B gene were excavated, and thereafter verified using a comparative genome and polymerase chain reaction (PCR). The potential effects of phenotype, gene expression and functions related to RIPs were also explored. The results showed that 13 distinct RIPs were identified in introns of porcine BMPR1B. Among these, only BMPR1B-SINE-RIP9 and BMPR1B-LINE-RIP13 displayed a close relationship with the growth traits of Large White pigs. Moreover, the total number of BMPR1B-SINE+/+-RIP9 individuals born was found to be significantly higher than that of SINE−/− (p < 0.05). These two RIPs showed an obvious distribution pattern among Chinese indigenous breeds and Western commercial breeds. The expression of BMPR1B in ovaries of adult BMPR1B-SINE+/+-RIP9 Sushan pigs was found to be significantly higher in comparison to those of BMPR1B-SINE−/−-RIP9 (p < 0.05). SINE insertion of BMPR1B-SINE-RIP9 and LINE insertion of BMPR1B-LINE-RIP13 were observed to significantly increase the activity of Octamer binding transcription factor 4 (OCT4) minipromoter in CHO and C2C12 cells (p < 0.01). Therefore, these two RIPs could serve as useful molecular markers for modulating the growth or reproductive traits in assisted selection of pig breeding, while the mechanisms of the insertion function should be studied further. [ABSTRACT FROM AUTHOR]

The epigenetic memory stored in the dynamic modifications, such as base modifications of cytosine (C) in DNA, including methylation/hydroxymethylation/demethylation, causes heritable phenotypes via regulating gene expression without alteration of DNA sequence. The process from cytosine modification to the epigenetic effect is orchestrated by complicated machinery consisting of writers, erasers, readers, and other factors. The two major forms of cytosine modification include methylcytosine (5-mC) and hydroxymethylcytosine (5-hmC). DNA methyltransferases (DNMTs) including DNMT1, DNMT3A, and DNMT3B function as writers for 5-mC. The ten-eleven translocation proteins (TET) including TET1, TET2, and TET3 in the mammalian genome are responsible for hydroxymethylation of 5-mC to generate 5-hmC, 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC). The 5-mC and 5-hmC have become the two most extensively investigated epigenetic markers, and the dynamic balance of these two markers shape the landscape of the epigenome, functioning as a platform to regulate gene expression epigenetically. The landscape of the 5-hmC in epigenome is precisely and tightly regulated during the development. Aberrant alterations of the epigenetic regulation may cause severe consequences such as phenotype change as well as initiation of disease. Progressively, significant achievements have been made in characterization of writers, erasers, and readers of 5-mC and 5-hmC, as well as the contribution of aberrant alteration of 5-hmC/5-mC landscape to the pathogenesis of human diseases, such as cancers and neurological disorders. This article will highlight the research advances in the distinct contribution of TET proteins as suppressors or promoters to the pathogenesis of tumorigenesis and progression. Furthermore, this article also discusses the challenges and the directions for research in the future.

Purpose Deregulation of SNCA encoding α-synuclein (α-SYN) has been associated with both the familial and sporadic forms of Parkinson disease (PD). Epigenetic regulation plays a crucial role in PD. The intron1 of SNCA harbors a large unmethylated CpG island. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1), a CpG island binding protein, can repress gene expression by occupying hypomethylated CpG-rich promoters, and therefore SNCA could be a target for TET1. We investigated whether TET1 binds to SNCA-intron1 and regulates gene expression. Methods The dopaminergic neuronal cell line, ReNcell VM, was used. Reverse transcription-polymerase chain reaction (RT-PCR), real time-quantitative PCR, Western blot, dot-blot, and Chromatin immunoprecipitation were conducted. The substantia nigra tissues of postmortem PD samples were used to confirm the level of TET1 expression. Results In the human dopaminergic cell line, ReNcell VM, overexpression of the DNA-binding domain of TET1 (TET1-CXXC) led to significant repression of α-SYN. On the contrary, knocking down of TET1 led to significantly higher expression of α-SYN. However, overexpression of the DNA-hydroxymethylating catalytic domain of TET1 failed to change the expression of α-SYN. Altogether, we showed that TET1 is a repressor for SNCA, and a CXXC domain of TET1 is the primary mediator for this repressive action independent of its hydroxymethylation activity. TET1 levels in PD patients are significantly lower than that in the controls. Conclusions We identified that TET1 acts as a repressor for SNCA by binding the intron1 regions of the gene. As a high level of α-SYN is strongly implicated in the pathogenesis of PD, discovering a repressor for the gene encoding α-SYN is highly important for developing novel therapeutic strategies for the disease.

PPARs are essential regulators of mammalian fatty acid and lipid metabolism. Although the effects of genetic variations, including single nucleotide polymorphisms (SNPs) in PPARs genes on the phenotype of domestic animals have been investigated, there is limited information on the impact of retrotransposon insertion polymorphisms (RIPs). In this study, a combined comparative genome and polymerase chain reaction (PCR) was used to excavate the RIPs in porcine PPARs. We also investigated the potential effects of retrotransposon insertion on phenotype and expression patterns. This study identified the two RIPs in PPARs genes, namely an ERV in intron 1 of PPARα and a combined retrotransposon in intron 2 of PPARγ, designated as PPARα-ERV-RIP and PPARγ-COM-RIP, respectively. These RIPs exhibited different distribution patterns among Chinese indigenous breeds and Western commercial breeds. Individuals with the PPARα-ERV-RIP+/+ genotype (+/+ indicated homozygous with insertion) among Large White pigs had significantly higher (p < 0.05) corrected backfat thickness compared to those with the other two genotypes. Similarly, those with the PPARγ-COM-RIP−/− genotype had significantly higher (p < 0.05) corrected backfat thickness than those with the other two genotypes in Large White pigs. Moreover, in 30-day-old Sujiang piglets, the PPARγ gene expression in the backfat of those with the PPARγ-COM-RIP−/− genotype (−/− indicated homozygous without insertion) was significantly greater (p < 0.01) than those with other genotypes. The dual luciferase reporter gene assay demonstrated that the combined retrotransposon insertion significantly reduced the activity of the MYC promoter in both C2C12 and 3T3-L1 cells (p < 0.01). Therefore, the combined retrotransposon insertion could function as a repressor to decrease the expression of PPARγ, making PPARγ-COM-RIP a valuable molecular marker for assisted selection of backfat thickness in pig breeding.

Summary: Higher plants adapt to different light intensities by altering hypocotyl elongation, stomatal density, seed size, and flowering time. Despite the importance of this developmental plasticity, knowledge of the underlying genetic and molecular mechanisms modulating and coordinating responses to light intensity remains incomplete. Here, I report that in Arabidopsis the PEAPOD (PPD) repressors PPD1 and PPD2 prevent exaggerated responses to light intensity.Genetic and transcriptome analyses, of a ppd deletion mutant and a PPD1 overexpression genotype, were used to identify how PPD repressors modulate the light signalling network.A ppd1/ppd2 deletion mutant has elongated hypocotyls, elevated stomatal density, enlarged seed, and delayed flowering, whereas overexpression of PPD1 results in the reverse. Transcription of both PPD1 and PPD2, upregulated in low light and downregulated in higher light, is activated by PHYTOCHROME INTERACTING FACTOR 4. I found PPDs modulate light signalling by negative regulation of SUPPRESSOR OF phyA‐105 (SPA1) transcription. Whereas PPDs coordinate many of the responses to light intensity – hypocotyl elongation, flowering time, and stomatal density – by repression/de‐repression of SPA1, PPD regulation of seed size occurs independent of SPA1.In conclusion PPD repressors modulate and coordinate developmental responses to light intensity by altering light signal transduction. [ABSTRACT FROM AUTHOR]

Retrotransposon is an important component of the mammalian genome. Previous studies have shown that the expression of protein-coding genes was affected by the insertion of retrotransposon into the proximal genes, and the phenotype variations would be related to the retrotransposon insertion polymorphisms (RIPs). In this study, leptin (LEP), leptin receptor (LEPR), and leptin receptor overlapping transcript (LEPROT), which play important roles in the regulation of fat synthesis and body weight, were screened to search for the RIPs and their effect on phenotype and gene expression, as well as to further study the function of the insertion. The results showed that three RIPs located in intron 1 of LEPROT and intron 2 and 21 of LEPR were identified, and they were all SINEA1, which was one type of retrotransposon. The SINE insertion at the LEPROT was the dominant allele in native pig breeds. The age of 100 kg body weight of SINE+/+ Large White individuals was significantly higher than those of SINE+/− and SINE−/− individuals (p < 0.05). The LEPROT gene expression in the liver and suet of 30-day-old SINE−/− Sujiang piglets were significantly higher than those of SINE+/+ and SINE+/− piglets (p < 0.01). The dual-luciferase reporter gene assay showed that SINE insertion in PK15 and 3T3-L1 cells significantly reduced the promoter activity of the LEPROT gene (p < 0.01). Therefore, SINE insertion can be a repressor to reduce the expression of LEPROT and could be a useful molecular marker for assisted selection of growth traits in pig breeding. [ABSTRACT FROM AUTHOR]

HucR is a MarR family protein of Deinococcus radiodurans , which binds tightly to the intergenic region of HucR and the uricase gene to inhibit their expression. Urate (or uric acid) antagonizes the repressor function of HucR by binding to HucR to impede its association with the cognate DNA. The previously reported crystal structure of HucR was without the bound urate showing significant structural homology to other MarR structures. In this paper, we report the crystal structure of HucR determined with the urate bound. However, despite the fact that the urate is found at a site well-known to harbor ligands in other MarR family proteins, the overall HucR structure indicates that no significant change in structure takes place with the urate bound. Structure analysis further suggests that the urate interaction in HucR is mediated by histidine/glutamate side chains and ordered water molecules stabilized by various residues. Such interaction is quite unique compared to other known structural interactions between urate and its binding proteins. Furthermore, structural comparison of the apo- and the urate bound forms allows us to hypothesize that the Trp20-mediated water network in the apo-form stabilizes the proper HucR fold for cognate DNA binding, and that urate binding, also via Trp20, and the consequent reorganization of water molecules in the binding pocket, likely disrupts the DNA binding configuration to result in the attenuated DNA binding. • The MarR family Deinococcus radiodurans HucR acts as a transcriptional repressor. • However, urate (uric acid) antagonizes the repression by releasing the cognate DNA. • Here, we report the crystal structure of HucR with the bound urate molecule. • HucR's specific urate interaction is not observed in other urate binding proteins. • The structure suggests a mechanism for urate-induced attenuation of DNA binding. [ABSTRACT FROM AUTHOR]

Summary: Anthocyanins are visual cues for pollination and seed dispersal. Fruit containing anthocyanins also appeals to consumers due to its appearance and health benefits. In kiwifruit (Actinidia spp.) studies have identified at least two MYB activators of anthocyanin, but their functions in fruit and the mechanisms by which they act are not fully understood.Here, transcriptome and small RNA high‐throughput sequencing were used to comprehensively identify contributors to anthocyanin accumulation in kiwifruit.Stable overexpression in vines showed that both 35S::MYB10 and MYB110 can upregulate anthocyanin biosynthesis in Actinidia chinensis fruit, and that MYB10 overexpression resulted in anthocyanin accumulation which was limited to the inner pericarp, suggesting that repressive mechanisms underlie anthocyanin biosynthesis in this species. Furthermore, motifs in the C‐terminal region of MYB10/110 were shown to be responsible for the strength of activation of the anthocyanic response. Transient assays showed that both MYB10 and MYB110 were not directly cleaved by miRNAs, but that miR828 and its phased small RNA AcTAS4‐D4(−) efficiently targeted MYB110. Other miRNAs were identified, which were differentially expressed between the inner and outer pericarp, and cleavage of SPL13, ARF16, SCL6 and F‐box1, all of which are repressors of MYB10, was observed.We conclude that it is the differential expression and subsequent repression of MYB activators that is responsible for variation in anthocyanin accumulation in kiwifruit species. [ABSTRACT FROM AUTHOR]

Vaccinium berries are regarded as "superfoods" owing to their high concentrations of anthocyanins, flavonoid metabolites that provide pigmentation and positively affect human health. Anthocyanin localization differs between the fruit of cultivated highbush blueberry (V. corymbosum) and wild bilberry (V. myrtillus), with the latter having deep red flesh coloration. Analysis of comparative transcriptomics across a developmental series of blueberry and bilberry fruit skin and flesh identified candidate anthocyanin regulators responsible for this distinction. This included multiple activator and repressor transcription factors (TFs) that correlated strongly with anthocyanin production and had minimal expression in blueberry (non-pigmented) flesh. R2R3 MYB TFs appeared key to the presence and absence of anthocyanin-based pigmentation; MYBA1 and MYBPA1.1 co-activated the pathway while MYBC2.1 repressed it. Transient overexpression of MYBA1 in Nicotiana benthamiana strongly induced anthocyanins, but this was substantially reduced when co-infiltrated with MYBC2.1. Co-infiltration of MYBC2.1 with MYBA1 also reduced activation of DFR and UFGT , key anthocyanin biosynthesis genes, in promoter activation studies. We demonstrated that these TFs operate within a regulatory hierarchy where MYBA1 activated the promoters of MYBC2.1 and bHLH2. Stable overexpression of VcMYBA1 in blueberry elevated anthocyanin content in transgenic plants, indicating that MYBA1 is sufficient to upregulate the TF module and activate the pathway. Our findings identify TF activators and repressors that are hierarchically regulated by SG6 MYBA1, and fine-tune anthocyanin production in Vaccinium. The lack of this TF module in blueberry flesh results in an absence of anthocyanins. [ABSTRACT FROM AUTHOR]

Bacillus subtilis is a gram-positive bacterium that has developed to coordinate gene expression and to survive against changes of nutrients and toxic chemicals. Flavonoids are exuded by plant cells and are abundant in the soil. To counteract the antibacterial effects of flavonoids, B. subtilis expresses flavonoid-detoxifying enzymes, and their expression is negatively regulated by transcription factors, including YetL. YetL was shown to control B. subtilis growth through the promoter regions of yetL and yetM genes in response to some flavonoids. Despite the functional significance of the YetL transcription factor in bacterial survival, no structural information is available for YetL. Here, we report the crystal structure of YetL and propose a flavonoid-induced regulatory mechanism. The YetL structure contains the canonical winged helix-turn-helix motif of the MarR superfamily but distinctly presents an additional N-terminal helix. In the dimeric assembly of YetL, the H1 helix intersects the YetL dimer and contributes to extensive intersubunit interactions. As a transcription factor, YetL recognizes a 28-mer operator of double-stranded DNA that contains a palindromic sequence. Moreover, our comparative structural analysis of YetL and other MarR members allows us to propose a flavonoid-induced transcription regulatory mechanism that is used for bacterial adaptation to environmental changes and stresses. • The crystal structure of YetL from B. subtilis was determined and analyzed. • YetL recognizes a 28-mer operator dsDNA. • YetL distinctly features an N-terminal helix from the MarR superfamily. [ABSTRACT FROM AUTHOR]

The Aux/IAA proteins are auxin-sensitive repressors that mediate diverse physiological and developmental processes in plants [1, 2]. There are 29 Aux/IAA genes in Arabidopsis that exhibit unique but partially overlapping patterns of expression [3]. Although some studies have suggested that individual Aux/IAA genes have specialized function, genetic analyses of the family have been limited by the scarcity of loss-of-function phenotypes [4]. Furthermore, with a few exceptions, our knowledge of the factors that regulate Aux/IAA expression is limited [1, 5]. We hypothesize that transcriptional control of Aux/IAA genes plays a central role in the establishment of the auxin-signaling pathways that regulate organogenesis, growth, and environmental response. Here, we describe a screen for transcription factors (TFs) that regulate the Aux/IAA genes. We identify TFs from 38 families, including 26 members of the DREB/CBF family. Several DREB/CBF TFs directly promote transcription of the IAA5 and IAA19 genes in response to abiotic stress. Recessive mutations in these IAA genes result in decreased tolerance to stress conditions, demonstrating a role for auxin in abiotic stress. Our results demonstrate that stress pathways interact with the auxin gene regulatory network (GRN) through transcription of the Aux/IAA genes. We propose that the Aux/IAA genes function as hubs that integrate genetic and environmental information to achieve the appropriate developmental or physiological outcome.

ABSTRACT Streptococcus suis is an important pathogen in both pigs and humans. Although the diseases associated with S. suis can typically be treated with antibiotics, such use has resulted in a sustained increase in drug resistance. Bacteria can sense and respond to antibiotics via two-component systems (TCSs). In this study, the TCS CiaRH was identified as playing an important role in the susceptibility of S. suis to fluoroquinolones (FQs). We found that a ΔciaRH mutant possessed lower susceptibility to FQs than the wild-type strain, with no observed growth defects at the tested concentrations and lower levels of intracellular drugs and dye. Proteomic data revealed that the levels of SatA and SatB expression were upregulated in the ΔciaRH mutant compared with their levels in the wild-type strain. The satA and satB genes encode a narrow-spectrum FQ efflux pump. The phenomena associated with combined ciaRH-and-satAB deletion mutations almost returned the ΔciaRH ΔsatAB mutant to the phenotype of the wild-type strain compared to the phenotype of the ΔciaRH mutant, suggesting that the resistance of the ΔciaRH strain to FQs could be attributed to satAB overexpression. Moreover, SatAB expression was regulated by CiaR (a response regulator of CiaRH) and SatR (a regulator of the MarR family). The ciaRH genes were consistently downregulated in response to antibiotic stress. The results of electrophoretic mobility shift assays (EMSAs) and affinity assays revealed that both regulator proteins directly controlled the ABC transporter proteins SatAB. Together, the results show that cascade-mediated regulation of antibiotic export by CiaRH is crucial for the ability of S. suis to adapt to conditions of antibiotic pressure. Our study may provide a new target for future antibiotic research and development. IMPORTANCE Streptococcus suis is a zoonotic pathogen with high incidence and mortality rates in both swine and humans. Following antibiotic treatment, the organism has evolved many resistance mechanisms, among which efflux pump overexpression can promote drug extrusion from the cell. This study clarified the role of CiaRH in fluoroquinolone resistance. A mutant with the ciaRH genes deleted showed decreased susceptibility to the antibiotics tested, an invariant growth rate, and reduced intracellular efflux pump substrates. This research also demonstrated that overexpression of the efflux pump SatAB was the main cause of ΔciaRH resistance. In addition, CiaR could combine with the promoter region of satAB to further directly suppress target gene transcription. Simultaneously, satAB was also directly regulated by SatR. Our findings may provide novel insights for the development of drug targets and help to exploit corresponding inhibitors to combat bacterial multidrug resistance.

The mild hypothermia response (MHR) maintains organismal homeostasis during cold exposure and is thought to be critical for the neuroprotection documented with therapeutic hypothermia. To date, little is known about the transcriptional regulation of the MHR. We utilize a forward CRISPR-Cas9 mutagenesis screen to identify the histone lysine methyltransferase SMYD5 as a regulator of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia. This repression correlates with temperature-dependent levels of histone H3 lysine 26 trimethylation (H3K36me3) at the SP1 locus and globally, indicating that the mammalian MHR is regulated at the level of histone modifications. We have identified 37 additional SMYD5-regulated temperature-dependent genes, suggesting a broader MHR-related role for SMYD5. Our study provides an example of how histone modifications integrate environmental cues into the genetic circuitry of mammalian cells and provides insights that may yield therapeutic avenues for neuroprotection after catastrophic events. [Display omitted] • SMYD5 represses the neuroprotective gene SP1 at 37°C by depositing H3K36me3 • Mild hypothermia reduces SMYD5 levels both in vitro and in vivo • Reduction in SMYD5 levels during hypothermia occurs via proteasomal degradation • SMYD5 similarly regulates 37 other genes indicating a broader role in the response Here, Rafnsdottir et al. demonstrate that under normothermia SMYD5 represses SP1 and other genes participating in the mild hypothermia response by H3K36me3 histone modification. Under mild hypothermia, SMYD5 is degraded via proteasomal degradation, and the repression caused by SMYD5 is lifted. SMYD5 also regulates 37 other genes in the same way. [ABSTRACT FROM AUTHOR]

Saro_0803 is a transcriptional factor modulating the transcription of the stilbene-degrading enzyme gene nov1 in Novosphingobium aromaticivorans DSM 12444. Reportedly, Saro_0803 undergoes resveratrol-mediated dissociation from the nov1 promotor and distinguishes resveratrol from its precursors, p-coumaric acid and trans-cinnamic acid, enabling the transcriptional factor to serve as a biosensor component for regulating resveratrol biosynthesis. However, little is known about the molecular mechanisms underlying the Saro_0803 interactions with either the nov1 promotor gene or resveratrol, which undermines the potential for Saro_0803 to be further modified for improved biosynthetic performance and other applications. Here, we report the discovery of the 22 bp A/T-rich Saro_0803 binding site near the −10 box of the nov1 promotor (named nov1p22bp). As validated by molecular docking-guided mutagenesis and binding affinity assays, the Saro_0803 binding of its target DNA sequence relies on charge-predominating interactions between several typical positively charged residues and nucleic acid. Furthermore, we semi-quantified the influence of resveratrol presence on Saro_0803–nov1p22bp interaction and identified a bilateral hydrophobic pocket within Saro_0803 comprising four aromatic residues that are crucial to maintaining the resveratrol binding capability of the transcriptional factor. Our data are beneficial to understanding saro_0803′s structural and functional properties, and could provide theoretical clues for future adaptations of this transcriptional factor.

Genetic alterations leading to the constitutive upregulation of specific efflux pumps contribute to antibacterial resistance in multidrug resistant bacteria. The identification of such resistance markers remains one of the most challenging tasks of genome-level resistance predictors. In this study, 487 non-redundant genetic events were identified in upstream zones of three operons coding for resistance-nodulation-division (RND) efflux pumps of 4,130 Acinetobacter baumannii isolates. These events included insertion sequences, small indels, and single nucleotide polymorphisms. In some cases, alterations explicitly modified the expression motifs described for these operons, such as the promoter boxes, operators, and Shine-Dalgarno sequences. In addition, changes in DNA curvature and mRNA secondary structures, which are structural elements that regulate expression, were also calculated. According to their influence on RND upregulation, the catalog of upstream modifications were associated with "experimentally verified," "presumed," and "probably irrelevant" degrees of certainty. For experimental verification, DNA of upstream sequences independently carrying selected markers, three for each RND operon, were fused to a luciferase reporter plasmid system. Five out of the nine selected markers tested showed significant increases in expression with respect to the wild-type sequence control. In particular, a 25-fold expression increase was observed with the IS Aba 1 insertion sequence upstream the adeABC pump. Next, overexpression of each of the three multi-specific RND pumps was linked to their respective antibacterial substrates by a deep A. baumannii literature screen. Consequently, a data flow framework was then developed to link genomic upregulatory RND determinants to potential antibiotic resistance. Assignment of potential increases in minimal inhibitory concentrations at the "experimentally verified" level was permitted for 42 isolates to 7–8 unrelated antibacterial agents including tigecycline, which is overlooked by conventional resistome predictors. Thus, our protocol may represent a time-saving filter step prior to laborious confirmation experiments for efflux-driven resistance. Altogether, a computational-experimental pipeline containing all components required for identifying the upstream regulatory resistome is proposed. This schema may provide the foundational stone for the elaboration of tools approaching antibiotic efflux that complement routine resistome predictors for preventing antimicrobial therapy failure against difficult-to-threat bacteria. [ABSTRACT FROM AUTHOR]

The phospholipid acyl chains of Enterococcus faecalis can be derived either by de novo synthesis or by incorporation of exogenous fatty acids through the fatty acid kinase complex (Fak)-phosphate acyltransferase (PlsX) pathway. Exogenous fatty acids suppress fatty acid synthesis through the transcriptional repressor FabT, the loss of which eliminated regulation of de novo fatty acid biosynthesis and resulted in decreased incorporation of exogenous unsaturated fatty acids. Purified FabT bound to the promoters of several fatty acid synthesis genes that contain a specific palindromic sequence and binding was enhanced by acylated derivatives of acyl carrier protein B (acyl-AcpB). The loss of the PlsX pathway blocked FabT-dependent transcriptional repression in the presence of oleic acid. Transcriptional repression was partially retained in a E. faecalis Δ acpB strain which showed decreased fatty acid biosynthesis in the presence of exogenous unsaturated fatty acids. The FabT-dependent activity remaining in the Δ acpB strain indicates that acylated derivatives of AcpA were weak enhancers of FabT binding although AcpA is believed to primarily function in de novo fatty acid synthesis. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a psychrotrophic food-borne pathogenic bacterium that causes listeriosis. Due to its unusual adaptation, an ability to grow at extended temperatures ranging from 4 to 45 °C, L. monocytogenes is notoriously hard to control in food-manufacturing processes. In addition, the growing number of antibiotic-resistant L. monocytogenes strains have made listeriosis steadily refractory to clinical treatments and can lead to serious life-threatening diseases, such as sepsis and meningitis, in immunocompromised persons and neonates. Transcription factors that belong to the PadR family play a key role in bacterial survival against unfavorable environmental stresses. The LltR protein from L. monocytogenes was identified as a PadR-type transcription factor and was shown to be required for bacterial growth adaptation at low temperatures. Despite the functional significance of LltR in listeria survival and pathogenesis, our molecular understanding of the LltR-mediated transcriptional regulation is highly limited. Here, we report the crystal structure of LltR and reveal the operator DNA recognition mechanism used by LltR. LltR dimerizes into an isosceles triangle-like shape and requires a winged helix-turn-helix motif for dsDNA recognition. Indeed, LltR and putative operator dsDNA binding was observed and suggests a transcriptional repression of the llfR-lmo0600-lmo0601 operon by direct interaction between the LltR transcription factor and its promoter region. Structure-based comparative and mutational analyses showed that LltR interacts with dsDNA via a unique strategy that combines both LltR-specific and PadR family-common mechanisms. • The crystal structure of LltR from L. monocytogenes was determined and analyzed. • LltR recognizes an operator site in its own promoter region. • LltR exhibits the canonical but distinct features of the PadR family. [ABSTRACT FROM AUTHOR]

Chinese cabbage (Brassica rapa L.) leaves are purple in color due to anthocyanin accumulation and have nutritional and aesthetic value, as well as antioxidant properties. Here, we identified the R3 MYB transcription factor BrMYBL2.1 as a key negative regulator of anthocyanin biosynthesis. A Chinese cabbage cultivar with green leaves harbored a functional BrMYBL2.1 protein, designated BrMYBL2.1-G, with transcriptional repressor activity of anthocyanin biosynthetic genes. By contrast, BrMYBL2.1 from a Chinese cabbage cultivar with purple leaves carried a poly(A) insertion in the third exon of the gene, resulting in the insertion of multiple lysine residues in the predicted protein, designated BrMYBL2.1-P. Although both BrMYBL2.1 variants localized to the nucleus, only BrMYBL2.1-G interacted with its cognate partner BrTT8. Transient infiltration assays in tobacco leaves revealed that BrMYBL2.1-G, but not BrMYBL2.1-P, actively represses pigment accumulation by inhibiting the transcription of anthocyanin biosynthetic genes. Transient promoter activation assay in Arabidopsis protoplasts verified that BrMYBL2.1-G, but not BrMYBL2.1-P, can repress transcriptional activation of BrCHS and BrDFR, which was activated by co-expression with BrPAP1 and BrTT8. We determined that BrMYBL2.1-P may be more prone to degradation than BrMYBL2.1-G via ubiquitination. Taken together, these results demonstrate that BrMYBL2.1-G blocks the activity of the MBW complex and thus represses anthocyanin biosynthesis, whereas the variant BrMYBL2.1-P from purple Chinese cabbage cannot, thus leading to higher anthocyanin accumulation. [ABSTRACT FROM AUTHOR]

Genetic alterations leading to the constitutive upregulation of specific efflux pumps contribute to antibacterial resistance in multidrug resistant bacteria. The identification of such resistance markers remains one of the most challenging tasks of genome-level resistance predictors. In this study, 487 non-redundant genetic events were identified in upstream zones of three operons coding for resistance-nodulation-division (RND) efflux pumps of 4,130 Acinetobacter baumannii isolates. These events included insertion sequences, small indels, and single nucleotide polymorphisms. In some cases, alterations explicitly modified the expression motifs described for these operons, such as the promoter boxes, operators, and Shine-Dalgarno sequences. In addition, changes in DNA curvature and mRNA secondary structures, which are structural elements that regulate expression, were also calculated. According to their influence on RND upregulation, the catalog of upstream modifications were associated with “experimentally verified,” “presumed,” and “probably irrelevant” degrees of certainty. For experimental verification, DNA of upstream sequences independently carrying selected markers, three for each RND operon, were fused to a luciferase reporter plasmid system. Five out of the nine selected markers tested showed significant increases in expression with respect to the wild-type sequence control. In particular, a 25-fold expression increase was observed with the ISAba1 insertion sequence upstream the adeABC pump. Next, overexpression of each of the three multi-specific RND pumps was linked to their respective antibacterial substrates by a deep A. baumannii literature screen. Consequently, a data flow framework was then developed to link genomic upregulatory RND determinants to potential antibiotic resistance. Assignment of potential increases in minimal inhibitory concentrations at the “experimentally verified” level was permitted for 42 isolates to 7–8 unrelated antibacterial agents including tigecycline, which is overlooked by conventional resistome predictors. Thus, our protocol may represent a time-saving filter step prior to laborious confirmation experiments for efflux-driven resistance. Altogether, a computational-experimental pipeline containing all components required for identifying the upstream regulatory resistome is proposed. This schema may provide the foundational stone for the elaboration of tools approaching antibiotic efflux that complement routine resistome predictors for preventing antimicrobial therapy failure against difficult-to-threat bacteria.