ResearchPad - promoter-regions https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[How global DNA unwinding causes non-uniform stress distribution and melting of DNA]]> https://www.researchpad.co/article/elastic_article_14712 DNA unwinding is an important process that controls binding of proteins, gene expression and melting of double-stranded DNA. In a series of all-atom MD simulations on two DNA molecules containing a transcription start TATA-box sequence we demonstrate that application of a global restraint on the DNA twisting dramatically changes the coupling between helical parameters and the distribution of deformation energy along the sequence. Whereas only short range nearest-neighbor coupling is observed in the relaxed case, long-range coupling is induced in the globally restrained case. With increased overall unwinding the elastic deformation energy is strongly non-uniformly distributed resulting ultimately in a local melting transition of only the TATA box segment during the simulations. The deformation energy tends to be stored more in cytidine/guanine rich regions associated with a change in conformational substate distribution. Upon TATA box melting the deformation energy is largely absorbed by the melting bubble with the rest of the sequences relaxing back to near B-form. The simulations allow us to characterize the structural changes and the propagation of the elastic energy but also to calculate the associated free energy change upon DNA unwinding up to DNA melting. Finally, we design an Ising model for predicting the local melting transition based on empirical parameters. The direct comparison with the atomistic MD simulations indicates a remarkably good agreement for the predicted necessary torsional stress to induce a melting transition, for the position and length of the melted region and for the calculated associated free energy change between both approaches.

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<![CDATA[Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium]]> https://www.researchpad.co/article/5c61e8cfd5eed0c48496f1be

The Cpx-envelope stress system regulates the expression of virulence factors in many Gram-negative pathogens. In Salmonella enterica serovar Typhimurium deletion of the sensor kinase CpxA but not of the response regulator CpxR results in the down regulation of the key regulator for invasion, HilA encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we provide evidence that cpxA deletion interferes with dephosphorylation of CpxR resulting in increased levels of active CpxR and consequently in misregulation of target genes. 14 potential operons were identified to be under direct control of CpxR. These include the virulence determinants ecotin, the omptin PgtE, and the SPI-2 regulator SsrB. The Tat-system and the PocR regulator that together promote anaerobic respiration of tetrathionate on 1,2-propanediol are also under direct CpxR control. Notably, 1,2-propanediol represses hilA expression. Thus, our work demonstrates for the first time the involvement of the Cpx system in a complex network mediating metabolism and virulence function.

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<![CDATA[Analysis of a new begomovirus unveils a composite element conserved in the CP gene promoters of several Geminiviridae genera: Clues to comprehend the complex regulation of late genes]]> https://www.researchpad.co/article/5c521848d5eed0c484797a14

A novel bipartite begomovirus, Blechum interveinal chlorosis virus (BleICV), was characterized at the genome level. Comparative analyses revealed that BleICV coat protein (CP) gene promoter is highly divergent from the equivalent region of other begomoviruses (BGVs), with the single exception of Tomato chino La Paz virus (ToChLPV) with which it shares a 23-bp phylogenetic footprint exhibiting dyad symmetry. Systematic examination of the homologous CP promoter segment of 132 New World BGVs revealed the existence of a quasi-palindromic DNA segment displaying a strongly conserved ACTT-(N7)-AAGT core. The spacer sequence between the palindromic motifs is constant in length, but its sequence is highly variable among viral species, presenting a relaxed consensus (TT)GGKCCCY, which is similar to the Conserved Late Element or CLE (GTGGTCCC), a putative TrAP-responsive element. The homologous CP promoter region of Old World BGVs exhibited a distinct organization, with the putative TATA-box overlapping the left half of the ACTT-N7 composite element. Similar CP promoter sequences, dubbed “TATA-associated composite element” or TACE, were found in viruses belonging to different Geminiviridae genera, hence hinting unsuspected evolutionary relationships among those lineages. To get cues about the TACE function, the regulatory function of the CLE was explored in distinct experimental systems. Transgenic tobacco plants harboring a GUS reporter gene driven by a promoter composed by CLE multimers expressed high beta-glucuronidase activity in absence of viral factors, and that expression was increased by begomovirus infection. On the other hand, the TrAP-responsiveness of a truncated CP promoter of Tomato golden mosaic virus (TGMV) was abolished by site-directed mutation of the only CLE present in it, whereas the artificial addition of one CLE to the -125 truncated promoter strongly enhanced the transactivation level in tobacco protoplasts. These results indicate that the CLE is a TrAP-responsive element, hence providing valuable clues to interpret the recurrent association of the CLE with the TACE. On the basis of the aforesaid direct evidences and the insights afforded by the extensive comparative analysis of BleICV CP promoter, we propose that the TACE might be involved in the TrAP-mediated derepression of CP gene in vascular tissues.

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<![CDATA[Long-distance communication: Looping of human papillomavirus genomes regulates expression of viral oncogenes]]> https://www.researchpad.co/article/5c06f060d5eed0c484c6d84e

High-risk human papillomaviruses (HPVs) are a major cause of cancers. HPVs infect epithelial cells, and viral oncogenes disrupt several cellular processes, including cell division, differentiation, and apoptosis. Expression of these oncogenes is relatively low in undifferentiated epithelial cells but increases in differentiating cells by unknown mechanisms. In a new study, Parish and colleagues unveil how two cellular proteins, CCCTC-binding factor (CTCF) and Yin Yang 1 (YY1), mediate looping of the HPV18 genome, which regulates expression of viral oncogenes in both dividing and differentiating epithelial cells.

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<![CDATA[Negative feedback loop of bone resorption by NFATc1-dependent induction of Cadm1]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc8fc

Trimethylation of histone H3 lysine 4 and lysine 27 (H3K4me3 and H3K27me3) at gene promoter regions critically regulates gene expression. Key developmental genes tend to exhibit changes in histone modification patterns from the H3K4me3/H3K27me3 bivalent pattern to the H3K4me3 monovalent pattern. Using comprehensive chromatin immunoprecipitation followed by sequencing in bone marrow-derived macrophages (BMMs) and mature osteoclasts, we found that cell surface adhesion molecule 1 (Cadm1) is a direct target of nuclear factor of activated T cells 1 (NFATc1) and exhibits a bivalent histone pattern in BMMs and a monovalent pattern in osteoclasts. Cadm1 expression was upregulated in BMMs by receptor activator of nuclear factor kappa B ligand (RANKL), and blocked by a calcineurin/NFATc1 inhibitor, FK506. Cadm1-deficient mice exhibited significantly reduced bone mass compared with wild-type mice, which was due to the increased osteoclast differentiation, survival and bone-resorbing activity in Cadm1-deficient osteoclasts. These results suggest that Cadm1 is a direct target of NFATc1, which is induced by RANKL through epigenetic modification, and regulates osteoclastic bone resorption in a negative feedback manner.

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<![CDATA[Classification of Promoters Based on the Combination of Core Promoter Elements Exhibits Different Histone Modification Patterns]]> https://www.researchpad.co/article/5989da20ab0ee8fa60b7e99a

Four different histones (H2A, H2B, H3, and H4; two subunits each) constitute a histone octamer, around which DNA wraps to form histone-DNA complexes called nucleosomes. Amino acid residues in each histone are occasionally modified, resulting in several biological effects, including differential regulation of transcription. Core promoters that encompass the transcription start site have well-conserved DNA motifs, including the initiator (Inr), TATA box, and DPE, which are collectively called the core promoter elements (CPEs). In this study, we systematically studied the associations between the CPEs and histone modifications by integrating the Drosophila Core Promoter Database and time-series ChIP-seq data for histone modifications (H3K4me3, H3K27ac, and H3K27me3) during development in Drosophila melanogaster via the modENCODE project. We classified 96 core promoters into four groups based on the presence or absence of the TATA box or DPE, calculated the histone modification ratio at the core promoter region, and transcribed region for each core promoter. We found that the histone modifications in TATA-less groups were static during development and that the core promoters could be clearly divided into three types: i) core promoters with continuous active marks (H3K4me3 and H3K27ac), ii) core promoters with a continuous inactive mark (H3K27me3) and occasional active marks, and iii) core promoters with occasional histone modifications. Linear regression analysis and non-linear regression by random forest showed that the TATA-containing groups included core promoters without histone modifications, for which the measured RNA expression values were not predictable accurately from the histone modification status. DPE-containing groups had a higher relative frequency of H3K27me3 in both the core promoter region and transcribed region. In summary, our analysis showed that there was a systematic link between the existence of the CPEs and the dynamics, frequency and influence on transcriptional activity of histone modifications.

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<![CDATA[Characterization of salA, syrF, and syrG Genes and Attendant Regulatory Networks Involved in Plant Pathogenesis by Pseudomonas syringae pv. syringae B728a]]> https://www.researchpad.co/article/5989da49ab0ee8fa60b8c76d

Pseudomonas syringae pv. syringae B728a, causal agent of brown spot on bean, is an economically important plant pathogen that utilizes extracellular signaling to initiate a lifestyle change from an epiphyte to a pathogen. LuxR regulatory proteins play an important role in the transcriptional regulation of a variety of biological processes involving two-component signaling, quorum sensing, and secondary metabolism. Analysis of the B728a genome identified 24 LuxR-like proteins, three of which are encoded by salA, syrF, and syrG located adjacent to the syringomycin gene cluster. The LuxR-like proteins encoded by these three genes exhibit a domain architecture that places them in a subfamily of LuxR-like proteins associated with regulation of secondary metabolism in B728a. Deletion mutants of salA, syrF, and syrG failed to produce syringomycin and displayed reduction of virulence on bean. The transcriptional start sites of salA, syrG, and syrF were located 63, 235, and 498 bp upstream of the start codons, respectively, using primer extension analysis. The predicted -10/-35 promoter regions of syrF and syrG were confirmed using site-directed mutagenesis and GFP reporters that showed conserved promoter sequences around the -35 promoter region. Overexpression analysis and GFP reporters identified SyrG as an upstream transcriptional activator of syrF, where both SyrG and SyrF activate promoters of syringomycin biosynthesis genes. This study shows that syrG and syrF encode important transcriptional regulators of syringomycin biosynthesis genes.

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<![CDATA[Relation between Established Glioma Risk Variants and DNA Methylation in the Tumor]]> https://www.researchpad.co/article/5989da46ab0ee8fa60b8bd22

Genome-wide association studies and candidate gene studies have identified several genetic variants that increase glioma risk. The majority of these variants are non-coding and the mechanisms behind the increased risk in carriers are not known. In this study, we hypothesize that some of the established glioma risk variants induce aberrant DNA methylation in the developing tumor, either locally (gene-specific) or globally (genome-wide). In a pilot data set including 77 glioma patients, we used Illumina beadchip technology to analyze genetic variants in blood and DNA methylation in matched tumor samples. To validate our findings, we used data from the Cancer Genome Atlas, including 401 glioblastoma patients. Consensus clustering identified the glioma CpG island methylator phenotype (gCIMP) and two additional subgroups with distinct patterns of global DNA methylation. In the pilot dataset, gCIMP was associated with two genetic variants in CDKN2B-AS1, rs1412829 and rs4977756 (9p21.3, p = 8.1 x 10−7 and 4.8 x 10−5, respectively). The association was in the same direction in the TCGA dataset, although statistically significant only when combining individuals with AG and GG genotypes. We also investigated the relation between glioma risk variants and DNA methylation in the promoter region of genes located within 30 kb of each variant. One association in the pilot dataset, between the TERT risk variant rs2736100 and lower methylation of cg23827991 (in TERT; p = 0.001), was confirmed in the TCGA dataset (p = 0.001). In conclusion, we found an association between rs1412829 and rs4977756 (9p21.3, CDKN2B-AS1) and global DNA methylation pattern in glioma, for which a trend was seen also in the TCGA glioblastoma dataset. We also found an association between rs2736100 (in TERT) and levels of methylation at cg23827991 (localized in the same gene, 3.3 kbp downstream of the risk variant), which was validated in the TCGA dataset. Except for this one association, we did not find strong evidence for gene-specific DNA methylation mediated by glioma risk variants.

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<![CDATA[CpxR Activates MexAB-OprM Efflux Pump Expression and Enhances Antibiotic Resistance in Both Laboratory and Clinical nalB-Type Isolates of Pseudomonas aeruginosa]]> https://www.researchpad.co/article/5989da7cab0ee8fa60b98c91

Resistance-Nodulation-Division (RND) efflux pumps are responsible for multidrug resistance in Pseudomonas aeruginosa. In this study, we demonstrate that CpxR, previously identified as a regulator of the cell envelope stress response in Escherichia coli, is directly involved in activation of expression of RND efflux pump MexAB-OprM in P. aeruginosa. A conserved CpxR binding site was identified upstream of the mexA promoter in all genome-sequenced P. aeruginosa strains. CpxR is required to enhance mexAB-oprM expression and drug resistance, in the absence of repressor MexR, in P. aeruginosa strains PA14. As defective mexR is a genetic trait associated with the clinical emergence of nalB-type multidrug resistance in P. aeruginosa during antibiotic treatment, we investigated the involvement of CpxR in regulating multidrug resistance among resistant isolates generated in the laboratory via antibiotic treatment and collected in clinical settings. CpxR is required to activate expression of mexAB-oprM and enhances drug resistance, in the absence or presence of MexR, in ofloxacin-cefsulodin-resistant isolates generated in the laboratory. Furthermore, CpxR was also important in the mexR-defective clinical isolates. The newly identified regulatory linkage between CpxR and the MexAB-OprM efflux pump highlights the presence of a complex regulatory network modulating multidrug resistance in P. aeruginosa.

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<![CDATA[Upregulation of TPX2 by STAT3: Identification of a Novel STAT3 Binding Site]]> https://www.researchpad.co/article/5989da63ab0ee8fa60b9167f

TPX2, a protein involved in mitosis, is considered a good marker for actively proliferating tissues, highly expressed in a number of cancer cells. We show the presence of high-affinity binding site for STAT3 in the 5′-flanking region of the Tpx2 gene, which is in vivo bound by activated STAT3. A specific STAT3 peptide inhibitor represses the expression of the Tpx2 gene and inhibits the binding of STAT3 to its consensus sequence in human cell lines where STAT3 is activated. These results indicate that activated STAT3 contributes to the over-expression of Tpx2 through the binding to an enhancer site.

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<![CDATA[Aberrant Expression of TIMP-2 and PBEF Genes in the Placentae of Cloned Mice Due to Epigenetic Reprogramming Error]]> https://www.researchpad.co/article/5989daf1ab0ee8fa60bc12af

Cloned mice derived from somatic or ES cells show placental overgrowth (placentomegaly) at term. We had previously analyzed cloned and normal mouse placentae by using two-dimensional gel electrophoresis and mass spectrometry to identify differential protein expression patterns. Cloned placentae showed upregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2), which is involved in extracellular matrix degradation and tissue remodeling, and downregulation of pre-B cell colony enhancing factor 1 (PBEF), which inhibits apoptosis and induces spontaneous labor. Here, we used Western blotting to further analyze the protein expression levels of TIMP-2 and PBEF in cloned placentae derived from cumulus cells, TSA-treated cumulus cells, intracytoplasmic sperm injection (ICSI), and natural mating (NM control). Cloned and TSA-treated cloned placentae had higher expression levels of TIMP-2 compared with NM control and ICSI-derived placentae, and there was a positive association between TIMP-2 expression and the placental weight of cloned mouse concepti. Conversely, PBEF protein expression was significantly lower in cloned and ICSI placentae compared to NM controls. To examine whether the observed differences were due to abnormal gene expression caused by faulty epigenetic reprogramming in clones, we investigated DNA methylation and histone modification in the promoter regions of the genes encoding TIMP-2 and PBEF. Sodium bisulfite sequencing did not reveal any difference in DNA methylation between cloned and NM control placentae. However, ChIP assays revealed that the level of H3-K9/K14 acetylation at the TIMP-2 locus was higher in cloned placentae than in NM controls, whereas acetylation of the PBEF promoter was lower in cloned and ICSI placenta versus NM controls. These results suggest that cloned placentae appear to suffer from failure of histone modification-based reprogramming in these (and potentially other) developmentally important genes, leading to aberrant expression of their protein products. These changes are likely to be involved in generating the abnormalities seen in cloned mouse placentae, including enlargement and/or a lack of proper placental function.

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<![CDATA[PEG3 control on the mammalian MSL complex]]> https://www.researchpad.co/article/5989db5eab0ee8fa60be0b17

Peg3 (paternally expressed gene 3) encodes a DNA-binding protein that functions as a transcriptional repressor. Recent studies revealed that PEG3 binds to Msl1 (male-specific lethal 1) and Msl3, the two main components of the MSL complex. In the current study, we investigated potential roles of Peg3 in controlling its downstream genes through H4K16ac, the histone modification by the MSL complex. According to the results, complete removal of PEG3 resulted in up-regulation of Msl1 and Msl3, and subsequently an increase in the global levels of H4K16ac, confirming PEG3 as a transcriptional repressor for MSL during mammalian development. Genome-wide analyses further revealed that about 10% of the entire gene catalogue was affected in the MEF cells lacking PEG3, displaying the increased levels of H4K16ac in their promoter regions. The expression levels of a small subset of the affected genes were up-regulated in the MEF cells lacking PEG3. Interestingly, three Hox clusters also exhibited changes in the levels of H4K16ac, suggesting potential roles of PEG3 and MSL in the regulation of Hox clusters. Overall, the current study reports that Peg3 may control its downstream genes through mammalian MSL.

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<![CDATA[Usefulness of DNA Methylation Levels in COASY and SPINT1 Gene Promoter Regions as Biomarkers in Diagnosis of Alzheimer’s Disease and Amnestic Mild Cognitive Impairment]]> https://www.researchpad.co/article/5989dab9ab0ee8fa60badd87

In order to conduct early therapeutic interventions for Alzheimer’s disease (AD), convenient, early diagnosis markers are required. We previously reported that changes in DNA methylation levels were associated with amnestic mild cognitive impairment (aMCI) and AD. As the results suggested changes in DNA methylation levels in the COASY and SPINT1 gene promoter regions, in the present study we examined DNA methylation in these regions in normal controls (NCs, n = 30), aMCI subjects (n = 28) and AD subjects (n = 30) using methylation-sensitive high resolution melting (MS-HRM) analysis. The results indicated that DNA methylation in the two regions was significantly increased in AD and aMCI as compared to NCs (P < 0.0001, P < 0.0001, ANOVA). Further analysis suggested that DNA methylation in the COASY gene promoter region in particular could be a high sensitivity, high specificity diagnosis biomarker (COASY: sensitivity 96.6%, specificity 96.7%; SPINT1: sensitivity 63.8%, specificity 83.3%). DNA methylation in the COASY promoter region was associated with CDR Scale Sum of Boxes (CDR-SB), an indicator of dementia severity. In the SPINT1 promoter region, DNA methylation was negatively associated with age in NCs and elevated in aMCI and AD subjects positive for antibodies to Herpes simplex virus type 1 (HSV-1). These findings suggested that changes in DNA methylation in the COASY and SPINT1 promoter regions are influenced by various factors. In conclusion, DNA methylation levels in the COASY and SPINT1 promoter regions were considered to potentially be a convenient and useful biomarker for diagnosis of AD and aMCI.

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<![CDATA[Genomic variation in Plasmodium vivax malaria reveals regions under selective pressure]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf70b

Background

Although Plasmodium vivax contributes to almost half of all malaria cases outside Africa, it has been relatively neglected compared to the more deadly P. falciparum. It is known that P. vivax populations possess high genetic diversity, differing geographically potentially due to different vector species, host genetics and environmental factors.

Results

We analysed the high-quality genomic data for 46 P. vivax isolates spanning 10 countries across 4 continents. Using population genetic methods we identified hotspots of selection pressure, including the previously reported MRP1 and DHPS genes, both putative drug resistance loci. Extra copies and deletions in the promoter region of another drug resistance candidate, MDR1 gene, and duplications in the Duffy binding protein gene (PvDBP) potentially involved in erythrocyte invasion, were also identified. For surveillance applications, continental-informative markers were found in putative drug resistance loci, and we show that organellar polymorphisms could classify P. vivax populations across continents and differentiate between Plasmodia spp.

Conclusions

This study has shown that genomic diversity that lies within and between P. vivax populations can be used to elucidate potential drug resistance and invasion mechanisms, as well as facilitate the molecular barcoding of the parasite for surveillance applications.

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<![CDATA[Fibronectin Affects Transient MMP2 Gene Expression through DNA Demethylation Changes in Non-Invasive Breast Cancer Cell Lines]]> https://www.researchpad.co/article/5989dab2ab0ee8fa60babda6

Metastasis accounts for more than 90% of cancer deaths. Cells from primary solid tumors may invade adjacent tissues and migrate to distant sites where they establish new colonies. The tumor microenvironment is now recognized as an important participant in the signaling that induces cancer cell migration. An essential process for metastasis is extracellular matrix (ECM) degradation by metalloproteases (MMPs), which allows tumor cells to invade local tissues and to reach blood vessels. The members of this protein family include gelatinase A, or MMP-2, which is responsible for the degradation of type IV collagen, the most abundant component of the basal membrane, that separates epithelial cells in the stroma. It is known that fibronectin is capable of promoting the expression of MMP-2 in MCF7 breast cancer cells in culture. In addition, it was already shown that the MMP2 gene expression is regulated by epigenetic mechanisms. In this work, we showed that fibronectin was able to induce MMP2 expression by 30% decrease in its promoter methylation. In addition, a histone marker for an open chromatin conformation was significantly increased. These results indicate a new role for fibronectin in the communication between cancer cells and the ECM, promoting epigenetic modifications.

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<![CDATA[A σE-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in Vibrio alginolyticus]]> https://www.researchpad.co/article/5989daeaab0ee8fa60bbf099

In vibrios, the expression of virulence factors is often controlled by LuxR, the master quorum-sensing regulator. Here, we investigate the interplay between LuxR and σE, an alternative sigma factor, during the control of virulence-related gene expression and adaptations to temperature elevations in the zoonotic pathogen Vibrio alginolyticus. An rpoE null V. alginolyticus mutant was unable to adapt to various stresses and was survival-deficient in fish. In wild type V. alginolyticus, the expression of LuxR-regulated virulence factors increased as the temperature was increased from 22°C to 37°C, but mutants lacking σE did not respond to temperature, indicating that σE is critical for the temperature-dependent upregulation of virulence genes. Further analyses revealed that σE binds directly to -10 and -35 elements in the luxR promoter that drive its transcription. ChIP assays showed that σE binds to the promoter regions of luxR, rpoH and rpoE at high temperatures (e.g., 30°C and 37°C). However, at higher temperatures (42°C) that induce thermal stress, σE binding to the luxR promoter decreased, while its binding to the rpoH and rpoE promoters was unchanged. Thus, the temperature-dependent binding of σE to distinct promoters appears to underlie a σE-controlled switch between the expression of virulence genes and adaptation to thermal stress. This study illustrates how a conserved temperature response mechanism integrates into quorum-sensing circuits to regulate both virulence and stress adaptation.

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<![CDATA[Cuf2 Is a Transcriptional Co-Regulator that Interacts with Mei4 for Timely Expression of Middle-Phase Meiotic Genes]]> https://www.researchpad.co/article/5989da64ab0ee8fa60b919f8

The Schizosaccharomyces pombe cuf2+ gene encodes a nuclear regulator that is required for timely activation and repression of several middle-phase genes during meiotic differentiation. In this study, we sought to gain insight into the mechanism by which Cuf2 regulates meiotic gene expression. Using a chromatin immunoprecipitation approach, we demonstrate that Cuf2 is specifically associated with promoters of both activated and repressed target genes, in a time-dependent manner. In case of the fzr1+ gene whose transcription is positively affected by Cuf2, promoter occupancy by Cuf2 results in a concomitant increased association of RNA polymerase II along its coding region. In marked contrast, association of RNA polymerase II with chromatin decreases when Cuf2 negatively regulates target gene expression such as wtf13+. Although Cuf2 operates through a transcriptional mechanism, it is unable to perform its function in the absence of the Mei4 transcription factor, which is a member of the conserved forkhead protein family. Using coimmunoprecipitation experiments, results showed that Cuf2 is a binding partner of Mei4. Bimolecular fluorescence complementation experiments brought further evidence that an association between Cuf2 and Mei4 occurs in the nucleus. Analysis of fzr1+ promoter regions revealed that two FLEX-like elements, which are bound by the transcription factor Mei4, are required for chromatin occupancy by Cuf2. Together, results reported here revealed that Cuf2 and Mei4 co-regulate the timely expression of middle-phase genes during meiosis.

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<![CDATA[Determination and Analysis of the Putative AcaCD-Responsive Promoters of Salmonella Genomic Island 1]]> https://www.researchpad.co/article/5989da98ab0ee8fa60ba2cb4

The integrative genomic island SGI1 and its variants confer multidrug resistance in numerous Salmonella enterica serovariants and several Proteus mirabilis and Acinetobacter strains. SGI1 is mobilized by the IncA/C family plasmids. The island exploits not only the conjugation apparatus of the plasmid, but also utilizes the plasmid-encoded master regulator AcaCD to induce the excision and formation of its transfer-competent form, which is a key step in the horizontal transfer of SGI1. Triggering of SGI1 excision occurs via the AcaCD-dependent activation of xis gene expression. AcaCD binds in Pxis to an unusually long recognition sequence. Beside the Pxis promoter, upstream regions of four additional SGI1 genes, S004, S005, S012 and S018, also contain putative AcaCD-binding sites. Furthermore, SGI1 also encodes an AcaCD-related activator, FlhDCSGI1, which has no known function. Here, we have analysed the functionality of the putative AcaCD-dependent promoter regions and proved their activation by either AcaCD or FlhDCSGI1. Moreover, we provide evidence that both activators act on the same binding site in Pxis and that FlhDCSGI1 is able to complement the acaCD deletion of the IncA/C family plasmid R16a. We determined the transcription start sites for the AcaCD-responsive promoters and showed that orf S004 is expressed probably from a different start codon than predicted earlier. Additionally, expression of S003 from promoter PS004 was ruled out. Pxis and the four SGI1 promoters examined here also lack obvious -35 promoter box and their promoter profile is consistent with the class II-type activation pathway. Although the role of the four additionally analysed AcaCD/FlhDCSGI1-controlled genes in transfer and/or maintenance of SGI1 is not yet clear, the conservation of the whole region suggests the existence of some selection for their functionality.

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<![CDATA[Identification of Genetic Associations and Functional Polymorphisms of SAA1 Gene Affecting Milk Production Traits in Dairy Cattle]]> https://www.researchpad.co/article/5989da3dab0ee8fa60b88823

Our initial RNA sequencing (RNA-seq) revealed that the Serum amyloid A1 (SAA1) gene was differentially expressed in the mammary glands of lactating Holstein cows with extremely high versus low phenotypic values of milk protein and fat percentage. To further validate the genetic effect and potential molecular mechanisms of SAA1 gene involved in regulating milk production traits in dairy cattle, we herein performed a study through genotype-phenotype associations. Six identified SNPs were significantly associated with one or more milk production traits (0.00002< P < 0.0025), providing additional evidence for the potential role of SAA1 variants in milk production traits in dairy cows. Subsequently, both luciferase assay and electrophoretic mobility shift assay (EMSA) clearly demonstrated that the allele A of g.-963C>A increased the promoter activity by binding the PARP factor while allele C did not. Bioinformatics analysis indicated that the secondary structure of SAA protein changed by the substitution A/G in the locus c. +2510A>G. Our findings were the first to reveal the significant associations of the SAA1 gene with milk production traits, providing basis for further biological function validation, and two identified SNPs, g.-963C>A and c. +2510A>G, may be considered as genetic markers for breeding in dairy cattle.

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<![CDATA[Herpes Simplex Virus 1 (HSV-1) ICP22 Protein Directly Interacts with Cyclin-Dependent Kinase (CDK)9 to Inhibit RNA Polymerase II Transcription Elongation]]> https://www.researchpad.co/article/5989d9e3ab0ee8fa60b6a465

The Herpes Simplex Virus 1 (HSV-1)-encoded ICP22 protein plays an important role in viral infection and affects expression of host cell genes. ICP22 is known to reduce the global level of serine (Ser)2 phosphorylation of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 heptapeptide repeats comprising the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase (pol) II. Accordingly, ICP22 is thought to associate with and inhibit the activity of the positive-transcription elongation factor b (P-TEFb) pol II CTD Ser2 kinase. We show here that ICP22 causes loss of CTD Ser2 phosphorylation from pol II engaged in transcription of protein-coding genes following ectopic expression in HeLa cells and that recombinant ICP22 interacts with the CDK9 subunit of recombinant P-TEFb. ICP22 also interacts with pol II in vitro. Residues 193 to 256 of ICP22 are sufficient for interaction with CDK9 and inhibition of pol II CTD Ser2 phosphorylation but do not interact with pol II. These results indicate that discrete regions of ICP22 interact with either CDK9 or pol II and that ICP22 interacts directly with CDK9 to inhibit expression of host cell genes.

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