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Frontiers in Cellular and Infection... 2024Competence development is essential for bacterial transformation since it enables bacteria to take up free DNA from the surrounding environment. The regulation of...
Competence development is essential for bacterial transformation since it enables bacteria to take up free DNA from the surrounding environment. The regulation of teichoic acid biosynthesis is tightly controlled during pneumococcal competence; however, the mechanism governing this regulation and its impact on transformation remains poorly understood. We demonstrated that a defect in lipoteichoic acid ligase (TacL)-mediated lipoteichoic acids (LTAs) biosynthesis was associated with impaired pneumococcal transformation. Using a fragment of regulatory probe as bait in a DNA pulldown assay, we successfully identified several regulatory proteins, including ComE. Electrophoretic mobility shift assays revealed that phosphomimetic ComE, but not wild-type ComE, exhibited specific binding to the probe. DNase I footprinting assays revealed the specific binding sequences encompassing around 30 base pairs located 31 base pairs upstream from the start codon of . Expression of was found to be upregulated in the Δ strain, and the addition of exogenous competence-stimulating peptide repressed the transcription in the wild-type strain but not the Δ mutant, indicating that ComE exerted a negative regulatory effect on the transcription of . Mutation in the JH2 region of upstream regulatory sequence led to increased LTAs abundance and displayed higher transformation efficiency. Collectively, our work identified the regulatory mechanisms that control LTAs biosynthesis during competence and thereby unveiled a repression mechanism underlying pneumococcal transformation.
Topics: Teichoic Acids; Bacterial Proteins; Lipopolysaccharides; Gene Expression Regulation, Bacterial; Streptococcus pneumoniae; Transformation, Bacterial; Transcription, Genetic; Promoter Regions, Genetic; DNA Transformation Competence; Mutation; Protein Binding; Ligases
PubMed: 38779562
DOI: 10.3389/fcimb.2024.1375312 -
Cancer Research May 2024Genome-wide association studies (GWAS) have identified more than a hundred single nucleotide variants (SNVs) associated with the risk of gastroesophageal cancer (GEC)....
Genome-wide association studies (GWAS) have identified more than a hundred single nucleotide variants (SNVs) associated with the risk of gastroesophageal cancer (GEC). The majority of the identified SNVs map to noncoding regions of the genome. Uncovering the causal SNVs and the genes they modulate could help improve GEC prevention and treatment. Here, we used HiChIP against histone 3 lysine 27 acetylation (H3K27ac) to simultaneously annotate active promoters and enhancers, identify the interactions between them, and detect nucleosome free regions (NFRs) harboring potential causal SNVs in a single assay. Application of H3K27Ac HiChIP in GEC relevant models identified 61 potential functional SNVs that reside in NFRs and interact with 49 genes at 17 loci. The approach led to a 67% reduction in the number of SNVs in linkage disequilibrium at these 17 loci, and at seven loci a single putative causal SNV was identified. One SNV, rs147518036, located within the promoter of the UDP-glucuronate decarboxylase 1 (UXS1) gene appeared to underlie the GEC risk association captured by the rs75460256 index SNV. The rs147518036 SNV creates a GABPA DNA recognition motif, resulting in increased promoter activity, and CRISPR-mediated inhibition of the UXS1 promoter reduced viability of GEC cells. These findings provide a framework that simplifies the identification of potentially functional regulatory SNVs and target genes underlying risk-associated loci. In addition, the study implicates increased expression of the enzyme UXS1 and activation of its metabolic pathway as a predisposition to gastric cancer, which highlights potential therapeutic avenues to treat this disease.
PubMed: 38748784
DOI: 10.1158/0008-5472.CAN-23-2397 -
BioRxiv : the Preprint Server For... Apr 2024Cooperation between the circadian transcription factor (TF) CLOCK:BMAL1 and other TFs at -regulatory elements (CREs) is critical to daily rhythms of transcription. Yet,...
Cooperation between the circadian transcription factor (TF) CLOCK:BMAL1 and other TFs at -regulatory elements (CREs) is critical to daily rhythms of transcription. Yet, the modalities of this cooperation are unclear. Here, we analyzed the co-binding of multiple TFs on single DNA molecules in mouse liver using single molecule footprinting (SMF). We found that SMF reads clustered in stereotypic chromatin states that reflect distinguishable organization of TFs and nucleosomes, and that were remarkably conserved between all samples. DNA protection at CLOCK:BMAL1 binding motif (E-box) varied between CREs, from E-boxes being solely bound by CLOCK:BMAL1 to situations where other TFs competed with CLOCK:BMAL1 for E-box binding. SMF also uncovered CLOCK:BMAL1 cooperative binding at E-boxes separated by 250 bp, which structurally altered the CLOCK:BMAL1-DNA interface. Importantly, we discovered multiple nucleosomes with E-boxes at entry/exit sites that were removed upon CLOCK:BMAL1 DNA binding, thereby promoting the formation of open chromatin states that facilitate DNA binding of other TFs and that were associated with rhythmic transcription. These results demonstrate the utility of SMF for studying how CLOCK:BMAL1 and other TFs regulate stereotypical chromatin states at CREs to promote transcription.
PubMed: 38712031
DOI: 10.1101/2024.04.24.590818 -
Scientific Reports Apr 2024Transcription factors (TFs) are crucial epigenetic regulators, which enable cells to dynamically adjust gene expression in response to environmental signals....
Transcription factors (TFs) are crucial epigenetic regulators, which enable cells to dynamically adjust gene expression in response to environmental signals. Computational procedures like digital genomic footprinting on chromatin accessibility assays such as ATACseq can be used to identify bound TFs in a genome-wide scale. This method utilizes short regions of low accessibility signals due to steric hindrance of DNA bound proteins, called footprints (FPs), which are combined with motif databases for TF identification. However, while over 1600 TFs have been described in the human genome, only ~ 700 of these have a known binding motif. Thus, a substantial number of FPs without overlap to a known DNA motif are normally discarded from FP analysis. In addition, the FP method is restricted to organisms with a substantial number of known TF motifs. Here we present DENIS (DE Novo motIf diScovery), a framework to generate and systematically investigate the potential of de novo TF motif discovery from FPs. DENIS includes functionality (1) to isolate FPs without binding motifs, (2) to perform de novo motif generation and (3) to characterize novel motifs. Here, we show that the framework rediscovers artificially removed TF motifs, quantifies de novo motif usage during an early embryonic development example dataset, and is able to analyze and uncover TF activity in organisms lacking canonical motifs. The latter task is exemplified by an investigation of a scATAC-seq dataset in zebrafish which covers different cell types during hematopoiesis.
Topics: Transcription Factors; Animals; Zebrafish; Chromatin Immunoprecipitation Sequencing; Humans; Nucleotide Motifs; Binding Sites; Protein Binding; DNA Footprinting; Computational Biology; Chromatin
PubMed: 38654130
DOI: 10.1038/s41598-024-59989-2 -
Nucleic Acids Research May 2024Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for...
Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here, we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m6A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m6A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.
Topics: Chromatin; DNA Methylation; Nucleosomes; Sequence Analysis, DNA; Software; Genome, Fungal; High-Throughput Nucleotide Sequencing; Saccharomycetales; Adenosine
PubMed: 38634798
DOI: 10.1093/nar/gkae288 -
Methods in Enzymology 2024DNA secondary structures are essential elements of the genomic landscape, playing a critical role in regulating various cellular processes. These structures refer to...
DNA secondary structures are essential elements of the genomic landscape, playing a critical role in regulating various cellular processes. These structures refer to G-quadruplexes, cruciforms, Z-DNA or H-DNA structures, amongst others (collectively called 'non-B DNA'), which DNA molecules can adopt beyond the B conformation. DNA secondary structures have significant biological roles, and their landscape is dynamic and can rearrange due to various factors, including changes in cellular conditions, temperature, and DNA-binding proteins. Understanding this dynamic nature is crucial for unraveling their functions in cellular processes. Detecting DNA secondary structures remains a challenge. Conventional methods, such as gel electrophoresis and chemical probing, have limitations in terms of sensitivity and specificity. Emerging techniques, including next-generation sequencing and single-molecule approaches, offer promise but face challenges since these techniques are mostly limited to only one type of secondary structure. Here we describe an updated version of a technique permanganate/S1 nuclease footprinting, which uses potassium permanganate to trap single-stranded DNA regions as found in many non-B structures, in combination with S1 nuclease digest and adapter ligation to detect genome-wide non-B formation. To overcome technical hurdles, we combined this method with direct adapter ligation and sequencing (PDAL-Seq). Furthermore, we established a user-friendly pipeline available on Galaxy to standardize PDAL-Seq data analysis. This optimized method allows the analysis of many types of DNA secondary structures that form in a living cell and will advance our knowledge of their roles in health and disease.
Topics: DNA; G-Quadruplexes; Oxides; Manganese Compounds; Oligonucleotides
PubMed: 38521584
DOI: 10.1016/bs.mie.2023.12.003 -
International Journal of Biological... May 2024Alkaline protease AprE, produced by Bacillus licheniformis 2709 is an important edible hydrolase, which has potential applications in nutrient acquisition and medicine....
Alkaline protease AprE, produced by Bacillus licheniformis 2709 is an important edible hydrolase, which has potential applications in nutrient acquisition and medicine. The expression of AprE is finely regulated by a complex transcriptional regulation system. However, there is little study on transcriptional regulation mechanism of AprE biosynthesis in Bacillus licheniformis, which limits system engineering and further enhancement of AprE. Here, the severely depressed expression of aprE in degU and degS deletion mutants illustrated that the regulator DegU and its phosphorylation played a crucial part in AprE biosynthesis. Further electrophoretic mobility shift assay (EMSA) in vitro indicated that phosphorylated DegU can directly bind to the regulatory region though the DNase I foot-printing experiments failed to observe protected region. The plasmid-mediated overexpression of degU32 (Hy) obviously improved the yield of AprE by 41.6 % compared with the control strain, which demonstrated the importance of phosphorylation state of DegU on the transcription of aprE in vivo. In this study, the putative binding sequence of aprE (5'-TAAAT……AAAAT…….AACAT…TAAAA-3') located upstream -91 to -87 bp, -101 to -97 bp, -195 to -191 bp, -215 to -211 bp of the transcription start site (TSS) in B. licheniformis was computationally identified based on the DNA-binding sites of DegU in Bacillus subtilis. Overall, we systematically investigated the influence of the interplay between phosphorylated DegU and its cognate DNA sequence on expression of aprE, which not only contributes to the further AprE high-production in a genetically modified host in the future, but also significantly increases our understanding of the aprE transcription mechanism.
Topics: Bacillus licheniformis; Bacterial Proteins; Gene Expression Regulation, Bacterial; Endopeptidases; Phosphorylation; Promoter Regions, Genetic; Membrane Transport Proteins
PubMed: 38479659
DOI: 10.1016/j.ijbiomac.2024.130818 -
BioRxiv : the Preprint Server For... Feb 2024The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer...
The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer sequence to TF binding, promoter state, and gene expression levels remain opaque. We applied single-molecule footprinting (SMF) to measure the simultaneous occupancy of TFs, nucleosomes, and components of the transcription machinery on engineered enhancer/promoter constructs with variable numbers of TF binding sites for both a synthetic and an endogenous TF. We find that activation domains enhance a TF's capacity to compete with nucleosomes for binding to DNA in a BAF-dependent manner, TF binding on nucleosome-free DNA is consistent with independent binding between TFs, and average TF occupancy linearly contributes to promoter activation rates. We also decompose TF strength into separable binding and activation terms, which can be tuned and perturbed independently. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the binding microstates observed at the enhancer and subsequent time-dependent gene expression. This work provides a template for quantitative dissection of distinct contributors to gene activation, including the activity of chromatin remodelers, TF activation domains, chromatin acetylation, TF concentration, TF binding affinity, and TF binding site configuration.
PubMed: 38352517
DOI: 10.1101/2024.02.02.578660 -
Development (Cambridge, England) Mar 2024The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre...
The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.
Topics: Animals; Mice; Cell Differentiation; Chromatin; Gene Expression; Gene Expression Regulation, Developmental; Mesoderm; Somites; DNA-Binding Proteins; Transcription Factors
PubMed: 38345319
DOI: 10.1242/dev.202649 -
Applied and Environmental Microbiology Feb 2024Dental caries is the most common chronic infectious disease around the world and disproportionately affects the marginalized socioeconomic group. , considered a primary...
Dental caries is the most common chronic infectious disease around the world and disproportionately affects the marginalized socioeconomic group. , considered a primary etiological agent of caries, depends on the coordinated physiological response to tolerate the oxidative stress generated by commensal species within dental plaque, which is a critical aspect of its pathogenicity. Here, we identified and characterized a novel tetracycline repressor family regulator, SMU_1361c, which appears to be acquired by the bacteria via horizontal gene transfer. Surprisingly, functions as a negative transcriptional regulator to regulate gene expression outside its operon and is involved in the oxidative stress response of . The overexpression strain UA159/pDL278- was more susceptible to oxidative stress and less competitive against hydrogen peroxide generated by commensal species and . Transcriptomics analysis revealed that overexpression resulted in the significant downregulation of 22 genes, mainly belonging to three gene clusters responsible for the oxidative stress response. The conversed DNA binding motif of SMU_1361c was determined by electrophoretic mobility shift and DNase I footprinting assay with purified SMU_1361c protein; therefore, is directly involved in gene transcription related to the oxidative stress response. Crucially, our finding provides a new understanding of how deals with the oxidative stress that is required for pathogenesis and will facilitate the development of new and improved therapeutic approaches for dental caries.IMPORTANCE is the major organism associated with the development of dental caries, which globally is the most common chronic disease. To persist and survive in biofilms, must compete with commensal species that occupy the same ecological niche. Here, we uncover a novel molecular mechanism of how tetracycline repressor family regulator is involved in the oxidative stress response through transcriptomics analysis, electrophoretic mobility shift assay, and DNase I footprinting assay. Furthermore, we demonstrated that mediates sensitivity to oxidative stress and competitiveness with commensal streptococci. Therefore, this study has revealed a previously unknown regulation between and genes outside its operon and demonstrated the importance of _ in the oxidative stress response and the fitness of within the plaque biofilms, which can be exploited as a new therapy to modulate ecological homeostasis and prevent dental caries.
Topics: Humans; Streptococcus mutans; Bacterial Proteins; Dental Caries; Biofilms; Oxidative Stress; Tetracyclines; Deoxyribonuclease I
PubMed: 38299814
DOI: 10.1128/aem.01871-23