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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 -
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 -
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 -
BioRxiv : the Preprint Server For... Nov 2023Recent 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 mA 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 mA 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.
PubMed: 38076871
DOI: 10.1101/2023.11.28.569045 -
BioRxiv : the Preprint Server For... Oct 2023DNA looping is vital for establishing many enhancer-promoter interactions. While CTCF is known to anchor many cohesin-mediated loops, the looped chromatin fiber appears...
DNA looping is vital for establishing many enhancer-promoter interactions. While CTCF is known to anchor many cohesin-mediated loops, the looped chromatin fiber appears to predominantly exist in a poorly characterized actively extruding state. To better characterize extruding chromatin loop structures, we used CTCF MNase HiChIP data to determine both CTCF binding at high resolution and 3D contact information. Here we present , a tool that identifies CTCF binding sites at near base-pair resolution. We leverage this substantial advance in resolution to determine that the fully extruded (CTCF-CTCF) state is rare genome-wide with locus-specific variation from ~1-10%. We further investigate the impact of chromatin state on loop extrusion dynamics, and find that active enhancers and RNA Pol II impede cohesin extrusion, facilitating an enrichment of enhancer-promoter contacts in the partially extruded loop state. We propose a model of topological regulation whereby the transient, partially extruded states play active roles in transcription.
PubMed: 37961446
DOI: 10.1101/2023.10.20.563340 -
Genome Biology Nov 2023Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing...
BACKGROUND
Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing their origin to evolutionary innovations first made in their archaeal ancestors. One of the most notable such features is nucleosomal chromatin, although archaeal histones and chromatin differ significantly from those of eukaryotes, not all archaea possess histones and it is not clear if histones are a main packaging component for all that do. Despite increased interest in archaeal chromatin in recent years, its properties have been little studied using genomic tools.
RESULTS
Here, we adapt the ATAC-seq assay to archaea and use it to map the accessible landscape of the genome of the euryarchaeote Haloferax volcanii. We integrate the resulting datasets with genome-wide maps of active transcription and single-stranded DNA (ssDNA) and find that while H. volcanii promoters exist in a preferentially accessible state, unlike most eukaryotes, modulation of transcriptional activity is not associated with changes in promoter accessibility. Applying orthogonal single-molecule footprinting methods, we quantify the absolute levels of physical protection of H. volcanii and find that Haloferax chromatin is similarly or only slightly more accessible, in aggregate, than that of eukaryotes. We also evaluate the degree of coordination of transcription within archaeal operons and make the unexpected observation that some CRISPR arrays are associated with highly prevalent ssDNA structures.
CONCLUSIONS
Our results provide the first comprehensive maps of chromatin accessibility and active transcription in Haloferax across conditions and thus a foundation for future functional studies of archaeal chromatin.
Topics: Chromatin; Histones; Haloferax volcanii; Nucleosomes; Biological Evolution; Eukaryota; Archaeal Proteins
PubMed: 37932847
DOI: 10.1186/s13059-023-03095-5 -
Frontiers in Microbiology 2023is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The...
is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The two-component regulatory system BvrR/BvrS modulates the expression of genes required to transition from extracellular to intracellular lifestyles. However, few regulatory regions of BvrR direct target genes have been studied. In this study, we characterized the regulatory region of , a gene encoding an outer membrane protein that is positively regulated by TCS BvrR/BvrS. By - reporter fusions and β-galactosidase activity assays, we found that the region between-262 and + 127 is necessary for transcriptional activity, particularly a 111-bp long fragment located from-262 to -152. In addition, we demonstrated the binding of P-BvrR to three sites within the -140 to +1 region. Two of these sites were delimited between -18 to +1 and - 99 to -76 by DNase I footprinting and called DNA regulatory boxes 1 and 2, respectively. The third binding site (box 3) was delimited from -140 to -122 by combining EMSA and fluorescence anisotropy results. A molecular docking analysis with HDOCK predicted BvrR-DNA interactions between 11, 13, and 12 amino acid residue-nucleotide pairs in boxes 1, 2, and 3, respectively. A manual sequence alignment of the three regulatory boxes revealed the presence of inverted and non-inverted repeats of five to eight nucleotides, partially matching DNA binding motifs previously described for BvrR. We propose that P-BvrR binds directly to up to three regulatory boxes and probably interacts with other transcription factors to regulate expression. This gene regulation model could apply to other BvrR target genes and to orthologs of the TCS BvrR/BvrS and Omp25 in phylogenetically closed .
PubMed: 37779712
DOI: 10.3389/fmicb.2023.1241143