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Nature Structural & Molecular Biology Oct 2023Nearly all essential nuclear processes act on DNA packaged into arrays of nucleosomes. However, our understanding of how these processes (for example, DNA replication,...
Nearly all essential nuclear processes act on DNA packaged into arrays of nucleosomes. However, our understanding of how these processes (for example, DNA replication, RNA transcription, chromatin extrusion and nucleosome remodeling) occur on individual chromatin arrays remains unresolved. Here, to address this deficit, we present SAMOSA-ChAAT: a massively multiplex single-molecule footprinting approach to map the primary structure of individual, reconstituted chromatin templates subject to virtually any chromatin-associated reaction. We apply this method to distinguish between competing models for chromatin remodeling by the essential imitation switch (ISWI) ATPase SNF2h: nucleosome-density-dependent spacing versus fixed-linker-length nucleosome clamping. First, we perform in vivo single-molecule nucleosome footprinting in murine embryonic stem cells, to discover that ISWI-catalyzed nucleosome spacing correlates with the underlying nucleosome density of specific epigenomic domains. To establish causality, we apply SAMOSA-ChAAT to quantify the activities of ISWI ATPase SNF2h and its parent complex ACF on reconstituted nucleosomal arrays of varying nucleosome density, at single-molecule resolution. We demonstrate that ISWI remodelers operate as density-dependent, length-sensing nucleosome sliders, whose ability to program DNA accessibility is dictated by single-molecule nucleosome density. We propose that the long-observed, context-specific regulatory effects of ISWI complexes can be explained in part by the sensing of nucleosome density within epigenomic domains. More generally, our approach promises molecule-precise views of the essential processes that shape nuclear physiology.
Topics: Animals; Mice; Nucleosomes; Chromatin; Histones; DNA; Chromatin Assembly and Disassembly; Adenosine Triphosphatases; Mammals
PubMed: 37696956
DOI: 10.1038/s41594-023-01093-6 -
Journal of Dental Research Aug 2021is considered the primary etiological agent of human dental caries. Glucosyltransferases (Gtfs) from play important roles in the formation of biofilm matrix and the...
is considered the primary etiological agent of human dental caries. Glucosyltransferases (Gtfs) from play important roles in the formation of biofilm matrix and the development of cariogenic oral biofilm. Therefore, Gtfs are considered an important target to prevent the development of dental caries. However, the role of transcription factors in regulating expression is not yet clear. Here, we identify a MarR (multiple antibiotic resistance regulator) family transcription factor named EpsR (exopolysaccharide synthesis regulator), which negatively regulates expression and exopolysaccharide (EPS) production in . The in-frame deletion strain grew slowly, aggregated more easily in the presence of dextran, and displayed different colony morphology and biofilm structure. Notably, deletion resulted in altered 3-dimensional biofilm architecture, increased water-insoluble EPS production, and upregulated GtfB protein content and activity. In addition, global gene expression profiling revealed differences in the expression levels of 69 genes in which was markedly upregulated. The conserved DNA motif for EpsR binding was determined by electrophoretic mobility shift assay and DNase I footprinting assays. Moreover, analysis of β-galactosidase activity suggested that EpsR acted as a repressor and inhibited expression. Taken together, our findings indicate that EpsR is an important transcription factor that regulates expression and EPS production in . These results add new aspects to the complexity of regulating the expression of genes involved in the cariogenicity of , which might lead to novel strategies to prevent the formation of cariogenic biofilm that may favor diseases.
Topics: Bacterial Proteins; Biofilms; Extracellular Polymeric Substance Matrix; Glucosyltransferases; Streptococcus mutans
PubMed: 33749354
DOI: 10.1177/00220345211000668 -
Journal of Proteome Research Apr 2022During tumorigenesis, DNA mutations in protein coding sequences can alter amino acid sequences which can change the structures of proteins. While the 3D structure of...
During tumorigenesis, DNA mutations in protein coding sequences can alter amino acid sequences which can change the structures of proteins. While the 3D structure of mutated proteins has been studied with atomic resolution, the precise impact of somatic mutations on the 3D proteome during malignant transformation remains unknown because methods to reveal protein structures in high throughput are limited. Here, we measured the accessibility of the lysine ε-amine for chemical modification across proteomes using covalent protein painting (CPP) to indirectly determine alterations in the 3D proteome. CPP is a novel, high-throughput quantitative mass spectrometric method that surveyed a total of 8052 lysine sites across the 60 cell lines of the well-studied anticancer cell line panel (NCI60). Overall, 5.2 structural alterations differentiated any cancer cell line from the other 59. Structural aberrations in 98 effector proteins correlated with the selected presence of 90 commonly mutated proteins in the NCI60 cell line panel, suggesting that different tumor genotypes reshape a limited set of effector proteins. We searched our dataset for druggable conformational aberrations and identified 49 changes in the cancer conformational landscape that correlated with the growth inhibition profiles of 300 drug candidates out of 50,000 small molecules. We found that alterations in heat shock proteins are key predictors of anticancer drug efficacy, which implies that the proteostasis network may have a general but hitherto unrecognized role in maintaining malignancy. Individual lysine sites may serve as biomarkers to guide drug selection or may be directly targeted for anticancer drug development.
Topics: Carcinogenesis; Humans; Mass Spectrometry; Neoplasms; Proteome; Proteostasis
PubMed: 35271278
DOI: 10.1021/acs.jproteome.1c00906 -
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 -
Bio-protocol Oct 2020Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In...
Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one with the 5' DNA end labeled and one with the 3' DNA end labeled. While homogeneous 5'-radiolabeling can be accomplished using a single kinase-catalyzed phosphorylation step, existing procedures for 3'-radiolabeling often result in probe heterogeneity, prohibiting precise DNA fragment identification in downstream experiments. We present here a new protocol to efficiently attach a P-phosphate to the 3' end of a DNA oligonucleotide of arbitrary sequence, relying on inexpensive DNA oligonucleotide modifications (2'-O-methylribonucleotide and ribonucleotide sugar substitutions), two enzymes (T4 polynucleotide kinase and T4 RNA ligase 2), and the differential susceptibility of DNA and RNA to hydroxide treatment. Radioactive probe molecules produced by this protocol are homogeneous and oxidant-compatible, and they can be used for precise cleavage-site mapping in the context of both DNase enzyme characterization and DNA footprinting assays. Graphic abstract.
PubMed: 33659442
DOI: 10.21769/BioProtoc.3787 -
Cell Genomics Nov 2022Gene expression is controlled by transcription factors (TFs) that bind cognate DNA motif sequences in -regulatory elements (CREs). The combinations of DNA motifs acting...
Gene expression is controlled by transcription factors (TFs) that bind cognate DNA motif sequences in -regulatory elements (CREs). The combinations of DNA motifs acting within homeostasis and disease, however, are unclear. Gene expression, chromatin accessibility, TF footprinting, and H3K27ac-dependent DNA looping data were generated and a random-forest-based model was applied to identify 7,531 cell-type-specific -regulatory modules (CRMs) across 15 diploid human cell types. A co-enrichment framework within CRMs nominated 838 cell-type-specific, recurrent heterotypic DNA motif combinations (DMCs), which were functionally validated using massively parallel reporter assays. Cancer cells engaged DMCs linked to neoplasia-enabling processes operative in normal cells while also activating new DMCs only seen in the neoplastic state. This integrative approach identifies cell-type-specific -regulatory combinatorial DNA motifs in diverse normal and diseased human cells and represents a general framework for deciphering -regulatory sequence logic in gene regulation.
PubMed: 36742369
DOI: 10.1016/j.xgen.2022.100191 -
Molecular Microbiology Apr 2020CodY is a global transcriptional regulator that controls, directly or indirectly, the expression of dozens of genes and operons in Listeria monocytogenes. We used in...
CodY is a global transcriptional regulator that controls, directly or indirectly, the expression of dozens of genes and operons in Listeria monocytogenes. We used in vitro DNA affinity purification combined with massively parallel sequencing (IDAP-Seq) to identify genome-wide L. monocytogenes chromosomal DNA regions that CodY binds in vitro. The total number of CodY-binding regions exceeded 2,000, but they varied significantly in their strengths of binding at different CodY concentrations. The 388 strongest CodY-binding regions were chosen for further analysis. A strand-specific analysis of the data allowed pinpointing CodY-binding sites at close to single-nucleotide resolution. Gel shift and DNase I footprinting assays confirmed the presence and locations of several CodY-binding sites. Surprisingly, most of the sites were located within genes' coding regions. The binding site within the beginning of the coding sequence of the prfA gene, which encodes the master regulator of virulence genes, has been previously implicated in regulation of prfA, but this site was weaker in vitro than hundreds of other sites. The L. monocytogenes CodY protein was functionally similar to Bacillus subtilis CodY when expressed in B. subtilis cells. Based on the sequences of the CodY-binding sites, a model of CodY interaction with DNA is proposed.
Topics: Bacterial Proteins; Binding Sites; DNA, Bacterial; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Listeria monocytogenes; Protein Binding; Transcription Factors; Virulence Factors
PubMed: 31944451
DOI: 10.1111/mmi.14449 -
Bioinformatics (Oxford, England) Jul 2021High-throughput chromatin immunoprecipitation (ChIP) sequencing-based assays capture genomic regions associated with the profiled transcription factor (TF). ChIP-exo is...
MOTIVATION
High-throughput chromatin immunoprecipitation (ChIP) sequencing-based assays capture genomic regions associated with the profiled transcription factor (TF). ChIP-exo is a modified protocol, which uses lambda exonuclease to digest DNA close to the TF-DNA complex, in order to improve on the positional resolution of the TF-DNA contact. Because the digestion occurs in the 5'-3' orientation, the protocol produces directional footprints close to the complex, on both sides of the double stranded DNA. Like all ChIP-based methods, ChIP-exo reports a mixture of different regions associated with the TF: those bound directly to the TF as well as via intermediaries. However, the distribution of footprints are likely to be indicative of the complex forming at the DNA.
RESULTS
We present ExoDiversity, which uses a model-based framework to learn a joint distribution over footprints and motifs, thus resolving the mixture of ChIP-exo footprints into diverse binding modes. It uses no prior motif or TF information and automatically learns the number of different modes from the data. We show its application on a wide range of TFs and organisms/cell-types. Because its goal is to explain the complete set of reported regions, it is able to identify co-factor TF motifs that appear in a small fraction of the dataset. Further, ExoDiversity discovers small nucleotide variations within and outside canonical motifs, which co-occur with variations in footprints, suggesting that the TF-DNA structural configuration at those regions is likely to be different. Finally, we show that detected modes have specific DNA shape features and conservation signals, giving insights into the structure and function of the putative TF-DNA complexes.
AVAILABILITY AND IMPLEMENTATION
The code for ExoDiversity is available on https://github.com/NarlikarLab/exoDIVERSITY.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Binding Sites; Chromatin Immunoprecipitation; DNA; DNA Footprinting; Exonucleases; Protein Binding; Sequence Analysis, DNA
PubMed: 34252930
DOI: 10.1093/bioinformatics/btab274 -
Methods in Molecular Biology (Clifton,... 2022The specificity and strength of protein-DNA complexes rely on tight interactions between side- and main chain atoms of amino acid residues and phosphates, sugars, and...
The specificity and strength of protein-DNA complexes rely on tight interactions between side- and main chain atoms of amino acid residues and phosphates, sugars, and base-specific groups. Various (in-gel) footprinting methods (for more information, see Chapter 11 ) allow the identification of the global-binding region but do not provide details on the contribution to complex formation of individual sequence-specific constituents of the DNA-binding site. Here, we describe how various chemicals can be used to randomly and sparingly modify specific bases or phosphates and allow the identification of those residues that are specifically protected against modification upon protein binding (protection studies) or interfere with complex formation when modified or removed prior to protein binding (premodification-binding interference). Each one of these complementary approaches has its advantages and shortcomings and results have to be interpreted with caution, having in mind the precise chemistry of the modification. However, used in combination, these methods provide an accurate and high-resolution image of the protein-DNA contacts.
Topics: Base Sequence; Binding Sites; DNA; Phosphates; Protein Binding
PubMed: 35922629
DOI: 10.1007/978-1-0716-2413-5_12 -
International Journal of Molecular... Jan 2023Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by formation of small intranucleosomal DNA loops (i-loops). The i-loops form more efficiently...
Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by formation of small intranucleosomal DNA loops (i-loops). The i-loops form more efficiently in the presence of single-strand breaks or gaps in a non-template DNA strand (NT-SSBs) and induce arrest of transcribing RNAP, thus allowing detection of NT-SSBs by the enzyme. Here we examined the role of histone tails and extranucleosomal NT-SSBs in i-loop formation and arrest of RNAP during transcription of promoter-proximal region of nucleosomal DNA. NT-SSBs present in linker DNA induce arrest of RNAP +1 to +15 bp in the nucleosome, suggesting formation of the i-loops; the arrest is more efficient in the presence of the histone tails. Consistently, DNA footprinting reveals formation of an i-loop after stalling RNAP at the position +2 and backtracking to position +1. The data suggest that histone tails and NT-SSBs present in linker DNA strongly facilitate formation of the i-loops during transcription through the promoter-proximal region of nucleosomal DNA.
Topics: Nucleosomes; Histones; Transcription, Genetic; RNA Polymerase II; DNA Breaks, Single-Stranded; DNA-Directed RNA Polymerases; DNA; DNA, Single-Stranded
PubMed: 36768621
DOI: 10.3390/ijms24032295