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Nature Protocols Dec 2021Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements. We recently developed single-molecule footprinting to... (Review)
Review
Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements. We recently developed single-molecule footprinting to simultaneously resolve the occupancy of multiple proteins including transcription factors, RNA polymerase II and nucleosomes on single DNA molecules genome-wide. The technique combines the use of cytosine methyltransferases to footprint the genome with bisulfite sequencing to resolve transcription factor binding patterns at cis-regulatory elements. DNA footprinting is performed by incubating permeabilized nuclei with recombinant methyltransferases. Upon DNA extraction, whole-genome or targeted bisulfite libraries are prepared and loaded on Illumina sequencers. The protocol can be completed in 4-5 d in any laboratory with access to high-throughput sequencing. Analysis can be performed in 2 d using a dedicated R package and requires access to a high-performance computing system. Our method can be used to analyze how transcription factors cooperate and antagonize to regulate transcription.
Topics: Animals; Cell Nucleus; DNA; DNA Footprinting; DNA Modification Methylases; Gene Expression Regulation; Gene Library; Genome; High-Throughput Nucleotide Sequencing; Humans; Mice; Mouse Embryonic Stem Cells; Nucleosomes; RNA Polymerase II; Sequence Analysis, DNA; Single Molecule Imaging; Software; Transcription Factors
PubMed: 34773120
DOI: 10.1038/s41596-021-00630-1 -
G3 (Bethesda, Md.) Sep 2020This study has taken advantage of the availability of the assembled genomic sequence of flies, mosquitos, ants and bees to explore the presence of ultraconserved...
This study has taken advantage of the availability of the assembled genomic sequence of flies, mosquitos, ants and bees to explore the presence of ultraconserved sequence elements in these phylogenetic groups. We compared non-coding sequences found within and flanking developmental genes to homologous sequences in and Many of the conserved sequence blocks (CSBs) that constitute -regulatory DNA, recognized by alignment protocols, are also conserved in and Also conserved is the position but not necessarily the orientation of many of these ultraconserved CSBs (uCSBs) with respect to flanking genes. Using the mosquito algorithm, we have also identified uCSBs shared among distantly related mosquito species. Side by side comparison of bee and ant of selected developmental genes identify uCSBs shared between these two Hymenoptera, as well as less conserved CSBs in either one or the other taxon but not in both. Analysis of uCSBs in these dipterans and Hymenoptera will lead to a greater understanding of their evolutionary origin and function of their conserved non-coding sequences and aid in discovery of core elements of enhancers.This study applies the phylogenetic footprinting program to detection of ultraconserved non-coding sequence elements in Diptera, including flies and mosquitos, and Hymenoptera, including ants and bees. outputs an interspecies comparison as a single sequence in terms of the input reference sequence. Ultraconserved sequences flanking known developmental genes were detected in and when compared with species, in and when compared with , and between ants and bees. Our methods are useful in detecting and understanding the core evolutionarily hardened sequences required for gene regulation.
Topics: Animals; Bees; Conserved Sequence; DNA; Diptera; Drosophila; Hymenoptera; Phylogeny
PubMed: 32601058
DOI: 10.1534/g3.120.401502 -
PloS One 2015The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA....
The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.
Topics: Animals; Cell Line; Chromatin; Chromatin Assembly and Disassembly; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Nucleosomes; Protein Binding
PubMed: 26496704
DOI: 10.1371/journal.pone.0140076 -
Trends in Genetics : TIG Sep 2021About 7% of the human genome encodes cis-regulatory elements (CREs) that function as regulatory switches to modulate the expression of genes. These short genetic... (Review)
Review
About 7% of the human genome encodes cis-regulatory elements (CREs) that function as regulatory switches to modulate the expression of genes. These short genetic sequences control the complex transcriptional changes necessary for organismal development. A topical challenge in the field is to understand how transcription factors (TFs) read and translate this information into gene expression patterns. Here, I review how the development of single-molecule footprinting (SMF) that resolves the genome occupancy of TFs on individual DNA molecules resolution contributes to our ability to establish how the regulatory genetic information is interpreted at the mechanistic level. I further discuss how future developments in the nascent field of single-molecule genomics (SMG) could impact our understanding of gene regulation mechanisms.
Topics: DNA; Gene Expression Regulation; Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Regulatory Elements, Transcriptional; Single Molecule Imaging; Transcription Factors
PubMed: 33892959
DOI: 10.1016/j.tig.2021.03.008 -
Biochemical and Biophysical Research... Dec 2020In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its...
In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.
Topics: Argonaute Proteins; Bacterial Proteins; Base Sequence; DNA; Models, Molecular; Nucleic Acid Conformation; Nucleic Acid Denaturation; Protein Binding; Rhodobacter sphaeroides; RNA, Guide, CRISPR-Cas Systems
PubMed: 33333714
DOI: 10.1016/j.bbrc.2020.10.051 -
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 -
Nature Communications Aug 2017Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a...
Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a challenge, particularly for small drugs where labeling or sequencing methods do not perform well. Here, we present a fast and accurate method based on parallelized single molecule magnetic tweezers to detect the sequence selectivity and characterize the thermodynamics and kinetics of binding in a single assay. Mechanical manipulation of DNA hairpins with an engineered sequence is used to detect ligand binding as blocking events during DNA unzipping, allowing determination of ligand selectivity both for small drugs and large proteins with nearly base-pair resolution in an unbiased fashion. The assay allows investigation of subtle details such as the effect of flanking sequences or binding cooperativity. Unzipping assays on hairpin substrates with an optimized flat free energy landscape containing all binding motifs allows determination of the ligand mechanical footprint, recognition site, and binding orientation.Mapping the sequence specificity of DNA ligands remains a challenge, particularly for small drugs. Here the authors develop a parallelized single molecule magnetic tweezers approach using engineered DNA hairpins that can detect sequence selectivity, thermodynamics and kinetics of binding for small drugs and large proteins.
Topics: Base Sequence; Binding Sites; DNA; DNA Footprinting; Kinetics; Ligands; Magnetics; Models, Genetic; Nucleic Acid Conformation; Optical Tweezers; Thermodynamics
PubMed: 28824174
DOI: 10.1038/s41467-017-00379-w