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Methods in Molecular Biology (Clifton,... 2023The bulk of gene expression regulation in most organisms is accomplished through the action of transcription factors (TFs) on cis-regulatory elements (CREs). In...
The bulk of gene expression regulation in most organisms is accomplished through the action of transcription factors (TFs) on cis-regulatory elements (CREs). In eukaryotes, these CREs are generally characterized by nucleosomal depletion and thus higher physical accessibility of DNA. Many methods exploit this property to map regions of high average accessibility, and thus putative active CREs, in bulk. However, these techniques do not provide information about coordinated patterns of accessibility along the same DNA molecule, nor do they map the absolute levels of occupancy/accessibility. SMF (Single-Molecule Footprinting) fills these gaps by leveraging recombinant DNA cytosine methyltransferases (MTase) to mark accessible locations on individual DNA molecules. In this chapter, we discuss current methods and important considerations for performing SMF experiments.
Topics: Chromatin; Nucleosomes; Sequence Analysis, DNA; DNA Methylation; Transcription Factors; High-Throughput Nucleotide Sequencing
PubMed: 36807067
DOI: 10.1007/978-1-0716-2899-7_8 -
Nature Genetics Mar 2021The advent of single-cell chromatin accessibility profiling has accelerated the ability to map gene regulatory landscapes but has outpaced the development of scalable...
The advent of single-cell chromatin accessibility profiling has accelerated the ability to map gene regulatory landscapes but has outpaced the development of scalable software to rapidly extract biological meaning from these data. Here we present a software suite for single-cell analysis of regulatory chromatin in R (ArchR; https://www.archrproject.com/ ) that enables fast and comprehensive analysis of single-cell chromatin accessibility data. ArchR provides an intuitive, user-focused interface for complex single-cell analyses, including doublet removal, single-cell clustering and cell type identification, unified peak set generation, cellular trajectory identification, DNA element-to-gene linkage, transcription factor footprinting, mRNA expression level prediction from chromatin accessibility and multi-omic integration with single-cell RNA sequencing (scRNA-seq). Enabling the analysis of over 1.2 million single cells within 8 h on a standard Unix laptop, ArchR is a comprehensive software suite for end-to-end analysis of single-cell chromatin accessibility that will accelerate the understanding of gene regulation at the resolution of individual cells.
Topics: Animals; Chromatin; Cluster Analysis; Gene Expression Regulation; Genome; Humans; Mice; Sequence Analysis, RNA; Single-Cell Analysis; Software; Transcription Factors; User-Computer Interface; Web Browser
PubMed: 33633365
DOI: 10.1038/s41588-021-00790-6 -
Bio-protocol Dec 2020DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to...
DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use traditional footprinting methods to determine protein-DNA contacts.
PubMed: 33659492
DOI: 10.21769/BioProtoc.3843 -
Frontiers in Cell and Developmental... 2022ATR is a PI3K-like kinase protein, regulating checkpoint responses to DNA damage and replication stress. Apart from its checkpoint function in the nucleus, ATR actively...
ATR is a PI3K-like kinase protein, regulating checkpoint responses to DNA damage and replication stress. Apart from its checkpoint function in the nucleus, ATR actively engages in an antiapoptotic role at mitochondria following DNA damage. The different functions of ATR in the nucleus and cytoplasm are carried out by two prolyl isomeric forms of ATR: - and -ATR, respectively. The isomerization occurs at the Pin1 Ser428-Pro429 motif of ATR. Here, we investigated the structural basis of the subcellular location-specific functions of human ATR. Using a mass spectrometry-based footprinting approach, the surface accessibility of ATR lysine residues to sulfo-NHS-LC-biotin modification was monitored and compared between the - and the -isomers. We have identified two biotin-modified lysine residues, K459 and K469, within the BH3-like domain of -ATR that were not accessible in -ATR, indicating a conformational change around the BH3 domain between and -ATR. The conformational alteration also involved the N-terminal domain and the middle HEAT domain. Moreover, experimental results from an array of complementary assays show that -ATR with the accessible BH3 domain was able to bind to tBid while -ATR could not. In addition, both - and -ATR can directly form homodimers their C-terminal domains without ATRIP, while nuclear (-ATR) in the presence of ATRIP forms dimer-dimer complexes involving both N- and C-termini of ATR and ATRIP after UV. Structural characteristics around the Ser428-Pro429 motif and the BH3 domain region are also analyzed by molecular modeling and dynamics simulation. In support, conformation was found to be significantly more energetically favorable than at the Ser428-Pro429 bond in a 20-aa wild-type ATR peptide. Taken together, our results suggest that the isomerization-induced structural changes of ATR define both its subcellular location and compartment-specific functions and play an essential role in promoting cell survival and DNA damage responses.
PubMed: 35721505
DOI: 10.3389/fcell.2022.826576 -
Microbiology Spectrum Aug 2022Streptococcus mutans is a primary cariogenic pathogen in humans. Arginine metabolism is required for bacterial growth. In S. mutans, however, the involvement of...
Streptococcus mutans is a primary cariogenic pathogen in humans. Arginine metabolism is required for bacterial growth. In S. mutans, however, the involvement of transcription factors in regulating arginine metabolism is unclear. The purpose of this study was to investigate the function and mechanism of ArgR family transcription factors in S. mutans. Here, we identified an ArgR (arginine repressor) family transcription factor named AhrC, which negatively regulates arginine biosynthesis and biofilm formation in S. mutans. The in-frame deletion strain exhibited slow growth and significantly increased intracellular arginine content. The strain overexpressing showed reduced intracellular arginine content, decreased biofilm biomass, reduced production of water-insoluble exopolysaccharides (EPS), and different biofilm structures. Furthermore, global gene expression profiles revealed differential expression levels of 233 genes in the -deficient strain, among which genes related to arginine biosynthesis (, , , , , , ) were significantly upregulated. In the overexpression strain, there are 89 differentially expressed genes, mostly related to arginine biosynthesis. The conserved DNA patterns bound by AhrC were identified by electrophoretic mobility shift assay (EMSA) and DNase I footprinting. In addition, the analysis of β-galactosidase activity showed that AhrC acted as a negative regulator. Taken together, our findings suggest that AhrC is an important transcription factor that regulates arginine biosynthesis gene expression and biofilm formation in S. mutans. These findings add new aspects to the complexity of regulating the expression of genes involved in arginine biosynthesis and biofilm formation in S. mutans. Arginine metabolism is essential for bacterial growth. The regulation of intracellular arginine metabolism in Streptococcus mutans, one of the major pathogens of dental caries, is unclear. In this study, we found that the transcription factor AhrC can directly and negatively regulate the expression of -acetyl-gamma-glutamyl-phosphate reductase (), thus regulating arginine biosynthesis in S. mutans. In addition, the overexpression strain exhibited a significant decrease in biofilm and water-insoluble extracellular polysaccharides (EPS). This study adds new support to our understanding of the regulation of intracellular arginine metabolism in S. mutans.
Topics: Arginine; Bacterial Proteins; Biofilms; Dental Caries; Gene Expression Regulation, Bacterial; Humans; Streptococcus mutans; Transcription Factors; Water
PubMed: 35938859
DOI: 10.1128/spectrum.00721-22 -
Methods in Molecular Biology (Clifton,... 2020Transposons are found in a wide variety of forms throughout the prokaryotic world where they actively contribute to the adaptive strategies of bacterial communities and...
Transposons are found in a wide variety of forms throughout the prokaryotic world where they actively contribute to the adaptive strategies of bacterial communities and hence, to the continuous emergence of new multiresistant pathogens. Contrasting with their biological and societal impact, only a few bacterial transposons have been the subject of detailed molecular studies. In this chapter, we propose a set of reliable biochemical methods as a primary route for studying new transposition mechanisms. These methods include (a) a straightforward approach termed "thermal shift induction" to produce the transposase in a soluble and properly folded configuration prior to its purification, (b) an adaptation of classical electrophoretic mobility shift assays (EMSA) combined to fluorescently labeled DNA substrates to determine the DNA content of different complexes assembled by the transposase, and (c) a highly sensitive "in-gel" DNA footprinting assay to further characterize these complexes at the base pair resolution level. A combination of these approaches was recently applied to decipher the molecular organization of key intermediates in the Tn3-family transposition pathway, a mechanism that has long been refractory to biochemical studies.
Topics: Bacteria; Bacterial Physiological Phenomena; DNA Transposable Elements; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Gene Expression; Macromolecular Substances; Protein Binding; Staining and Labeling; Temperature; Transposases
PubMed: 31584162
DOI: 10.1007/978-1-4939-9877-7_12 -
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 -
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