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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.

DNA–protein interaction study has always been a topic of interest for scientists as it is crucial for the cell to survive, differentiate, and divide. It helps the cell to conduct different major activities like replication, transcription, translation, DNA repair and recombination, and RNA processing. A lot of research has already been conducted to understand how the DNA–protein interaction occurs inside the cell. In this chapter, we discuss the major requirements for DNA–protein interaction to occur, several detection methods for DNA–protein interaction, their limitations, and advantages. This study will give a better understanding of the vital biological processes occurring inside the cell to understand the process of cell growth, development, and disease.

ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) provides an efficient way to analyze nucleosome-free regions and has been applied widely to identify transcription factor footprints. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq in our study. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at https://github.com/tzhu-bio/UMI-ATAC-seq., Tao Zhu et al. present UMI-ATAC-seq, an improved ATAC-seq technique that uses unique molecular identifiers to avoid filtering out true independent - but identical - Tn5 insertion events that would otherwise be treated as PCR duplicates. They apply UMI-ATAC-seq to rice panicle samples and show that their approach improves the quantification of chromatin accessibility and footprinting.

The in vivo footprinting method identifies protein-targeted DNA regions under different conditions such as carbon sources. Dimethyl sulfate (DMS) generates methylated purine bases at DNA sites which are not bound by proteins or transcription factors. The DNA is cleaved by HCl, and the resulting DNA fragments are 5'-end [6-FAM]-labeled by a linker-mediated PCR (LM-PCR). Fluorescent fragments are separated and analyzed on a capillary sequencer, followed by automated data analysis using the software tool ivFAST.

Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this damage, DNA models that mimic the in vivo situation should be utilized. In a cellular context, genomic DNA constantly interacts with proteins and these interactions could influence both the primary radical processes (triggered by ionizing radiation) and secondary reactions, ultimately leading to DNA damage. However, this is seldom addressed in the literature. In this work, we propose a general approach to tackle these shortcomings. We synthesized a protein-DNA complex that more closely represents DNA in the physiological environment than oligonucleotides solution itself, while being sufficiently simple to permit further chemical analyses. Using click chemistry, we obtained an oligonucleotide-peptide conjugate, which, if annealed with the complementary oligonucleotide strand, forms a complex that mimics the specific interactions between the GCN4 protein and DNA. The covalent bond connecting the oligonucleotide and peptide constitutes a part of substituted triazole, which forms due to the click reaction between the short peptide corresponding to the specific amino acid sequence of GCN4 protein (yeast transcription factor) and a DNA fragment that is recognized by the protein. DNAse footprinting demonstrated that the part of the DNA fragment that specifically interacts with the peptide in the complex is protected from DNAse activity. Moreover, the thermodynamic characteristics obtained using differential scanning calorimetry (DSC) are consistent with the interaction energies calculated at the level of metadynamics. Thus, we present an efficient approach to generate a well-defined DNA-peptide conjugate that mimics a real DNA-peptide complex. These complexes can be used to investigate DNA damage under conditions very similar to those present in the cell.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes., The authors summarize the data produced by phase III of the Encyclopedia of DNA Elements (ENCODE) project, a resource for better understanding of the human and mouse genomes.

Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.

Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.

International audience; 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.

SNAIL1 is a key regulator of epithelial-mesenchymal transition (EMT) and its expression is associated with tumor progression and poor clinical prognosis of cancer patients. Compared to the studies of SNAIL1 stability and its transcriptional regulation, very limited knowledge is available regarding effective approaches to directly target SNAIL1. In this study, we revealed the potential regulation of SNAIL1 gene expression by G-quadruplex structures in its promoter. We first revealed that the negative strand of the SNAIL1 promoter contained a multi-G-tract region with high potential of forming G-quadruplex structures. In circular dichroism studies, the oligonucleotide based on this region showed characteristic molar ellipticity at specific wavelengths of G-quadruplex structures. We also utilized native polyacrylamide gel electrophoresis, gel-shift assays, immunofluorescent staining, dimethyl sulfate footprinting and chromatin immunoprecipitation studies to verify the G-quadruplex structures formed by the oligonucleotide. In reporter assays, disruption of G-quadruplex potential increased SNAIL1 promoter-mediated transcription, suggesting that G-quadruplexes played a negative role in SNAIL1 expression. In a DNA synthesis study, we detected G-quadruplex-mediated retardation in the SNAIL1 promoter replication. Consistently, we discovered that the G-quadruplex region of the SNAIL1 promoter is highly enriched for mutations, implicating the clinical relevance of G-quadruplexes to the altered SNAIL1 expression in cancer cells.

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 32P-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.

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.

G-quadruplexes (G4s) are four-stranded DNA structures comprising stacks of four guanines, are prevalent in genomes, and have diverse biological functions in various chromosomal structures. A conserved protein, Rap1-interacting factor 1 (Rif1) from fission yeast (Schizosaccharomyces pombe), binds to Rif1-binding sequence (Rif1BS) and regulates DNA replication timing. Rif1BS is characterized by the presence of multiple G-tracts, often on both strands, and their unusual spacing. Although previous studies have suggested generation of G4-like structures on duplex Rif1BS, its precise molecular architecture remains unknown. Using gel-shift DNA binding assays and DNA footprinting with various nuclease probes, we show here that both of the Rif1BS strands adopt specific higher-order structures upon heat denaturation. We observed that the structure generated on the G-strand is consistent with a G4 having unusually long loop segments and that the structure on the complementary C-strand does not have an intercalated motif (i-motif). Instead, we found that the formation of the C-strand structure depends on the G4 formation on the G-strand. Thus, the higher-order structure generated at Rif1BS involved both DNA strands, and in some cases, G4s may form on both of these strands. The presence of multiple G-tracts permitted the formation of alternative structures when some G-tracts were mutated or disrupted by deazaguanine replacement, indicating the robust nature of DNA higher-order structures generated at Rif1BS. Our results provide general insights into DNA structures generated at G4-forming sequences on duplex DNA.

Prion diseases are neurodegenerative disorders that affect many mammalian species. Mammalian prion proteins (PrPs) can misfold into many different aggregates. However, only a small subpopulation of these structures is infectious. One of the major unresolved questions in prion research is identifying which specific structural features of these misfolded protein aggregates are important for prion infectivity in vivo. Previously, two types of proteinase K–resistant, self-propagating aggregates were generated from the recombinant mouse prion protein in the presence of identical cofactors. Although these two aggregates appear biochemically very similar, they have dramatically different biological properties, with one of them being highly infectious and the other one lacking any infectivity. Here, we used several MS-based structural methods, including hydrogen–deuterium exchange and hydroxyl radical footprinting, to gain insight into the nature of structural differences between these two PrP aggregate types. Our experiments revealed a number of specific differences in the structure of infectious and noninfectious aggregates, both at the level of the polypeptide backbone and quaternary packing arrangement. In particular, we observed that a high degree of order and stability of β-sheet structure within the entire region between residues ∼89 and 227 is a primary attribute of infectious PrP aggregates examined in this study. By contrast, noninfectious PrP aggregates are characterized by markedly less ordered structure up to residue ∼167. The structural constraints reported here should facilitate development of experimentally based high-resolution structural models of infectiosus mammalian prions.

Agrobacterium tumefaciens AcrR is the transcriptional repressor of the acrABR operon. The AcrAB efflux pump confers resistance to various toxic compounds, including antibiotics [ciprofloxacin (CIP), nalidixic acid (NAL), novobiocin (NOV) and tetracycline (TET)], a detergent [sodium dodecyl sulfate (SDS)] and a biocide [triclosan (TRI)]. The sequence to which AcrR specifically binds in the acrA promoter region was determined by EMSA and DNase I footprinting. The AcrR-DNA interaction was abolished by adding NAL, SDS and TRI. Quantitative real time-PCR analysis showed that induction of the acrA transcript occurred when wild-type cells were exposed to NAL, SDS and TRI. Indole is a signaling molecule that increases the antibiotic resistance of bacteria, at least in part, through activation of efflux pumps. Expression of the A. tumefaciens acrA transcript was also inducible by indole in a dose-dependent manner. Indole induced protection against CIP, NAL and SDS but enhanced susceptibility to NOV and TRI. Additionally, the TET resistance of A. tumefaciens was not apparently modulated by indole. A. tumefaciens AcrAB played a dominant role and was required for tolerance to high levels of the toxic compounds. Understanding the regulation of multidrug efflux pumps and bacterial adaptive responses to intracellular and extracellular signaling molecules for antibiotic resistance is essential. This information will be useful for the rational design of effective treatments for bacterial infection to overcome possible multidrug-resistant pathogens.

MprAB and PhoPR are important two-component systems (TCSs) in Mycobacterium tuberculosis, and both regulate EspR, a key regulator of the ESX-1 secretion system. Although previous studies suggest that the response regulator PhoP does not directly regulate mprA, the interplay between MprAB and PhoPR remains unclear. In this study, we found that the response regulator MprA can bind to the phoP promoter. Four repeat motifs, D1-D4, constituting two predicted binding sites, were located in the region protected by MprA in DNA footprinting. D1-D4 lack the reported conserved MprA binding sequences, indicating that MprA can recognize a greater range of target sites. Interestingly, D1-D2 overlap a previously reported PhoP binding site, and mutation of D1-D2 inhibited PhoP binding, whereas the D3-D4 site, but not the D1-D2 site, was required for MprA binding. EMSA assays also suggest that MprA and PhoP compete to bind to the phoP promoter. The results of the transcriptional and western blot assays are consistent with a model in which MprA positively controls the phoP expression, which in turn upregulates the expression of espR. These findings reveal complex regulation of a major mycobacterial TCS by dual TCSs.

Hydroxyl radical footprinting (HRF) of proteins with mass spectrometry (MS) is a widespread approach for assessing protein structure. Hydroxyl radicals react with a wide variety of protein side chains, and the ease with which radicals can be generated (by radiolysis or photolysis) has made the approach popular with many laboratories. As some side chains are less reactive and thus cannot be probed, additional specific and nonspecific labeling reagents have been introduced to extend the approach. At the same time, advances in liquid chromatography and MS approaches permit an examination of the labeling of individual residues, transforming the approach to high resolution. Lastly, advances in understanding of the chemistry of the approach have led to the determination of absolute protein topologies from HRF data. Overall, the technology can provide precise and accurate measures of side-chain solvent accessibility in a wide range of interesting and useful contexts for the study of protein structure and dynamics in both academia and industry. Expected final online publication date for the Annual Review of Biophysics Volume 47 is May 20, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Significance One-tenth of human genes produce proteins called transcription factors (TFs) that bind to our genome and read the local DNA sequence. They work together to regulate the degree to which each gene is expressed. The affinity with which DNA is bound by a particular TF can vary more than a thousand-fold with different DNA sequences. This study presents the first computational method able to quantify the sequence-affinity relationship almost perfectly over the full affinity range. It achieves this by analyzing data from experiments that use massively parallel DNA sequencing to comprehensively probe protein–DNA interactions. Strikingly, it can accurately predict the effect in vivo of DNA mutations on gene expression levels in fly embryos even for very-low-affinity binding sites., Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Aberrant transcription factors (TFs) activities are closely related to the occurrence and development of various diseases. Herein, we presented a fluorescence-encoded microsphere-based approach for TFs detection coupling with common DNA footprinting assay. Target TFs specifically bound the binding sites of double-stranded DNA (dsDNA) probes which were conjugated to microspheres. Thus, the probes were protected from being hydrolyzed by exonuclease III (Exo III). Afterwards, biotins labeled on the probes reacted with streptavidin-phycoerythrin (SA-PE) to produce fluorescent signal; however, in the absence of target TFs, the dsDNA probes would be hydrolyzed by Exo III resulting in biotins falling off and thus fluorescence signal was not generated. This strategy can be used to detect nuclear factor-kappa B p50 (NF-κB p50) with a detection limit of 0.2 nM. The steric hindrance of microspheres overcome the disadvantage of Exo III that can nibble into the protein-bound DNA region. Meanwhile, the fluorescent label of microsphere was specific to each TF, enabling multiplex detection could be achieved by changing specific protein binding site of corresponding dsDNA probe. This method has been successfully applied for simultaneous detection of NF-κB p50, AP-1 and CREB in nuclear extract isolated from HeLa cells stimulated or unstimulated by TNF-α, showing great potential for biomedical researches and precise disease diagnosis.

If unrepaired, damage to genomic DNA can cause mutations and/or be cytotoxic. Single base lesions are repaired via the base excision repair (BER) pathway. The first step in BER is the recognition and removal of the nucleobase lesion by a glycosylase enzyme. For example, human oxoguanine glycosylase 1 (hOGG1) is responsible for removal of the prototypic oxidatively damaged nucleobase, 8-oxo-7,8-dihydroguanine (8-oxoG). To date, most studies of glycosylases have used free duplex DNA substrates. However, cellular DNA is packaged as repeating nucleosome units, with 145 base pair segments of DNA wrapped around histone protein octamers. Previous studies revealed inhibition of hOGG1 at the nucleosome dyad axis and in the absence of chromatin remodelers. In this study, we reveal that even in the absence of chromatin remodelers or external cofactors, hOGG1 can initiate BER at positions off the dyad axis and that this activity is facilitated by spontaneous and transient unwrapping of DNA from the histones. Additionally, we find that solution accessibility as determined by hydroxyl radical footprinting is not fully predictive of glycosylase activity and that histone tails can suppress hOGG1 activity. We therefore suggest that local nuances in the nucleosome environment and histone-DNA interactions can impact glycosylase activity.

Summary Protein-DNA interactions provide the basis for chromatin structure and gene regulation. Comprehensive identification of protein-occupied sites is thus vital to an in-depth understanding of genome function. Dimethyl sulfate (DMS) is a chemical probe that has long been used to detect footprints of DNA-bound proteins in vitro and in vivo. Here, we describe a genomic footprinting method, dimethyl sulfate sequencing (DMS-seq), which exploits the cell-permeable nature of DMS to obviate the need for nuclear isolation. This feature makes DMS-seq simple in practice and removes the potential risk of protein re-localization during nuclear isolation. DMS-seq successfully detects transcription factors bound to cis -regulatory elements and non-canonical chromatin particles in nucleosome-free regions. Furthermore, an unexpected preference of DMS confers on DMS-seq a unique potential to directly detect nucleosome centers without using genetic manipulation. We expect that DMS-seq will serve as a characteristic method for genome-wide interrogation of in vivo protein-DNA interactions.

The eponymous DNA-binding domain of ETS (E26 transformation specific) transcription factors binds a single sequence-specific site as a monomer over a single helical turn. Following our previous observation by titration calorimetry that the ETS member PU.1 dimerizes sequentially at a single sequence-specific DNA binding site to form a 2:1 complex, we have carried out an extensive spectroscopic and biochemical characterization of site-specific PU.1 ETS complexes. While 10 bp of DNA was sufficient to support PU.1 binding as a monomer, additional flanking bases were required to invoke sequential dimerization of the bound protein. NMR spectroscopy revealed a marked loss of signal intensity in the 2:1 complex, and mutational analysis implicated the distal surface away from the bound DNA as the dimerization interface. Hydroxyl radical DNA footprinting indicated that the site-specifically bound PU.1 dimers occupied an extended DNA interface downstream from the 5'-GGAA-3' core consensus relative to its 1:1 counterpart, thus explaining the apparent site size requirement for sequential dimerization. The site-specifically bound PU.1 dimer resisted competition from nonspecific DNA and showed with affinities similar to other functionally significant PU.1 interactions. As sequential dimerization did not occur with the ETS domain of Ets-1, a close structural homolog of PU.1, 2:1 complex formation may represent an alternative auto-inhibitory mechanism in the ETS family at the protein-DNA level.

DNA sequences that are guanine-rich have received considerable attention because of their potential to fold into a secondary, four-stranded DNA structure termed G-quadruplex (G4), which has been implicated in genomic instability and some human diseases. We have previously identified positive coactivator of transcription (PC4), a single-stranded DNA (ssDNA)-binding protein, as a novel G4 interactor. Here, to expand on these previous observations, we biochemically and biophysically characterized the interaction between PC4 and G4DNA. PC4 can bind alternative G4DNA topologies with a low nanomolar Kd value of ∼2 nm, similar to that observed for ssDNA. In consideration of the different structural features between G4DNA and ssDNA, these binding data indicated that PC4 can interact with G4DNA in a manner distinct from ssDNA. The stoichiometry of the PC4-G4 complex was 1:1 for PC4 dimer:G4 substrate. PC4 did not enhance the rate of folding of G4DNA, and formation of the PC4-G4DNA complex did not result in unfolding of the G4DNA structure. We assembled a G4DNA structure flanked by duplex DNA. We find that PC4 can interact with this G4DNA, as well as the complementary C-rich strand. Molecular docking simulations and DNA footprinting experiments suggest a model where a PC4 dimer accommodates the DNA with one monomer on the G4 strand and the second monomer bound to the C-rich strand. Collectively, these data provide a novel mode of PC4 binding to a DNA secondary structure that remains within the framework of the model for binding to ssDNA. Additionally, consideration of the PC4-G4DNA interaction could provide insight into the biological functions of PC4, which remain incompletely understood.

RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginning productive elongation. This pause is a key component of metazoan gene expression regulation. Some promoters have a strong disposition for Pol II pausing and often mediate faster, more synchronous changes in expression. This requires multiple rounds of transcription and thus cannot rely solely on pause release. However, it is unclear how pausing affects the initiation of new transcripts during consecutive rounds of transcription. Using our recently developed ChIP-nexus method, we find that Pol II pausing inhibits new initiation. We propose that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise.

Mass spectrometry-based protein footprinting emerged as a useful technology to understand protein ligand interactions in vitro. We have previously demonstrated the application of footprinting in live E. coli cells. Here, we further optimized an ultrafast laser photolysis hydroxyl radical footprinting method and applied it to study the interaction of EGF and EGFR in live mammalian cells. This method used a nanosecond laser to photochemically generate a burst of hydroxyl radicals in situ in-cell suspension to oxidize the amino acids on the protein surface. Mass spectrometric analysis of the thus modified peptides was interpreted to probe the solvent-accessible surface areas of the protein in its native biological state with and without EGF activation. Our footprinting data agreed with the two relevant EGFR crystal structures, indicating that this in-cell laser photolysis footprinting technique is a valid approach to study the structural properties of integral membrane proteins directly in the native environment.

Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei.

Protein footprinting mediated by mass spectrometry has evolved over the last 30 years from proof of concept to commonplace biophysics tool, with unique capabilities for assessing structure and dynamics of purified proteins in physiological states in solution. This review outlines the history and current capabilities of two major methods of protein footprinting: reversible hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (HRF), an irreversible covalent labeling approach. Technological advances in both approaches now permit high-resolution assessments of protein structure including secondary and tertiary structure stability mediated by backbone interactions (measured via HDX) and solvent accessibility of side chains (measured via HRF). Applications across many academic fields and in biotechnology drug development are illustrated including: detection of protein interfaces, identification of ligand/drug binding sites, and monitoring dynamics of protein conformational changes along with future prospects for advancement of protein footprinting in structural biology and biophysics research.

AR9 is a giant Bacillus subtilis phage whose uracil-containing double-stranded DNA genome encodes distant homologs of β and β’ subunits of bacterial RNA polymerase (RNAP). The products of these genes are thought to assemble into two non-canonical multisubunit RNAPs - a virion RNAP (vRNAP) that is injected into the host along with phage DNA to transcribe early phage genes, and a non-virion RNAP (nvRNAP), which is synthesized during the infection and transcribes late phage genes. We purified the AR9 nvRNAP from infected B. subtilis cells and characterized its transcription activity in vitro. The AR9 nvRNAP requires uracils rather than thymines at specific conserved positions of late viral promoters. Uniquely, the nvRNAP recognizes the template strand of its promoters and is capable of specific initiation of transcription from both double- and single-stranded DNA. While the AR9 nvRNAP does not contain homologs of bacterial RNAP α subunits, it contains, in addition to the β and β’-like subunits, a phage protein gp226. The AR9 nvRNAP lacking gp226 is catalytically active but unable to bind to promoter DNA. Thus, gp226 is required for promoter recognition by the AR9 nvRNAP and may represent a new group of transcription initiation factors.

The alaE gene in Escherichia coli encodes an l -alanine exporter that catalyzes the active export of l -alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l -alanyl- l -alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less β-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ -deficient and harbored this reporter plasmid showed significant induction of β-galactosidase activity (approximately 17-fold) in the presence of 6 mM l -alanine, l -leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, lrp -deficient cells showed almost no β-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide −161 to −83 with a physiologically relevant affinity (apparent K D , 288.7 ± 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis -element was increased by l -alanine and l -leucine, but not by Ala-Ala and d -alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of l -alanine, which eventually leads to intracellular homeostasis of l -alanine concentrations.

Background Fibroblast growth factor receptor 2 (FGFR2) is overexpressed in breast cancer tissues and cells, and has been shown to be a susceptibility factor for breast cancer. In this study, we found that the G-rich sequences in the FGFR2 promoter region can form G-quadruplexes, which could be the target and inhibitor of the FGFR2 gene. Methods Initially, the formation of G-quadruplexes was confirmed by ESI-MS and CD, and DMS footprinting experiments gave the folding pattern of the G-quadruplexes. After luciferase reporter assays revealed that the G-quadruplex could inhibit the activity of the FGFR2 promoter, MS and SPR showed binding affinity and selectivity of the ligand. Then cell culture experiments and ChIP assay showed the bioactivity of the ligand. Results We found that three G-rich sequences (S1–S3) in the FGFR2 promoter region can form G-quadruplex structures. And a natural alkaloid, liensinine, was found to bind to the S1 G-quadruplex with relative high affinity and selectivity. Cell culture experiments showed that liensinine inhibits FGFR2 activity at both the transcriptional and translational levels. Moreover, chromatin immunoprecipitation assay (ChIP) results showed that liensinine blocks the binding of E2F1 at the transcription factor binding site (TFBS) in the S1 sequence, which is the mechanism through which liensinine inhibits the FGFR2 gene. Conclusions A natural alkaloid was discovered to selectively bind to the S1 G-quadruplex with relative high affinity, and therefore inhibited FGFR2 transcription and translation. General significance Our discovery offers a useful strategy to inhibit FGFR2 transcription, i.e., targeting the G-quadruplex with a natural alkaloid.

The mitochondrial transcription initiation machinery in humans consists of three proteins: the RNA polymerase (POLRMT) and two accessory factors, transcription factors A and B2 (TFAM and TFB2M, respectively). This machinery is required for the expression of mitochondrial DNA and the biogenesis of the oxidative phosphorylation system. Previous experiments suggested that TFB2M is required for promoter melting, but conclusive experimental proof for this effect has not been presented. Moreover, the role of TFB2M in promoter unwinding has not been discriminated from that of TFAM. Here we used potassium permanganate footprinting, DNase I footprinting, and in vitro transcription from the mitochondrial light-strand promoter to study the role of TFB2M in transcription initiation. We demonstrate that a complex composed of TFAM and POLRMT was readily formed at the promoter but alone was insufficient for promoter melting, which only occurred when TFB2M joined the complex. We also show that mismatch bubble templates could circumvent the requirement of TFB2M, but TFAM was still required for efficient initiation. Our findings support a model in which TFAM first recruits POLRMT to the promoter, followed by TFB2M binding and induction of promoter melting.

Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro. This technique is mainly dependent on antibody-antigen immunity, simple and convenient, while radioactive isotope labeling and optimization of partial degradation by deoxyribonuclease (DNase) are avoided. As an example, the specific binding sequence of a target promoter by DdrO (a DNA damage response protein from Deinococcus radiodurans) in vitro was determined by the developed method. The central sequence of the binding site could be easily located using this technique.

DNA replication requires that the duplex genomic DNA strands be separated; a function that is implemented by ring-shaped hexameric helicases in all Domains. Helicases are composed of two domains, an N- terminal DNA binding domain (NTD) and a C- terminal motor domain (CTD). Replication is controlled by loading of helicases at origins of replication, activation to preferentially encircle one strand, and then translocation to begin separation of the two strands. Using a combination of site-specific DNA footprinting, single-turnover unwinding assays, and unique fluorescence translocation monitoring, we have been able to quantify the binding distribution and the translocation orientation of Saccharolobus (formally Sulfolobus) solfataricus MCM on DNA. Our results show that both the DNA substrate and the C-terminal winged-helix (WH) domain influence the orientation but that translocation on DNA proceeds N-first.

Transcription is tightly regulated by cis-regulatory DNA elements where transcription factors can bind. Thus, identification of transcription factor binding sites is key to understanding gene expression and whole regulatory networks within a cell. The standard approaches for transcription factor binding sites (TFBSs) prediction such as position weight matrices (PWMs) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) are widely used but have their drawbacks such as high false positive rates and limited antibody availability, respectively. Several computational footprinting algorithms have been developed to detect TFBSs by investigating chromatin accessibility patterns, but also have their limitations. To improve on these methods, we have developed a footprinting method to predict Transcription factor footpRints in Active Chromatin Elements (TRACE). Trace incorporates DNase-seq data and PWMs within a multivariate Hidden Markov Model (HMM) to detect footprint-like regions with matching motifs. Trace is an unsupervised method that accurately annotates binding sites for specific TFs automatically with no requirement on pre-generated candidate binding sites or ChIP-seq training data. Compared to published footprinting algorithms, TRACE has the best overall performance with the distinct advantage of targeting multiple motifs in a single model.

Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed “remosomes”. Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.

In vitro transcription of double-stranded DNA (dsDNA) induces a formation of two types of G-quadruplexes, intramolecular DNA G-quadruplexes or DNA:RNA hybrid G-quadruplexes, in clusters of guanine tracts. The formation of G-quadruplexes can be characterized by native PEG polyacrylamide gel electrophoresis, DMS footprinting, and ligand-induced photocleavage footprinting. The type of G-quadruplexes can be distinguished by transcription with 7-deaza-GTP (dzGTP) or 4-thio-uridine-5'-triphosphate (4

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes., Author summary Cholera remains a devastating diarrhoeal disease responsible for millions of cases, thousands of deaths, and a $3 billion financial burden every year. Although notorious for causing human disease, the microorganism responsible for cholera is predominantly a resident of aquatic environments. Here, the organism survives in densely packed communities on the surfaces of crustaceans. Remarkably, in this situation, the microbe can feast on neighbouring cells and acquire their DNA. This provides a useful food source and an opportunity to obtain new genetic information. In this paper, we have investigated how acquisition of DNA from the local environment is regulated. We show that a “switch” within the microbial cell, known to activate disease processes in the human host, also controls DNA uptake. Our results explain why DNA scavenging only occurs in suitable environments and illustrates how interactions between common regulatory switches affords precise control of microbial behaviours.

Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.

Type VI secretion systems (T6SSs) are highly conserved and complex protein secretion systems that deliver effector proteins into eukaryotic hosts or other bacteria. T6SSs are regulated precisely by a variety of regulatory systems, which enables bacteria to adapt to varied environments. A T6SS within Salmonella pathogenicity island 6 (SPI-6) is activated during infection, and it contributes to the pathogenesis, as well as interbacterial competition, of Salmonella enterica serovar Typhimurium (S. Typhimurium). However, the regulation of the SPI-6 T6SS in S. Typhimurium is not well understood. In this study, we found that the SPI-6 T6SS core gene clpV was significantly upregulated in response to the iron-depleted condition and during infection. The global ferric uptake regulator (Fur) was shown to repress the clpV expression in the iron-replete medium. Moreover, electrophoretic mobility shift and DNase I footprinting assays revealed that Fur binds directly to the clpV promoter region at multiple sites spanning the transcriptional start site. We also observed that the relieving of Fur-mediated repression on clpV contributed to the interbacterial competition activity and pathogenicity of S. Typhimurium. These findings provide insights into the direct regulation of Fur in the expression and functional activity of SPI-6 T6SS in S. Typhimurium and thus help to elucidate the mechanisms of bacterial adaptability and virulence.

Fast photochemical oxidation of proteins (FPOP) is a MS-based method that has proved useful in studies of protein structures, interactions, conformations, and protein folding. The success of this method relies on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. FPOP generates these radicals through laser-induced photolysis of hydrogen peroxide. The data obtained provide residue-level resolution of protein structures and interactions on the microsecond timescale, enabling investigations of fast processes such as protein folding and weak protein–protein interactions. An extensive comparison between FPOP and other footprinting techniques gives insight on their complementarity as well as the robustness of FPOP to provide unique structural information once unattainable. The versatility of this method is evidenced by both the heterogeneity of samples that can be analyzed by FPOP and the myriad of applications for which the method has been successfully used: from proteins of varying size to intact cells. This review discusses the wide applications of this technique and highlights its high potential. Applications including, but not limited to, protein folding, membrane proteins, structure elucidation, and epitope mapping are showcased. Furthermore, the use of FPOP has been extended to probing proteins in cells and in vivo. These promising developments are also presented herein.

[Image: see text] Protein footprinting coupled with mass spectrometry is being increasingly used for the study of protein interactions and conformations. The hydroxyl radical footprinting method, fast photochemical oxidation of proteins (FPOP), utilizes hydroxyl radicals to oxidatively modify solvent accessible amino acids. Here, we describe the further development of FPOP for protein structural analysis in vivo (IV-FPOP) with Caenorhabditis elegans. C. elegans, part of the nematode family, are used as model systems for many human diseases. The ability to perform structural studies in these worms would provide insight into the role of structure in disease pathogenesis. Many parameters were optimized for labeling within the worms including the microfluidic flow system and hydrogen peroxide concentration. IV-FPOP was able to modify several hundred proteins in various organs within the worms. The method successfully probed solvent accessibility similarily to in vitro FPOP, demonstrating its potential for use as a structural technique in a multiorgan system. The coupling of the method with mass spectrometry allows for amino-acid-residue-level structural information, a higher resolution than currently available in vivo methods.