scholarly journals Alignment and quantification of ChIP-exo crosslinking patterns reveal the spatial organization of protein-DNA complexes

2019 ◽  
Author(s):  
Naomi Yamada ◽  
Matthew J. Rossi ◽  
Nina Farrell ◽  
B. Franklin Pugh ◽  
Shaun Mahony
Keyword(s):  
Spatial Organization ◽  
Regulatory Protein ◽  
Physical Distance ◽  
Trna Genes ◽  
Ribosomal Protein Genes ◽  
Dna Complexes ◽  
Regulatory Complex ◽  
Dna Crosslinking ◽  
Exonuclease Digestion ◽  
Dna Complex

AbstractThe ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5′ to 3′ exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between the subunits of a regulatory complex. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. We demonstrate that our approach can recover aspects of regulatory complex spatial organization when applied to collections of ChIP-exo data that profile regulatory machinery at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein-DNA complex organization across conditions by applying our approach to data profiling Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.

scholarly journals Alignment and quantification of ChIP-exo crosslinking patterns reveal the spatial organization of protein–DNA complexes

2020 ◽  
Vol 48 (20) ◽  
pp. 11215-11226
Author(s):  
Naomi Yamada ◽  
Matthew J Rossi ◽  
Nina Farrell ◽  
B Franklin Pugh ◽  
Shaun Mahony
Keyword(s):  
Spatial Organization ◽  
Regulatory Protein ◽  
Physical Distance ◽  
Trna Genes ◽  
Ribosomal Protein Genes ◽  
Dna Complexes ◽  
Regulatory Complex ◽  
Dna Crosslinking ◽  
Exonuclease Digestion ◽  
Dna Complex

Abstract The ChIP-exo assay precisely delineates protein–DNA crosslinking patterns by combining chromatin immunoprecipitation with 5′ to 3′ exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein–DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between its subunits. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein–DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein–DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. Applying our approach to collections of ChIP-exo data, we demonstrate that it can recover aspects of regulatory complex spatial organization at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein–DNA complex organization across conditions by applying our approach to analyze Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein–DNA complexes.

scholarly journals DNA-Dependent Oligomerization of Herpes Simplex Virus Type 1 Regulatory Protein ICP4

2007 ◽  
Vol 81 (17) ◽  
pp. 9230-9237 ◽  
Author(s):  
Ruhul H. Kuddus ◽  
Neal A. DeLuca
Keyword(s):  
Herpes Simplex ◽  
Binding Site ◽  
Binding Sites ◽  
Regulatory Protein ◽  
Dna Probes ◽  
Dna Complexes ◽  
Weak Binding ◽  
Simplex Virus ◽  
Dna Complex

ABSTRACT The human herpes simplex virus type 1 regulatory protein ICP4 binds DNA as a dimer and forms a single protein-DNA complex (A complex) with short DNA probes. ICP4 oligomerized in a DNA-dependent manner, forming two or more protein-DNA complexes with longer DNA fragments containing a single DNA binding site. When resolved electrophoretically, one or more low-mobility DNA-protein complexes follow the fast-moving A complex. The major protein-DNA complex (B complex) formed by ICP4 with long DNA probes migrates just behind the A complex in the electric field, implying the oligomerization of ICP4 on the DNA. Binding experiments with circularly permutated DNA probes containing one ICP4 binding site revealed that about 70 bp of nonspecific DNA downstream of the cognate ICP4 binding site was required for efficient B complex formation. In addition, the C-terminal domain of ICP4 was found to be required for DNA-dependent oligomerization and B complex formation. Gel mobility shift analysis of protein-DNA complexes, combined with supershift analysis using different monoclonal antibodies, indicated that the B complex contained two ICP4 dimers. DNase I footprinting of ICP4-DNA complexes showed that one ICP4 dimer contacts the specific binding site and another ICP4 dimer contacts nonspecific DNA in the B complex. DNA-dependent oligomerization increased the affinity of ICP4 for relatively weak binding sites on large DNA molecules. The results of this study suggest how ICP4 may use multiple weak binding sites to aid in transcription activation.

Identification of a 150-kilodalton polypeptide that copurifies with yeast TFIIIC and binds specifically to tRNA genes

1989 ◽  
Vol 9 (5) ◽  
pp. 2018-2024
Author(s):  
D L Johnson ◽  
S L Wilson
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Iii ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Trna Genes ◽  
Dna Binding Activity ◽  
Transcription In Vitro ◽  
Affinity Interaction ◽  
Dna Complex

The transcription in vitro of eucaryotic tRNA genes by RNA polymerase III requires two transcription factors, designated TFIIIB and TFIIIC. One of the critical functions of TFIIIC in the transcription of tRNA genes is that it interacts directly and specifically with the two internal promoter elements of these genes. We have partially purified Saccharomyces cerevisiae TFIIIC by chromatography on Bio-Rex 70, DEAE-cellulose, and phosphocellulose resins. A 150-kilodalton (kDa) DNA-binding polypeptide copurified with TFIIIC activity. This 150-kDa protein coeluted with the DNA-binding activity of TFIIIC after rechromatography of TFIIIC on phosphocellulose and its elution with a linear salt gradient. The stable and high-affinity interaction of this protein with tRNA genes was demonstrated by the maintenance of a protein-DNA complex under conditions of high ionic strength. Finally, we showed by two criteria that the interaction of this protein with tRNA genes was specific. First, the protein-DNA complex was competed with only by DNA-containing tRNA genes; second, the protein preferentially bound to DNA fragments containing a tRNA gene. These results strongly suggest that the DNA-binding domain of the yeast TFIIIC is contained within this 150-kDa polypeptide.

scholarly journals Paramyxovirus V Proteins Interact with the RIG-I/TRIM25 Regulatory Complex and Inhibit RIG-I Signaling

2018 ◽  
Vol 92 (6) ◽  
Author(s):  
Maria T. Sánchez-Aparicio ◽  
Leighland J. Feinman ◽  
Adolfo García-Sastre ◽  
Megan L. Shaw
Keyword(s):  
Signaling Pathway ◽  
Measles Virus ◽  
Sendai Virus ◽  
Regulatory Protein ◽  
Nipah Virus ◽  
Antiviral Signaling ◽  
V Protein ◽  
Protein Functions ◽  
Regulatory Complex ◽  
Mitochondrial Antiviral Signaling

ABSTRACT Paramyxovirus V proteins are known antagonists of the RIG-I-like receptor (RLR)-mediated interferon induction pathway, interacting with and inhibiting the RLR MDA5. We report interactions between the Nipah virus V protein and both RIG-I regulatory protein TRIM25 and RIG-I. We also observed interactions between these host proteins and the V proteins of measles virus, Sendai virus, and parainfluenza virus. These interactions are mediated by the conserved C-terminal domain of the V protein, which binds to the tandem caspase activation and recruitment domains (CARDs) of RIG-I (the region of TRIM25 ubiquitination) and to the SPRY domain of TRIM25, which mediates TRIM25 interaction with the RIG-I CARDs. Furthermore, we show that V interaction with TRIM25 and RIG-I prevents TRIM25-mediated ubiquitination of RIG-I and disrupts downstream RIG-I signaling to the mitochondrial antiviral signaling protein. This is a novel mechanism for innate immune inhibition by paramyxovirus V proteins, distinct from other known V protein functions such as MDA5 and STAT1 antagonism. IMPORTANCE The host RIG-I signaling pathway is a key early obstacle to paramyxovirus infection, as it results in rapid induction of an antiviral response. This study shows that paramyxovirus V proteins interact with and inhibit the activation of RIG-I, thereby interrupting the antiviral signaling pathway and facilitating virus replication.

scholarly journals Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes

2020 ◽  
Vol 295 (27) ◽  
pp. 9012-9020
Author(s):  
Carel Fijen ◽  
Mariam M. Mahmoud ◽  
Meike Kronenberg ◽  
Rebecca Kaup ◽  
Mattia Fontana ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Conformational Changes ◽  
Dna Complexes ◽  
Single Molecule Fret ◽  
Nucleotide Incorporation ◽  
Eukaryotic Dna ◽  
Cellular Dna ◽  
Dna Complex ◽  
Gapped Dna

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as “fingers closing.” Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

scholarly journals Structural basis for DNA recognition by the transcription regulator MetR

2016 ◽  
Vol 72 (6) ◽  
pp. 417-426 ◽  
Author(s):  
Avinash S. Punekar ◽  
Jonathan Porter ◽  
Stephen B. Carr ◽  
Simon E. V. Phillips
Keyword(s):  
Molecular Level ◽  
Dna Recognition ◽  
Data Driven ◽  
Structural Basis ◽  
Methionine Biosynthesis ◽  
Dna Complexes ◽  
Winged Helix ◽  
Target Dna ◽  
Operator Sequence ◽  
Dna Complex

MetR, a LysR-type transcriptional regulator (LTTR), has been extensively studied owing to its role in the control of methionine biosynthesis in proteobacteria. A MetR homodimer binds to a 24-base-pair operator region of themetgenes and specifically recognizes the interrupted palindromic sequence 5′-TGAA-N5-TTCA-3′. Mechanistic details underlying the interaction of MetR with its target DNA at the molecular level remain unknown. In this work, the crystal structure of the DNA-binding domain (DBD) of MetR was determined at 2.16 Å resolution. MetR-DBD adopts a winged-helix–turn–helix (wHTH) motif and shares significant fold similarity with the DBD of the LTTR protein BenM. Furthermore, a data-driven macromolecular-docking strategy was used to model the structure of MetR-DBD bound to DNA, which revealed that a bent conformation of DNA is required for the recognition helix α3 and the wing loop of the wHTH motif to interact with the major and minor grooves, respectively. Comparison of the MetR-DBD–DNA complex with the crystal structures of other LTTR-DBD–DNA complexes revealed residues that may confer operator-sequence binding specificity for MetR. Taken together, the results show that MetR-DBD uses a combination of direct base-specific interactions and indirect shape recognition of the promoter to regulate the transcription ofmetgenes.

scholarly journals RRS1, a Conserved Essential Gene, Encodes a Novel Regulatory Protein Required for Ribosome Biogenesis inSaccharomyces cerevisiae

2000 ◽  
Vol 20 (6) ◽  
pp. 2066-2074 ◽  
Author(s):  
Akiko Tsuno ◽  
Keita Miyoshi ◽  
Rota Tsujii ◽  
Tokichi Miyakawa ◽  
Keiko Mizuta
Keyword(s):  
Ribosomal Protein ◽  
Signaling Pathway ◽  
Transcriptional Repression ◽  
Ribosome Biogenesis ◽  
Essential Gene ◽  
Regulatory Protein ◽  
Rrna Genes ◽  
Amino Acid Residues ◽  
Ribosomal Protein Genes ◽  
Gene Encoding

ABSTRACT A secretory defect causes specific and significant transcriptional repression of both ribosomal protein and rRNA genes (K. Mizuta and J. R. Warner, Mol. Cell. Biol. 14:2493–2502, 1994), suggesting the coupling of plasma membrane and ribosome syntheses. In order to elucidate the molecular mechanism of the signaling pathway, we isolated a cold-sensitive mutant with a mutation in a gene termedRRS1 (regulator of ribosome synthesis), which appeared to be defective in the signaling pathway. The rrs1-1 mutation greatly reduced transcriptional repression of both rRNA and ribosomal protein genes that is caused by a secretory defect. RRS1 is a novel, essential gene encoding a nuclear protein of 203 amino acid residues that is conserved in eukaryotes. A conditionalrrs1-null mutant was constructed by placingRRS1 under the control of the GAL1 promoter. Rrs1p depletion caused defects in processing of pre-rRNA and assembly of ribosomal subunits.

Determining the topology of stable protein–DNA complexes

2013 ◽  
Vol 41 (2) ◽  
pp. 601-605 ◽  
Author(s):  
Isabel K. Darcy ◽  
Mariel Vazquez
Keyword(s):  
Present Article ◽  
Experimental Technique ◽  
Topological Structure ◽  
Reaction Pathway ◽  
Dna Supercoiling ◽  
Dna Complexes ◽  
Nucleoprotein Complex ◽  
Mu Transpososome ◽  
Dna Complex

Difference topology is an experimental technique that can be used to unveil the topological structure adopted by two or more DNA segments in a stable protein–DNA complex. Difference topology has also been used to detect intermediates in a reaction pathway and to investigate the role of DNA supercoiling. In the present article, we review difference topology as applied to the Mu transpososome. The tools discussed can be applied to any stable nucleoprotein complex.

scholarly journals Characterizing protein-DNA binding event subtypes in ChIP-exo data

2018 ◽  
Author(s):  
Naomi Yamada ◽  
William K.M. Lai ◽  
Nina Farrell ◽  
B. Franklin Pugh ◽  
Shaun Mahony
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Regulatory Protein ◽  
Distribution Patterns ◽  
Dna Interaction ◽  
Binding Modes ◽  
Dna Motifs ◽  
Multiple Protein ◽  
Binding Event ◽  
Dna Crosslinking

AbstractMotivationRegulatory proteins associate with the genome either by directly binding cognate DNA motifs or via protein-protein interactions with other regulators. Each recruitment mechanism may be associated with distinct motifs and may also result in distinct characteristic patterns in high-resolution protein-DNA binding assays. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5’ → 3’ exonuclease digestion. Since different regulatory complexes will result in different protein-DNA crosslinking signatures, analysis of ChIP-exo tag enrichment patterns should enable detection of multiple protein-DNA binding modes for a given regulatory protein. However, current ChIP-exo analysis methods either treat all binding events as being of a uniform type or rely on motifs to cluster binding events into subtypes.ResultsTo systematically detect multiple protein-DNA interaction modes in a single ChIP-exo experiment, we introduce the ChIP-exo mixture model (ChExMix). ChExMix probabilistically models the genomic locations and subtype memberships of binding events using both ChIP-exo tag distribution patterns and DNA motifs. We demonstrate that ChExMix achieves accurate detection and classification of binding event subtypes using in silico mixed ChIP-exo data. We further demonstrate the unique analysis abilities of ChExMix using a collection of ChIP-exo experiments that profile the binding of key transcription factors in MCF-7 cells. In these data, ChExMix identifies possible recruitment mechanisms of FoxA1 and ERα, thus demonstrating that ChExMix can effectively stratify ChIP-exo binding events into biologically meaningful subtypes.AvailabilityChExMix is available from https://github.com/seqcode/[email protected]

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