ABSTRACT
Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.
Subject(s)
Alternative Splicing , Cell Differentiation , Chromatin , Heterogeneous-Nuclear Ribonucleoproteins , Neurons , Polypyrimidine Tract-Binding Protein , Transcription Factors , Alternative Splicing/genetics , Polypyrimidine Tract-Binding Protein/metabolism , Polypyrimidine Tract-Binding Protein/genetics , Animals , Cell Differentiation/genetics , Chromatin/metabolism , Mice , Neurons/metabolism , Neurons/cytology , Transcription Factors/metabolism , Transcription Factors/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Transcription, Genetic , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/cytology , Exons/genetics , Humans , Cell Self Renewal/geneticsABSTRACT
Inducible nucleosome remodeling at hundreds of latent enhancers and several promoters shapes the transcriptional response to Toll-like receptor 4 (TLR4) signaling in macrophages. We aimed to define the identities of the transcription factors that promote TLR-induced remodeling. An analysis strategy based on ATAC-seq and single-cell ATAC-seq that enriched for genomic regions most likely to undergo remodeling revealed that the transcription factor nuclear factor κB (NF-κB) bound to all high-confidence peaks marking remodeling during the primary response to the TLR4 ligand, lipid A. Deletion of NF-κB subunits RelA and c-Rel resulted in the loss of remodeling at high-confidence ATAC-seq peaks, and CRISPR-Cas9 mutagenesis of NF-κB-binding motifs impaired remodeling. Remodeling selectivity at defined regions was conferred by collaboration with other inducible factors, including IRF3- and MAP-kinase-induced factors. Thus, NF-κB is unique among TLR4-activated transcription factors in its broad contribution to inducible nucleosome remodeling, alongside its ability to activate poised enhancers and promoters assembled into open chromatin.
Subject(s)
NF-kappa B , Toll-Like Receptor 4 , NF-kappa B/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Nucleosomes , Signal Transduction , Gene Expression Regulation , Transcription Factor RelA/metabolismABSTRACT
Head-on (HO) collisions between the DNA replication machinery and RNA polymerase over R-loop forming sequences (RLFS) are genotoxic, leading to replication fork blockage and DNA breaks. Current models suggest that HO collisions are avoided through replication initiation site (RIS) positioning upstream of active genes, ensuring co-orientation of replication fork movement and genic transcription. However, this model does not account for pervasive transcription, or intragenic RIS. Moreover, pervasive transcription initiation and CG-rich DNA is a feature of RIS, suggesting that HO transcription units (HO TUs) capable of forming R-loops might occur. Through mining phased GRO-seq data, and developing an informatics strategy to stringently identify RIS, we demonstrate that HO TUs containing RLFS occur at RIS in MCF-7 cells, and are downregulated at the G1/S phase boundary. Our analysis reveals a novel spatiotemporal relationship between transcription and replication, and supports the idea that HO collisions are avoided through transcriptional regulatory mechanisms.
ABSTRACT
Knowledge of how Mediator and TFIID cross-talk contributes to promoter-enhancer (P-E) communication is important for elucidating the mechanism of enhancer function. We conducted an shRNA knockdown screen in murine embryonic stem cells to identify the functional overlap between Mediator and TFIID subunits on gene expression. Auxin-inducible degrons were constructed for TAF12 and MED4, the subunits eliciting the greatest overlap. Degradation of TAF12 led to a dramatic genome-wide decrease in gene expression accompanied by destruction of TFIID, loss of Pol II preinitiation complex (PIC) at promoters, and significantly decreased Mediator binding to promoters and enhancers. Interestingly, loss of the PIC elicited only a mild effect on P-E looping by promoter capture Hi-C (PCHi-C). Degradation of MED4 had a minor effect on Mediator integrity but led to a consistent twofold loss in gene expression, decreased binding of Pol II to Mediator, and decreased recruitment of Pol II to the promoters, but had no effect on the other PIC components. PCHi-C revealed no consistent effect of MED4 degradation on P-E looping. Collectively, our data show that TAF12 and MED4 contribute mechanistically in different ways to P-E communication but neither factor appears to directly control P-E looping, thereby dissociating P-E communication from physical looping.
Subject(s)
RNA Polymerase II , Transcription Factor TFIID , Animals , Mediator Complex/genetics , Mediator Complex/metabolism , Mice , Promoter Regions, Genetic/genetics , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Transcription Factor TFIID/genetics , Transcription, GeneticABSTRACT
The proper coordination of transcription with DNA replication and repair is central for genomic stability. We investigate how the INO80C chromatin remodeling enzyme might coordinate these genomic processes. We find that INO80C co-localizes with the origin recognition complex (ORC) at yeast replication origins and is bound to replication initiation sites in mouse embryonic stem cells (mESCs). In yeast, INO80C recruitment requires origin sequences but does not require ORC, suggesting that recruitment is independent of pre-replication complex assembly. In both yeast and ESCs, INO80C co-localizes at origins with Mot1 and NC2 transcription factors, and genetic studies suggest that they function together to promote genome stability. Interestingly, nascent transcript sequencing demonstrates that INO80C and Mot1 prevent pervasive transcription through origin sequences, and absence of these factors leads to formation of new DNA double-strand breaks. We propose that INO80C and Mot1/NC2 function through distinct pathways to limit origin transcription, maintaining genomic stability.
Subject(s)
ATPases Associated with Diverse Cellular Activities/genetics , Chromatin/metabolism , DNA-Binding Proteins/genetics , Genomic Instability/genetics , Replication Origin/genetics , Transcription Factors/metabolism , HumansABSTRACT
Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.
Subject(s)
Copper/metabolism , Histones/chemistry , Oxidoreductases/chemistry , Protein Multimerization , Animals , Biocatalysis , Catalytic Domain/genetics , Gain of Function Mutation , Histones/genetics , Histones/metabolism , Mitochondria/metabolism , Nuclear Proteins/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Superoxide Dismutase-1/chemistry , Transcription Factors/metabolism , Xenopus laevisABSTRACT
Mediator 12 (MED12) and MED13 are components of the Mediator multi-protein complex, that facilitates the initial steps of gene transcription. Here, in an Arabidopsis mutant screen, we identify MED12 and MED13 as positive gene regulators, both of which contribute broadly to morc1 de-repressed gene expression. Both MED12 and MED13 are preferentially required for the expression of genes depleted in active chromatin marks, a chromatin signature shared with morc1 re-activated loci. We further discover that MED12 tends to interact with genes that are responsive to environmental stimuli, including light and radiation. We demonstrate that light-induced transient gene expression depends on MED12, and is accompanied by a concomitant increase in MED12 enrichment during induction. In contrast, the steady-state expression level of these genes show little dependence on MED12, suggesting that MED12 is primarily required to aid the expression of genes in transition from less-active to more active states.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant , Repressor Proteins/metabolism , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Chromatin/metabolism , DNA Methylation/genetics , DNA Methylation/radiation effects , Epigenesis, Genetic/radiation effects , Gene Expression Regulation, Plant/radiation effects , Genes, Plant , Genes, Suppressor , Genetic Loci , Green Fluorescent Proteins/metabolism , Light , Plants, Genetically Modified , Repressor Proteins/genetics , Up-Regulation/genetics , Up-Regulation/radiation effectsABSTRACT
Metazoan chromosomes are sequentially partitioned into topologically associating domains (TADs) and then into smaller sub-domains. One class of sub-domains, insulated neighborhoods, are proposed to spatially sequester and insulate the enclosed genes through self-association and chromatin looping. However, it has not been determined functionally whether promoter-enhancer interactions and gene regulation are broadly restricted to within these loops. Here, we employed published datasets from murine embryonic stem cells (mESCs) to identify insulated neighborhoods that confine promoter-enhancer interactions and demarcate gene regulatory regions. To directly address the functionality of these regions, we depleted estrogen-related receptor ß (Esrrb), which binds the Mediator co-activator complex, to impair enhancers of genes within 222 insulated neighborhoods without causing mESC differentiation. Esrrb depletion reduces Mediator binding, promoter-enhancer looping, and expression of both nascent RNA and mRNA within the insulated neighborhoods without significantly affecting the flanking genes. Our data indicate that insulated neighborhoods represent functional regulons in mammalian genomes.
Subject(s)
Chromosomes, Mammalian , Enhancer Elements, Genetic , Insulator Elements , Mouse Embryonic Stem Cells/physiology , Promoter Regions, Genetic , Transcription, Genetic , Animals , Binding Sites , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Databases, Genetic , Down-Regulation , Mice , Protein Binding , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , CohesinsABSTRACT
Regulation of transcription in eukaryotic cells is a dynamic interplay between chromatin structure and recruitment of a plethora of transcription factors to enhancers, upstream activator sequences, and proximal promoter elements. These factors serve to recruit RNA polymerase to the core promoter for productive transcription. In this Thematic Minireview Series on chromatin and transcription, five reviews summarize current knowledge of diverse aspects of transcriptional regulation and the role of chromatin structure in transcription and development.
Subject(s)
Chromatin/genetics , Transcription, Genetic , Animals , Gene Expression Regulation , HumansABSTRACT
Oxidative stress affects the contractile behavior of smooth muscle resulting in complications during labor. Toxicants such as lindane and ferric chloride (FeCl3)/adenosine diphosphate (ADP) cause oxidative stress and have previously been shown to inhibit smooth muscle contraction. In this study we examined the effects of the oxygen species scavengers, ascorbic acid and N-acetylcysteine on lindane and FeCl3/ADP's inhibition of spontaneous myometrial contractions in rat and human myometrium. Lindane and FeCl3/ADP gave rise to concentration-dependent reductions in rat (EC50 11.8×10-6M and 0.9×10-3M) and human myometrial contractions (EC50 16.3×10-6M and 1.1×10-3M, respectively). Pre-treatment with N-acetylcysteine significantly increased the EC50 for the effects of lindane on motility index of human tissue and reduced the maximum inhibitory effect of FeCl3/ADP on contractions in both rat and human myometrium. Ascorbic acid reduced the effects of FeCl3/ADP in rat tissue only. In conclusion pre-treatment with specific antioxidants may protect both rat and human myometrium from the inhibitory effects of lindane and FeCl3/ADP.
Subject(s)
Acetylcysteine/pharmacology , Adenosine Diphosphate/analogs & derivatives , Antioxidants/pharmacology , Hexachlorocyclohexane/toxicity , Insecticides/toxicity , Iron Chelating Agents/toxicity , Myometrium/drug effects , Adenosine Diphosphate/toxicity , Adult , Animals , Female , Humans , Myometrium/physiology , Rats, Wistar , Uterine Contraction/drug effectsABSTRACT
Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.
Subject(s)
Adenosine Triphosphatases/metabolism , Embryonic Stem Cells/enzymology , Phosphoproteins/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , TATA-Binding Protein Associated Factors/metabolism , Transcription Factors/metabolism , Transcription, Genetic , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/genetics , Binding Sites , Cell Line , DNA-Binding Proteins , Euchromatin/genetics , Euchromatin/metabolism , Gene Expression Regulation, Fungal , Gene Silencing , Genotype , Heterochromatin/genetics , Heterochromatin/metabolism , Phenotype , Phosphoproteins/genetics , Promoter Regions, Genetic , Protein Binding , RNA Interference , Repressor Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , TATA-Binding Protein Associated Factors/genetics , TATA-Box Binding Protein/genetics , TATA-Box Binding Protein/metabolism , Transcription Factor TFIID , Transcription Factors/genetics , TransfectionABSTRACT
The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) is a protein complex that physically tethers the two organelles to each other and creates the physical basis for communication between them. ERMES functions in lipid exchange between the ER and mitochondria, protein import into mitochondria, and maintenance of mitochondrial morphology and genome. Here, we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of known ERMES roles in calcium regulation, phospholipid biosynthesis, or effects on mitochondrial morphology. A mutation in the vacuolar protein sorting 13 (VPS13) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our findings reveal that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels.
Subject(s)
Endoplasmic Reticulum/metabolism , Iron/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Models, Biological , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Alleles , Amino Acid Substitution , Endoplasmic Reticulum/chemistry , Energy Metabolism , Gene Deletion , Gene Expression Regulation, Fungal , Homeostasis , Iron/analysis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mitochondria/chemistry , Point Mutation , Protein Transport , RNA, Fungal/chemistry , RNA, Fungal/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Analysis, RNA , Spectrophotometry, AtomicABSTRACT
BACKGROUND: Ergometrine is a uterotonic agent that is recommended in the prevention and management of postpartum hemorrhage. Despite its long-standing use, the mechanism by which it acts in humans has never been elucidated fully. The objective of this study was to investigate the role of adrenoreceptors in ergometrine's mechanism of action in human myometrium. The study examined the hypothesis that α-adrenoreceptor antagonism would result in the reversal of the uterotonic effects of ergometrine. METHODS: Myometrial samples were obtained from women undergoing elective cesarean delivery. The samples were then dissected into strips and mounted in organ bath chambers. After the generation of an ergometrine concentration-response curve (10 to 10 M), strips were treated with increasing concentrations of ergometrine (10 to 10 M) alone and ergometrine (10 to 10 M) in the presence of phentolamine (10 M), prazosin (10 M), propranolol (10 M), or yohimbine (10 M). The effects of adding ergometrine and the effect of drug combinations were analyzed using linear mixed effects models with measures of amplitude (g), frequency (contractions/10 min), and motility index (g×contractions/10 min). RESULTS: A total of 157 experiments were completed on samples obtained from 33 women. There was a significant increase in the motility index (adding 0.342 g × counts/10 min/µM; 95% confidence interval [CI], 0.253-0.431, P < .001), amplitude (0.078 g/µM; 95% CI, 0.0344-0.121, P = 5e-04), and frequency (0.051 counts/10 min/µM; 95% CI, 0.038-0.063, P < .001) in the presence of ergometrine. The α-adrenergic antagonist phentolamine and the more selective α1-adrenergic antagonist prazosin inhibited the ergometrine mediated increase in motility index, amplitude, and frequency (-1.63 g × counts/10 min/µM and -16.70 g × counts/10 min/µM for motility index, respectively). CONCLUSIONS: These results provide novel evidence for a role for α-adrenergic signaling mechanisms in the action of ergometrine on human myometrial smooth muscle in the in vitro setting. Information that sheds light on the mechanism of action of ergometrine may have implications for the development of further uterotonic agents.
Subject(s)
Ergonovine/pharmacology , Myometrium/drug effects , Oxytocics/pharmacology , Receptors, Adrenergic, alpha/drug effects , Uterus/drug effects , Adrenergic alpha-Antagonists/pharmacology , Adrenergic beta-Antagonists/pharmacology , Adult , Cesarean Section , Dose-Response Relationship, Drug , Drug Interactions , Female , Humans , In Vitro Techniques , Pregnancy , Uterine Contraction/drug effectsABSTRACT
Gene activation in metazoans is accompanied by the presence of histone variants H2AZ and H3.3 within promoters and enhancers. It is not known, however, what protein deposits H3.3 into chromatin or whether variant chromatin plays a direct role in gene activation. Here we show that chromatin containing acetylated H2AZ and H3.3 stimulates transcription in vitro. Analysis of the Pol II pre-initiation complex on immobilized chromatin templates revealed that the E1A binding protein p400 (EP400) was bound preferentially to and required for transcription stimulation by acetylated double-variant chromatin. EP400 also stimulated H2AZ/H3.3 deposition into promoters and enhancers and influenced transcription in vivo at a step downstream of the Mediator complex. EP400 efficiently exchanged recombinant histones H2A and H3.1 with H2AZ and H3.3, respectively, in a chromatin- and ATP-stimulated manner in vitro. Our data reveal that EP400 deposits H3.3 into chromatin alongside H2AZ and contributes to gene regulation after PIC assembly.
Subject(s)
Chromatin Assembly and Disassembly , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Histones/metabolism , Promoter Regions, Genetic , Transcriptional Activation , Acetylation , Adenosine Triphosphate/metabolism , Binding Sites , Cell Line, Tumor , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Genes, Reporter , Histones/genetics , Humans , RNA Interference , RNA Polymerase II/metabolism , Time Factors , TransfectionABSTRACT
In this issue of Molecular Cell, Kubik et al. (2015) describe how the RSC chromatin remodeling complex collaborates with two DNA sequence motifs and sequence-specific general regulatory factors to assemble fragile nucleosomes at highly transcribed yeast Pol II promoters, and they distinguish these from promoters bearing stable nucleosomes.
Subject(s)
Gene Expression Regulation, Fungal/physiology , Nucleosomes/metabolism , Promoter Regions, Genetic/physiology , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae/metabolismABSTRACT
Here we show that the Ino80 chromatin remodeling complex (Ino80C) directly prevents euchromatin from invading transcriptionally silent chromatin within intergenic regions and at the border of euchromatin and heterochromatin. Deletion of Ino80C subunits leads to increased H3K79 methylation and noncoding RNA polymerase II (Pol II) transcription centered at the Ino80C-binding sites. The effect of Ino80C is direct, as it blocks H3K79 methylation by Dot1 in vitro. Heterochromatin stimulates the binding of Ino80C in vitro and in vivo. Our data reveal that Ino80C serves as a general silencing complex that restricts transcription to gene units in euchromatin.
Subject(s)
Chromatin/genetics , Euchromatin/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Binding Sites , Euchromatin/genetics , Gene Expression Regulation, Fungal , Gene Silencing , Histone-Lysine N-Methyltransferase/metabolism , Methylation , Nuclear Proteins/metabolism , Protein Binding , RNA Polymerase II/metabolismABSTRACT
During eukaryotic evolution, genome size has increased disproportionately to nuclear volume, necessitating greater degrees of chromatin compaction in higher eukaryotes, which have evolved several mechanisms for genome compaction. However, it is unknown whether histones themselves have evolved to regulate chromatin compaction. Analysis of histone sequences from 160 eukaryotes revealed that the H2A N-terminus has systematically acquired arginines as genomes expanded. Insertion of arginines into their evolutionarily conserved position in H2A of a small-genome organism increased linear compaction by as much as 40%, while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated in vitro with nucleosomal arrays using unmodified histones, indicating that the H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine-DNA contacts to enable tighter nucleosomal packing. Our findings reveal a novel evolutionary mechanism for regulation of chromatin compaction and may explain the frequent mutations of the H2A N-terminus in cancer.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/chemistry , Evolution, Molecular , Histones/chemistry , Animals , Arginine/chemistry , Cell Line, Tumor , Genome, Fungal , HEK293 Cells , Humans , Neoplasms/genetics , Nucleosomes/chemistry , Nucleosomes/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Xenopus laevisABSTRACT
Unlike traditional site-directed mutagenesis, this protocol requires only a single PCR step using full plasmid amplification to generate point mutants. The method can introduce small mutations into promoter sites and is even better suited for introducing single or double mutations into proteins. It is elegant in its simplicity and can be applied quite easily in any laboratory using standard protein expression vectors and commercially available reagents.
Subject(s)
Mutagenesis, Site-Directed/methods , Point Mutation , Polymerase Chain Reaction/methods , Open Reading Frames , Plasmids , Promoter Regions, GeneticABSTRACT
In an electrophoretic mobility-shift assay (EMSA, or simply "gel shift"), a (32)P-labeled DNA fragment containing a specific DNA site is incubated with a cognate DNA-binding protein. The protein-DNA complexes are separated from free (unbound) DNA by electrophoresis through a nondenaturing polyacrylamide gel. The protein retards the mobility of the DNA fragments to which it binds. Thus, the free DNA will migrate faster than the DNA-protein complex. An image of the gel is used to reveal the positions of the free and bound radiolabeled DNAs.
Subject(s)
Electrophoretic Mobility Shift Assay/methods , Binding Sites , DNA/metabolism , DNA-Binding Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Isotope Labeling , Phosphorus Radioisotopes/analysis , Protein BindingABSTRACT
DNase I footprinting has found a wide following for both identifying and characterizing DNA-protein interactions, particularly because of its simplicity. The concept is that a partial digestion by DNase I of a uniquely (32)P-end-labeled fragment will generate a ladder of fragments, whose mobilities on a denaturing acrylamide gel and whose positions in a subsequent autoradiograph will represent the distance from the end label to the points of cleavage. Bound protein prevents binding of DNase I in and around its binding site and thus generates a "footprint" in the cleavage ladder. The distance from the end label to the edges of the footprint represents the position of the protein-binding site on the DNA fragment. The position of the binding site can be determined by electrophoresing a DNA sequencing ladder alongside the footprint. DNase I cannot bind directly adjacent to a DNA-bound protein because of steric hindrance. Hence, the footprint gives a broad indication of the binding site, generally 8-10 base pairs (bp) larger than the site itself.