DNA-binding Repressor Research Articles

A robust ultrasensitive transcriptional switch in noisy cellular environments

Ultrasensitive transcriptional switches enable sharp transitions between transcriptional on and off states and are essential for cells to respond to environmental cues with high fidelity. However, conventional switches, which rely on direct repressor-DNA binding, are extremely noise-sensitive, leading to unintended changes in gene expression. Here, through model simulations and analysis, we discovered that an alternative design combining three indirect transcriptional repression mechanisms, sequestration, blocking, and displacement, can generate a noise-resilient ultrasensitive switch. Although sequestration alone can generate an ultrasensitive switch, it remains sensitive to noise because the unintended transcriptional state induced by noise persists for long periods. However, by jointly utilizing blocking and displacement, these noise-induced transitions can be rapidly restored to the original transcriptional state. Because this transcriptional switch is effective in noisy cellular contexts, it goes beyond previous synthetic transcriptional switches, making it particularly valuable for robust synthetic system design. Our findings also provide insights into the evolution of robust ultrasensitive switches in cells. Specifically, the concurrent use of seemingly redundant indirect repression mechanisms in diverse biological systems appears to be a strategy to achieve noise-resilience of ultrasensitive switches.

Sustained MYB Activity Is Necessary for Oncogenic Transcription in KMT2A-Rearranged Acute Lymphoblastic Leukemia through Enhancer-Promoter Interactions and Epigenetic Modifications at Enhancers

Aberrant enhancer activity is a hallmark of many cancers including KMT2A-rearranged acute lymphoblastic leukemia (ALL), an aggressive leukemia subtype with an event-free survival of 19-45% in infant-ALL (<1 year of age) and 50-59% in childhood-ALL cases. Targeting enhancer activity may provide a novel therapeutic strategy for this hard-to-treat disease. Transcription factors are key effectors of enhancer activity. They recognise and bind to specific DNA sequences at enhancers, driving the expression of target genes from these distal loci. MYB is an important hematopoietic transcription factor that is commonly overexpressed in KMT2A-rearranged leukemias and is required for leukemia survival, where loss of MYB has been shown to abrogate the leukemia phenotype. However, the precise mechanism by which MYB contributes to enhancer function, and the stages of enhancer activation at which it acts are unknown. We employed a well-characterized chromatin anchoring system to interrogate MYB function and determine whether it can recruit specific enhancer activities de novo. By fusing the MYB transactivation domain (MYB TA) to the Tet repressor (TetR) DNA binding domain, MYB TA is directed to an array of Tet operator ( TetO) sequences incorporated into a neutral genomic region devoid of native gene regulatory elements. We found that MYB TA alone is sufficient to recruit key enhancer-associated proteins, including P300, Brd4 and Mediator to the TetO locus. We observed MYB TA-dependent deposition of H3K27ac, increased chromatin accessibility and transcription not just at the TetO locus, but remarkably, also at regions more than 50kb away. Using the chromosome conformation capture (3C) technique NG Capture-C, we observed MYB TA-induced DNA interactions between TetO and the distal sites of transcription, as well as more long-range interactions up to 400kb distal to where MYB TA is bound. Together, these observations suggest that MYB TA binding alone is sufficient to establish an enhancer-like regulatory element, which activates distant cryptic promoters. This activity is dependent on the continued presence of MYB TA, as disruption of MYB TA binding results in a rapid loss of transcription and acquired chromatin features, implicating MYB in enhancer maintenance as well as initiation. To confirm our observations in a disease context, we tested the requirement for MYB at endogenous enhancers in SEM cells, a model of KMT2A-rearranged ALL. By stably introducing a degradation tag (FKBP12 F36V) to the endogenous MYB gene, we were able to rapidly induce protein degradation and observe the immediate effects on chromatin features. Using transient transcriptome sequencing, we noted significant transcriptional downregulation of known MYB target genes, including BCL2, MYC, LMO4 and CDK6. We also observed decreases in enhancer H3K27ac levels and eRNA transcription, most marked at MYB-bound loci. To investigate the requirement for MYB in maintaining physical proximity between enhancer and promoter, we performed the base pair-resolution 3C technique Micro-Capture-C (MCC). MCC revealed highly punctate interactions between enhancer elements and their target promoters. At BCL2 and MYC, MYB degradation results in a reduction in interactions with the promoter at discrete regions within the enhancers. This argues for highly localised MYB-dependent activity at enhancers, driving interaction with and activation of distal target promoters. Overall, our data argue for a role for MYB in establishing enhancers de novo, indicating that MYB overexpression may directly result in onco-enhancer activity at key genes in vivo. At a subset of MYB-bound enhancers, the continued presence of MYB is absolutely required to maintain enhancer activity and oncogene upregulation. Further exploration of associated co-factors that mediate MYB-dependent enhancer activity may identify novel targets for therapeutic exploitation.

Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B.

Purine-rich element binding protein B (Purβ) is a single-stranded DNA (ssDNA) and RNA-binding protein that functions as a transcriptional repressor of genes encoding certain muscle-restricted contractile proteins in the setting of cellular stress or tissue injury. A prior report from our laboratory implicated specific basic amino acid residues in the physical and functional interaction of Purβ with the smooth muscle-α actin gene (Acta2) promoter. Independent structural analysis of fruit fly Purα uncovered a role for several aromatic residues in the binding of this related protein to ssDNA. Herein, we examine the functional importance of a comparable set of hydrophobic residues that are positionally conserved in the repeat I (Y59), II (F155), and III (F256) domains of murine Purβ. Site-directed Y/F to alanine substitutions were engineered, and the resultant Purβ point mutants were tested in various biochemical and cell-based assays. None of the mutations affected the cellular expression, structural stability, or dimerization capacity of Purβ. However, the Y59A and F155A mutants demonstrated weaker Acta2 repressor activity in transfected fibroblasts and reduced binding affinity for the purine-rich strand of an Acta2 cis-regulatory element in vitro. Mutation of Y59 and F155 also altered the multisite binding properties of Purβ for ssDNA and diminished the interaction of Purβ with Y-box binding protein 1, a co-repressor of Acta2. Collectively, these findings suggest that some of the same aromatic residues, which govern the specific and high-affinity binding of Purβ to ssDNA, also mediate certain heterotypic protein interactions underlying the Acta2 repressor function of Purβ.

Abstract 2266: Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B

Abstract 2266: Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B

Conformational Change of Transcription Factors from Search to Specific Binding: A lac Repressor Case Study.

In a process known as facilitated diffusion, DNA-binding proteins find their target sites by combining three-dimensional diffusion and one-dimensional scanning of the DNA. Following the trade-off between speed and stability, agile exploration of DNA requires loose binding, whereas, at the DNA target site, the searching protein needs to establish tight interactions with the DNA. To enable both efficient search and stable binding, DNA-binding proteins and DNA often switch conformations upon recognition. Here, we study the one-dimensional diffusion and DNA binding of the dimeric lac repressor (LacI), which was reported to adopt two different conformations when binding different conformations of DNA. Using coarse-grained molecular dynamic simulations, we studied the diffusion and the sequence-specific binding of these conformations of LacI, as well as their truncated or monomeric variants, with two DNA conformations: straight and bent. The simulations were compared to experimental observables. This study supports that linear diffusion along DNA combines tight rotation-coupled groove tracking and rotation-decoupled hopping, where the protein briefly dissociates and reassociates just a few base pairs away. Tight groove tracking is crucial for target-site recognition, while hopping speeds up the overall search process. We investigated the diffusion of different LacI conformations on DNA and show how the flexibility of LacI's hinge regions ensures agility on DNA as well as faithful groove tracking. If the hinge regions instead form α-helices at the protein-DNA interface, tight groove tracking is not possible. On the contrary, the helical hinge region is essential for tight binding to bent, specific DNA, for the formation of the specific complex. Based on our study of different encounter complexes, we argue that the conformational change in LacI and DNA bending are somewhat coupled. Our findings underline the importance of two distinct protein conformations for facilitated diffusion and specific binding, respectively.

Super-enhancer hypermutation alters oncogene expression in B cell lymphoma.

Diffuse large Bcell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets1-5. However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6, BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.

GFI1 regulates hair cell differentiation by acting as an off-DNA transcriptional co-activator of ATOH1, and a DNA-binding repressor

GFI1 is a zinc finger transcription factor that is necessary for the differentiation and survival of hair cells in the cochlea. Deletion of Gfi1 in mice significantly reduces the expression of hundreds of hair cell genes: this is a surprising result, as GFI1 normally acts as a transcriptional repressor by recruiting histone demethylases and methyltransferases to its targets. To understand the mechanisms by which GFI1 promotes hair cell differentiation, we used CUT&RUN to identify the direct targets of GFI1 and ATOH1 in hair cells. We found that GFI1 regulates hair cell differentiation in two distinct ways—first, GFI1 and ATOH1 can bind to the same regulatory elements in hair cell genes, but while ATOH1 directly binds its target DNA motifs in many of these regions, GFI1 does not. Instead, it appears to enhance ATOH1’s transcriptional activity by acting as part of a complex in which it does not directly bind DNA. Second, GFI1 can act in its more typical role as a direct, DNA-binding transcriptional repressor in hair cells; here it represses non-hair cell genes, including many neuronal genes. Together, our results illuminate the function of GFI1 in hair cell development and hair cell reprogramming strategies.

Differential functions of TLE1 and TLE3 depending on a specific phosphorylation site

Mammalian Transducin-like enhancer of split (TLE) confer global repression of numerous target genes in conjunction with a myriad of DNA-binding repressors. These factors have a major role in the regulation of multiple signal transduction pathways. Evidence have been obtained regarding the possible role of some of these proteins in cancer. TLE3 was suggested as a marker for increased chemosensitivity from pathological studies. Here we demonstrate, using the TCGA data base, differences in expression of this gene compared to TLE1 in several cancers. In-vitro transduction of a retrovirus encoding TLE3 to A549 lung cancer cells increased paclitaxel effectivity while TLE1 introduction to these cells decreased it. While TLE1 and TLE3 share ∼80% amino acid identity, we show that mutating or reconstituting an amino-terminal phosphorylation site, which is present only in TLE1 but absent from TLE3, and is evolutionary conserved, converts the activity of TLE1 to that of TLE3 like and vice versa. We repeated these results in an adipocytes differentiation system. Our results reveal how a single phosphorylation site can confer distinct qualitative or quantitative activities on highly homologous transcriptional regulators.

Evolution of a New Function by Fusion between Phage DNA and a Bacterial Gene.

Mobile genetic elements, such as plasmids, phages, and transposons, are important sources for evolution of novel functions. In this study, we performed a large-scale screening of metagenomic phage libraries for their ability to suppress temperature-sensitivity in Salmonella enterica serovar Typhimurium strain LT2 mutants to examine how phage DNA could confer evolutionary novelty to bacteria. We identified an insert encoding 23 amino acids from a phage that when fused with a bacterial DNA-binding repressor protein (LacI) resulted in the formation of a chimeric protein that localized to the outer membrane. This relocalization of the chimeric protein resulted in increased membrane vesicle formation and an associated suppression of the temperature sensitivity of the bacterium. Both the host LacI protein and the extracellular 23-amino acid stretch are necessary for the generation of the novel phenotype. Furthermore, mutational analysis of the chimeric protein showed that although the native repressor function of the LacI protein is maintained in this chimeric structure, it is not necessary for the new function. Thus, our study demonstrates how a gene fusion between foreign DNA and bacterial DNA can generate novelty without compromising the native function of a given gene.

Regulation of lactate metabolism in the acetogenic bacterium Acetobacterium woodii.

Acetogenic bacteria compete in an energy-limited environment by coupling different metabolic routes to their central metabolism of CO2 fixation. The underlying regulatory mechanisms are often still not understood. In this work, we analysed how lactate metabolism is regulated in the model acetogen Acetobacterium woodii. Construction of a ΔlctCDEF mutant and growth analyses demonstrated that the genes are essential for growth on lactate. Subsequent bridging PCR and quantitative PCR analyses revealed that the lctBCDEF genes form an operon that was expressed only during lactate metabolism. The lctA gene was cloned, expressed in Escherichia coli and purified. LctA bound to the intergenic DNA region between lctA and the lct operon in electromobility shift assays, and binding was revoked in the presence of lactate. Further restriction site protection analyses consolidated the lactate-dependent binding of LctA and identified the binding site within the DNA. Cells grew mixotrophically on lactate and another energy source and showed no diauxic growth. From these data, we conclude that the catabolic lactate metabolism is encoded by the lct operon and its expression is negatively regulated by the DNA-binding repressor LctA.

Evolutionary potential of transcription factors for gene regulatory rewiring.

Gene regulatory networks evolve through rewiring of individual components-that is, through changes in regulatory connections. However, the mechanistic basis of regulatory rewiring is poorly understood. Using a canonical gene regulatory system, we quantify the properties of transcription factors that determine the evolutionary potential for rewiring of regulatory connections: robustness, tunability and evolvability. In vivo repression measurements of two repressors at mutated operator sites reveal their contrasting evolutionary potential: while robustness and evolvability were positively correlated, both were in trade-off with tunability. Epistatic interactions between adjacent operators alleviated this trade-off. A thermodynamic model explains how the differences in robustness, tunability and evolvability arise from biophysical characteristics of repressor-DNA binding. The model also uncovers that the energy matrix, which describes how mutations affect repressor-DNA binding, encodes crucial information about the evolutionary potential of a repressor. The biophysical determinants of evolutionary potential for regulatory rewiring constitute a mechanistic framework for understanding network evolution.

Tuning Transcriptional Regulation through Signaling: A Predictive Theory of Allosteric Induction.

SUMMARYAllosteric regulation is found across all domains of life, yet we still lack simple, predictive theories that directly link the experimentally tunable parameters of a system to its input-output response. To that end, we present a general theory of allosteric transcriptional regulation using the Monod-Wyman-Changeux model. We rigorously test this model using the ubiquitous simple repression motif in bacteria by first predicting the behavior of strains that span a large range of repressor copy numbers and DNA binding strengths and then constructing and measuring their response. Our model not only accurately captures the induction profiles of these strains, but also enables us to derive analytic expressions for key properties such as the dynamic range and [EC50]. Finally, we derive an expression for the free energy of allosteric repressors that enables us to collapse our experimental data onto a single master curve that captures the diverse phenomenology of the induction profiles.

Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.

Mechanisms of Groucho-mediated repression revealed by genome-wide analysis of Groucho binding and activity

BackgroundThe transcriptional corepressor Groucho (Gro) is required for the function of many developmentally regulated DNA binding repressors, thus helping to define the gene expression profile of each cell during development. The ability of Gro to repress transcription at a distance together with its ability to oligomerize and bind to histones has led to the suggestion that Gro may spread along chromatin. However, much is unknown about the mechanism of Gro-mediated repression and about the dynamics of Gro targeting.ResultsOur chromatin immunoprecipitation sequencing analysis of temporally staged Drosophila embryos shows that Gro binds in a highly dynamic manner primarily to clusters of discrete (<1 kb) segments. Consistent with the idea that Gro may facilitate communication between silencers and promoters, Gro binding is enriched at both cis-regulatory modules, as well as within the promotors of potential target genes. While this Gro-recruitment is required for repression, our data show that it is not sufficient for repression. Integration of Gro binding data with transcriptomic analysis suggests that, contrary to what has been observed for another Gro family member, Drosophila Gro is probably a dedicated repressor. This analysis also allows us to define a set of high confidence Gro repression targets. Using publically available data regarding the physical and genetic interactions between these targets, we are able to place them in the regulatory network controlling development. Through analysis of chromatin associated pre-mRNA levels at these targets, we find that genes regulated by Gro in the embryo are enriched for characteristics of promoter proximal paused RNA polymerase II.ConclusionsOur findings are inconsistent with a one-dimensional spreading model for long-range repression and suggest that Gro-mediated repression must be regulated at a post-recruitment step. They also show that Gro is likely a dedicated repressor that sits at a prominent highly interconnected regulatory hub in the developmental network. Furthermore, our findings suggest a role for RNA polymerase II pausing in Gro-mediated repression.

A Synthetic Hybrid Promoter for Xylose-Regulated Control of Gene Expression in Saccharomyces Yeasts.

Metabolism of non-glucose carbon sources is often highly regulated at the transcriptional and post-translational levels. This level of regulation is lacking in Saccharomyces cerevisiae strains engineered to metabolize xylose. To better control transcription in S. cerevisiae, the xylose-dependent, DNA-binding repressor (XylR) from Caulobacter crescentus was used to block transcription from synthetic promoters based on the constitutive Ashbya gossypii TEF promoter. The new hybrid promoters were repressed in the absence of xylose and showed up to a 25-fold increase in the presence of xylose. Activation of the promoter was highly sensitive to xylose with activity seen at concentrations below 2μM xylose. These new xylose-inducible promoters allow improved control of gene expression for engineered strains of Saccharomyces yeasts.

A yeast library-hybrid assay to screen maize-Rhizoctonia transcription factors and protein-protein interactions in one experimental pipeline

Yeast one- (Y1H) and two-hybrid (Y2H) assays are widely used to study transcription factors (TFs) and protein-protein interactions (PPIs), respectively. Here we combined the Y1H and Y2H into a yeast library-hybrid (YLH) assay, which can systematically screen transcription factors (TFs) and PPIs in one experimental pipeline. In typical TFs, the DNA-binding domain (DBD) and activation domain (AD) evolved separately, but were covalently linked. In the YLH assay, TFs are identified based on functionally conserved ADs, whereas in various Y1H assays TFs are identified based on DBDs. Using the YLH method, we isolated 51 pairs of maize-Rhizoctonia PPIs and 38 novel Rhizoctonia solani TFs. TFs and PPIs related to pathogen virulence and plant resistance responses were isolated by the YLH assay. Our results show that 57.75% of isolated TFs contain typical DBDs such as Zn2Cys6, nucleic acid-binding OB-fold, winged helix repressor DNA-binding, and zinc finger CCHC-type. Key PPI pairs related to major functional categories such as metabolism and cellular signalling were obtained. The percentage of verified PPIs is 69.39%. We proved that common TFs have nonspecific or broad-spectrum activities in the yeast plasmid gene expression system. YLH screening can be conducted on library scales to systematically reveal possible TFs and PPIs at the same time.

736. Silencing Substituting the RHODODPSIN Gene to Treat Blinding Disorders

Silencing and replacement strategy is a promising strategy to overcome mutational heterogeneity of genetic defects. In autosomal dominant retinitis pigmentosa (adRP) approximately 200 different mutations have been associated with rhodopsin gene (RHO). We recently described a system based on the delivery of a synthetic transcriptional repressor (DNA-binding-mediated silencing; ZF6-DBD), which upon Adeno-associated viral (AAV) vector delivery to the porcine retina, demonstrated selective somatic ablation of RHO in mutation independent manner, thus silencing both the wild-type and mutated alleles. To determine the actual therapeutic impact of DNA-binding-mediated silencing, we carried out the silencing-replacement strategy by coupling ZF6-DBD with RHO replacement (human RHO, hRHO CDS). To warrant simultaneous photoreceptor transduction of both ZF6-DBD and hRHO, we enclosed two expression cassettes into a single vector (DNA-binding repression and replacement, DBR-R). To generate the construct we evaluated key variables for balanced simultaneous RHO repression and replacement, which are the vector dose and promoter strength. To ensure high and rod-specific hRHO replacement, we opted for a high vector dose and the strength of the GNAT1 promoter elements. To decrease ZF6-DB expression levels at high vector dose, while keeping rod-specificity, we both shortened the human RHO promoter and deleted the 5’ sequence of the ZF6-DB target. We used 1×1012 gc of vector of DBR-R (AAV8-RHOΔ-ZF6-DB-GNAT1-hRHO) to administer to porcine retina. Administration of the DBR-R vector resulted in concomitant rod-specific transcriptional repression of the porcine Rho (35%) and in balanced replacement with the exogenous hRHO (45%), as assessed by transcripts levels, protein expression and integrity of photoreceptor outer segments. The data support pre-clinical development including a dose-response and long-term efficacy and toxicity.

Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor

DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92-198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant.

Streptococcus mutans copper chaperone, CopZ, is critical for biofilm formation and competitiveness.

The oral cavity is a dynamic environment characterized by hundreds of bacterial species, saliva, and an influx of nutrients and metal ions such as copper. Although there is a physiologic level of copper in the saliva, the oral cavity is often challenged with an influx of copper ions. At high concentrations copper is toxic and must therefore be strictly regulated by pathogens for them to persist and cause disease. The cariogenic pathogen Streptococcus mutans manages excess copper using the copYAZ operon that encodes a negative DNA-binding repressor (CopY), the P1-ATPase copper exporter (CopA), and the copper chaperone (CopZ). These hypothetical roles of the copYAZ operon in regulation and copper transport to receptors led us to investigate their contribution to S.mutans virulence. Mutants defective in the copper chaperone CopZ, but not CopY or CopA, were impaired in biofilm formation and competitiveness against commensal streptococci. Characterization of the CopZ mutant biofilm revealed a decreased secretion of glucosyltransferases and reduced expression of mutacin genes. These data suggest that the function of copZ on biofilm and competitiveness is independent of copper resistance and CopZ is a global regulator for biofilm and other virulence factors. Further characterization of CopZ may lead to the identification of new biofilm pathways.

Mechanism of Iron-Dependent Repressor (IdeR) Activation and DNA Binding: A Molecular Dynamics and Protein Structure Network Study.

Metalloproteins form a major class of enzymes in the living system that are involved in crucial biological functions such as catalysis, redox reactions and as ‘switches’ in signal transductions. Iron dependent repressor (IdeR) is a metal-sensing transcription factor that regulates free iron concentration in Mycobacterium tuberculosis. IdeR is also known to promote bacterial virulence, making it an important target in the field of therapeutics. Mechanistic details of how iron ions modulate IdeR such that it dimerizes and binds to DNA is not understood clearly. In this study, we have performed molecular dynamic simulations and integrated it with protein structure networks to study the influence of iron on IdeR structure and function. A significant structural variation between the metallated and the non-metallated system is observed. Our simulations clearly indicate the importance of iron in stabilizing its monomeric subunit, which in turn promotes dimerization. However, the most striking results are obtained from the simulations of IdeR-DNA complex in the absence of metals, where at the end of 100ns simulations, the protein subunits are seen to rapidly dissociate away from the DNA, thereby forming an excellent resource to investigate the mechanism of DNA binding. We have also investigated the role of iron as an allosteric regulator of IdeR that positively induces IdeR-DNA complex formation. Based on this study, a mechanistic model of IdeR activation and DNA binding has been proposed.