Cooperative DNA Binding Research Articles

Cooperative DNA-Binding Effect through DNA Allostery

Allostery is well-documented for proteins but less recognized for DNA-protein interactions, in which DNA has been often considered as a mere template providing recognition sequences. Here we report that for two proteins bound on DNA at a separation of tens of base pairs, their DNA binding affinities can be significantly altered. This coupling effect oscillates between positive and negative cooperativity, depending on the separation distance between the two proteins on the DNA. With a DNA hairpin experiment, we provide definitive evidence for the structural basis of DNA allostery. We prove this effect is not due to protein-protein interactions but originates from the distortion of the inter-helical distance along the linker DNA. The oscillation has a periodicity of ∼10 base pairs, the helical pitch of the B-form DNA, and a characteristic decay length of ∼15 base pairs. In the theoretical analysis, we elucidate the relation between the mechanical structural distortion of DNA induced by protein-binding and the free energy coupling measured thermodynamically, providing a complete picture for the origin of DNA allostery. The allosteric coupling between two DNA-bound proteins is found to be ubiquitous, regardless of proteins’ properties, implying its general roles in gene regulation. We demonstrate such DNA allostery affects gene expression levels in live E.coli cells. Pertinent to eukaryotic gene expression, we show that the binding affinity of a transcription factor depends on its separation from nearby nucleosomes. This work provides the first comprehensive study of allostery through DNA, with the understanding of its physical underpinning and ubiquity and biological relevance.

Opposite Orientations of a Transcription Factor Heterodimer Bind DNA Cooperatively with Interaction Partners but Have Different Effects on Interferon-β Gene Transcription

ATF2-Jun, IRF3, and HMGI recognize a composite regulatory element within the interferon-β enhancer (IFNb). Cooperative ATF2-Jun-IRF3 complex formation at IFNb has been proposed to require a fixed orientation of ATF2-Jun binding. Our results show that ATF2-Jun heterodimers bound IFNb in both orientations alone and in association with IRF3 and HMGI. Two sets of symmetrically located amino acid residues in ATF2 and Jun facilitated the interactions between heterodimers bound in opposite orientations and IRF3 at IFNb. IRF3 and HMGI bound IFNb in association with both orientations of ATF2-Jun heterodimers with the same cooperativity. ATF2-Jun heterodimers that bound IFNb in opposite orientations in vitro had different effects on interferon-β gene transcription when they were co-expressed with IRF3 in cultured cells. These heterodimers had different transcriptional activities at different endogenous genes. Different regions of ATF2 and Jun mediated their orientation-dependent transcriptional activities at different genes. These studies revealed that cooperative DNA binding does not require a unique nucleoprotein complex configuration, and that transcription factor complexes that bind the same enhancer in different configurations can have different transcriptional activities.

Disentangling the Many Layers of Eukaryotic Transcriptional Regulation

Regulation of gene expression in eukaryotes is an extremely complex process. In this review, we break down several critical steps, emphasizing new data and techniques that have expanded current gene regulatory models. We begin at the level of DNA sequence where cis-regulatory modules (CRMs) provide important regulatory information in the form of transcription factor (TF) binding sites. In this respect, CRMs function as instructional platforms for the assembly of gene regulatory complexes. We discuss multiple mechanisms controlling complex assembly, including cooperative DNA binding, combinatorial codes, and CRM architecture. The second section of this review places CRM assembly in the context of nucleosomes and condensed chromatin. We discuss how DNA accessibility and histone modifications contribute to TF function. Lastly, new advances in chromosomal mapping techniques have provided increased understanding of intra- and interchromosomal interactions. We discuss how these topological maps influence gene regulatory models.

The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis

DnaA is an AAA+ ATPase and the conserved replication initiator in bacteria. Bacteria control the timing of replication initiation by regulating the activity of DnaA. DnaA binds to multiple sites in the origin of replication (oriC) and is required for recruitment of proteins needed to load the replicative helicase. DnaA also binds to other chromosomal regions and functions as a transcription factor at some of these sites. Bacillus subtilis DnaD is needed during replication initiation for assembly of the replicative helicase at oriC and during replication restart at stalled replication forks. DnaD associates with DnaA at oriC and at other chromosomal regions bound by DnaA. Using purified proteins, we found that DnaD inhibited the ability of DnaA to bind cooperatively to DNA and caused a decrease in the apparent dissociation constant. These effects of DnaD were independent of the ability of DnaA to bind or hydrolyze ATP. Other proteins known to regulate B. subtilis DnaA also affect DNA binding, whereas much of the regulation of Escherichia coli DnaA affects nucleotide hydrolysis or exchange. We found that the rate of nucleotide exchange for B. subtilis DnaA was high and not affected by DnaD. The rapid exchange is similar to that of Staphylococcus aureus DnaA and in contrast to the low exchange rate of Escherichia coli DnaA. We suggest that organisms in which DnaA has a high rate of nucleotide exchange predominantly regulate the DNA binding activity of DnaA and that those with low rates of exchange regulate hydrolysis and exchange.

Ultrafast Kinetic DNA Hybridization Assay Based on the Visualization of Threshold Turbidity

We report herein the development of an ultrafast kinetic DNA hybridization assay system based on the visualization of threshold turbidity associated with the assembly of polystyrene nanospheres. Initial testing of our diagnostic protocol on a sequence associated with the anthrax lethal factor indicates that a visually identifiable, turbidity-definitive, and kinetic threshold state could be reached at a time as short as 1 min. The assay scheme allows for both target concentration quantification and differentiation of single base mismatches through registry of the threshold turbidity onset time. The positively charged environment on nanospheres not only contributes to expedited signal generation but also imparts cooperative DNA binding properties. The kinetic visual protocol complements conventionally used thermodynamic strategies and provides an entry point for the circumvention of assay issues associated with ill-defined thermodynamic end points.

Competition for cofactor-dependent DNA binding underlies Hox phenotypic suppression

Hox transcription factors exhibit an evolutionarily conserved functional hierarchy, termed phenotypic suppression, in which the activity of posterior Hox proteins dominates over more anterior Hox proteins. Using directly regulated Hox targeted reporter genes in Drosophila, we show that posterior Hox proteins suppress the activities of anterior ones by competing for cofactor-dependent DNA binding. Furthermore, we map a motif in the posterior Hox protein Abdominal-A (AbdA) that is required for phenotypic suppression and facilitates cooperative DNA binding with the Hox cofactor Extradenticle (Exd). Together, these results suggest that Hox-specific motifs endow posterior Hox proteins with the ability to dominate over more anterior ones via a cofactor-dependent DNA-binding mechanism.

Function and disruption of DNA Methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein–DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.

Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways

Gene expression is regulated by DNA as well as histone modifications but the crosstalk and mechanistic link between these epigenetic signals are still poorly understood. Here we investigate the multi-domain protein Uhrf2 that is similar to Uhrf1, an essential cofactor of maintenance DNA methylation. Binding assays demonstrate a cooperative interplay of Uhrf2 domains that induces preference for hemimethylated DNA, the substrate of maintenance methylation, and enhances binding to H3K9me3 heterochromatin marks. FRAP analyses revealed that localization and binding dynamics of Uhrf2 in vivo require an intact tandem Tudor domain and depend on H3K9 trimethylation but not on DNA methylation. Besides the cooperative DNA and histone binding that is characteristic for Uhrf2, we also found an opposite expression pattern of uhrf1 and uhrf2 during differentiation. While uhrf1 is mainly expressed in pluripotent stem cells, uhrf2 is upregulated during differentiation and highly expressed in differentiated mouse tissues. Ectopic expression of Uhrf2 in uhrf1−/− embryonic stem cells did not restore DNA methylation at major satellites indicating functional differences. We propose that the cooperative interplay of Uhrf2 domains may contribute to a tighter epigenetic control of gene expression in differentiated cells.

Mutations That Probe the Cooperative Assembly of O6-Alkylguanine-DNA Alkyltransferase Complexes

O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O(6)-alkylguanine and O(4)-alkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNA cooperatively, and models of cooperative complexes predict that residues 1-7 of one protein molecule and residues 163-169 of a neighboring protein are closely juxtaposed. To test these models, we used directed mutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six of eight designed mutants expressed AGT at detectable levels. All mutant AGTs that were expressed were folded compactly, bound DNA with stoichiometries equivalent to that of the wild-type protein, and were able to protect Escherichia coli to varying degrees from the potent alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). All mutations attenuated DNA binding cooperativity, but unexpectedly, they also reduced the affinity of AGT for DNA. This suggests that the protein-protein and protein-DNA interactions of AGT are strongly coupled. When normalized for differences in AGT expression, cells expressing mutants KDC(3-5)-AAA, DCE(4-6)-AAA, and KEW(165-167)-AAA were significantly more susceptible to MNNG than wild-type cells. This is the first evidence, to the best of our knowledge, of a role for residues at the protein-protein interface and, by implication, cooperative protein-protein interactions in the cell-protective mechanisms of AGT.

Quantitative models of the mechanisms that control genome-wide patterns of transcription factor binding during early Drosophila development.

Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6–0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription factor binding may be used to predict the binding landscape of any animal transcription factor with significant precision.

Improving a Designed Photocontrolled DNA-Binding Protein

Photocontrolled transcription factors could be powerful tools for probing the roles of transcriptional processes in a variety of settings. Previously, we designed a photocontrolled DNA-binding protein based on a fusion between the bZIP region of GCN4 and photoactive yellow protein from Halorhodospira halophila [Morgan, S. A., et al. (2010) J. Mol. Biol. 399, 94-112]. Here we report a structure-based attempt to improve the degree of photoswitching observed with this chimeric protein. Using computational design tools PoPMuSiC 2.0, Rosetta, Eris, and bCIPA, we identified a series of single- and multiple-point mutations that were expected to stabilize the folded dark state of the protein and thereby enhance the degree of photoswitching. While a number of these mutations, particularly those that introduced a hydrophobic residue at position 143, did significantly enhance dark-state protein stability as judged by urea denaturation studies, dark-state stability did not correlate directly with the degree of photoswitching. Instead, the influence of mutations on the degree of photoswitching was found to be related to their effects on the degree to which DNA binding slowed the pB to pG transition in the PYP photocycle. One mutant, K143F, caused an ∼10-fold slowing of the photocycle and also showed the largest difference in the apparent K(d) for DNA binding, 3.5-fold lower, upon irradiation. This change in the apparent K(d) causes a 12-fold enhancement in the fraction bound DNA upon irradiation due to the cooperativity of DNA binding by this family of proteins. The results highlight the strengths and weaknesses of current approaches to a practical problem in protein design and suggest strategies for further improvement of designed photocontrolled transcription factors.

Mode of DNA binding by SopA protein of Escherichia coli F plasmid

The binding of SopA to the promoter region of its own gene, in which four copies of SopA's recognition sequence, 5'-CTTTGC-3', are arrayed asymmetrically, was examined in vitro. Titration using electrophoretic mobility shift assay showed that the stoichiometry of SopA protomers to the promoter-region DNA is 4 and that the binding is highly co-operative. The co-operativity was corroborated by EMSA and DNase I footprinting for a number of mutant DNA fragments in which 5'-CTTTGC-3' was changed to 5'-CTTACG-3'. EMSA in the style of circular permutation showed that SopA bends DNA. Mutation at either outermost binding site had a different effect on DNA bending by SopA, reflecting the asymmetry in the arrangement of the binding sites, for which the results of DNase I footprinting were in agreement. Gel filtration chromatography and analytical ultracentrifugation of free SopA showed that the protein can exist as a monomer and oligomers in the absence of ATP. Hence, the results indicate that the co-operativity in SopA's DNA binding is based on its intrinsic protein-protein interaction modified by DNA interaction.

Crystal structure of TtgV in complex with its DNA operator reveals a general model for cooperative DNA binding of tetrameric gene regulators

The majority of bacterial gene regulators bind as symmetric dimers to palindromic DNA operators of 12-20 base pairs (bp). Multimeric forms of proteins, including tetramers, are able to recognize longer operator sequences in a cooperative manner, although how this is achieved is not well understood due to the lack of complete structural information. Models, instead of structures, of complete tetrameric assembly on DNA exist in literature. Here we present the crystal structures of the multidrug-binding protein TtgV, a gene repressor that controls efflux pumps, alone and in complex with a 42-bp DNA operator containing two TtgV recognition sites at 2.9 Å and 3.4 Å resolution. These structures represent the first full-length functional tetrameric protein in complex with its intact DNA operator containing two continuous recognition sites. TtgV binds to its DNA operator as a highly asymmetric tetramer and induces considerable distortions in the DNA, resulting in a 60° bend. Upon binding to its operator, TtgV undergoes large conformational changes at the monomeric, dimeric, and tetrameric levels. The structures here reveal a general model for cooperative DNA binding of tetrameric gene regulators and provide a structural basis for a large body of biochemical data and a reinterpretation of previous models for tetrameric gene regulators derived from partial structural data.

The latch modulates nucleotide and DNA binding to the helicase-like domain of Thermotoga maritima reverse gyrase and is required for positive DNA supercoiling

Reverse gyrase is the only topoisomerase that can introduce positive supercoils into DNA in an ATP-dependent process. It has a modular structure and harnesses a helicase-like domain to support a topoisomerase activity, thereby creating the unique function of positive DNA supercoiling. The isolated topoisomerase domain can relax negatively supercoiled DNA, an activity that is suppressed in reverse gyrase. The isolated helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain. Inter-domain communication thus appears central for the functional cooperation of the two domains. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the influence of the latch on nucleotide and DNA binding to the helicase-like domain, and on DNA supercoiling by reverse gyrase. We find that the latch is required for positive DNA supercoiling. It is crucial for the cooperativity of DNA and nucleotide binding to the helicase-like domain. The latch contributes to DNA binding, and affects the preference of reverse gyrase for ssDNA. Thus, the latch coordinates the individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domain.

Elevated O-LinkedN-Acetylglucosamine Correlated with Reduced Sp1 Cooperative DNA Binding with Its Collaborating Factorsin Vivo

O-Linked N-acetylglucosamine (O-GlcNAc), a single GlcNAc modification of proteins, is abundant in nucleocytoplasmic proteins of eukaryotes. Most nuclear transcriptional regulator proteins carry O-GlcNAc, implicating O-GlcNAc in gene regulation. This study suggested the possibility that O-GlcNAc regulates cooperative binding of Sp1 and its collaborating transcription factors, Oct1 and Elf-1, onto DNA templates in vivo. Chromatin immunoprecipitation assays on cells in which O-GlcNAc was modulated pharmacologically revealed that Sp1-Oct1- and Sp1-Elf-1-paired occupancies of previously known target promoter regions were suppressed by elevated O-GlcNAc modification. Since these pairs of transcription factors bind the target promoters cooperatively and DNA binding of Sp1 alone is not affected by O-GlcNAc, our results imply that O-GlcNAc weakens the DNA binding of Sp1 and its cooperative binding partners by inhibiting stable interaction on DNA templates.

Protein Complex Binding to Promoter DNA: Immobilized Template Assay

INTRODUCTIONLarge multiprotein complexes involved in transcription generally cannot be resolved by the methods conventionally used to study cooperative DNA binding by smaller proteins. The immobilized template recruitment assay can be used to circumvent this limitation. In this procedure, a biotinylated DNA fragment is immobilized on streptavidin-coated magnetic beads. DNA-binding proteins are incubated with the immobilized template, and proteins not specifically assembled in a complex are removed by separating the immobilized template with a magnetic particle concentrator and subsequent washing of the magnetic bead/template pellet. Bound proteins are detected by immunoblotting. To detect cooperative binding, subsaturating amounts of two or more proteins or protein complexes are added to the immobilized template. Template binding of a protein in concert with its cooperative binding partner is then compared to the binding of each individual protein or protein complex alone. In addition to studying the binding of protein complexes, the immobilized promoter template can also be used in functional assays such as in vitro transcription, allowing a correlation of complex assembly with function.

DNA Binding by the ETS Protein TEL (ETV6) Is Regulated by Autoinhibition and Self-association

The ETS protein TEL, a transcriptional repressor, contains a PNT domain that, as an isolated fragment in vitro, self-associates to form a head-to-tail polymer. How such polymerization might affect the DNA-binding properties of full-length TEL is unclear. Here we report that monomeric TEL binds to a consensus ETS site with unusually low affinity (K(d) = 2.8 x 10(-8) M). A deletion analysis demonstrated that the low affinity was caused by a C-terminal inhibitory domain (CID) that attenuates DNA binding by approximately 10-fold. An NMR spectroscopically derived structure of a TEL fragment, deposited in the Protein Data Bank, revealed that the CID consists of two alpha-helices, one of which appears to block the DNA binding surface of the TEL ETS domain. Based on this structure, we substituted two conserved glutamic acids (Glu-431 and Glu-434) with alanines and found that this activated DNA binding and enhanced trypsin sensitivity in the CID. We propose that TEL displays a conformational equilibrium between inhibited and activated states and that electrostatic interactions involving these negatively charged residues play a role in stabilizing the inhibited conformation. Using a TEL dimer as a model polymer, we show that self-association facilitates cooperative binding to DNA. Cooperativity was observed on DNA duplexes containing tandem consensus ETS sites at variable spacing and orientations, suggesting flexibility in the region of TEL linking its self-associating PNT domain and DNA-binding ETS domain. We speculate that TEL compensates for the low affinity, which is caused by autoinhibition, by binding to DNA as a cooperative polymer.

DNA Binding Cooperativity of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis

p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.

Cooperative DNA-binding and sequence-recognition mechanism of aristaless and clawless

To achieve accurate gene regulation, some homeodomain proteins bind cooperatively to DNA to increase those site specificities. We report a ternary complex structure containing two homeodomain proteins, aristaless (Al) and clawless (Cll), bound to DNA. Our results show that the extended conserved sequences of the Cll homeodomain are indispensable to cooperative DNA binding. In the Al-Cll-DNA complex structure, the residues in the extended regions are used not only for the intermolecular contacts between the two homeodomain proteins but also for the sequence-recognition mechanism of DNA by direct interactions. The residues in the extended N-terminal arm lie within the minor groove of DNA to form direct interactions with bases, whereas the extended conserved region of the C-terminus of the homeodomain interacts with Al to stabilize and localize the third alpha helix of the Cll homeodomain. This structure suggests a novel mode for the cooperativity of homeodomain proteins.

Over Expression, Purification and Crystallization of the PTF1‐J and PTF‐L Complexes

Students in Biomedical Research Studies at Hillcrest High School in Dallas, Texas, participate in a ongoing research program at the University of Texas Southwestern Medical Center to study the structure and function of a mammalian transcription factor complex. Ptf1a is a transcription factor that is crucial to the development of the embryonic pancreas. Its functional form is in a trimeric complex composed of a common E‐box binding protein (E12/47, HEB, or TCF12), Ptf1a and either Rbpj or Rbpjl. The Rbpj form of the complex (PTF1‐J) is required for the early stage of pancreatic development. Subsequently, the Rbpjl‐form (PTF1‐L) is required for the formation of mature acinar cells. PTF1‐L is involved in an auto‐regulatory loop for the maintenance of transcription of both Ptf1a and Rbpjl genes. We have utilized PCR to clone full length and truncated forms of Ptf1a, HEB, E12/47, Rbpj and Rbpjl. These cDNAs have been subcloned into the bacterial expression vector pSMT3. The over expressed proteins will be purified and crystallized in order to perform structural determination of PTF1‐J and PTF1‐L complexes. Determination of the structures for the PTF1‐J and PTF1‐L complexes will elucidate the structural motifs required for the protein‐protein interactions found in the trimeric complex, as well as the mechanism and the structural determinants for the strong cooperative DNA binding of the complexes.