Cognate DNA Binding Site Research Articles

The HP1α protein is mandatory to repress the circadian clock and its output genes during the 12 h period of transcriptional repression

The transcriptional repressions driven by the circadian core clock repressors RevErbα, E4BP4, and CRY1/PER1 involve feedback loops which are mandatory for generating the circadian rhythms. These repressors are known to bind to cognate DNA binding sites, but how their circadian bindings trigger the cascade of events leading to these repressions remain to be elucidated. Through molecular and genetic analyses, we now demonstrate that the chromatin protein HP1α plays a key role in these transcriptional repressions of both the circadian clock (CC) genes and their cognate output genes (CCGs). We show that these CC repressors recruit the HP1α protein downstream from a repressive cascade, and that this recruitment is mandatory for the maintenance of both the CC integrity and the expression of the circadian genes. We further show that the presence of HP1α is critical for both the repressor-induced chromatin compaction and the generation of "transcriptionally repressed biomolecular hydrophobic condensates" and demonstrates that HP1α is mandatory within the CC output genes for both the recruitment of DNA methylating enzymes on the intronic deoxyCpG islands and their subsequent methylation.

Sulfane Sulfur Is an Intrinsic Signal for the Organic Peroxide Sensor OhrR of Pseudomonas aeruginosa.

Sulfane sulfur, including organic persulfide and polysulfide, is a normal cellular component, and its level varies during growth. It is emerging as a signaling molecule in bacteria, regulating the gene regulator MarR in Escherichia coli, MexR in Pseudomonas aeruginosa, and MgrA of Staphylococcus aureus. They are MarR-family regulators and are often repressors for multiple antibiotic resistance genes. Here, we report that another MarR-type regulator OhrR that represses the expression of itself and a thiol peroxidase gene ohr in P. aeruginosa PAO1 also responded to sulfane sulfur. PaOhrR formed disulfide bonds between three Cys residues within a dimer after polysulfide treatment. The modification reduced its affinity to its cognate DNA binding site. An Escherichia coli reporter system, in which mKate was under the repression of OhrR, showed that PaOhrR derepressed its controlled gene when polysulfide was added, whereas the mutant PaOhrR with two Cys residues changed to Ser residues did not respond to polysulfide. The expression of the PaOhrR-repressed mKate was significantly increased when the cells enter the late log phase when cellular sulfane sulfur reached a maximum, but the mKate expression under the control of the PaOhrR-C9SC19S double mutant was not increased. Furthermore, the expression levels of ohrR and ohr in P. aeruginosa PAO1 were significantly increased when cellular sulfane sulfur was high. Thus, PaOhrR senses both exogenous and intrinsic sulfane sulfur to derepress its controlled genes. The finding also suggests that sulfane sulfur may be a common inducer of the MarR-type regulators, which may confer the bacteria to resist certain stresses without being exposed to the stresses.

Flexibility of flanking DNA is a key determinant of transcription factor affinity for the core motif

Selective gene regulation is mediated by recognition of specific DNA sequences by transcription factors (TFs). The extremely challenging task of searching out specific cognate DNA binding sites among several million putative sites within the eukaryotic genome is achieved by complex molecular recognition mechanisms. Elements of this recognition code include the core binding sequence, the flanking sequence context, and the shape and conformational flexibility of the composite binding site. To unravel the extent to which DNA flexibility modulates TF binding, in this study, we employed experimentally guided molecular dynamics simulations of ternary complex of closely related Hox heterodimers Exd-Ubx and Exd-Scr with DNA. Results demonstrate that flexibility signatures embedded in the flanking sequences impact TF binding at the cognate binding site. A DNA sequence has intrinsic shape and flexibility features. While shape features are localized, our analyses reveal that flexibility features of the flanking sequences percolate several basepairs and allosterically modulate TF binding at the core. We also show that lack of flexibility in the motif context can render the cognate site resistant to protein-induced shape changes and subsequently lower TF binding affinity. Overall, this study suggests that flexibility-guided DNA shape, and not merely the static shape, is a key unexplored component of the complex DNA-TF recognition code.

Protein‐DNA Interactomes of NKX2‐5 and TBX5 Mutants Identified in Congenital Heart Defects

Congenital Heart Defects (CHDs) are the most common birth defect from children to adults in the United States. CHDs are characterized by malformations in the heart’s chambers, walls, and great vessels, sometimes leading to embryonic death. Cardiac Transcription Factors (cardiac TFs) such as NKX2‐5 and TBX5 play a critical role in developmental stages and physiological processes of the heart by regulating gene expression through DNA‐protein interactions. Genetic analyses have identified various non‐synonymous mutations within the homeodomain (HD) DNA‐binding domain (DBD) of NKX2‐5 and the (Tbox) DBD of TBX5 in associated with CHD. We hypothesize that non‐synonymous mutations in cardiac TFs DBD will alter their DNA‐binding properties ultimately affecting transcriptional pathways necessary for proper heart development. To address this hypothesis, we studied how the cardiac TFs DNA‐protein interactomes are affected by non‐synonymous mutations found in CHD patients. We expressed and purified the DBD of NKX2‐5 (HD), TBX5 (Tbox), also NKX2‐5 and TBX5 non‐synonymous mutations (A148E, E154G, R161P, T178M, Q181H, R190C, Y191C), and (I54T, M74V, G80R, I101F, R113K, R237Q, R237W) respectively. Functional analysis using Electrophoretic Mobility Shift Assay (EMSA) confirms HD and Tbox binding to cognate DNA binding sites. DNA‐binding activity as assessed by EMSA, shows retention of DNA activity in the case of NKX2‐5 and TBX5 mutants A148E, R161P, Y191C and I54T, R113K respectively. Mutants T178M, Q181H, R190C, M74V, G80R, I101F, R237Q, R237W showed complete loss in DNA binding activity towards their specific cognate sites. We measured the comprehensive protein‐DNA interactomes of NKX2‐5 and TBX5 non‐synonymous mutants using High‐Throughput Systematic Evolution of Ligands and Exponential Enrichment (SELEX‐seq) to identify changes in DNA targets between wild‐type and mutant TFs. Mutants A148E, R161P and Y191C bind to similar binding sites as NKX2‐5 (HD). Interestingly, mutant Y191C showed an altered DNA binding site significantly different from NKX2‐5 (HD). Mutants I54T, R113K bind to similar binding sites as TBX5 (Tbox). Mutants E154G, T178M, Q181H, R190C, M74V, G80R, I101F, R237Q and R237W showed no significant enrichment for specific DNA binding sites. Integrative analysis with available NKX2‐5 and TBX5 genomic binding sites data (ChIP‐seq), human and mouse cardiac enhancers and promoters catalogued in the literature will facilitate the identification of putative genes mutant cardiac TFs are regulating and how different are from their wild‐type TFs.

Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

BackgroundDNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators.ResultsThis study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions.ConclusionKfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

Single-gene imaging links genome topology, promoter-enhancer communication and transcription control.

Transcription activation by distal enhancers is essential for cell-fate specification and maintenance of cellular identities. How long-range gene regulation is physically achieved, especially within complex regulatory landscapes of non-binary enhancer-promoter configurations, remains elusive. Recent nanoscopy advances quantitatively linked promoter kinetics and ~100–200 nm-sized clusters of enhancer-associated regulatory factors (RFs) at important developmental genes. Here, we further dissect mechanisms of RF clustering and transcription activation in mouse embryonic stem cells. RF recruitment into clusters involves specific molecular recognition of cognate DNA and chromatin binding sites, suggesting underlying cis-element clustering. Strikingly, imaging tagged genomic loci, with ≤1 kilobase and ≈20 nanometer precision, in live cells, reveals distal enhancer clusters over the extended locus in frequent close proximity to target genes - within RF clustering distances. These high-interaction-frequency enhancer cluster “super-clusters” create nano-environments wherein clustered RFs activate target genes, providing a structural framework for relating genome organization, focal RF accumulation and transcription activation.

Crystal structure of TraN, a regulator of conjugative DNA transfer, in complex with its cognate DNA binding site

Crystal structure of TraN, a regulator of conjugative DNA transfer, in complex with its cognate DNA binding site

Characterization of Small Molecules Inhibiting the Pro-Angiogenic Activity of the Zinc Finger Transcription Factor Vezf1.

Discovery of inhibitors for endothelial-related transcription factors can contribute to the development of anti-angiogenic therapies that treat various diseases, including cancer. The role of transcription factor Vezf1 in vascular development and regulation of angiogenesis has been defined by several earlier studies. Through construction of a computational model for Vezf1, work here has identified a novel small molecule drug capable of inhibiting Vezf1 from binding to its cognate DNA binding site. Using structure-based design and virtual screening of the NCI Diversity Compound Library, 12 shortlisted compounds were tested for their ability to interfere with the binding of Vezf1 to DNA using electrophoretic gel mobility shift assays. We identified one compound, T4, which has an IC50 of 20 μM. Using murine endothelial cells, MSS31, we tested the effect of T4 on endothelial cell viability and angiogenesis by using tube formation assay. Our data show that addition of T4 in cell culture medium does not affect cell viability at concentrations lower or equal to its IC 50 but strongly inhibits the network formation by MSS31 in the tube formation assays. Given its potential efficacy, this inhibitor has significant therapeutic potential in several human diseases.

Selective Distance Measurements Using Triple Spin Labeling with Gd3+, Mn2+, and a Nitroxide.

Distance measurements by pulse electron paramagnetic resonance techniques, such as double electron-electron resonance (DEER, also called PELDOR), have become an established tool to explore structural properties of biomacromolecules and their assemblies. In such measurements a pair of spin labels provides a single distance constraint. Here we show that by employing three different types of spin labels that differ in their spectroscopic and spin dynamics properties it is possible to extract three independent distances from a single sample. We demonstrate this using the Antennapedia homeodomain orthogonally labeled with Gd3+ and Mn2+ tags in complex with its cognate DNA binding site labeled with a nitroxide.

Abstract NTOC-111: TARGETING COMPONENTS OF THE STAT3 SIGNALING PATHWAY FOR OVARIAN CANCER THERAPY: IMPLICATIONS FROM 3D CULTURE SYSTEMS

Abstract High grade serous ovarian cancer remains deadly for countless women; therefore, better treatment options are necessary. To identify new targeted therapies for this disease, it is necessary to delineate critical signaling pathways that are driving the pathogenesis of ovarian cancer. Since ovarian cancer in patients grows as three dimensional clusters, particularly within the peritoneum, we examined signaling pathways that were activated with 3D culture in vitro. Comparing the growth of ovarian cancer cell lines in 2D and 3D, we found that the transcription factor STAT3 consistently becomes activated in 3D culture systems. Ovarian cancer cells that lack STAT3 activation when grown in 2D acquire STAT3 activation when grown in 3D. Additionally, ovarian cancer cell lines that have constitutively active STAT3 when grown in 2D display enhanced STAT3 activation when these cells are grown in 3D. STAT3 is often activated through autocrine or paracrine mechanisms via cytokine receptors interacting with the transmembrane signal transducing molecule GP130 and intracellular Jak kinases. Once activated, these kinases tyrosine phosphorylate STAT3, which then translocates to the nucleus, binds to cognate DNA binding sites, and regulates genes that are involved in migration, invasion, and survival. We have found that not only is STAT3 tyrosine phosphorylated in cells grown in 3D, but that STAT3 is functionally active such that STAT3 DNA binding and target gene expression is enhanced in ovarian cancer cells grown in 3D versus 2D. We next determined if STAT3 signaling was necessary for ovarian cancer cell growth in 3D. Using RNA interference directed to STAT3 or GP130, we found that both proteins are necessary for the 3D growth of ovarian cancer cells. In addition, we have found that cells grown in 3D are more resistant to cytotoxic drugs; therefore, we wanted to determine if inhibiting STAT3 would enhance the response to conventional chemotherapy. Using both chemical biology and computational approaches, we have identified small molecule inhibitors of STAT3 that target a number of distinct points in the STAT3 activation pathway. We have found that a subset of these compounds disrupt the morphology of ovarian cancer spheroids. Furthermore, combination of these inhibitors with paclitaxel leads to enhanced loss of viability of cells grown in 3D, raising the possibility of using STAT3 inhibitors alone or in combination with chemotherapeutic drugs for the treatment of ovarian cancer. Citation Format: Sarah R. Walker, Yixi Zhang, Suhu Liu, Zachary T. Giaccone, and David A. Frank. TARGETING COMPONENTS OF THE STAT3 SIGNALING PATHWAY FOR OVARIAN CANCER THERAPY: IMPLICATIONS FROM 3D CULTURE SYSTEMS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-111.

Chemical principles additive model aligns low consensus DNA targets of p53 tumor suppressor protein

Computational prediction of the interaction between protein transcription factors and their cognate DNA binding sites in genomic promoters constitutes a formidable challenge in two situations: when the number of discriminatory interactions is small compared to the length of the binding site, and when DNA binding sites possess a poorly conserved consensus binding motif. The transcription factor p53 tumor suppressor protein and its target DNA exhibit both of these issues. From crystal structure analysis, only three discriminatory amino acid side chains contact the DNA for a binding site spanning ten base pairs. Furthermore, our analysis of a dataset of genome wide fragments binding to p53 revealed many sequences lacking the expected consensus. The low information content leads to an overestimation of binding sites, and the lack of conservation equates to a computational alignment problem. Within a fragment of DNA known to bind to p53, computationally locating the position of the site equates to aligning the DNA with the binding interface. From a molecular perspective, that alignment implies a specification of which DNA bases are interacting with which amino acid side chains, and aligning many sequences to the same protein interface concomitantly produces a multiple sequence alignment. From this vantage, we propose to cast prediction of p53 binding sites as an alignment to the protein binding surface with the novel approach of optimizing the alignment of DNA fragments to the p53 binding interface based on chemical principles. A scoring scheme based on this premise was successfully implemented to score a dataset of biological DNA fragments known to contain p53 binding sites. The results illuminate the mechanism of recognition for the protein-DNA system at the forefront of cancer research. These findings implicate that p53 may recognize its target binding sites via several different mechanisms which may include indirect readout.

Identification of Position-Specific Correlations between DNA-Binding Domains and Their Binding Sites. Application to the MerR Family of Transcription Factors

The large and increasing volume of genomic data analyzed by comparative methods provides information about transcription factors and their binding sites that, in turn, enables statistical analysis of correlations between factors and sites, uncovering mechanisms and evolution of specific protein-DNA recognition. Here we present an online tool, Prot-DNA-Korr, designed to identify and analyze crucial protein-DNA pairs of positions in a family of transcription factors. Correlations are identified by analysis of mutual information between columns of protein and DNA alignments. The algorithm reduces the effects of common phylogenetic history and of abundance of closely related proteins and binding sites. We apply it to five closely related subfamilies of the MerR family of bacterial transcription factors that regulate heavy metal resistance systems. We validate the approach using known 3D structures of MerR-family proteins in complexes with their cognate DNA binding sites and demonstrate that a significant fraction of correlated positions indeed form specific side-chain-to-base contacts. The joint distribution of amino acids and nucleotides hence may be used to predict changes of specificity for point mutations in transcription factors.

Genome-Wide Organization of GATA1 and TAL1 Determined at High Resolution.

Erythroid development and differentiation from multiprogenitor cells into red blood cells requires precise transcriptional regulation. Key erythroid transcription factors, GATA1 and TAL1, cooperate, along with other proteins, to regulate many aspects of this process. How GATA1 and TAL1 are juxtaposed along the DNA and their cognate DNA binding site across the mouse genome remains unclear. We applied high-resolution ChIP-exo (chromatin immunoprecipitation followed by 5′-to-3′ exonuclease treatment and then massively parallel DNA sequencing) to GATA1 and TAL1 to study their positional organization across the mouse genome during GATA1-dependent maturation. Two complementary methods, MultiGPS and peak pairing, were used to determine high-confidence binding locations by ChIP-exo. We identified ∼10,000 GATA1 and ∼15,000 TAL1 locations, which were essentially confirmed by ChIP-seq (chromatin immunoprecipitation followed by massively parallel DNA sequencing). Of these, ∼4,000 locations were bound by both GATA1 and TAL1. About three-quarters of them were tightly linked to a partial E-box located 7 or 8 bp upstream of a WGATAA motif. Both TAL1 and GATA1 generated distinct characteristic ChIP-exo peaks around WGATAA motifs that reflect their positional arrangement within a complex. We show that TAL1 and GATA1 form a precisely organized complex at a compound motif consisting of a TG 7 or 8 bp upstream of a WGATAA motif across thousands of genomic locations.

Structural determinants of DNA recognition by plant MADS-domain transcription factors

Plant MADS-domain transcription factors act as key regulators of many developmental processes. Despite the wealth of information that exists about these factors, the mechanisms by which they recognize their cognate DNA-binding site, called CArG-box (consensus CCW6GG), and how different MADS-domain proteins achieve DNA-binding specificity, are still largely unknown. We used information from in vivo ChIP-seq experiments, in vitro DNA-binding data and evolutionary conservation to address these important questions. We found that structural characteristics of the DNA play an important role in the DNA binding of plant MADS-domain proteins. The central region of the CArG-box largely resembles a structural motif called ‘A-tract’, which is characterized by a narrow minor groove and may assist bending of the DNA by MADS-domain proteins. Periodically spaced A-tracts outside the CArG-box suggest additional roles for this structure in the process of DNA binding of these transcription factors. Structural characteristics of the CArG-box not only play an important role in DNA-binding site recognition of MADS-domain proteins, but also partly explain differences in DNA-binding specificity of different members of this transcription factor family and their heteromeric complexes.

Correlated Evolution of Positions within Mammalian cis Elements

Transcriptional regulation critically depends on proper interactions between transcription factors (TF) and their cognate DNA binding sites. The widely used model of TF-DNA binding – the Positional Weight Matrix (PWM) – presumes independence between positions within the binding site. However, there is evidence to show that the independence assumption may not always hold, and the extent of interposition dependence is not completely known. We hypothesize that the interposition dependence should partly be manifested as correlated evolution at the positions. We report a Maximum-Likelihood (ML) approach to infer correlated evolution at any two positions within a PWM, based on a multiple alignment of 5 mammalian genomes. Application to a genome-wide set of putative cis elements in human promoters reveals a prevalence of correlated evolution within cis elements. We found that the interdependence between two positions decreases with increasing distance between the positions. The interdependent positions tend to be evolutionarily more constrained and moreover, the dependence patterns are relatively similar across structurally related transcription factors. Although some of the detected mutational dependencies may be due to context-dependent genomic hyper-mutation, notably CG to TG, the majority is likely due to context-dependent preferences for specific nucleotide combinations within the cis elements. Patterns of evolution at individual nucleotide positions within mammalian TF binding sites are often significantly correlated, suggesting interposition dependence. The proposed methodology is also applicable to other classes of non-coding functional elements. A detailed investigation of mutational dependencies within specific motifs could reveal preferred nucleotide combinations that may help refine the DNA binding models.

Unzip Single Protein Zippers using Optical Tweezers

Basic leucine zippers (bZIPs) are dimeric alpha-helical domains found in many transcription factors. They comprise coiled coil regions (zippers) that mediate specific protein dimerization, and basic regions that undergo disorder-to-order transitions to grip their cognate DNA binding sites. It remains unclear how folding of the two regions and binding of DNA are coupled. We investigated the detailed folding kinetics of a model bZIP domain derived from GCN4 transcriptional factor, using high-resolution optical tweezers. When pulled from the same amino- or carboxyl-terminals, the coiled coil alone folds and unfolds rapidly in a two-state manner, with little energy barrier. The folding kinetics of the full GCN4 bZIP domain is similar to the coiled coil alone. However, the presence of its binding site (AP1) induces a third new state corresponding to the bZIP-DNA complex. The probability of this new state critically depends upon the AP1 concentration, which reaches a maximum at an intermediate AP1 concentration. Results from our single-molecule manipulation experiments reveal a complex folding energy landscape even for a small protein domain that can be further tuned by their binding ligands.

Development of a High-Throughput Screen for Inhibitors of Epstein-Barr Virus EBNA1

Latent infection with Epstein-Barr virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small-molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work, the authors describe the development of a biochemical high-throughput screening (HTS) method using a homogeneous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. The authors demonstrate that EBNA1 binding to a fluorescent-labeled DNA probe provides a robust assay with a Z factor consistently greater than 0.6. A pilot screen of a small-molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1 but not Zta. All 3 compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof of concept that small-molecule inhibitors of EBNA1 can be identified by biochemical HTS of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.

Electrophoretic mobility shift assay of zinc finger proteins: Competition for Zn 2+ bound to Sp1 in protocols including EDTA

The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA–protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn 2+ and readily competes with zinc finger peptides for Zn 2+ resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein–DNA adducts. This paper examines the chemistry that permits the detection of zinc finger–DNA complexes in the presence of EDTA, using Zn 3-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1–GC1 was shown to depend on three properties: the inertness of Zn-Sp1–GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn 3-Sp1 under the conditions of the assay that permit some Zn 3-Sp1–GC1 to form. Inquiring about the mechanism of stabilization of Zn 3-Sp1 by GC1, EDTA readily reacted with Zn 3-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn 3-Sp1 failed to compete for zinc bound in the Zn 3-Sp1–GC-1 adduct. On the basis of these, other results indicated that the stability of Zn 3-Sp1–GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.

Conservation and Evolution in and among SRF- and MEF2-Type MADS Domains and Their Binding Sites

Serum response factor (SRF) and myocyte enhancer factor 2 (MEF2) represent two types of members of the MCM1, AGAMOUS, DEFICIENS, and SRF (MADS)-box transcription factor family present in animals and fungi. Each type has distinct biological functions, which are reflected by the distinct specificities of the proteins bound to their cognate DNA-binding sites and activated by their respective cofactors. However, little is known about the evolution of MADS domains and their DNA-binding sites. Here, we report on the conservation and evolution of the two types of MADS domains with their cognate DNA-binding sites by using phylogenetic analyses. First, there are great similarities between the two types of proteins with amino acid positions highly conserved, which are critical for binding to the DNA sequence and for the maintenance of the 3D structure. Second, in contrast to MEF2-type MADS domains, distinct conserved residues are present at some positions in SRF-type MADS domains, determining specificity and the configuration of the MADS domain bound to DNA sequences. Furthermore, the ancestor sequence of SRF- and MEF2-type MADS domains is more similar to MEF2-type MADS domains than to SRF-type MADS domains. In the case of DNA-binding sites, the MEF2 site has a T-rich core in one DNA sequence and an A-rich core in the reverse sequence as compared with the SRF site, no matter whether where either A or T is present in the two complementary sequences. In addition, comparing SRF sites in the human and the mouse genomes reveals that the evolution rate of CArG-boxes is faster in mouse than in human. Moreover, interestingly, a CArG-like sequence, which is probably functionless, could potentially mutate to a functional CArG-box that can be bound by SRF and vice versa. Together, these results significantly improve our knowledge on the conservation and evolution of the MADS domains and their binding sites to date and provide new insights to investigate the MADS family, which is not only on evolution of MADS factors but also on evolution of their binding sites and even on coevolution of MADS factors with their binding sites.

Estrogen Receptors Recruit SMRT and N-CoR Corepressors through Newly Recognized Contacts between the Corepressor N Terminus and the Receptor DNA Binding Domain

Estrogen receptors (ERs) are hormone-regulated transcription factors that regulate key aspects of reproduction and development. ERs are unusual in that they do not typically repress transcription in the absence of hormone but instead possess otherwise cryptic repressive functions that are revealed upon binding to certain hormone antagonists. The roles of corepressors in the control of these aspects of ER function are complex and incompletely understood. We report here that ERs recruit SMRT through an unusual mode of interaction involving multiple contact surfaces. Two surfaces of SMRT, located at the N- and C-terminal domains, contribute to the recruitment of the corepressor to ERs in vitro and are crucial for the corepressor modulation of ER transcriptional activity in cells. These corepressor surfaces contact the DNA binding domain of the receptor, rather than the hormone binding domain previously elucidated for other corepressor/nuclear receptor interactions, and are modulated by the ER's recognition of cognate DNA binding sites. Several additional nuclear receptors, and at least one other corepressor, N-CoR, share aspects of this novel mode of corepressor recruitment. Our results highlight a molecular mechanism that helps explain several previously paradoxical aspects of ER-mediated transcriptional antagonism, which may have a broader significance for an understanding of target gene repression by other nuclear receptors.