Cognate DNA Binding Site Research Articles

Chimeras of the Flp and Cre recombinases: tests of the mode of cleavage by flp and cre

The Flp and Cre recombinases are members of the integrase family of tyrosine recombinases. Each protein consists of a 13 kDa NH2-terminal domain and a larger COOH-terminal domain that contains the active site of the enzyme. The COOH-terminal domain also contains the major determinants for the binding specificity of the recombinase to its cognate DNA binding site. All family members cleave the DNA by the attachment of a conserved nucleophilic tyrosine residue to the 3′-phosphate group at the sites of cleavage.In order to gain further insights into the determinants of the binding specificity and modes of cleavage of Flp and Cre, we have made chimeric proteins in which we have fused the NH2-terminal domain of Flp to the COOH-terminal domain of Cre (“Fre”) and the NH2-terminal domain of Cre to the COOH-terminal domain of Flp (“Clp”). These chimeras have novel binding specificities in that they bind strongly to hybrid sites containing elements from both the Flp and Cre DNA targets but poorly to the native target sites.In this study we have taken advantage of the unique binding specificities of Fre and Clp to examine the mode of cleavage by Cre, Flp, Fre and Clp. We find that the COOH-terminal domain of the recombinases determines their mode of cleavage. Thus Flp and Clp cleave in trans whereas Cre and Fre cleave in cis. These results agree with the studies of Flp and with the cocrystal structure of Cre bound to its DNA target site. They disagree with our previous findings that Cre could carry out trans cleavage. We discuss the variations in the experimental approaches in order to reconcile the different results.

Molecular targets of a human HNF1 alpha mutation responsible for pancreatic beta-cell dysfunction.

The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1 alpha (HNF1 alpha)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1 alpha-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1 alpha for the cognate DNA-binding site. HNF1 alpha controls multiple genes implicated in pancreatic beta-cell function and notably in metabolism- secretion coupling. In addition to reduced expression of the genes encoding insulin, glucose transporter-2, L-pyruvate kinase, aldolase B and 3-hydroxy-3-methylglutaryl coenzyme A reductase, induction of HNF1 alpha-P291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [(14)C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1 alpha-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1 alpha-P291fsinsC also inhibits insulin secretory responses to glucose and leucine, correlated with impaired nutrient-evoked mitochondrial ATP production and mitochondrial membrane hyperpolarization. These unprecedented results suggest the molecular mechanism of HNF1 alpha-P291fsinsC causing beta-cell dysfunction.

Role of the N- and C- Terminal Dimer Interfaces of 434 Repressor in Recognizing Sequence-Dependent DNA Structure

The binding of proteins to specific DNA sequences plays a central role in the regulation of gene expression. Crucial to understanding how these proteins exert their effects is insight into the structure and flexibility of the protein-DNA complex. Over the past several years much has been learned about how the intimate contacts made between proteins and DNA enable proteins to recognize and bind with high specificity only to their cognate DNA binding sites. Studies conducted in our laboratory have shown that sequence-specific binding of DNA by proteins not only involves the close approach of amino acids and base pairs in the binding site, but also that base pairs not in contact with the protein affect binding and specificity through sequence-specific effects on DNA structure. The direct reading of the DNA sequence by proteins occurs by chemical complementarity between the interacting groups. Proper alignment of the interacting surfaces of functional groups on the protein and DNA molecules is crucial to the formation of stable and specific protein-DNA complexes. In many cases, the appropriate juxtaposition of the chemically complementary groups requires mutual adjustments in the structure of protein and DNA. Failure to do so can result in loss of affinity, loss of specificity or both. Together, the dimer interface and noncontacted bases within or adjacent to the binding site direct the structural complementarity between the functional groups on the protein and DNA.

Multiple Modes of Repression by the Smad Transcriptional Corepressor TGIF

TGIF is a DNA-binding homeodomain protein that has been demonstrated to play a role in transforming growth factor beta-regulated transcription and implicated in the control of retinoid-responsive transcription. We investigated the intrinsic transcriptional activity of TGIF fused to a heterologous DNA-binding domain. Our results demonstrate that TGIF is a transcriptional repressor able to repress transcription from several different promoters. Repression by TGIF is insensitive to the distance at which it is bound from the promoter. Moreover, the wild type TGIF effectively represses transcription when bound to its cognate DNA-binding site via its homeodomain. Deletion analysis revealed the presence of at least two separable repression domains within TGIF. Repression by one of these is dependent on the activity of histone deacetylases (HDACs), whereas the other appears not to require HDAC activity. Finally, we demonstrate that TGIF interacts with HDACs via its carboxyl-terminal repression domain. Together, these results suggest that TGIF is a multifunctional transcriptional repressor, which acts in part by recruiting HDAC activity.

The recognition of distorted DNA structures by HMG-D: a footprinting and molecular modelling study

The high mobility group (HMG) domain is a DNA binding motif found in some eukaryotic chromosomal proteins and transcription factors. This domain binds in the minor groove of DNA inducing a sharp bend and also preferentially binds to certain distorted DNA structures. Although structures of sequence-specific HMG domains with their cognate double-helical DNA binding sites have been solved, the nature of the interaction of the domain with distorted DNA remains to be established. In this study we have investigated the interaction of HMG-D, a Drosophilacounterpart of the vertebrate HMG1, with a DNA oligomer containing a bulge of two adenine residues. We show by footprinting that HMG-D binds preferentially on one side of the bulged DNA. Based on these data and on the published NMR structures of the HMG domain of HMG-D and the LEF-1-DNA complex, we modelled the HMG-D - bulged DNA complex. This model predicts that two residues, Val32 and Thr33, in the loop between α-helices I and II are inserted deep into the “hole” in the DNA formed by the two missing bases on one strand of the DNA bulge. Mutation of these residues confirmed that both are required for the efficient binding and bending of DNA by HMG-D. We discuss both the role of this loop in the recognition of distorted DNA structures by non-sequence specific HMG domain proteins and that of the basic tail in stabilising the induced DNA bend.

Modulation of Human Heat Shock Factor Trimerization by the Linker Domain

Heat shock transcription factors (HSFs) are stress-responsive proteins that activate the expression of heat shock genes and are highly conserved from bakers' yeast to humans. Under basal conditions, the human HSF1 protein is maintained as an inactive monomer through intramolecular interactions between two coiled-coil domains and interactions with heat shock proteins; upon environmental, pharmacological, or physiological stress, HSF1 is converted to a homotrimer that binds to its cognate DNA binding site with high affinity. To dissect regions of HSF1 that make important contributions to the stability of the monomer under unstressed conditions, we have used functional complementation in bakers' yeast as a facile assay system. Whereas wild-type human HSF1 is restrained as an inactive monomer in yeast that is unable to substitute for the essential yeast HSF protein, mutations in the linker region between the DNA binding domain and the first coiled-coil allow HSF1 to homotrimerize and rescue the viability defect of a hsfDelta strain. Fine mapping by functional analysis of HSF1-HSF2 chimeras and point mutagenesis revealed that a small region in the amino-terminal portion of the HSF1 linker is required for maintenance of HSF1 in the monomeric state in both yeast and in transfected human 293 cells. Although linker regions in transcription factors are known to modulate DNA binding specificity, our studies suggest that the human HSF1 linker plays no role in determining HSF1 binding preferences in vivo but is a critical determinant in regulating the HSF1 monomer-trimer equilibrium.

Corepressor required for adenovirus E1B 55,000-molecular-weight protein repression of basal transcription.

Adenovirus E1B 55,000-molecular-weight protein (55K) binds to host cell p53, stabilizing it, greatly increasing its affinity for its cognate DNA-binding site, and converting it from a regulated activator to a constitutive repressor. Here we analyzed the mechanism of repression by the p53-E1B 55K complex. E1B 55K repression requires that 55K be tethered to the promoter by binding directly to DNA-bound p53. Transcription from an assembled, p53-activated preinitiation complex was not repressed by the subsequent addition of E1B 55K, suggesting that either sites of 55K interaction with p53 or targets of 55K in the preinitiation complex are blocked. Specific E1B 55K repression was observed in reactions lacking TFIIA and with recombinant TATA-binding protein in place of TFIID, conditions under which p53 does not activate transcription. Thus, E1B 55K does not simply inhibit a p53-specific activation mechanism but rather blocks basal transcription. As a consequence, E1B 55K may repress transcription from any promoter with an associated p53-binding site, no matter what other activators associate with the promoter. E1B 55K did not repress basal transcription in reactions with recombinant and highly purified general transcription factors and RNA polymerase II but rather required a corepressor that copurifies with the polymerase.

Testis-specific TTF-D Binds to Single-stranded DNA in the c-mos and Odf1 Promoters and ActivatesOdf1

We recently identified testis-specific nuclear factor binding sites in the testis-specific promoters of the c-mos gene and the Odf1 gene, which are 80% identical. Here we characterize a testis-specific nuclear factor, TTF-D, which is able to complex with both binding sites and stimulates Odf1 promoter activity. TTF-D is detectable in mouse testis as early as day 11 postpartum and contains three peptides of 22, 25, and 35 kDa in size. Surprisingly, TTF-D binds specifically to its cognate double-stranded DNA binding site as well as to its single-stranded DNA binding site. Both double-stranded and single-stranded binding site oligonucleotide DNA can specifically repress Odf1 promoter activity. Our results suggest that TTF-D is involved in positive transcription regulation of a pre-meiotic and a post-meiotic gene in the testis.

The SV40 large T oncoprotein disrupts DNA-binding of the cell cycle-regulated transcriptional repressor CDF

A hallmark of neoplastic transformation by DNA tumor viruses is the deregulation of cell cycle genes. At least in some genes, this deregulation appears to be due to the oncoprotein-mediated disruption of complexes between E2F and pocket proteins and the ensuing generation of transcriptionally active free E2F. In the present study, we have analysed the effect of the SV40 large T oncoprotein (SV-LT) on the function of a different cell cycle-regulated transcriptional repressor, CDF, which is the principal regulator of the cdc25C, cyclin A and cdc2 genes. As shown by genomic footprinting of sorted G1 and G2 cell populations, transformation by SV-LT completely abrogated protection of the CDF binding site (CDE-CHR) in the cdc25C promoter. In agreement with this observation, expression of the SV-LT in fibroblasts led to a dramatic up-regulation of the cdc25C promoter in cells synchronized in G0. These findings indicate that the oncoprotein-mediated dissociation of the CDF repressor protein from its cognate DNA-binding site is a major mechanism in virus-induced transcriptional deregulation.

A novel DNA-binding motif in MarA: the first structure for an AraC family transcriptional activator.

A crystal structure for a member of the AraC prokaryotic transcriptional activator family, MarA, in complex with its cognate DNA-binding site is described. MarA consists of two similar subdomains, each containing a helix-turn-helix DNA-binding motif. The two recognition helices of the motifs are inserted into adjacent major groove segments on the same face of the DNA but are separated by only 27 A thereby bending the DNA by approximately 35 degrees. Extensive interactions between the recognition helices and the DNA major groove provide the sequence specificity.

Sequence Specific Collective Motions in a Winged Helix DNA Binding Domain Detected by 15N Relaxation NMR

The recognition between transcription factors and their DNA binding sites is a highly dynamic process. During transcriptional regulation, transcription factors must bind to or dissociate from their cognate DNA binding sites. The winged helix DNA binding motif is one of many highly conserved DNA binding motifs identified in transcription factors. Backbone dynamics has been studied on the 15N- and 2H-enriched winged helix family member Genesis. Our data show that the overall motions of the single domain Genesis are better described by more than two autocorrelation times (taum). Our data also demonstrate that Genesis shows structure specific conformation exchange characterized by Rex. Therefore, our results indicate that the structure of Genesis is highly dynamic and that secondary structure elements in Genesis have collective motions in the nanosecond to millisecond time scale. Since the winged helix DNA binding motif is highly conserved, this unique dynamic property observed in Genesis is also likely to be conserved in other winged helix family members and important in DNA binding.

Overlapping RNA and DNA binding domains of the wt1 tumor suppressor gene product.

The Wilms' tumour suppressor gene (wt1) is mutated in a subset of patients with Wilms' tumour and has a critical role in urogenital development. wt1 encodes a zinc finger transcription factor which regulates expression of several genes involved in cellular proliferation and differentiation. Although a number of studies have characterized the DNA binding properties of the WT1 protein, recent evidence has suggested that WT1 may also have a role in RNA metabolism. We have used an RNA selection method to identify WT1 binding ligands from a random RNA pool. Three groups of RNA ligands specifically recognized by WT1 were identified. Mutational analysis pinpointed ribonucleotide sequences critical for binding. Analysis of truncated WT1 proteins demonstrated that three of four zinc fingers were necessary for RNA-protein interaction. The naturally occurring WT1 isoforms with insertion of lysine, threonine and serine between zinc fingers three and four were unable to bind the selected RNAs. The selected RNA ligands competed with the cognate WT1 DNA binding site for complex formation with WT1. Our findings suggest potential cellular RNA target sequences for WT1 and provide tools for studying the structural and functional properties of this tumour suppressor protein.

The Hepatitis B Virus X Protein Enhances the DNA Binding Potential and Transcription Efficacy of bZip Transcription Factors

The hepatitis B virus X protein interacts with the basic-region, leucine zipper protein (bZip) domain of cAMP response element-binding protein increasing its affinity for the cAMP response element site in vitro and its transcriptional efficacy in vivo (Williams, J. S., and Andrisani, O. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3819-3823). Here we examine pX interactions with bZip transcription factors ATF3, gadd153/Chop10, ICER IIgamma, and NF-IL6. We demonstrate direct interactions in vitro between pX and the bZip proteins tested. In contrast MyoD and Gal4(1-147) fail to interact with pX. We also demonstrate by the mammalian two-hybrid assay the direct interaction of pX with cAMP response element- binding protein, ICER IIgamma, ATF3, and NF-IL6 in hepatocytes. In addition, pX increases the DNA binding potential of bZip proteins for their cognate DNA-binding site in vitro. In transient transfections in hepatocytes (AML12 cell line), pX increases the transcriptional efficacy of the bZip transcription factors. NF-IL6-mediated transcriptional activation is enhanced 3-fold by pX. Most interestingly, pX augments the repression mediated by bZip repressors ATF3 and ICER IIgamma, by 6- and 7-fold, respectively, demonstrating for the first time the involvement of pX in gene repression. We conclude that pX is an enhancer of the DNA binding potential of bZip transcription factors, thereby increasing the transactivation or repression efficacy of bZip-responsive genes.

Proliferation of hepatic stellate cells is inhibited by phosphorylation of CREB on serine 133.

Proliferating, activated, hepatic stellate cells have a high level of collagen type I expression. Therefore, stellate cell proliferation is a critical step in hepatic fibrosis. Here we show that proliferation of activated primary rat stellate cells was blocked by elevation of cAMP with 8 Br-cAMP or isomethylbutyl xanthine, a phosphodiesterase inhibitor, and by stimulation of Ca2+ fluxes with the Ca2+ ionophore A-23187. Because phosphorylation of CREB on Ser133 is an important mediator of cAMP-protein kinase (PKA) and Ca2+-calmodulin kinase II (CAMK-II) activation, we tested whether CREB-PSer133 was essential for stellate cell quiescence. Nuclear extracts from quiescent, but not from activated, stellate cells contained CREB-PSer133. Moreover, the phosphorylation of CREB on Ser133 was stimulated in activated cells by inducing the activity of PKA or CAMK-II. In addition, coexpression of CREB and either a constitutively active PKA or a constitutively active CAMK-II inhibited the proliferation of activated stellate cells. In contrast, expression of CREB alone, PKA or CAMK-II alone, CREB-Ala 133 (which lacks the Ser133 phosphoacceptor) with PKA or CAMK-II, or CREB with inactive PKA or CAMK-II mutants did not affect stellate cell proliferation, suggesting that CREB-PSer133 is necessary for blocking the stellate cell cycle. Conversely, expression of a trans-dominant negative CREB-Ala 133 mutant (which competes with CREB/CREB-PSer133 for cognate DNA binding sites and presumably for protein interactions) induced a greater than fivefold entry into S-phase of quiescent stellate cells, compared with control cells expressing either beta-galactosidase or wt CREB, indicating that CREB-PSer133 may be indispensable for the quiescent stellate cell phenotype. This study suggests that PKA and CAMK-II play an essential role on stellate cell activation through the induction of CREB phosphorylation on Ser133, and provides potential approaches for the treatment of hepatic fibrogenesis in patients with chronic liver diseases.

Aerobic glycolysis by proliferating cells: protection against oxidative stress at the expense of energy yield.

Primary cultures of mitogen-activated rat thymocytes were used to study energy metabolism, gene expression of glycolytic enzymes, and production of reactive oxygen species during cell cycle progression. During transition from the resting to the proliferating state a 7- to 10-fold increase of glycolytic enzyme induction occurs which enables the cells to meet the enhanced energy demand by increased aerobic glycolysis. Cellular redox changes have been found to regulate gene expression of glycolytic enzymes by reversible oxidative inactivation of Sp1-binding to the cognate DNA-binding sites in the promoter region. In contrast to nonproliferating cells, production of phorbol 12-myristate 13-acetate (PMA)-primed reactive oxygen species (ROS) in proliferating rat thymocytes and HL-60 cells is nearly abolished. Pyruvate, a product of aerobic glycolysis, is an effective scavenger of ROS, which could be shown to be generated mainly at the site of complex III of the mitochondrial respiratory chain. Aerobic glycolysis by proliferating cells is discussed as a means to minimize oxidative stress during the phases of the cell cycle when maximally enhanced biosynthesis and cell division do occur.

The DNA binding domain of the initiator protein DnaA.

The 94 C-terminal amino acids of the initiator protein DnaA of Escherichia coli are required and sufficient for specific binding to the cognate DNA binding site. The binding domain contains two potential amphipathic alpha-helices and a third alpha-helix. It represents a new DNA binding motif so far not found in other DNA binding proteins. Temperature-sensitive mutations in the binding motif, dnaA204, dnaA205 and dnaA211, abolish DNA binding. In the solid-phase DNA binding assay, applicable to other DNA binding proteins, fusions of domains of DnaA protein to beta-galactosidase are reacted with biotinylated anti-beta-galactosidase antibody. These are coupled to streptavidin-coated magnetic beads. The DNA binding domain is able to selectively remove the DNA target (oriC) from the liquid phase. Alternatively, the DNA binding domain is fused to a peptide containing a target sequence which is naturally biotinylated in vivo in E.coli. This fusion protein can be coupled directly to streptavidin-coated magnetic beads. Homologies between DnaA protein and transcription factors of the NtrC family are discussed.

Transcriptional activation by the orphan nuclear receptor ARP-1.

ARP-1 is a ubiquitous orphan nuclear receptor that binds to a site (site A) in the apolipoprotein AI (apoAI) liver-specific enhancer and represses its transcriptional activity in hepatoblastoma HepG2 cells. Electrophoretic mobility shift analysis of HepG2 cell nuclear extracts showed that in addition to ARP-1, site A also binds the orphan nuclear receptors Ear-2 and HNF-4. In in vitro transcription assays, Hela cell nuclear extracts which contain ARP-1 had no effect on transcription from a basal promoter linked to multiple copies of site A. However, supplementation of these extracts with excess amounts of recombinant ARP-1 resulted in significant stimulation. Supplementation of the extracts with purified polypeptides representing fusions between the ARP-1 N- or C-terminal domains and the yeast activator GAL4 DNA binding domain also stimulated transcription from a basal promoter linked to multiple GAL4 DNA binding sites. Co-immunoprecipitation assays using ARP-1-selective antibodies revealed specific physical interactions between ARP-1 and the basal transcription factor TFIIB. We conclude that ARP-1 possesses intrinsic transcription activation potential which is modulated, at least in part, by the intracellular balance of other nuclear receptors that also bind to its cognate DNA binding site.

The replication terminator protein of the gram-positive bacterium Bacillus subtilis functions as a polar contrahelicase in gram-negative Escherichia coli.

The replication terminator protein (RTP) of Bacillus subtilis is a dimer with a monomeric molecular mass of 14.5 kDa. The protein terminates DNA replication at a specific binding site. Although the protein has been crystallized and its crystal structure has been solved, the lack of an in vitro replication system in B. subtilis has been a serious impediment to the analysis of the mechanism of action of this protein. We have discovered that the protein is functional in the Gram-negative bacterium Escherichia coli in vivo and in vitro. RTP blocked replication forks initiated from a ColE1 replication origin at the cognate DNA-binding site (BS3) in a polar mode. The protein did not block rolling circle replication initiated from the pT181 origin in cell extracts of Staphylococcus aureus. RTP antagonized the helicase activity of DnaB but not that of helicase II of E. coli. Thus, RTP functioned as a polar contrahelicase blocking a helicase that participates in symmetric DNA replication but it did not impede rolling circle replication nor the action of a helicase involved in DNA repair.

Transforming growth factor-beta stimulates alpha 2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site.

Transforming growth factor-beta 1 (TGF-beta) is a strong and rapid inducer of several genes coding for extracellular matrix components, such as type I collagen. We report here that TGF-beta stimulates transcription of the human alpha 2(I) collagen gene (COL1A2) promoter by increasing the affinity of an Sp1-containing protein complex for its cognate DNA-binding site. Cell transfection experiments mapped the TGF-beta-responsive element (TbRE) of the COL1A2 promoter to a 131 bp region that contains at least two cis-acting elements. Insertion of the TbRE upstream of the thymidine kinase promoter conferred TGF-beta inducibility to the otherwise unresponsive thymidine kinase promoter. Footprinting assays revealed that the TbRE contains two neighboring protein-bound sequences, termed Box 3A and Box B. Within Box 3A is an Sp1 recognition sequence whose structural integrity is required for nuclear protein binding in vitro, and for promoter inducibility in vivo. Gel mobility shift assays documented increased binding to the TbRE of nuclear proteins from TGF-beta-treated cells compared with those from TGF-beta-untreated fibroblasts. There was, however, no binding increase with Box 3A alone or with an Sp1 oligonucleotide. Thus, the results strongly suggest a functional interaction between Sp1 and other components of the TbRE complex in mediating TGF-beta stimulation of COL1A2 gene expression.

Biophysical characteristics of Tus, the replication arrest protein of Escherichia coli.

Tus, a DNA-binding protein, mediates arrest of DNA replication in Escherichia coli. Tus binds to DNA sequences called Ter sites, located in the terminus region of the chromosome, and forms replication-arrest complexes that block movement of DNA replication forks in a polar fashion. We have analyzed Tus to determine some of its physical parameters and biochemical characteristics. Native Tus had an 8(20,w) of 3.2, a Stokes' radius of 23 A, an axial ratio of 2, and a molar absorption coefficient of 39,700 M-1 cm-1. The data also indicated that Tus existed as a monomeric protein in solution and when complexed with its cognate DNA binding site. Secondary structure estimated from the circular dichroism spectrum suggested that Tus consisted of 40% alpha-helix, 0% beta-sheet, 15% turn, and 45% aperiodic structure. The isoelectric point of native Tus (pH 7.5) was significantly different than that calculated from its amino acid sequence (pH 10.1), possibly because the tertiary structure of Tus perturbs the ionization of several residues. In addition, partial proteolytic digests of free Tus protein did not produce a subfragment of Tus that retained DNA binding activity, but did demonstrate that Tus was resistant to proteolysis when complexed with a Ter site.