DNA-binding Transcriptional Regulators Research Articles

TATA binding protein associated factor 3 (TAF3) interacts with p53 and inhibits its function

BackgroundThe tumour suppressor protein p53 is a sequence specific DNA-binding transcription regulator, which exerts its versatile roles in genome protection and apoptosis by affecting the expression of a large number of genes. In an attempt to obtain a better understanding of the mechanisms by which p53 transcription function is regulated, we studied p53 interactions.ResultsWe identified BIP2 (Bric-à-brac interacting protein 2), the fly homolog of TAF3, a histone fold and a plant homeodomain containing subunit of TFIID, as an interacting partner of Drosophila melanogaster p53 (Dmp53). We detected physical interaction between the C terminus of Dmp53 and the central region of TAF3 both in yeast two hybrid assays and in vitro. Interestingly, DmTAF3 can also interact with human p53, and mammalian TAF3 can bind to both Dmp53 and human p53. This evolutionarily conserved interaction is functionally significant, since elevated TAF3 expression severely and selectively inhibits transcription activation by p53 in human cell lines, and it decreases the level of the p53 protein as well.ConclusionWe identified TAF3 as an evolutionarily conserved negative regulator of p53 transcription activation function.

Isolation and functional characterization of Spirulina D6D gene promoter: Role of a putative GntR transcription factor in transcriptional regulation of D6D gene expression

Δ6-Desaturase (D6D) is a key enzyme that catalyzes the synthesis of γ-linolenic acid (GLA), an essential polyunsaturated fatty acid. We report here the isolation and first functional characterization of the D6D gene promoter from Spirulina platensis C1. Functional analysis of this isolated promoter showed that the Spirulina promoter was functional in Escherichia coli. Site-specific mutation studies demonstrated that the −10 sequence (TATAAT), located at −33 bp relative to the translation start site, was essential for D6D promoter function. Temperature responsive deletion analysis studies identified the minimal core promoter within the region −51 to +1, which was sufficient for basal D6D promoter activity, and several cold-shock responsive cis-acting elements with activating and repressing activity. Electrophoretic mobility shift assay and LC–MS/MS studies demonstrated that an ‘AT-rich Inverted Repeat’ (−192 to −164) served as a target-binding site for a transcriptional regulator (probably a member of the GntR family) from Spirulina. Western blot analysis studies revealed that the DNA-binding transcriptional regulator underwent phosphorylation after a temperature downshift and possibly associated with transcriptional regulation of D6D gene expression. Taken together, our results suggest complex regulation of D6D gene expression in Spirulina.

Determinants of Oncogenic Transformation in Acute Promyelocytic Leukemia: The Hetero-Union Makes the Force

Acute promyelocytic leukemia (APL) is caused by chromosomal translocations that involve the retinoic acid receptor alpha (RAR) and several other genes to yield X-RAR fusion proteins. Unlike wild-type RARs, which require heterodimerization with the retinoid X receptor (RXR) for their function as DNA-binding transcriptional regulators, X-RAR fusion proteins bind DNA and deregulate transcription as homo-oligomers. In this issue of Cancer Cell, however, Zeisig et al. and Zhu et al. show that RXR recruitment is a critical determinant for the transforming potential of oligomeric X-RAR fusion proteins and explore the possibility for targeted interventions in APL with either RAR or RXR ligands.

Catabolite Activator Protein in Aqueous Solution: A Molecular Simulation Study

The homodimeric catabolite activator protein (CAP) is a bacterial DNA binding transcription regulator whose activity is controlled by the binding of the intracellular mediator cyclic adenosine monophosphate (cAMP). Each CAP subunit consists of a cyclic nucleotide and a DNA binding domain. Here, we investigate the structural features of the ligand-bound CAP in aqueous solution by molecular dynamics simulations based on the available X-ray structures (Passner et al. J. Mol. Biol. 2000, 304, 847-859 and Chen et al. J. Mol. Biol. 2001, 314, 63-74). Our calculations suggest that the homodimer in solution assumes a symmetric arrangement in which both DNA binding domains are separated from the respective cyclic nucleotide binding domains by a cleft. This contrasts with the X-ray structure, which exhibits instead an asymmetric conformation. On the basis of electrostatics calculations, we propose that the symmetric structure in solution may be an important feature for DNA molecular recognition.

The transcriptional regulatory network of the amino acid producer Corynebacterium glutamicum

The complete nucleotide sequence of the Corynebacterium glutamicum ATCC 13032 genome was previously determined and allowed the reliable prediction of 3002 protein-coding genes within this genome. Using computational methods, we have defined 158 genes, which form the minimal repertoire for proteins that presumably act as transcriptional regulators of gene expression. Most of these regulatory proteins have a direct role as DNA-binding transcriptional regulator, while others either have less well-defined functions in transcriptional regulation or even more general functions, such as the sigma factors. Recent advances in genome-wide transcriptional profiling of C. glutamicum generated a huge amount of data on regulation of gene expression. To understand transcriptional regulation of gene expression from the perspective of systems biology, rather than from the analysis of an individual regulatory protein, we compiled the current knowledge on the defined DNA-binding transcriptional regulators and their physiological role in modulating transcription in response to environmental signals. This comprehensive data collection provides a solid basis for database-guided reconstructions of the gene regulatory network of C. glutamicum, currently comprising 56 transcriptional regulators that exert 411 regulatory interactions to control gene expression. A graphical reconstruction revealed first insights into the functional modularity, the hierarchical architecture and the topological design principles of the transcriptional regulatory network of C. glutamicum.

Versatility and Connectivity Efficiency of Bipartite Transcription Networks

The modulation of promoter activity by DNA-binding transcription regulators forms a bipartite network between the regulators and genes, in which a smaller number of regulators control a much lager number of genes. To facilitate representation of gene expression data with the simplest possible network structure, we have characterized the ability of bipartite networks to describe data. This has led to the classification of two types of bipartite networks, versatile and nonversatile. Versatile networks can describe any data of the same rank, and are indistinguishable from one another. Nonversatile networks require constraints to be present in data they describe, which may be used to distinguish between different network topologies. By quantifying the ability of bipartite networks to represent data we were able to define connectivity efficiency, which is a measure of how economic the use of connections is within a network with respect to data representation and generation. We postulated that it may be desirable for an organism to maximize its gene expression range per network edge, since development of a regulatory connection may have some evolutionary cost. We found that the transcriptional regulatory networks of both Saccharomyces cerevisiae and Escherichia coli lie close to their respective connectivity efficiency maxima, suggesting that connectivity efficiency may have some evolutionary influence.

Dlx homeobox gene control of mammalian limb and craniofacial development

The Dlx homeobox gene family is of ancient origin and crucial for embryonic development in invertebrates and vertebrates. The Dlx proteins are thought to function as DNA-binding transcriptional regulators, likely controlling large numbers of downstream effector genes. In mammals gene expression analysis of the six members of the Dlx gene family has been demonstrated in the nervous system, neural crest derivatives, branchial arches, and developing appendages. Through genetic approaches the Dlx genes have been implicated in patterning and development of the brain, craniofacial structures, and the axial and appendicular skeleton. Substantial functional redundancy within the Dlx gene family has prevented the analysis of single gene mutations from demonstrating the full developmental control exerted by the Dlx proteins. Here, we will discuss data from recent combined loss and gain-of-function genetic mutations, which have given greater insight into the role of Dlx homeobox genes in craniofacial, limb, and bone development.

An eh1-Like Motif in Odd-skipped Mediates Recruitment of Groucho and Repression In Vivo

Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined. First, WRPW and related tetrapeptide motifs have been well characterized in several repressors. Second, a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. Here we describe a yeast two-hybrid screen that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. We show that one of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation. Comparison between diverse eh1 motifs reveals a bias for the phosphoacceptor amino acids serine and threonine at a fixed position, and a mutational analysis of Odd-skipped indicates that these residues are critical for efficient interactions with Groucho and for repression in vivo. Our data suggest that phosphorylation of these phosphomeric residues, if it occurs, will down-regulate Groucho binding and therefore repression, providing a mechanism for posttranslational control of Groucho-mediated repression.

A Novel Platinum Compound Inhibits Constitutive Stat3 Signaling and Induces Cell Cycle Arrest and Apoptosis of Malignant Cells

Previous studies have established constitutive activation of Stat3 protein as one of the molecular changes required for tumorigenesis. To develop novel therapeutics for tumors harboring constitutively active Stat3, compounds from the NCI 2000 diversity set were evaluated for inhibition of Stat3 DNA-binding activity in vitro. Of these, a novel platinum (IV) compound, IS3 295, interacted with Stat3 and inhibited its binding to specific DNA-response elements. Further analysis suggested noncompetitive-type kinetics for the inhibition of Stat3 binding to DNA. In human and mouse tumor cell lines with constitutively active Stat3, IS3 295 selectively attenuated Stat3 signaling, thereby inducing cell growth arrest at G0/G1 phase and apoptosis. Moreover, in transformed cells, IS3 295 repressed expression of cyclin D1 and bcl-xL, two of the known Stat3-regulated genes that are overexpressed in malignant cells, suggesting that IS3 295 mediates anti-tumor cell activity in part by blocking Stat3-mediated sub-version of cell growth and apoptotic signals. Together, our findings provide evidence for the inhibition of Stat3 activity and biological functions by IS3 295 through interaction with Stat3 protein. This study represents a significant advance in small molecule-based approaches to target Stat3 and suggests potential new applications for platinum (IV) complexes as modulators of the Stat3 pathway for cancer therapy.

The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences

BackgroundThe genus Corynebacterium includes Gram-positive microorganisms of great biotechnologically importance, such as Corynebacterium glutamicum and Corynebacterium efficiens, as well as serious human pathogens, such as Corynebacterium diphtheriae and Corynebacterium jeikeium. Although genome sequences of the respective species have been determined recently, the knowledge about the repertoire of transcriptional regulators and the architecture of global regulatory networks is scarce. Here, we apply a combination of bioinformatic tools and a comparative genomic approach to identify and characterize a set of conserved DNA-binding transcriptional regulators in the four corynebacterial genomes.ResultsA collection of 127 DNA-binding transcriptional regulators was identified in the C. glutamicum ATCC 13032 genome, whereas 103 regulators were detected in C. efficiens YS-314, 63 in C. diphtheriae NCTC 13129 and 55 in C. jeikeium K411. According to amino acid sequence similarities and protein structure predictions, the DNA-binding transcriptional regulators were grouped into 25 regulatory protein families. The common set of DNA-binding transcriptional regulators present in the four corynebacterial genomes consists of 28 proteins that are apparently involved in the regulation of cell division and septation, SOS and stress response, carbohydrate metabolism and macroelement and metal homeostasis.ConclusionThis work describes characteristic features of a set of conserved DNA-binding transcriptional regulators present within the corynebacterial core genome. The knowledge on the physiological function of these proteins should not only contribute to our understanding of the regulation of gene expression but will also provide the basis for comprehensive modeling of transcriptional regulatory networks of these species.

Regulation of α-Synuclein Expression by Poly (ADP Ribose) Polymerase-1 (PARP-1) Binding to the NACP-Rep1 Polymorphic Site Upstream of the SNCA Gene

Alleles at NACP-Rep1, the polymorphic microsatellite repeat located approximately 10 kb upstream of the alpha -synuclein gene (SNCA), are associated, in some reports, with differing risks of sporadic Parkinson disease (PD). We showed previously that NACP-Rep1 acts as a negative modulator of SNCA transcription, with an effect that varied threefold among different NACP-Rep1 alleles. Given that duplications and triplications of SNCA have been implicated in familial Parkinson disease (PD), even a 1.5-2-fold increase in alpha -synuclein expression may, over many decades, contribute to PD. Thus, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of SNCA. Here we aimed to identify the factor(s) that bind to NACP-Rep1 and potentially contribute to SNCA transcriptional modulation, by pulling down proteins that bind to NACP-Rep1 and identifying them by mass spectrometry. One of these proteins was poly-(ADP-ribose) transferase/polymerase-1 (PARP-1), a DNA-binding protein and transcriptional regulator. Electrophoresis mobility shift and chromatin immunoprecipitation assays showed specific binding of PARP-1 to NACP-Rep1. Inhibition of PARP-1's catalytic domain increased the endogenous SNCA mRNA levels in cultured SH-SY5Y cells. Furthermore, PARP-1 binding to NACP-Rep1 specifically reduced the transcriptional activity of the SNCA promoter/enhancer in luciferase reporter assays. This down-regulation effect of PARP-1 depended on NACP-Rep1 being present in the construct and was abrogated by inhibiting PARP-1's catalytic activity with 3-aminobenzamide. The association of different NACP-Rep1 alleles with PD may be mediated, in part, by the effect of PARP-1, as well as other factors, on SNCA expression.

Three-pronged genomic analysis reveals yeast cell-type regulation circuitry

The phenotypic and physiological potential of a cell is determined by its transcription program, which, in turn, is determined by the array of transcription factors present in the cell. A sophisticated and detailed understanding of the role of a particular transcription factor requires that all of its target genes be identified. This task is a daunting one, but it has become feasible with the availability of complete genome sequences, coupled with genome-wide analytical methods such as transcription profiling and chromatin immunoprecipitation (ChIP) assays. In a recent study, Harbison et al. (1) used ChIP analysis, together with predicted sequence-recognition motifs, to determine the genomic occupancy of 203 DNA-binding transcription regulators in yeast. Because of the scope of that study, the question of whether all of the targets of a particular transcription regulator function in a particular physiological setting were identified was left unanswered. In a recent issue of PNAS, Galgoczy et al. (2) focus on a specific biological question with the goal of identifying the complete set of target genes for the transcription regulators that determine cell type in the budding yeast Saccharomyces cerevisiae . Cell-type specification in yeast has been studied intensely in a number of laboratories over the last 20 years, and the information gained from those studies served as a touchstone for the current work; that is, certain targets could be anticipated. However, there is ample room for surprise. Some target genes may have eluded discovery, and it is conceivable that the cell-type regulators also make connections to other physiological processes. Indeed, both possibilities are realized in the current study. In their work, Galgoczy et al. (2) used ChIP analysis, as had Harbison et al. (1), but, …

A Fully Recombinant System for Activator-dependent Archaeal Transcription

The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from the in vitro assembly of the 12-subunit RNA polymerase from the hyperthermophile Methanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.

A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B. cereus haemolysin II

Haemolysin II, HlyII, is one of several cytotoxic proteins produced by Bacillus cereus, an opportunistic human pathogen that causes food poisoning. The hlyII gene confers haemolytic activity to Escherichia coli cells. Here a new B. cereus gene, hlyIIR, which is located immediately downstream of hlyII and regulates hlyII expression, is reported. The deduced amino acid sequence of HlyIIR is similar to prokaryotic DNA-binding transcriptional regulators of the TetR/AcrA family. Measurements of haemolytic activity levels and of hlyII promoter activity levels using gene fusions and primer-extension assays demonstrated that, in E. coli, hlyII transcription decreased in the presence of hlyIIR. Recombinant HlyIIR binds to a 22 bp inverted DNA repeat centred 48 bp upstream of the hlyII promoter transcription initiation point. In vitro transcription studies showed that HlyIIR inhibits transcription from the hlyII promoter by binding to the 22 bp repeat and RNA polymerase, and by decreasing the formation of the catalytically competent open promoter complex.

Transcriptional regulatory code of a eukaryotic genome.

DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators.

The embryo MADS domain protein AGAMOUS-Like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism.

AGL15 (for AGAMOUS-Like 15) is a member of the MADS domain family of DNA binding transcriptional regulators that accumulates to its highest amounts during embryo development. To better understand how AGL15 functions, a chromatin immunoprecipitation approach was used to identify directly regulated genes. One DNA fragment that coprecipitated with AGL15 corresponded to a portion of the regulatory region of a gene named DTA1 (for Downstream Target of AGL15-1). The expression of DTA1 was positively correlated with AGL15 abundance during embryogenesis. In this report, a cis element for response to AGL15 was identified, and the activity of DTA1 as a gibberellin (GA) 2-oxidase was confirmed. DTA1 corresponds to AtGA2ox6 and was renamed to indicate this identity. Further experiments related the function of AtGA2ox6 to regulation by AGL15. Constitutive expression of AGL15 and of AtGA2ox6 altered endogenous GA amounts and caused GA-deficient phenotypes in Arabidopsis thaliana that could be at least partially rescued by application of biologically active GA. The phenotype of plants with decreased expression of AtGA2ox6 was the converse of plants overexpressing AtGA2ox6 in terms of seed germination attributes and effects on somatic embryo production.

T-box genes in early embryogenesis.

The T-box gene family, encoding related DNA-binding transcriptional regulators, plays an essential role in controlling many aspects of embryogenesis in a wide variety of organisms. The T-box genes exhibit diverse patterns of spatial and temporal expression in the developing embryo, and both genetic and molecular embryological studies have demonstrated their importance in regulating cell fate decisions that establish the early body plan, and in later processes underlying organogenesis. Despite these studies, little is known of either the regulation of the T-box genes or the identities of their transcriptional targets. The aim of this review is to examine the diverse yet conserved roles of several T-box genes in regulating early patterning in chordates and to discuss possible mechanisms through which this functional diversity might arise. Developmental Dynamics 229:201-218, 2004.

Acetylation of androgen receptor enhances coactivator binding and promotes prostate cancer cell growth.

Modification by acetylation occurs at epsilon-amino lysine residues of histones and transcription factors. Unlike phosphorylation, a direct link between transcription factor acetylation and cellular growth or apoptosis has not been established. We show that the nuclear androgen receptor (AR), a DNA-binding transcriptional regulator, is acetylated in vivo. The acetylation of the AR is induced by ligand dihydrotestosterone and by histone deacetylase (HDAC) inhibitors in living cells. Direct AR acetylation augmented p300 binding in vitro. Constructs mimicking neutral polar substitution acetylation (AR(K630Q), AR(K630T)) enhanced p300 binding and reduced N-CoR/HDAC/Smad3 corepressor binding, whereas charged residue substitution (AR(K630R)) reduced p300 binding and enhanced corepressor binding. The AR acetylation mimics promoted cell survival and growth of prostate cancer cells in soft agar and in nude mice and augmented transcription of a subset of growth control target gene promoters. Thus, transcription factor acetylation regulates coactivator/corepressor complex binding, altering expression of specific growth control genes to promote aberrant cellular growth in vivo.

The Krüppel-like transcription factor 6 gene in sporadic pituitary tumours.

The oncogenes and/or tumour suppressor genes which may be involved in the transformation process for the vast majority of pituitary tumours remain unknown. There is substantial evidence for derangement of cell cycle control in such tumours, but cell cycle protein mutations identified in other human malignancies are restricted to only a very small subset of sporadic pituitary neoplasms. Krüppel-like factors are DNA-binding transcriptional regulators with diverse effects including the upregulation of the cell cycle protein p21(WAF1/CIP1). It has been reported that the Krüppel-like transcription factor 6 (KLF6) gene is mutated in a proportion (15-55%) of human prostate cancers, and more recent data are emerging regarding mutated KLF6 in nasopharyngeal carcinomas, astrocytoid gliomas and colorectal cancer. We therefore speculated that other tumours such as pituitary adenomas might also harbour such mutations that may be involved in the control of cell proliferation in the pituitary. The aim of the current study was thus to analyse the KLF6 gene for mutations in sporadic pituitary tumours. We analysed 60 pituitary adenomas (15 GH-, four ACTH-, two PRL-secreting and 39 non-functioning) with direct sequence analysis of exons 2 and 3 of the KLF6 gene, the region where most of the previously described mutations are located. Three non-functioning pituitary adenomas of the 60 pituitary tumours (5%) had two identical sequence changes in exon 2 (missense mutation Val165Met, 523G-->A and a silent substitution in Ser77Ser codon 261C-->T). Analysis of genomic DNA extracted from peripheral lymphocytes in one patient confirmed these changes to be present in the germline and they therefore probably represent polymorphisms, although we cannot exclude the possibility that these are predisposing germline mutations. We conclude that mutations of the KLF6 gene are unlikely to play an important role in sporadic pituitary tumorigenesis.

A rapidly evolving MYB-related protein causes species isolation in Drosophila.

Matings among different species of animals or plants often result in sterile or lethal hybrids. Identifying the evolutionary forces that create hybrid incompatibility alleles is fundamental to understanding the process of speciation, but very few such alleles have been identified, particularly in model organisms that are amenable to experimental manipulation. We report here the cloning of the first, to our knowledge, Drosophila melanogaster gene involved in hybrid incompatibilities, Hybrid male rescue (Hmr). Hmr causes lethality and female sterility in hybrids among D. melanogaster and its sibling species. We have found that Hmr encodes a protein with homology to a family of MYB-related DNA-binding transcriptional regulators. The HMR protein has evolved both amino acid substitutions and insertions and deletions at an extraordinarily high rate between D. melanogaster and its sibling species, including in its predicted DNA-binding domain. Our results suggest that hybrid lethality may result from disruptions in gene regulation, and we also propose that rapid evolution may be a hallmark of speciation genes in general.