Composite Module Analyst: identification of transcription factor binding site combinations using genetic algorithm.

Composite Module Analyst (CMA) is a novel software tool aiming to identify promoter-enhancer models based on the composition of transcription factor (TF) binding sites and their pairs. CMA is closely interconnected with the TRANSFAC database. In particular, CMA uses the positional weight matrix (PWM) library collected in TRANSFAC and therefore provides the possibility to search for a large variety of different TF binding sites. We model the structure of the long gene regulatory regions by a Boolean function that joins several local modules, each consisting of co-localized TF binding sites. Having as an input a set of co-regulated genes, CMA builds the promoter model and optimizes the parameters of the model automatically by applying a genetic-regression algorithm. We use a multicomponent fitness function of the algorithm which includes several statistical criteria in a weighted linear function. We show examples of successful application of CMA to a microarray data on transcription profiling of TNF-alpha stimulated primary human endothelial cells. The CMA web server is freely accessible at http://www.gene-regulation.com/pub/programs/cma/CMA.html. An advanced version of CMA is also a part of the commercial system ExPlaintrade mark (www.biobase.de) designed for causal analysis of gene expression data.

TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes.

The TRANSFAC database on transcription factors, their binding sites, nucleotide distribution matrices and regulated genes as well as the complementing database TRANSCompel on composite elements have been further enhanced on various levels. A new web interface with different search options and integrated versions of Match and Patch provides increased functionality for TRANSFAC. The list of databases which are linked to the common GENE table of TRANSFAC and TRANSCompel has been extended by: Ensembl, UniGene, EntrezGene, HumanPSD and TRANSPRO. Standard gene names from HGNC, MGI and RGD, are included for human, mouse and rat genes, respectively. With the help of InterProScan, Pfam, SMART and PROSITE domains are assigned automatically to the protein sequences of the transcription factors. TRANSCompel contains now, in addition to the COMPEL table, a separate table for detailed information on the experimental EVIDENCE on which the composite elements are based. Finally, for TRANSFAC, in respect of data growth, in particular the gain of Drosophila transcription factor binding sites (by courtesy of the Drosophila DNase I footprint database) and of Arabidopsis factors (by courtesy of DATF, Database of Arabidopsis Transcription Factors) has to be stressed. The here described public releases, TRANSFAC 7.0 and TRANSCompel 7.0, are accessible under http://www.gene-regulation.com/pub/databases.html.

Evaluation of sequence motifs found in scaffold/matrix-attached regions (S/MARs).

Based on the contents of the database S/MARt DB, the most comprehensive data collection of scaffold/matrix-attached regions (S/MARs) publicly available thus far, we initiated a systematic evaluation of the stored data. By analyzing the 245 S/MAR sequences presently described in this database, we found that the S/MARs contained in this collection are generally AT-rich, with certain significant exceptions. Comparative analyses showed that most of the AT-rich motifs which were found to be enriched in S/MARs are also enriched in randomized S/MAR sequences of the same AT content. Some sequence patterns previously suggested to be characteristic for S/MARs were also investigated, among them potential binding sites for homeodomain transcription factors. Even though hexanucleotides containing the core motif of homeodomain factors were frequently observed in S/MARs, only a few potential binding sites for these factors were found enriched when compared with regulatory regions or exon sequences. All our analyses indicated that, on average, the observed frequency of motifs in S/MAR elements is largely influenced by the AT content. Our results can serve as a guideline for further improvements in the definition of S/MARs, which are now believed to constitute the functional coordinate system for genomic regulatory regions.

MATCH: A tool for searching transcription factor binding sites in DNA sequences.

Match is a weight matrix-based tool for searching putative transcription factor binding sites in DNA sequences. Match is closely interconnected and distributed together with the TRANSFAC database. In particular, Match uses the matrix library collected in TRANSFAC and therefore provides the possibility to search for a great variety of different transcription factor binding sites. Several sets of optimised matrix cut-off values are built in the system to provide a variety of search modes of different stringency. The user may construct and save his/her specific user profiles which are selected subsets of matrices including default or user-defined cut-off values. Furthermore a number of tissue-specific profiles are provided that were compiled by the TRANSFAC team. A public version of the Match tool is available at: http://www.gene-regulation.com/pub/programs.html#match. The same program with a different web interface can be found at http://compel.bionet.nsc.ru/Match/Match.html. An advanced version of the tool called Match Professional is available at http://www.biobase.de.

The TRANSFAC system on gene expression regulation.

The TRANSFAC database on transcription factors and their DNA-binding sites and profiles (http://www.gene-regulation.de/) has been quantitatively extended and supplemented by a number of modules. These modules give information about pathologically relevant mutations in regulatory regions and transcription factor genes (PathoDB), scaffold/matrix attached regions (S/MARt DB), signal transduction (TRANSPATH) and gene expression sources (CYTOMER). Altogether, these distinct database modules constitute the TRANSFAC system. They are accompanied by a number of program routines for identifying potential transcription factor binding sites or for localizing individual components in the regulatory network of a cell.

TRANSFAC: transcriptional regulation, from patterns to profiles.

The TRANSFAC database on eukaryotic transcriptional regulation, comprising data on transcription factors, their target genes and regulatory binding sites, has been extended and further developed, both in number of entries and in the scope and structure of the collected data. Structured fields for expression patterns have been introduced for transcription factors from human and mouse, using the CYTOMER database on anatomical structures and developmental stages. The functionality of Match, a tool for matrix-based search of transcription factor binding sites, has been enhanced. For instance, the program now comes along with a number of tissue-(or state-)specific profiles and new profiles can be created and modified with Match Profiler. The GENE table was extended and gained in importance, containing amongst others links to LocusLink, RefSeq and OMIM now. Further, (direct) links between factor and target gene on one hand and between gene and encoded factor on the other hand were introduced. The TRANSFAC public release is available at http://www.gene-regulation.com. For yeast an additional release including the latest data was made available separately as TRANSFAC Saccharomyces Module (TSM) at http://transfac.gbf.de. For CYTOMER free download versions are available at http://www.biobase.de:8080/index.html.

Database-assisted promoter analysis.

The analysis of regulatory sequences is greatly facilitated by database-assisted bioinformatic approaches. The TRANSFAC database contains information on transcription factors and their origins, functional properties and sequence-specific binding activities. Software tools enable us to screen the database with a given DNA sequence for interacting transcription factors. If a regulatory function is already attributed to this sequence then the database-assisted identification of binding sites for proteins or protein classes and subsequent experimental verification might establish functionally relevant sites within this sequence. The binding transcription factors and interacting factors might already be present in the database.

Transcription complexes for various class III genes differ in parameters of formation and stability towards salt.

RNA polymerase III faithfully transcribes the genes for ribosomal 5 S RNA, tRNA(1Met) or adenovirus VA RNA in vitro in the presence of required transcription factors. These genes display distinct differences in the kinetics of transcription complex formation and in their response to excess template. In contrast to tRNA and VA RNA synthesis, 5 S RNA synthesis displays a lag phase of 15 minutes before the onset of transcription and is clearly inhibited by high concentrations of template. Once formed, transcription complexes for the RNA polymerase III genes listed can be isolated by glycerol gradient centrifugation and display a remarkable stability against transient treatment with high salt concentrations. Complexes for 5 S RNA and tRNA remain functionally active up to 2.5 M-KCl. The activity of transcription complexes for VA RNA, however, is significantly diminished after treatment with high salt concentrations. This effect is shown to be due to an irreversible loss of transcription factors. RNA polymerase III is dissociated by high salt concentrations from all the transcription complexes studied but remains part of these complexes during the normal reinitiation cycle at 60 mM-KCl. An additional method for the purification of partial transcription complexes was developed that involves equilibrium centrifugation on cesium sulfate gradients. This method completely releases TFIIIB from 5 S complexes and a core complex, composed of the 5 S RNA gene, factors IIIA and IIIC, is retained. In the case of tRNA and VA RNA, core complexes are obtained that remain partly associated with TFIIIC and TFIIIB. These results indicate a qualitatively and/or quantitatively different interaction of individual factors in different polymerase III transcription complexes.

Recognition of NFATp/AP-1 composite elements within genes induced upon the activation of immune cells.

Composite elements are regulatory modules of promoters or enhancers that consist of binding sites of two different but synergizing transcription factors. A well-studied example is nuclear factors of activated T-cell (NFAT) sites which are composite elements of a NFATp/c and an activating protein 1 (AP-1) binding site. We have developed a computational approach to identify potential NFAT target genes which (a) comprises an improved method to scan for individual NFAT composite elements; (b) considers positional effects relative to transcription start sites; and (c) involves cluster analysis of potential NFAT composite elements. All three steps progressively helpX?ed to discriminate T-cell-specific promoter sequences against other functional regions (coding and intronic sequences) of the same genes, against promoters of muscle-specific genes or against random sequences. Using this approach, we identified potential NFAT composite elements in promoters of cytokine genes and their receptors as well as in promoters of genes for AP-1 family members, Ca2+-binding proteins and some other components of the regulatory network operating in activated T-cells and other immune cells. The method developed can be adapted to characterize and identify other composite elements as well. The program for recognition NFAT composite elements is available through the World Wide Web (http://compel.bionet.nsc.ru/FunSite/CompelScan. html and http://transfac.gbf.de/dbsearch/funsitep/s _comp.html).

The human immunodeficiency virus type 1 encoded Vpu protein is phosphorylated by casein kinase-2 (CK-2) at positions Ser52 and Ser56 within a predicted alpha-helix-turn-alpha-helix-motif.

The human immunodeficiency virus type 1 (HIV-1) encoded Vpu is a small integral membrane phosphoprotein that functions in the enhancement of viral particle release and has more recently been shown to cause degradation of CD4 at the endoplasmic reticulum. We have demonstrated earlier that Vpu is phosphorylated by the ubiquitous casein kinase-2 (CK-2) in HIV-1 infected cells. The phosphoacceptor sites targeted by CK-2 in Vpu, however, have not been demonstrated and it was unclear whether Vpu was phosphorylated at one or more of its four serine residues. In this study we characterized the CK-2 phosphoacceptor sites in Vpu using recombinant CK-2 for in vitro phosphorylation of recombinant Vpu protein as well as synthetic peptides of Vpu. Phosphorylation of both Ser52 and Ser56 was demonstrated by in vitro phosphorylation using three 54-residue peptides comprising the entire hydrophilic part of Vpu and containing single serine to asparagine transitions in either position 52 or 56. The Km values of CK-2 to these peptides were established, revealing a preferential phosphorylation of Ser56. The Km values are: Ser56 = 31 microM; Ser 52 = 156 microM; wild type = 27 microM. In addition, we studied phosphorylation of Vpu by endogenous CK-2 following in vitro translation in rabbit reticulocyte lysate of wild-type Vpu or a mutant, Vpum2/6, carrying serine to asparagine changes at amino acid positions 52 and 56. The in vivo phosphorylation of Vpu was studied in transiently transfected human embryonic kidney (293) cells. In this system, the mutant Vpum2/6 was not phosphorylated, indicating that the seryl residues of Vpu at amino acid positions 52 and 56, but not those at positions 23 and 61, are phosphorylated by CK-2. The two CK-2 phosphorylation sites are conserved in all known Vpu sequences and represent the consensus Ser52GlyAsn(Glu/Asp)Ser(Glu/Asp)Gly(Glu/Asp)59. Prediction of the secondary structure revealed a conserved alpha-helix-turn-alpha-helix motif for the hydrophilic C-terminal part of Vpu. A structural model for Vpu is proposed in which the membrane anchor precedes a region comprising two amphipathic alpha-helices of opposed polarity, joined by a strongly acidic turn that protrudes into the cytoplasm and contains the CK-2 phosphorylation sites. Possible functional and structural homologies of Vpu to the membrane channel-forming M2 protein of influenza A viruses are discussed.

Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo.

Computer-assisted identification of cell cycle-related genes: new targets for E2F transcription factors.

The processes that take place during development and differentiation are directed through coordinated regulation of expression of a large number of genes. One such gene regulatory network provides cell cycle control in eukaryotic organisms. In this work, we have studied the structural features of the 5' regulatory regions of cell cycle-related genes. We developed a new method for identifying composite substructures (modules) in regulatory regions of genes consisting of a binding site for a key transcription factor and additional contextual motifs: potential targets for other transcription factors that may synergistically regulate gene transcription. Applying this method to cell cycle-related promoters, we created a program for context-specific identification of binding sites for transcription factors of the E2F family which are key regulators of the cell cycle. We found that E2F composite modules are found at a high frequency and in close proximity to the start of transcription in cell cycle-related promoters in comparison with other promoters. Using this information, we then searched for E2F sites in genomic sequences with the goal of identifying new genes which play important roles in controlling cell proliferation, differentiation and apoptosis. Using a chromatin immunoprecipitation assay, we then experimentally verified the binding of E2F in vivo to the promoters predicted by the computer-assisted methods. Our identification of new E2F target genes provides new insight into gene regulatory networks and provides a framework for continued analysis of the role of contextual promoter features in transcriptional regulation. The tools described are available at http://compel.bionet.nsc.ru/FunSite/SiteScan.html.

Organization and sequence of the gene encoding the human acrosin-trypsin inhibitor (HUSI-II).

A complete cDNA encoding the acrosin-trypsin inhibitor, HUSI-II, was used as a probe to isolate genomic clones from a human placenta library. Three clones which cover the entire HUSI-II gene were isolated and characterized. The exon-intron organization of the gene was determined and found to be identical to other known Kazal-type inhibitor-encoding genes. The striking similarity in the amino acid sequences which was found previously in HUSI-II and glycoprotein hormone beta-subunits, is neither reflected in codon usage nor in the exon-intron arrangement of the genes. A 1.8-kb segment 5' of the gene was sequenced. The analysis of this sequence showed that HUSI-II contains a G + C-rich region upstream from the transcription start point (tsp) which fulfills the criteria for a CpG island. Furthermore, in the first intron, a potential glucocorticoid-responsive element was found as a half-palindrome flanked by two CACCC elements. Determination of the tsp by S1 mapping revealed that HUSI-II has multiple tsp. Genomic Southern hybridization was used to show that HUSI-II is a single-copy gene. The localization of the gene to chromosome 4 was determined by hybridization of a 5' genomic fragment to the DNA of a panel of somatic hybrids between human and rodent cells.

Complete synthesis and transcription in vitro of a gene coding for human ribosomal 5S RNA.

The gene coding for the major human ribosomal 5S RNA was chemically synthesized and cloned into a pUC13 vector. This approach was taken, because attempts to isolate the human 5S gene have thus far yielded either pseudogenes or variant 5S genes of unknown function. The synthetic human gene was transcribed by RNA polymerase III either in a crude HeLa cell extract or in a system reconstituted from partially purified transcription factors. Comparative studies with the Xenopus laevis somatic 5S gene show that the human gene is transcribed with similar fidelity and an efficiency of about 80% under optimal conditions. The time-course of transcription and optimal concentrations of template and transcription factors were found to be similar for both genes studied. The synthetic gene described may prove useful to study its interaction with human transcription factors in a homologous system.

TRANSFAC retrieval program: a network model database of eukaryotic transcription regulating sequences and proteins.

DNA sequences that are involved in the control of gene expression in eukaryotes have been collected in conjunction with the proteins binding to and acting through them (TRANSFAC data set). To make these data accessible, the TRANSFAC retrieval program (TRP) has been developed as a database management system which is based upon the network model. This database model possesses particular advantages for data management of a complex structure. The aim of TRP is to provide an easily handled statistical basis for a computational approach to transcription control.

Recognition of regulatory regions in genomic sequences.

For the functional interpretation of genomic sequences, effective algorithms have to be developed that will recognize regions of specific function and thus will suggest experiments for their verification. As a first step, relevant data have to be collected in an appropriate database from which suitable training sets can be extracted. In this paper, I discuss the requirements for a database that collects information about regulatory DNA sequences and describe the structure and contents of such a database (TRANSFAC). This compiled information will serve as a basis for comprehensive analysis of sites that regulate transcription, e.g., by statistical methods. It will thus facilitate the recognition of regulatory genomic sequence information and the assignment of the corresponding regulators. Moreover, it will provide all relevant data about the regulating proteins which will allow to trace back transcriptional control cascades to their origin.

Production of human parathyroid hormone by recombinant Escherichia coli TG1 on synthetic medium.

Production of human parathyroid hormone (hPTH) by Escherichia coli TG1:I52cIts was studied. The hPTH is expressed as a fusion protein under control of the bacteriophage lambda pR promoter. The organism grows on glucose/mineral salt medium and the expression of the gene product was investigated under variation of temperature and growth rate prior to and after induction. hPTH formation largely depends on cultivation temperature and is optimal for a temperature shift from 30 to 38 degrees C. Product expression is growth coupled and specific hPTH concentration is independent of growth rate. The results are compared with a previous study on E. coli N4830:pEX-PPTH grown on complex media.

Large-scale preparation and biological activity of recombinant human parathyroid hormone.

Human parathyroid hormone (hPTH) has been bacterially expressed in bioreactors as cro-beta-galactosidase-hPTH fusion protein. We have developed a large-scale purification scheme that exploits the pH-dependent differential solubility of hPTH and a two-step chromatographic procedure. We demonstrate that in a number of assay systems, the recombinant material obtained by this procedure is biologically active.

Expression of the RT6 mono(ADP-ribosyl)transferases is regulated by two promoter regions.

The structure of the RT6 mono(ADP-ribosyl)transferase gene was studied. Analysis of cDNA clones revealed eight exons and suggested two independent transcriptional start sites. The existence of the downstream initiation site was confirmed by S1-nuclease protection and localized to position +29 of exon 2. The corresponding 5' flanking regions were found to contain typical promoter structures such as TATA- and CCAAT-boxes. Comparison with sequences deposited in the TRANSFAC database of transcription factor binding sites revealed few putative regulatory elements in the region associated with exon 1 (promoter 1). In contrast, several elements contained in the regulatory regions of other T cell-specific genes, such as ets, lyf-1 and ikaros were found in in promoter 2. Analysis of RT6-transcripts showed this region to be the most active promoter in spleen cells of adult rats. Finally, transient transfection assays with reporter gene constructs showed promoter 2 to mediate T-cell specific transcription.

New perspectives in the differentiation of bone-forming cells.

Bone formation comprises a complex but ordered sequence of events which involves the proliferation and differentiation of chondrogenic and osteoblastic precursor cells ultimately leading to the formation of a calcified extracellular matrix. This process can be observed in vivo but under these conditions is difficult to study at the molecular level. A number of in vitro models have been developed which recapitulate discrete elements of this process. Using these models, detailed information has been obtained regarding the differentiation of bone forming cells and the molecular biology of the mineralization process. It has been shown that, in vitro, osteoblastic precursor cells can form a mineralized matrix similar to that seen in vivo. This calcification process was shown to consist of three interdependent phases: proliferation, matrix maturation and mineralization. Each of these phases was characterized by the expression of particular genes. Osteoblast precursors have been cloned and consequently shown to be able to differentiate in vitro into a number of other mesenchymal cells, supporting the theory that osteoblasts are derived from multipotent mesenchymal cells. It is possible that markers derived from these models could be used in the future to extend our knowledge of bone formation in vivo.