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Thomas Werner

Publications and source records attributed to Thomas Werner.

11 recordsLinked to original sources
PubMed2003

IFN-alpha induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3.

The anti-inflammatory cytokine IL-10 can be induced by type I IFNs, but the molecular mechanisms involved have remained elusive. With in silico analysis of the human IL-10 promoter we identified a module consisting of an IFN regulatory factor 1 (IRF-1) site and a Stat3 site. We demonstrate that IFN-alpha will induce the binding of IRF-1 and Stat3 to the respective motifs. Mutational analysis revealed that inactivation of the IRF-1 motif substantially reduces trans-activation from 5- to 2-fold and that inactivation of the Stat3 motif completely ablates trans-activation by IFN-alpha. The dominant role of Stat3 in this module was confirmed with the blockade of trans-activation by a dominant negative Stat3. By contrast, Stat1 contributes a minor proportion to the DNA binding to the Stat site, and overexpression will counteract Stat3-mediated trans-activation. The data show that IFN-alpha induces the IL-10 gene via a module consisting of interdependent IRF-1 and Stat3 motifs. Of note, LPS-induced trans-activation does not target this module, since it is independent of the IRF-1 motif but completely depends on Stat3.

Amino Acid Motifs↗
PubMed2003

Integrated functional and bioinformatics approach for the identification and experimental verification of RNA signals: application to HIV-1 INS.

Regulation of gene expression involves sequence elements in nucleic acids. In promoters, multiple sequence elements cooperate as functional modules, which in combination determine overall promoter activity. We previously developed computational tools based on this hierarchical structure for in silico promoter analysis. Here we address the functional organization of post-transcriptional control elements, using the HIV-1 genome as a model. Numerous mutagenesis studies demonstrate that expression of HIV structural proteins is restricted by inhibitory sequences (INS) in HIV mRNAs in the absence of the HIV-1 Rev protein. However, previous attempts to detect conserved sequence patterns of HIV-1 INS have failed. We defined four distinct sequence patterns for inhibitory motifs (weight matrices), which identified 22 out of the 25 known INS as well as several new candidate INS regions contained in numerous HIV-1 strains. The conservation of INS motifs within the HIV genome was not due to overall sequence conservation. The functionality of two candidate INS regions was analyzed with a new assay that measures the effect of non-coding mRNA sequences on production of red fluorescent reporter protein. Both new INS regions showed inhibitory activity in sense but not in antisense orientation. Inhibitory activity increased by combining both INS regions in the same mRNA. Inhibitory activity of known and new INS regions was overcome by co-expression of the HIV-1 Rev protein.

Computational Biology↗
PubMed2003

Functional diversity of the Drosophila PGRP-LC gene cluster in the response to lipopolysaccharide and peptidoglycan.

The peptidoglycan recognition protein PGRP-LC is a major activator of the imd/Relish pathway in the Drosophila immune response. Three transcripts are generated by alternative splicing of the complex PGRP-LC gene. The encoded transmembrane proteins share an identical intracellular part, but each has a separate extracellular PGRP-domain: x, y, or a. Here we show that two of these isoforms play unique roles in the response to different microorganisms. Using RNA interference in Drosophila mbn-2 cells, we found that PGRP-LCx is the only isoform required to mediate signals from Gram-positive bacteria and purified bacterial peptidoglycan. By contrast, the recognition of Gram-negative bacteria and bacterial lipopolysaccharide requires both PGRP-LCa and LCx. The third isoform, LCy, is expressed at lower levels and may be partially redundant. Two additional PGRP domains in the gene cluster, z and w, are both included in a single transcript of a separate gene, PGRP-LF. Suppression of this transcript does not block the response to any of the microorganisms tested.

Alternative Splicing↗
PubMed2003

Promoters can contribute to the elucidation of protein function.

Protein interaction maps are valuable tools in the understanding of the actions of genes and proteins in a real-life context. However, many proteins and genes interact functionally - without physical contact - via transcriptional coregulation of the genes, which is partially manifested in functional promoter structures called promoter modules. Here, the example of interleukin 9 (IL-9) induction by IL-1 and tumour necrosis factor (TNF) is used to demonstrate how comparative promoter analysis can identify a shared promoter module and thus indicate the crucial partners. This strategy can be used for genes from a small initial set of coregulated genes, such as can be derived from expression array data. The combination of expression and promoter analysis with proteomics should be considered as an important link in functional genomics.

Animals↗
PubMed2003

The state of the art of mammalian promoter recognition.

The draft sequences of whole genomes are being published at an ever-increasing pace, thus providing access to the human genomic sequence and, more recently, the mouse sequence. Genomes of the invertebrates are also becoming available. Now that the genomic DNA of mammalian species is available, an old problem can be tackled with renewed vigour mammalian promoter prediction. Gene promoters have proved elusive for more than a decade, despite their pivotal role in gene regulation. Recently, however, several new developments have made it possible to make meaningful large-scale predictions. This paper reviews the methods used for the prediction of mammalian, mostly human, promoters.

Animals↗
PubMed2003

Computer modeling of promoter organization as a tool to study transcriptional coregulation.

Understanding how the regulation of gene networks is orchestrated is an important challenge for characterizing complex biological processes. Gene transcription is regulated in part by nuclear factors that recognize short DNA sequence motifs, called transcription factor binding sites, in most cases located upstream of the gene coding sequence in promoter and enhancer regions. Genes expressed in the same tissue under similar conditions often share a common organization of at least some of these regulatory binding elements. In this way the organization of promoter motifs represents a "footprint" of the transcriptional regulatory mechanisms at work in a specific biologic context and thus provides information about signal and tissue specific control of expression. Analysis of promoters for organizational features as demonstrated here provides a crucial link between the static nucleotide sequence of the genome and the dynamic aspects of gene regulation and expression.

Animals↗
PubMed2002

Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila.

Components of microbial cell walls are potent activators of innate immune responses in animals. For example, the mammalian TLR4 signaling pathway is activated by bacterial lipopolysaccharide and is required for resistance to infection by Gram-negative bacteria. Other components of microbial surfaces, such as peptidoglycan, are also potent activators of innate immune responses, but less is known about how those components activate host defense. Here we show that a peptidoglycan recognition protein, PGRP-LC, is absolutely required for the induction of antibacterial peptide genes in response to infection in Drosophila and acts by controlling activation of the NF-kappaB family transcription factor Relish.

Active Transport, Cell Nucleus↗
PubMed2002

Regulatory context is a crucial part of gene function.

Information about the time and place of gene transcription, which until recently was only possible by extensive experimental analysis, can now be predicted through in silico analysis. Using the human RANTES/CCL5 promoter, we show that organizational features of promoters derived from promoter sequences contain information about the spatial and temporal 'functional context' of expression.

Animals↗
PubMed2002

In silico prediction of scaffold/matrix attachment regions in large genomic sequences.

Scaffold/matrix attachment regions (S/MARs) are essential regulatory DNA elements of eukaryotic cells. They are major determinants of locus control of gene expression and can shield gene expression from position effects. Experimental detection of S/MARs requires substantial effort and is not suitable for large-scale screening of genomic sequences. In silico prediction of S/MARs can provide a crucial first selection step to reduce the number of candidates. We used experimentally defined S/MAR sequences as the training set and generated a library of new S/MAR-associated, AT-rich patterns described as weight matrices. A new tool called SMARTest was developed that identifies potential S/MARs by performing a density analysis based on the S/MAR matrix library (http://www.genomatix.de/cgi-bin/smartest_pd/smartest.pl). S/MAR predictions were evaluated by using six genomic sequences from animal and plant for which S/MARs and non-S/MARs were experimentally mapped. SMARTest reached a sensitivity of 38% and a specificity of 68%. In contrast to previous algorithms, the SMARTest approach does not depend on the sequence context and is suitable to analyze long genomic sequences up to the size of whole chromosomes. To demonstrate the feasibility of large-scale S/MAR prediction, we analyzed the recently published chromosome 22 sequence and found 1198 S/MAR candidates.

Algorithms↗
PubMed2002

Finding and decrypting of promoters contributes to the elucidation of gene function.

The combination of full-scale genomic sequencing with high throughput expression analysis provides a new and largely unexploited basis for in silico functional genomics. Recent break through developments in locating and analyzing promoters now allow extending functional genomics in silico far beyond identification of protein sequences into the complex regulatory structures and mechanisms of the genome. However, only first examples of this new type of approach are emerging at present and intensive further developments of bioinformatics tools will be required before such analysis can become large-scale routine in genomic sequence analysis. Nevertheless, the door to a new dimension of functional analysis of the genomic sequence is open. Finally, only the tight integration of the enormous amount of knowledge gained from proteins sequence analysis with the complementary information about gene regulation will afford us with a more complete picture of the networks than constitute life.

Computational Biology↗