Biomedical subjects

David E Nowak

Publications and source records attributed to David E Nowak.

4 recordsLinked to original sources

PubMed2005

A TNF-induced gene expression program under oscillatory NF-kappaB control.

BACKGROUND: The cytokine tumor necrosis factor (TNF) initiates tissue inflammation, a process mediated by the NF-kappaB transcription factor. In response to TNF, latent cytoplasmic NF-kappaB is activated, enters the nucleus, and induces expression of inflammatory and anti-apoptotic gene expression programs. Recently it has been shown that NF-kappaB displays two distinct activation modes, monophasic and oscillatory, depending on stimulus duration. Characterization of temporal expression patterns for the NF-kappaB network and determination of those genes under monophasic- or oscillatory control has not been experimentally addressed. RESULTS: To identify the kinetics of NF-kappaB-dependent gene expression and determine whether these two types of NF-kappaB translocation modes control distinct gene programs, a detailed kinetic analysis of a validated microarray data set was performed on 74 unique NF-kappaB-dependent genes in response to TNF. Hierarchical clustering identified distinct expression profiles termed the "Early", "Middle", "Late" response groups, peaking 1, 3, and 6 h after stimulation, respectively. These expression patterns were validated by Quantitative Real Time PCR (Q-RT-PCR) and NF-kappaB binding was demonstrated by chromatin immunoprecipitation (ChIP) assays. Each response group was mapped to its molecular function; this analysis indicated that the Early group encodes cytokines or negative regulators of the IKK-NF-kappaB pathway, and the Late group encodes cell surface receptors, adhesion molecules and signal adapters. That similar coordinated sequential cascades of gene expression were also seen in response to stimulation by the cytokine IL-1, and expression patterns observed in MRC-5 fibroblasts indicated that the epithelial NF-kappaB program is relatively stimulus- and cell type-independent. Bioinformatic analysis of the Early and Late gene promoters indicates that although both groups contain similar patterns of NF-kappaB-binding sites, only the Early gene promoters contain NF-kappaB-binding sites located in phylogenetically conserved domains. Stimulation protocols designed to produce either monophasic or oscillatory NF-kappaB activation modes showed that the oscillatory mode is required only for expression of the Late genes. CONCLUSION: This analysis provides important insights into the TNF-regulated genetic response program in epithelial cells, where NF-kappaB controls sequential expression patterns of functionally distinct genes that depend on its oscillatory activation mode.

Cell Adhesion↗

PubMed2005

Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F)<0.001) and demonstrated a change in signal intensity of+/-3-fold relative to control. Of these, 28 were NF-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

Binding Sites↗

PubMed2005

Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.

The chromatin immunoprecipitation (ChIP) assay has recently been exploited as a powerful and versatile technique for probing protein-DNA interactions within the chromatin environment. In this method, intact cells are fixed with a reversible DNA-protein cross-linking agent (formaldehyde), and associated DNA is enriched by immunoprecipitating a target DNA binding protein. The bound DNA in the immune complexes is then used to identify that specific DNA binding protein's endogenous genomic targets. Nuclear factor kappaB (NF-kappaB) is a highly inducible transcription factor that controls genetic networks important for pathogen- or cytokine-induced inflammation, immune response, and cellular survival. In our studies of the genetic network under control of the inducible NF-kappaB transcription factor, we found that the conventional ChIP technique using a single formaldehyde cross-linking step did not reproducibly cross-link it to DNA. As a result, we have developed a novel ChIP assay using a two-step cross-linking procedure, incorporating N-hydroxysuccinimide (NHS)-ester-mediated protein-protein cross-linking prior to conventional DNA-protein cross-linking. We demonstrate that this technique is highly efficient, cross-linking virtually all NF-kappaB/Rel A into covalent complexes, resulting in quantitative and robust identification of inducible NF-kappaB family binding to a variety of validated NF-kappaB-dependent genomic targets. To demonstrate the general utility of this two-step cross-linking procedure, we performed enhanced capture of cytokine-inducible signal transducer and activator of transcription-3 (STAT3) binding to one of its known target genes. Our method represents a significant improvement in the efficiency of ChIP analysis in the study of endogenous targets for rare transcription factors.

Binding Sites↗

PubMed2004

Stochastic effects of multiple regulators on expression profiles in eukaryotes.

The stochastic nature of gene regulation still remains not fully understood. In eukaryotes, the stochastic effects are primarily attributable to the binary nature of genes, which are considered either switched "on" or "off" due to the action of the transcription factors binding to the promoter. In the time period when the gene is activated, bursts of mRNA transcript are produced. In the present paper, we investigate regulation of gene expression at the single cell level. We propose a mechanism of gene regulation, which is able to explain the observed distinct transcription profiles assuming the number of co-regulatory activities, without attempting to identify the specific proteins involved. The model is motivated by our experiments on NF-kappaB-dependent genes in HeLa cells. Our experimental data shows that NF-kappaB-dependent genes can be stratified into three characteristic groups according to their expression profiles: early, intermediate and late having maximum of expression at about 1, 3 and 6 h, respectively, from the beginning of TNF stimulation. We provide a tractable analytical approach, not only in the terms of expected expression profiles and their moments, which corresponds to the measurements on the cell population, but also in the terms of single cell behavior. Comparison between these two modes of description reveals that single cells behave qualitatively different from the cell population. This analysis provides insights useful for understanding of microarray experiments.

Animals↗