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Biomedical subjects

Ivan Sadowski

Publications and source records attributed to Ivan Sadowski.

9 recordsLinked to original sources
PubMed2005

TFII-I and USF (RBF-2) regulate Ras/MAPK-responsive HIV-1 transcription in T cells.

The HIV-1 long terminal repeat (LTR) is stringently controlled by T cell activation signals, and binds a variety of transcription factors whose activities are regulated downstream of the T cell receptor. One of the most highly conserved cis-elements on the LTR, designated RBEIII, binds the factor RBF-2 which is comprised of a USF-1/USF-2 heterodimer and a co-factor TFII-I. RBF-2 is necessary for transcription from the LTR in response to RAS-MAPK activation through T cell receptor engagement, but is also required for repression of viral expression in unstimulated cells. Considering the defined activities of USF and TFII-I, RBF-2 may be responsible for regulating promoter context by controlling chromatin organisation, thereby coordinating opportunity for transcriptional activation by additional factors bound to the enhancer region.

Cell Communication↗
PubMed2005

Identification of the mismatch repair genes PMS2 and MLH1 as p53 target genes by using serial analysis of binding elements.

The ability to determine the global location of transcription factor binding sites in vivo is important for a comprehensive understanding of gene regulation in human cells. We have developed a technology, called serial analysis of binding elements (SABE), involving subtractive hybridization of chromatin immunoprecipitation-enriched DNA fragments followed by the generation and analysis of concatamerized sequence tags. We applied the SABE technology to search for p53 target genes in the human genome, and have identified several previously described p53 targets in addition to numerous potentially novel targets, including the DNA mismatch repair genes MLH1 and PMS2. Both of these genes were determined to be responsive to DNA damage and p53 activation in normal human fibroblasts, and have p53-response elements within their first intron. These two genes may serve as a sensor in DNA repair mechanisms and a critical determinant for the decision between cell-cycle arrest and apoptosis. These results also demonstrate the potential for use of SABE as a broadly applicable means to globally identify regulatory elements for human transcription factors in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed2005

TFII-I regulates induction of chromosomally integrated human immunodeficiency virus type 1 long terminal repeat in cooperation with USF.

Human immunodeficiency virus type 1 (HIV-1) replication is coupled to T-cell activation through its dependence on host cell transcription factors. Despite the enormous sequence variability of these factors, several cis elements for host factors are highly conserved within the 5' long terminal repeats (LTRs) of viruses from AIDS patients; among these is the RBEIII upstream element for the Ras response element binding factor 2 (RBF-2). Here we show that RBF-2 is comprised of a USF1/USF2 heterodimer and TFII-I, which bind cooperatively to RBEIII. Recombinant USF1/USF2 binds to the RBEIII core sequence 160-fold less efficiently than it binds to an E box element, but the interaction with RBEIII is stimulated by TFII-I. Chromosomally integrated HIV-1 LTRs bearing an RBEIII mutation have slightly elevated basal transcription in unstimulated Jurkat cells but are unresponsive to cross-linking of the T-cell receptor or stimulation with phorbol myristate acetate (PMA) and ionomycin. Induction is inhibited by dominant interfering USF and TFII-I but not by the dominant negative I-kappaB protein. USF1, USF2, and TFII-I bind to the integrated wild-type LTR in unstimulated cells and become phosphorylated during the induction of transcription upon stimulation with PMA. These results demonstrate that USF1/USF2 and TFII-I interact cooperatively at the upstream RBEIII element and are necessary for the induction of latent HIV-1 in response to T-cell activation signals.

Animals↗
PubMed2003

Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system.

The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate. Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins. However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity. To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD. DDC (L-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo. Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide. Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected. Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression.

Amino Acid Sequence↗
PubMed2003

Mitogen-responsive expression of RhoB is regulated by RNA stability.

The small GTPase-encoding gene RhoB is strongly induced as part of the immediate early response of serum-stimulated fibroblasts. In this report, we have characterized the mechanism for growth factor responsiveness of RhoB in Rat-2 fibroblasts. By Northern blotting and ribonuclease protection, we observed low or barely detectable levels of RhoB mRNA in quiescent cells, but expression was transiently induced in response to serum stimulation, such that the mRNA peaked within 30 min and then declined over the next hour. Analysis of the rat promoter revealed cis-elements conserved with the mouse and human genes, including a pair of CEBP sites near the transcriptional start site. However, in contrast to the analysis of RNA, RhoB promoter fusions were constitutively expressed in quiescent cells in transient transfections, and were unaffected by serum. Similarly, stable RhoB promoter integrants were highly expressed in quiescent cells, and growth factor caused a slight decrease in activity. This indicates that growth factor-inducible RhoB expression cannot be mediated by transcriptional activation. We then examined decay of the RhoB mRNA and found that serum caused significant stabilization. Additionally, fusion of the 3' RhoB untranslated region (UTR) to a constitutively expressed reporter gene caused serum and growth factor as well as DNA damage-inducible expression. These observations are consistent with the view that RhoB mRNA is produced constitutively but its abundance is controlled in response to growth factors, and other signals including DNA damage, by stabilization through elements within the 3' UTR.

3T3 Cells↗
PubMed2003

Srb10/Cdk8 regulates yeast filamentous growth by phosphorylating the transcription factor Ste12.

The budding yeast Saccharomyces cerevisiae differentiates into filamentous invasively growing forms under conditions of nutrient limitation. This response is dependent on the transcription factor Ste12 and on the mating pheromone-response mitogen-activated protein (MAP) kinase cascade, but a mechanism for regulation of Ste12 by nutrient limitation has not been defined. Here we show that Ste12 function in filamentous growth is regulated by the cyclin-dependent kinase Srb10 (also known as Cdk8), which is associated with the RNA polymerase II holoenzyme. Srb10 inhibits filamentous growth in cells growing in rich medium by phosphorylating Ste12 and decreasing its stability. Under conditions of limiting nitrogen, loss of Srb10 protein and kinase activity occurs, with a corresponding loss of Ste12 phosphorylation. Mutation of the Srb10-dependent phosphorylation sites increases pseudohyphal development but has no effect on the pheromone response of haploid yeast. Srb10 kinase activity is also regulated independently of the mating pheromone-response pathway. This indicates that Srb10 controls Ste12 activity for filamentous growth in response to nitrogen limitation and is consistent with the hypothesis that Srb10 regulates gene-specific activators in response to physiological signals to coordinate gene expression with growth potential.

Cell Division↗
PubMedDate not supplied

Organization and expression of the Cyr61 gene in normal human fibroblasts.

We have examined the human Cyr61 gene and its expression in normal fibroblasts. The core promoter, second intron, and 3' untranslated region (UTR) are highly conserved between the human and mouse genes. Cyr61 expression was induced slightly slower but more transiently in human fibroblasts compared to Rat-2 fibroblasts. These differences may relate to the absence of a serum response element in the human Cyr61 promoter, and the presence of additional AU-rich elements within the 3' UTR. Cycloheximide causes accumulation of human Cyr61 RNA in the absence of growth factors, and EGF prevents decay of transcripts in actinomycin-D-treated cells, which suggests that induction by growth factors may partially involve mRNA stabilization. We detect an alternative RNA in serum-stimulated fibroblasts containing an in-frame deletion within exon 4 which disrupts the thrombospondin type 1 repeat. Constitutive expression of the full hCyr61 genomic DNA in rodent fibroblasts causes production of multiple protein species, whereas expression of hCyrDelta4 produces a single stable protein of the expected size. We also observed multiple hCyr61 protein species in normal fibroblasts following serum stimulation, indicating that Cyr61 may normally be produced as alternative isoforms.

Animals↗