Carl P. Simkevich

A Large Scale Genetic Analysis of c-Myc-regulated Gene Expression Patterns

The myc proto-oncogenes encode transcriptional regulators whose inappropriate expression is correlated with a wide array of human malignancies. Up-regulation of Myc enforces growth, antagonizes cell cycle withdrawal and differentiation, and in some situations promotes apoptosis. How these phenotypes are elicited is not well understood, largely because we lack a clear picture of the biologically relevant downstream effectors. We created a new biological system for the optimal profiling of Myc target genes based on a set of isogenic c-myc knockout and conditional cell lines. The ability to modulate Myc activity from essentially null to supraphysiological resulted in a significantly increased and reproducible yield of targets and revealed a large subset of genes that respond optimally to Myc in its physiological range of expression. The total extent of transcriptional changes that can be triggered by Myc is remarkable and involves thousands of genes. Although the majority of these effects are not direct, many of the indirect targets are likely to have important roles in mediating the elicited cellular phenotypes. Myc-activated functions are indicative of a physiological state geared toward the rapid utilization of carbon sources, the biosynthesis of precursors for macromolecular synthesis, and the accumulation of cellular mass. In contrast, the majority of Myc-repressed genes are involved in the interaction and communication of cells with their external environment, and several are known to possess antiproliferative or antimetastatic properties.

Genome‐scale expression profiling of Hutchinson–Gilford progeria syndrome reveals widespread transcriptional misregulation leading to mesodermal/mesenchymal defects and accelerated atherosclerosis

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease with widespread phenotypic features resembling premature aging. HGPS was recently shown to be caused by dominant mutations in the LMNA gene, resulting in the in‐frame deletion of 50 amino acids near the carboxyl terminus of the encoded lamin A protein. Children with this disease typically succumb to myocardial infarction or stroke caused by severe atherosclerosis at an average age of 13 years. To elucidate further the molecular pathogenesis of this disease, we compared the gene expression patterns of three HGPS fibroblast cell strains heterozygous for the LMNA mutation with three normal, age‐matched cell strains. We defined a set of 361 genes (1.1% of the approximately 33 000 genes analysed) that showed at least a 2‐fold, statistically significant change. The most prominent categories encode transcription factors and extracellular matrix proteins, many of which are known to function in the tissues severely affected in HGPS. The most affected gene, MEOX2/GAX, is a homeobox transcription factor implicated as a negative regulator of mesodermal tissue proliferation. Thus, at the gene expression level, HGPS shows the hallmarks of a developmental disorder affecting mesodermal and mesenchymal cell lineages. The identification of a large number of genes implicated in atherosclerosis is especially valuable, because it provides clues to pathological processes that can now be investigated in HGPS patients or animal models.

Sp1 Cooperates with the ets Transcription Factor, GABP, to Activate the CD18 (β2 Leukocyte Integrin) Promoter

CD18, the β chain of the leukocyte integrins, plays a crucial role in immune and inflammatory responses. CD18 is expressed exclusively by leukocytes, and it is transcriptionally regulated during the differentiation of myeloid cells. Theets factors, PU.1 and GABP, bind to three etssites in the CD18 promoter, which are essential for high level myeloid expression of CD18. We now identify two binding sites for the transcription factor, Sp1, that flank these ets sites. Sp1 is the only factor from myeloid cells that binds to these sites in a sequence-specific manner. Mutagenesis of these sites abrogates Sp1 binding and significantly reduces the activity of the transfected CD18 promoter in myeloid cells. Transfection of Sp1 intoDrosophila Schneider cells, which otherwise lack Sp1, activates the CD18 promoter dramatically. GABP also activates the CD18 promoter in Schneider cells. Co-transfection of Sp1 and GABP activates CD18 more than the sum of their individual effects, indicating that these factors cooperate to transcriptionally activate myeloid expression of CD18. These studies support a model of high level, lineage-restricted gene expression mediated by cooperative interactions between widely expressed transcription factors.

GABP and PU.1 compete for binding, yet cooperate to increase CD18 (beta 2 leukocyte integrin) transcription.

CD18 (β2 leukocyte integrin) is a leukocyte-specific adhesion molecule that plays a crucial role in immune and inflammatory responses. A 79-nucleotide fragment of the CD18 promoter is sufficient to direct myeloid transcription. The CD18 promoter is bound by the B lymphocyte- and myeloid-restricted ets factor, PU.1, and disruption of the PU.1-binding sites significantly reduces promoter activity. However, PU.1 alone cannot fully account for the leukocyte-specific and myeloid-inducible transcription of CD18. We identified a ubiquitously expressed nuclear protein complex of extremely low electrophoretic mobility that also binds to this region of the CD18 promoter. This binding complex is a heterotetramer composed of GABPα, an ets factor, and GABPβ, a subunit with homology to Drosophila Notch. GABPα competes with the lineage restricted factor, PU.1, for the same critical CD18 ets sites. The CD18 promoter is activated in myeloid cells by transfection with both GABPα and GABPβ together, but not by either factor alone. Transfection of non-hematopoietic cells with the two GABP subunits together with PU.1 is sufficient to activate CD18 transcription in otherwise non-permissive cells. Thus, GABP and PU.1 compete for the same binding sites but cooperate to activate CD18 transcription.

An Enhancer Located between the Neutrophil Elastase and Proteinase 3 Promoters Is Activated by Sp1 and an Ets Factor

The adjacent neutrophil elastase, proteinase 3, and azurocidin genes encode serine proteases expressed specifically in immature myeloid cells. Subclones of a 17-kilobase (kb) murine neutrophil elastase genomic clone were assessed for their ability to stimulate the neutrophil elastase promoter in 32D cl3 myeloid cells. Region −9.3 to −7.3 kb stimulated transcription 7-fold, whereas other genomic segments were inactive. This enhancer is located in the second intron of the proteinase-3 gene and so may regulate more than one gene in the myeloid protease cluster. Deletional analysis of the enhancer identified several segments which activated the neutrophil elastase and thymidine kinase promoters 3–6-fold. The most active segment was a 220-base pair region centered at −8.6 kb, which activated transcription 31-fold. This segment contains an Sp1 consensus site, which bound Sp1, flanked by two Ets family consensus sequences, which bound PU.1, GABP, and an Ets factor present in myeloid cell extracts. Mutation of the Sp1-binding site reduced enhancer activity 8-fold in 32D cl3 cells, and mutation of either or both Ets-binding sites reduced activity 3–4-fold. Sp1 activated the distal enhancer 5-fold, GABP 3-fold, and the combination 8-fold in Schneider cells.

NFAT4 Is Required for JC Virus Infection of Glial Cells

ABSTRACT The human polyomavirus JC virus (JCV) infects 70% of the population worldwide. In immunosuppressed patients, JCV infection can lead to progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS). The majority of PML cases occur in the setting of human immunodeficiency virus (HIV) infection, and it has been suggested that the link between HIV and the development of PML is in part related to the production of numerous cytokines in the CNS during HIV infection. To examine the link between the expression of inflammatory cytokines and JCV infection, we tested an anti-inflammatory compound, cyclosporine A (CsA), for its ability to block JCV infection of glial cells. We found that CsA inhibited JCV infection by preventing the activation of the transcription factor nuclear factor of activated T cells 4 (NFAT4). Luciferase reporter assays and chromatin immunoprecipitation assays revealed that NFAT4 directly bound the JCV promoter during infection and was important for the activation of both early and late transcription. In addition, the expression of the JCV early viral gene products increased NFAT activity to further aid viral transcription. The necessity of NFAT for JCV infection suggests that calcium signaling and the activation of NFAT in glial cells are required for JCV infection of the CNS.

Short-term arginine deprivation results in large-scale modulation of hepatic gene expression in both normal and tumor cells: microarray bioinformatic analysis

BackgroundWe have reported arginine-sensitive regulation of LAT1 amino acid transporter (SLC 7A5) in normal rodent hepatic cells with loss of arginine sensitivity and high level constitutive expression in tumor cells. We hypothesized that liver cell gene expression is highly sensitive to alterations in the amino acid microenvironment and that tumor cells may differ substantially in gene sets sensitive to amino acid availability. To assess the potential number and classes of hepatic genes sensitive to arginine availability at the RNA level and compare these between normal and tumor cells, we used an Affymetrix microarray approach, a paired in vitro model of normal rat hepatic cells and a tumorigenic derivative with triplicate independent replicates. Cells were exposed to arginine-deficient or control conditions for 18 hours in medium formulated to maintain differentiated function.ResultsInitial two-way analysis with a p-value of 0.05 identified 1419 genes in normal cells versus 2175 in tumor cells whose expression was altered in arginine-deficient conditions relative to controls, representing 9–14% of the rat genome. More stringent bioinformatic analysis with 9-way comparisons and a minimum of 2-fold variation narrowed this set to 56 arginine-responsive genes in normal liver cells and 162 in tumor cells. Approximately half the arginine-responsive genes in normal cells overlap with those in tumor cells. Of these, the majority was increased in expression and included multiple growth, survival, and stress-related genes. GADD45, TA1/LAT1, and caspases 11 and 12 were among this group. Previously known amino acid regulated genes were among the pool in both cell types. Available cDNA probes allowed independent validation of microarray data for multiple genes. Among genes downregulated under arginine-deficient conditions were multiple genes involved in cholesterol and fatty acid metabolism. Expression of low-density lipoprotein receptor was decreased in both normal and tumor cells.ConclusionArginine-sensitive regulation appears to be an important homeostatic mechanism to coordinate cell response and nutrient availability in hepatic cells. Genes predicted as arginine-responsive in stringent microarray data analysis were confirmed by Northern blot and RT-PCR. Although the profile of arginine-responsive genes is altered and increased, a considerable portion of the arginome is maintained upon neoplastic transformation.

Characterization and localization to chromosome 7 of ψhGABPα, a human processed pseudogene related to the ets transcription factor, hGABPα

Abstract GABP is a heteromeric transcription factor complex which consists of the ets related protein, GABPα, and the Notch-related protein, GABPβ. We isolated a human genomic DNA fragment which is highly homologous and colinear with human GABPα cDNA, but which lacks introns. This processed pseudogene, ψhGABPα , is expressed as RNA in U937 human myeloid cells, but a mutation at the site that corresponds to the ATG start methionine codon prevents its translation into protein. The pseudogene was localized to chromosome 7 using a somatic cell hybrid mapping panel and it is not syntenic with authentic GABPα , which was localized to chromosome 21. We have identified ψhGABPα , a novel, GABPα -related processed pseudogene which is expressed as a RNA transcript in human myeloid cells.