Pharmacologic profiling of transcriptional targets deciphers promoter logic.

The blueprint for cellular diversity and response to environmental change is encoded in the cis-acting regulatory sequences of most genes. Deciphering this 'cis-regulatory code' requires multivariate data sets that examine how these regions coordinate transcription in response to diverse environmental stimuli and therapeutic treatments. We describe a transcriptional approach that profiles the activation of multiple transcriptional targets against combinatorial arrays of therapeutic and signal transducing agents. Application of this approach demonstrates how cis-element composition and promoter context combine to influence transcription downstream of mitogen-induced signaling networks. Computational dissection of these transcriptional profiles in activated T cells uncovers a novel regulatory synergy between IGF-1 and CD28 costimulation that modulates NF-kappaB and AP1 pathways through signaling cascades sensitive to cyclosporin A and wortmannin. This approach provides a broader view of the hierarchical signal integration governing gene expression and will facilitate a practical design of combinatorial therapeutic strategies for exploiting critical control points in transcriptional regulation.

Human tear protein fractions during waking hours.

Reliable collection and analysis of human tear proteins presents considerable problems. The volume of unstimulated tear samples is necessarily very small, although stimulated tears may be easily provoked with a consequent change in composition. In the present study, 5-microliters samples of human tear fluid were carefully removed, over a 10 min period, from 10 male Caucasian subjects with the aid of a 5-microliter "microcap". The procedure was repeated at 2-h intervals between 08.00 and 22.00 hours. The collected samples were individually separated into their major protein fractions using gel column electrophoresis. The separated material was stained and scanned, and the concentration of proteins present estimated by comparison with known protein standards. Consistent values for protein concentrations were recorded for all participating subjects, and no variation during waking hours was demonstrated.

Deficient gene specific repair of cisplatin-induced lesions in Xeroderma pigmentosum and Fanconi's anemia cell lines.

Cisplatin is a chemotherapeutic agent known to cause DNA damage. The cytotoxicity of this drug is believed to result from the formation of DNA intrastrand adducts (IA) and DNA interstrand crosslinks (ICL). While there are many studies on DNA repair of cisplatin damage at the overall level of the genome in various human cell lines, there is little information on the gene-specific repair. In this report, we have measured the formation and repair of cisplatin induced DNA adducts in the dihydrofolate reductase (DHFR) and ribosomal RNA (rRNA) genes in three cell lines: normal human fibroblasts, Fanconi's anemia complementation group A (FAA) and Xeroderma pigmentosum complementation group A (XPA). It is generally thought that XPA cells lack nucleotide excision repair and that FAA cells are deficient in the repair of DNA ICL. We find that normal human fibroblast cells repair 84% of the ICL in the DHFR gene after 24 h, whereas XPA and FAA cell lines only repaired 32 and 50% of the ICL respectively. Furthermore, 69% of the cisplatin IA in the DHFR gene were repaired in 24 h in normal human fibroblasts compared to 22% for XPA and 24% for FAA cells. The repair of the rRNA gene was less efficient than in the DHFR gene, but the relative pattern between the different cell lines was similar to that of the DHFR gene. We thus find that FAA cells are deficient not only in the gene specific repair of cisplatin ICL, but also in the gene specific repair of the more common cisplatin IA. XPA cells are normally thought to be without any nucleotide excision repair capacity, but our data could support a slight ICL unhooking activity.

Targeting of p300 to the interleukin-2 promoter via CREB-Rel cross-talk during mitogen and oncogenic molecular signaling in activated T-cells.

In this report, we explore the mechanisms of targeting of p300 to the interleukin-2 (IL-2) promoter in response to mitogenic and oncogenic molecular signals. Recruitment of p300 by cAMP-responsive element-binding protein-Rel cross-talk at the composite CD28 response element (CD28RE)-TRE element of the IL-2 promoter is essential for promoter inducibility during T-cell activation, and CD28RE-TRE is the exclusive target of the human T-cell lymphotropic virus type I oncoprotein Tax. The intrinsic histone acetyltransferase activity of p300 is dispensable for activation of the IL-2 promoter, and the N-terminal 743 residues contain the minimal structural requirements for synergistic transactivation of the CD28RE-TRE, the IL-2 promoter, and endogenous IL-2 gene expression. Mutational analysis of p300 reveals differential structural requirements for the N-terminal p300 module by individual cis-elements within the IL-2 promoter. These findings provide evidence that p300 assembles at the IL-2 promoter to form an enhanceosome-like signal transduction target that is centrally integrated at the CD28RE-TRE element of the IL-2 promoter through specific protein module-targeted associations in activated T-cells.

Identification of residues critical for enzymatic activity in the domain encoded by exons 8 and 9 of the human inducible nitric oxide synthase.

Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of several diseases including airway inflammation of asthma. iNOS is active only as a homodimer. We previously demonstrated that the region encoded by exons 8 and 9 is critical for dimerization. In this study, alanine-scanning mutagenesis was used to identify critical amino acids in that region by expression of mutant proteins in human embryonic kidney 293 cells. All iNOS mutants yielded iNOS protein as detected by Western analysis. Four iNOS mutants with alanine replacing Trp260, Asn261, Tyr267, or Asp280 did not generate NO. Dimer formation was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 4 degrees C, followed by immunoblotting. Wild-type iNOS migrated both as monomers and dimers. iNOS mutants with alanine replacing Trp260, Asn261, or Tyr267, however, migrated only as monomers, suggesting that their inability to produce NO is related to a defect in dimer formation. Interestingly, the Asp280 mutant retained the ability to dimerize, indicating that it represents an inactive form of an iNOS dimer. These data identify four amino acids in exons 8 and 9 critical for iNOS activity, three of which also influence dimerization. These residues are strictly conserved among all NOS isforms and across species. Thus all NOS isoforms share general structural similarities, including specific amino acids critical for dimerization and catalytic activity. These data increase our understanding of the structural elements critical for NO synthesis and lay the groundwork for future studies aimed at downregulation of iNOS activity.

Alternative splicing of human inducible nitric-oxide synthase mRNA. tissue-specific regulation and induction by cytokines.

Human inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide synthesis in response to inflammatory mediators. The human iNOS gene, containing 26 exons, encodes a protein of 131 kDa. This study was aimed at investigating the presence of alternative splicing of human iNOS mRNA. Total RNA from human alveolar macrophages, nasal and bronchial epithelial cells, and several human tissues was transcribed to cDNA and analyzed using polymerase chain reaction with specific primers for segmental analysis of the iNOS gene. Four sites of alternative splicing were identified by sequence analysis; these included deletion of: (i) exon 5; (ii) exons 8 and 9; (iii) exons 9, 10, and 11; and (iv) exons 15 and 16. The deduced amino acid sequences of the novel iNOS cDNAs predict one truncated protein (resulting from exon 5 deletion) and three iNOS proteins with in-frame deletions. Southern analyses of polymerase chain reaction products were consistent with tissue-specific regulation of alternative splicing. In cultured cells, iNOS induction by cytokines and lipopolysaccharide was associated with an increase in alternatively spliced mRNA transcripts. Because iNOS is active as a dimer, the novel forms of alternatively spliced iNOS may be involved in regulation of nitric oxide synthesis.

Effect of aging on EGF-stimulated replication of specific genes in rat hepatocytes.

EGF-stimulated replication of specific genes was examined in primary hepatocyte cultures from mature (6 months) and senescent (24 months) rats. Basal and EGF-stimulated [3H]thymidine incorporation and DNA polymerase alpha activities, as well as total cellular DNA, were also assessed. The genes examined were dihydrofolate reductase (DHFR) and c-myc, as well as total mitochondrial DNA (mt DNA). Although [3H]thymidine incorporation, DNA polymerase alpha activity, total cellular DNA, DHFR, and c-myc gene specific DNA replication stimulated by EGF are reduced with age, mt DNA replication is not affected by either EGF or age. Chromosomal DNA replication is mediated mainly by DNA polymerase alpha while mt DNA replication is mediated by its own DNA polymerase gamma. Thus, the age-related decline in stimulated DNA replication appears to be associated mainly with the DNA polymerase alpha activation pathway.

Cloning and characterization of human inducible nitric oxide synthase splice variants: a domain, encoded by exons 8 and 9, is critical for dimerization.

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242-335 of the oxygenase domain. In this study, iNOS8(-)9(-) and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8(-)9(-) in 293 cell line resulted in generation of iNOS8(-)9(-) mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8(-)9(-) did not form dimers. Similar to iNOSFL, iNOS8(-)9(-) exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two "control" iNOS deletion mutants were synthesized, lacking either residues 45-52 of the oxygenase domain or residues 1131-1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.