Genome-wide differential gene expression in children exposed to air pollution in the Czech Republic.

The Teplice area in the Czech Republic is a mining district where elevated levels of air pollution including airborne carcinogens, have been demonstrated, especially during winter time. This environmental exposure can impact human health; in particular children may be more vulnerable. To study the impact of air pollution in children at the transcriptional level, peripheral blood cells were subjected to whole genome response analysis, in order to identify significantly modulated biological pathways and processes as a result of exposure. Using genome-wide oligonucleotide microarrays, we investigated differential gene expression in children from the Teplice area (n=23) and compared them with children from the rural control area of Prachatice (n=24). In an additional approach, individual gene expressions were correlated with individual peripheral blood lymphocyte micronuclei frequencies, in order to evaluate the linkage of individual gene expressions with an established biomarker of effect that is representative for increased genotoxic risk. Children from the Teplice area showed a significantly higher average micronuclei frequency than Prachatice children (p=0.023). For considerable numbers of genes, the expression differed significantly between the children from the two areas. Amongst these genes, considerable numbers of genes were observed to correlate significantly with the frequencies of micronuclei. The main biological process that appeared significantly affected overall was nucleosome assembly. This suggests an effect of air pollution on the primary structural unit of the condensed DNA. In addition, several other pathways were modulated. Based on the results of this study, we suggest that transcriptomic analysis represents a promising biomarker for environmental carcinogenesis.

Adenovirus vector designed for expression of toxic proteins.

To construct recombinant adenoviruses expressing biologically active proteins may be impossible, or result in a significant reduction in virus yield, if the protein expressed has an inhibitory effect on virus replication or cellular growth. To overcome this problem, we previously designed adenovirus vectors expressing foreign proteins from inducible promoters. However, during our work with a replication-deficient virus expressing the ASF/SF2 splicing factor from a progesterone antagonist-inducible gene cassette, we discovered that ASF/SF2 was expressed at a significant level in the 293 producer cell line, even in the absence of inducer. 293 cells code for adenovirus E1A and E1B proteins and thus support the growth of E1-deficient adenoviruses. Here we show that this background ASF/SF2 expression results from a low level of E1A-mediated transactivation of the basal promoter driving transgene expression. To overcome the problem of leaky expression, we reconstructed a novel gene cassette that combines an inducible promoter and a Lac repressor protein-based block to reduce transcriptional elongation. We show that this novel vector system dramatically reduced background transgene expression and therefore should be useful for the rescue and propagation of high-titer stocks of recombinant adenoviruses expressing toxic proteins.

Functional knockout of the corepressor CtBP by the second exon of adenovirus E1a relieves repression of transcription.

The C-terminal binding protein (CtBP) acts as a transcriptional corepressor upon recruitment to transcriptional regulators. In contrast, interaction between CtBP and the adenovirus E1A protein is required for efficient activation of E1A-responsive genes, suggesting that E1A might block CtBP-mediated repression. Recruitment of CtBP to a promoter, either as a Gal4CtBP fusion or through an interaction with a Gal4 fusion protein expressing the CtBP interacting domain (CID) of E1A, resulted in transcriptional repression. The second exon of E1A, containing the CID, alleviated repression by Gal4E1ACID-recruited CtBP, but not Gal4CtBP-mediated repression, suggesting that E1A prevented repression by blocking promoter recruitment of CtBP. E1ACID was also sufficient to derepress transcription from several cotransfected promoter constructs. Furthermore, inducible expression of E1ACID in established cell lines resulted in significant changes of endogenous gene expression, possibly by sequestration of CtBP. Together, these data indicated that CtBP might act as a wide-range regulator of transcription. Although CtBP was shown to interact with histone deacetylases (HDACs), transcriptional repression by a Gal4CtBP fusion protein was not sensitive to inhibition of HDACs by trichostatin A (TSA). In contrast, TSA eliminated E1ACID derepression of E1A second exon-responsive promoters. Although the reason for this difference remains to be experimentally verified, it is possible that the requirement for HDACs might differ depending on the mechanism by which CtBP becomes promoter recruited.