Use of cDNA microarrays to analyze dioxin-induced changes in human liver gene expression.

One mechanism by which cells adapt to environmental changes is by altering gene expression. Here, we have used cDNA microarrays to identify genes whose expression is altered by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The goal of our study was to enhance our understanding of toxicity mediated through the pathway by which TCDD stimulates gene expression. To model this toxicity response, we exposed human hepatoma (HepG2) cells to TCDD (10 nM for 18 h) and analyzed mRNA by two-color fluorescent hybridization to cDNA sequences immobilized on glass microscope slides (2.5 x 7.5 cm) covering a surface area of 2.25 cm(2). We analyzed approximately one-third of the genes expressed in HepG2 cells and found that TCDD up- or down-regulates 112 genes two-fold or more. Most changes are relatively subtle (two- to four-fold). We verified the regulation of protooncogene cot, XMP, and human enhancer of filamentation-1 (HEF1), genes involved in cellular proliferation, as well as metallothionein, plasminogen activator inhibitor (PAI1), and HM74, genes involved in cellular signaling and regeneration. To characterize the response in more detail, we performed time-course, dose-dependence studies, and cycloheximide experiments. We observed direct and indirect responses to TCDD implying that adaptation to TCDD (and other related environmental stimuli) is substantially more complex than we previously realized.

Mediator protein mutations that selectively abolish activated transcription.

Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4-VP16 in vitro. By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected. A different but overlapping Mediator subunit dependence was found for activation by Gcn4. The genetic requirements for activation in vivo were closely coincident with those in vitro. A whole genome expression profile of a Deltamed2 strain showed diminished transcription of a subset of inducible genes but only minor effects on "basal" transcription. These findings make an important connection between transcriptional activation in vitro and in vivo, and identify Mediator as a "global" transcriptional coactivator.

Template-directed interference footprinting of cytosine contacts in a protein-DNA complex: potent interference by 5-aza-2'-deoxycytidine.

In the template-directed interference (TDI) footprinting method (Hayashibara & Verdine, 1990), analogs of the naturally occurring DNA bases are incorporated into DNA enzymatically and assayed for interference of sequence-specific binding by a protein. Here we extend this method to include analysis of contacts of amino acid residues to the major groove surface of cytosine residues (TDI-C footprinting). The base analog 5-aza-2'-deoxycytidine, in which the hydrophobic 5-CH of cytosine is replaced by a hydrophilic aza nitrogen, was incorporated into DNA via the corresponding 5'-triphosphate. The analog was found to base pair with guanine during polymerization, resulting in substitution of 2'-deoxycytidine residues. TDI-C footprints of the lambda repressor-OL1 operator complex revealed apparent contacts to the cytosines at operator positions 7 and 8. Inspection of the high-resolution X-ray crystal structure of the lambda-OL1 complex (Clarke et al., 1992; Beamer & Pabo, 1992) revealed that C8 makes a hydrogen binding contact with the Lys3; C7, on the other hand, makes a previously unnoticed hydrophobic contact with the alkane side chain of Lys3. In only the consensus operator half-site was cytosine interference observed, suggesting that the nonconsensus arm binds DNA very differently if at all. The N-terminal arm represents the archetypal case of a sequence-specific peptide-DNA complex characterized at high resolution; thus, the present studies suggest strategies for design and screening of DNA binding peptides. The finding that 5-aza-2'-deoxycytidine inhibits sequence-specific DNA binding proteins may suggest an alternative rationale for the biological activities of this and related azapyrimidine nucleosides.