The overall program in the Comparative Genomics Group involves understanding contribution of environmental toxicants to the etiology of human diseases. We are currently focusing on how metal-responsive regulatory processes control the gene expression. The projects on-going in this group can be defined in two major categories: Determining the mechanism by-which the metal responsive transcription factor MTF-1 actives gene expression;and global genomic responses to metal exposure in multiple species. METAL RESPONSIVE TRANSCRIPTION FACTOR, MTF-1 Targeted site-directed mutagenesis of MTF-1- Prosite analysis predicts six PKC consensus phosphorylation sites in MTF-1. We generated a collection of site-directed MTF-1 mutants in-which combinations of five of the six the potential PKC phosphorylated residues have been modified (e.g., single, double, triple, etc. mutants). In collaboration with the lentivirus expression core a collection of virus strains containing: wild-type MTF-1, five individual PKC mutants, and a strain expressing MTF-1 containing all five PKC mutations have been produced. After transducing dko7 cells (MTF-1 nulls) with each strain of virus, the ability of cadmium and zinc to activate transcription was measured. Two mutations, T224A and S641A, were unable to activate metal-inducible transcription. The other mutations activated transcription to the level observed in dko7 cells transduced with wild-type MTF-1. Interestingly,a mutant in which all six PKC site were modified had wild-type activity. We have shown that cells expressing mutant MTF-1s are sensitive to metals and that these mutants can bind to MRE's with wildtype activity. We are currently examining their ability to translocate into the nucleus. We have also begun to generate a series of MTF-1 mutants with modified casein kinase 2 sites. GENOMIC RESPONSES TO TRANSITION METAL EXPOSURE 1. Response of retinoic acid metabolism to cadmium exposure - In the analysis of the genomic responses of C. elegans exposed to cadmium, we observed that the expression of genes encoding proteins involved in retinoic acid (RA) metabolism was affected. We translated the information from C. elegans to a mammalian system, hepa 1-6 cells, and found that exposure to cadmium increased the level of RA-responsive transcription. It also affected the steady-state levels of mRNAs encoding proteins that are involved in the synthesis and degradation of RA. Finally, preliminary results indicated that exposure to cadmium causes a significant increase the level of RA in hepa 1-6 cells. These results suggest a mechanism by-which cadmium causes birth defects. 2. Characterization of a novel metal-responsive gene from C. elegans - From the C. elegans microarray analysis we identified a novel cadmium-inducible gene, designated numr-1 (nuclear localized metal responsive). Subsequent genomic analysis identified a second gene numr-2 that is 99% identical, in both coding and regulatory regions, to numr-1. Both numrs are metal responsive and expressed in identical cells: constitutively in a sub-set of neurons in the head, vulva and tail;and in intestinal and pharyngeal cells following metal exposure. In addition, both NUMRs are targeted to punctate nuclear structures putatively identified as nuclear stress granules. Over expression of NUMR-1 caused increased resistance to metal toxicity and life-span. Likewise, knocking down NUMR-1/2 expression increased C. elegans sensitivity to metal exposure. We observed that calcium activated numr-1 transcription. This has lead to a reexamination of the relation between calcium and cadmium in transcription regulation. Much of the previous work suggesting that cadmium behaves as a calcium mimic was preformed at supra-toxicological metal concentrations (>LC70). We are examining the calcium/cadmium relation in cultured cells at metal concentrations that induce gene expression but are minimally toxic to the cell. Initial results suggests that calcium does not function as a second messenger for cadmium, until cells have begun to die. 3. Genomic responses to copper in HepG2 cells - We previously showed that copper was able to affect the activities of specific transcription factors, MTF-1, and AP-1, and their cognate signal transduction pathways. We have completed an analysis of transcriptome of HepG2 cells exposed to copper. Bioinformatic analysis of this data including Gene Ontology, Interactomes and Ingenuity has identified several novel transcription factors and signaling pathways responsible of copper-responsive gene expression. This work has been submitted for publication in Genome Biology. We examined the relation between copper exposure and the activity of the transcription factors NF-B, HNF4and NRF-1 using RNA interference and biochemical assays. 4. Genetic screens to identify regulators of metal-inducible transcription in C. elegans We have initiated a new project to identify regulators of metal-inducible transcription using a classic genetics approach. We created mtl-1::GFP, mtl-2::GFP, numr-1::GFP and cdr-1::GFP C. elegans reporter strains to screen for metal-responsive regulators and signaling pathways. We are currently mutagenizing these strains, and will isolate gain-of-function (high levels of transcription in the absence of cadmium) and loss-of-function (no or attenuated transcription in the presence of cadmium) mutants. Ultimately, the C. elegans gene(s) and cognate pathways responsible for the phenotypes will be identified, and the role of the homologous mammalian gene(s) in regulating metal-inducible transcription will be explored. 5. Affects of mercurials on the C. elegans transcriptome - We are investigating the effects of inorganic mercury (HgCl2) and methyl mercury on gene expression in C. elegans. A direct comparison between these two mercurials at equi-toxic concentrations, under identical exposure conditions (media, temperature, etc) in a single species has not been reported. This study will address the proposal that methyl mercury and inorganic mercury affect similar genes and signaling pathways. We have generated microarray data for each mercurial at three concentrations. Initial analysis indicates that the chemicals have few genes in common. Clustering and principal components analyses clearly differentiate between the two mercurials. These results contradict the hypothesis that the biological effects of methyl mercury are caused by ionic mercury, which is released during the metabolic degradation of organic mercury. This expression data will be compared to our previous results including the C. elegans-cadmium and the yeast-mercury transcriptomes
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