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 understanding how transition metals affect gene expression to ultimately influence development. This group is primarily interested in understanding how organisms respond on the molecular level to exposure to cadmium, copper, and mercury. Specifically, how metal-responsive regulatory processes control the gene expression. The disruption of these regulatory processes or the inability of an organism to effectively respond to metal exposure may lead to the development of pathologies. 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 Upon exposure to elevated levels of metals, MTF-1, a six zinc-finger, metal-responsive transcription factor, rapidly translocates into the nucleus and binds to metal responsive elements (MREs) in promoters. After MTF-1 binds to the MRE, it is then able to activate transcription of mammalian metallothionein and other stress responsive genes. However, we have clearly demonstrated that binding alone is not sufficient to activate transcription. Although many aspects of MTF-1s biochemical activity have been examined, the molecular mechanism by which MTF-1 responds to increased intracellular concentrations of transition metals has not been elucidated. There are currently two models that describe how metals affect MTF-1 to cause transcriptional activation. The first model proposes that MTF-1 is zinc sensor. Zinc binds to the zinc fingers, which results in a conformational change, allowing MTF-1 to bind to MREs to activate transcription. My group proposes that exposure metal affects multiple signal transduction pathways that ultimately converge at MTF-1, and it is the change in the level of MTF-1 phosphorylation that controls its transcriptional activity. To investigative our model, we are using mass spectrometry;to identify the phosphorylation sites;and site-directed mutagenesis;to determine the functional role of different domains of MTF-1 in controlling its activities (nuclear translocation, DNA binding, and transcriptional activation). 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 six 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. In addition, a mutant where all six PKC sites were modified demonstrated wild-type activity. We are currently measuring the ability of the three mutants to translocate into the nucleus, bind to DNA, and be a substrate for PKC. We are currently generating a series of phosphomimetic MTF-1 mutants at T224 and S641. Lentiviruses have been created and we are transducing dko7 cells. Once these lines have been established, the effects of cadmium and zinc on cell viability, ability to activate MT-1 and MT-2 transcription, nuclear translocation and DNA binding will be assessed. GENOMIC RESPONSES TO TRANSITION METALS When organisms are exposed to environmental toxicants, they defend themselves against intracellular damage by activating the transcription of genes that encode proteins that defend the host, repair the damage, or remove/metabolize the toxicant. Changes in expression of these genes following toxicant exposure is referred to as the stress-response. Many of the genes induced as part of the stress-response are evolutionarily conserved. Likewise, many of the signal transduction cascades that regulate the stress-response are conserved. We proposed that because of the conserved nature of the regulatory process controlling the stress-response;species from yeast to mammals would activate the transcription of a similar set of genes following toxicant exposure. Moreover, we hypothesized that discovery of this conserved set of responsive genes would focus our attention on genes and pathways that have greater biological relevance to disease etiology. 1.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. A first draft of a manuscript describing numr-1 and numr-2 has been prepared. I plan to submit it at the beginning of FY11. 2.Role of calcium in cadmium signaling. 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 have examined the calcium/cadmium relation in cultured cells at metal concentrations that induce gene expression but are minimally toxic to the cell. Using in vivo calcium sensors, we have shown that at concentrations that will induce transcription, cadmium does not affect intracellular calcium stores. Only at cadmium concentrations that are toxic to the cell are intracellular calcium levels affected. From these studies we conclude that calcium is not a second messenger for cadmium. 3.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, and cdr-1::GFP C. elegans reporter strains to screen for metal-responsive regulators and signaling pathways. Using a reverse genetic screen we have shown that akt-1 signaling is a regulator of mtl-1 transcription. This regulation is independent of the insulin signaling pathway (daf-16). We are currently examining other members of the akt-1 and daf-16 signaling pathway to genetically define this metal-responsive regulatory pathway. We have completed a forward mutagenesis screen and isolated seventeen gain-of-function (high levels of transcription in the absence of cadmium) mutants. The physical and genetic location of these mutations is currently being determined. 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.
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