METAL RESPONSIVE TRANSCRIPTION FACTOR, MTF-1 In addition to MTF-1, several other transcription factors have been found to bind to the upstream regulatory region of MT. Other groups reported that co-immunoprecipitation of Flag-tagged-MTF-1 from zinc-treated cells led to the identification of a multi-protein complex that contained MTF-1, histone acetyltransferase (p300/CBP), and Sp1. These results and the observations that the responsiveness of MTF-1-mediated transcription is modulated by other transcription factors suggest that additional MTF-1 interacting proteins may be present. Through the NIEHS contract with Myriad Genetics, a yeast two-hybrid screen was performed using mouse MTF-1 as bait. Twenty-four interacting partners were isolated that hybridized to the MTF-1 bait. The identification of multiple MTF-1 interacting partners has provided the exciting opportunity to investigate a relatively unexplored area of MTF-1 function and activity. Among the MTF-1 interacting protein, we focused on CARM1. CARM1 (co-activator-associated arginine methyltransferase 1) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to arginine residues in histone and non-histone proteins. We have completed our analysis of the interaction between CARM1 and MTF-1. We confirmed the interaction between these proteins through co-immunoprecipitation studies in cadmium-treated and non-treated cells. We also determined that MTF-1 and CARM1 co-localize to a subset of MREs in the human MT2A gene by ChIP analysis. Finally, we showed that CARM1 was essential for optimal expression of MT-1 mRNA in response to zinc and cadmium, and resistance to cadmium toxicity. GENOMIC RESPONSES TO TRANSITION METALS Mtl-1 and mtl-2 To identify regulatory factors and pathways that control metal-inducible C. elegans metallothionein-1 (mtl-1) transcription, we created integrated transgenic strains of C. elegans containing GFP under the control of the 5-regulatory region of mtl-1;pmtl-1::GFP. This strain constitutively expresses GFP in the pharynx and following cadmium exposure, express GFP in the intestine. Using the pmtl-1::GFP strains, genes involved in various stress response pathways were tested for their potential role in controlling mtl-1 expression using a candidate gene screen. Knockout of akt-1 or akt-2 does not affect GFP expression;however, the knockout of both genes simultaneously increases mtl-1 expression. AKT-1 is a serine/threonine kinase involved in the insulin signaling pathway and complexes with AKT-2 to regulate the activity of downstream factors. PDK-1 directly interacts with this complex and the knockout of pdk-1 results in an increase in expression of mtl-1. Interestingly, mtl-1 transcription is not affected when other insulin signaling pathway genes are knocked out. This suggests that PDK-1 and the AKT-1/2 complex act independently of this pathway to control mtl-1 transcription. To identify other transcriptional regulators, transcription factors involved in various MAPK pathways were tested. Knockout of atf-7, which is involved in the JNK/p38 pathway, results in an increase in GFP expression. Pathway analysis indicates that ATF-7 regulates cadmium-inducible MT transcription downstream of PDK-1 and AKT-1/2 and that this regulation is independent of the insulin signaling pathway. We also completed a forward mutagenesis screen and isolated seventeen gain-of-function(high levels of transcription in the absence of cadmium) mutants. The location of these mutations was determined by SNP mapping and the exact mutation by full genome sequencing. Among these mutants, a new allele of atf-7 was identified, confirming the candidate gene screen. We focused our efforts this year on defining the regulatory pathway linking ATF-7, PDK-1, and AKT-1/2 to cadmium-inducible mtl-1 expression. We have determined that cadmium inducible mtl-1 transcription is controlled via a negative regulatory process. In addition to the genes in this pathway, we have identified four genes in the candidate gene screen and ten genes in the forward screen, all of which are in non-insulin signaling pathways. Their role(s) in mtl-1 transcription will be explored. Cdr-1 Cdr-1 is expressed exclusively in intestinal cells of adult C. elegans. Loss of cdr-1 expression results in phenotypes associated with increased sensitivity to cadmium toxicity, such as slow growth, failure to develop and sterility. Although there are currently no known mammalian homologs of cdr-1, it is likely that components of regulatory pathways controlling cdr-1 transcription are evolutionarily conserved. We performed both candidate gene and forward genetic screens to identify members of the pathways regulating cdr-1 transcription in response to cadmium. For these screens, we utilized an integrated transgenic strain of C. elegans containing GFP under the control of the 5-regulatory region of cdr-1, pcdr-1::GFP. In the absence of cadmium, pcdr-1::GFP expression is not observed in adult nematodes. After exposure to 10M cadmium for 24 h however, pcdr-1::GFP expression is observed throughout the intestine. We have completed a candidate gene screen using available mutant strains and RNAi. Approximately 160 candidates have been examined using the pcdr-1::GFP transgenic strain and/or by measuring of cdr-1 mRNA levels. We identified the insulin signaling pathway as one of the regulatory pathways controlling cdr-1 transcription. In addition, genes encoding histone-modifying proteins and homeobox proteins have also been shown to affect cdr-1 expression. To identify additional genes involved in cdr-1 regulation, a forward genetic EMS screen was conducted. A total of 28 mutants that expressed reduced levels of pcdr-1::GFP and 23 mutants that expressed increased levels of pcdr-1::GFP were isolated. We are testing a new NextGen sequencing protocol that will simultaneously SNP map and identify the physical mutations in these strains. Once our current sequence data analysis software has been modified and the protocol has been tested, we will submit additional DNA for NextGen sequencing. Numr-1 and numr-2 From our 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 to numr-1, in both coding and regulatory regions. 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. We prevously found that changes in intracellular calcium significantly affected numr-1/-2 transcription. This observation suggests that numr-1/-2 are regulated by novel mechanisms. We began a candidate gene screen based on an analysis of numr-1/-2 regulatory regions from several Caenorhabdi species. The promoter analysis identified 17 potential transcription factor binding sites. We obtained viable C. elegans strains containing lof mutation in these candidate genes. The effect of cadmium on the expression of numr-1/-2 in these mutants led to the identification of three genes that affected cadmium-inducible numr-1/-2 mRNA levels: osm-9, ceh-20, and gem-20. Osm-9 and ceh-20 are expressed in a subset of osmosensory, mechanosensory and chemosensory neurons. These candidate genes are not part of the same signaling pathway;therefore, cadmium-inducible numr-1 transcription may be regulated by multiple signaling pathways. We are currently testing the candidate genes previously examined in the cdr-1 and mtl-1 screens for their effect on cadmium-inducible and basal numr-1/-2 expression.

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