Metal-Regulatory Factor 1 (MTF-1) Role in Development and Stress Response

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The metal response element-binding transcription factor-1 (MTF-1) is a ubiquitously expressed transcription factor that plays a primary role in the regulation of a variety of genes involved in metal homeostasis, embryonic development, tumor progression, and oxidative stress or hypoxia signaling. The overall objective ofthis research is to characterize the overlapping role of MTF-1 with NRF2, the primary transcription factor for regulating the oxidative stress response, in development and transcriptional co-regulation of stress responsive genes. The specific aims ofthis project are to (1) determine role of MTF-1 in zebrafish embryonic development, (2) determine phenotypic changes in expression of known stress response genes and identify novel target genes via MTF-1 and NRF2 'knockdowns', and (3) determine the interactive role of MTF-1 in coregulating transcriptional activation of NRF2 target genes. A variety of techniques and approaches will be employed to achieve these aims. These techniques will include development of transgenic zebrafish lines, morpholino knockdown of gene expression, gene expression profiling with oligonucleotide microarrays, and advanced bioinformatic techniques for microarray analysis and computational promoter analysis. A novel transgenic zebrafish strain that expresses a constitutively nuclear, dominant-negative MTF-1 isoform and three transgenic zebrafish lines that express green or yellow fluorescent protein in the gut, liver, pancreas and neurons will be used to visualize potential developmental phenotypes associated with the MTF-1 'knockdowns'. Second, oligonucleotide microarrays will be used to determine changes in gene expression after MTF-1 or NRF2 'knockdown' to discover novel target genes co-regulated by both transcription factors during development or in response to cadmium or tBHQ (oxidative stress). Third, the interactive role of MTF-1 with NRF2 in the transcriptional response to metals or oxidative stress will be further characterized by selecting target genes for computational promoter analysis to confirm the presence of MTF-1 and NRF2 recognition sites, and one gene will be selected for additional characterization by in vivo promoter analysis in response to cadmium or oxidative stress.