The human disease hemochromatosis is a genetic disorder caused by excess uptake of iron from the diet, resulting in multi-organ failure and, often, liver cancer. Although a candidate gene for this disorder was recently identified, the molecular events through which iron causes damage to cells are largely unknown. Hepatic iron overload is frequently associated with chronic hepatitis and other inflammatory diseases of the liver. Similarly, the molecular events leading to iron accumulation are unknown. We have designed a genetic model of iron overload in S. cerevisiae by using a dominant mutant allele of the iron-sensing transcription factor, AFT1. Expression of this allele, AFT1-1up, results in constitutive uptake of iron regardless of intracellular or extracellular iron abundance. In budding yeast, iron overload was associated with cell arrest in both the G1 and G2 phases of the cell cycle. The G1 arrest was not due to activation of a previously identified check point, but to decreased expression of the G1 cyclins, Cln1 and Cln2. We determined that this lower level of expression was due to impaired translation of cyclin mRNA, a newly described locus of cell cycle control. Current research efforts are directed at identifying the segments of the Cln2 transcript required for translational regulation and at identifying the components of the transcriptional apparatus that are regulated by iron. AFT1-1-up strains transferred to iron-rich media also exhibit a G2 arrest that is exacerbated in strains deficient in DNA repair. Our work in this area provides evidence indicating that iron intoxication leads to DNA damage, which activates a cell cycle checkpoint that results in a cell cycle arrest in G2. These data raise the intriguing possibility that the eukaryotic cell is regulated by iron in a specific fashion. These areas of research will provide new insights as to how environmental signals are integrated into the regulation of the cell cycle. A new area of research involves the use of whole-genome approaches to identify genes that are involved in iron metabolism in S. cerevisiae. In collaboration with the Department of Genetics at Stanford University, we have employed DNA microarray analysis to identify over 50 genes that are regulated by the transcription factor AFT1. All of the genes that were previously identified as being regulated by iron and AFT1 were identified with this technique, some known genes were found to be regulated by AFT1, and, in addition, many previously uncharacterized genes were identified. Using available genome and protein databases, these newly identified genes have been grouped into families and experiments are underway to evaluate the functions of these genes. Using this approach we hope to identify mammalian homologues of these proteins, and to define pathways of iron uptake, utilization, and detoxification in humans. This work will enable the design of rational therapeutic or preventative treatments for patients susceptible to iron overload.
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