Our research can be divided among three interrelated projects designed to study the mechanisms of regulation of gene expression by metal ions and the mechanisms of zinc homeostasis in mice.
Project 1. The overall long-term objective of these studies is to apply molecular, biochemical and transgenic approaches to study environmental health-related issues. Current studies focus on the mechanisms by which the toxic metal Cd, and the essential metal Zn regulate gene expression. Cd is a widespread environmental toxin that poses an increasing threat to public health. It is a common industrial pollutant that is also present in cigarette smoke. Cd poisoning causes damage to many major organ systems, leading to Itai-Itai disease, retardation of growth, sterility and cancer. In contrast, Zn is an essential metal which is required for the activity of hundreds of proteins, but is toxic when in high concentration. Specifically, our studies concentrate on metal-response element-binding transcription factor-1 (MTF-1), which coordinates the Cd- and Zn-induced transcription of several protective genes (e.g., the metal chelator metallothionein, the metal exporter zinc-transporter-1, and γ-glutamylcysteine synthetase heavy chain, the rate limiting step in glutathione synthesis). MTF-1 functions as a cellular metal-sensor, but the molecular mechanisms by which it senses different metals are not well understood. Our studies suggest that Cd and Zn utilize overlapping yet distinct mechanisms for activation of gene expression using MTF-1. Remarkably, the mouse MTF-1 gene is essential and homozygous knockout embryos die at midgestation, thus underscoring the importance of this transcription factor. Whether MTF-1 is an essential gene in other species remains to be determined, as does the function of this protein during development. The six zinc-finger domain of this factor is highly conserved from insects to mammals which indicates a conserved function. Therefore, the specific aims of this proposal are to: 1) Identify proteins which interact with MTF-1 specifically during Cd versus Zn induction, and examine their roles in MTF-1-regulation of gene expression; 2) Determine the roles of histone acetylation in the MTF-1 activation of gene expression; and 3) Develop the Zebrafish as a model system to reveal essential functions of MTF-1 during embryonic development. Proteins which interact with MTF-1 will be studied using Superose-6 HPLC, immunoprecipitation, binding-site chromatography and mass spectrometry of proteolytic peptides. Roles of histone acetylation will be examined using chromatin immunoprecipitation, co-transfection with HAT expression vectors, detection of specific HAT proteins in the MTF-1 complex, and analysis of MTF-1 functions in yeast with mutations in HAT genes. Functions of MTF-1 during development will be examined in Zebrafish using morpholino antisense oligonucleotides, whole mount in situ hybridization and morphometric analyses of developing embryos.
Project 2 The overall long-term objective of these studies is to elucidate the molecular mechanisms involved in zinc homeostasis in mice during pregnancy and embryonic development. Zinc deficiency profoundly affects reproductive processes and jeopardizes embryonic development, yet little is known about how zinc homeostasis is maintained. Specifically, we will study genes which encode zinc transporters. We will focus on three recently identified members of the mouse ZIP gene superfamily of metal transporters. Mouse ZIP1, 2 and 3 are closely related to each other and to recently identified human zinc transporters of the ZIP superfamily. These ZIP genes are responsible for zinc uptake and similar zinc transporters are up-regulated by zinc deficiency and display cell-specific expression in lower eukaryotes. The molecular biology of these mZIP genes in the reproductive tract and embryo will be examined. We will test the hypothesis that these zinc transporters play a central physiological role in zinc homeostasis in mammals. Therefore, the specific aims of this proposal are to; 1) clone and characterize these mouse ZIP genes and cDNAs, and examine their transport properties; 2) delineate the temporal-spatial patterns of expression of these mZIP genes in maternal and embryonic tissues, and examine their regulation by dietary zinc, and 3) determine the affects of targeted mutations of these mZIP genes on zinc homeostasis. This project represents a collaborative effort between the laboratories of Drs. Andrews and Dr. Eide at the University of Missouri.
Project 3 The overall objective of these studies is to elucidate the molecular mechanisms of regulation and functions of mouse metallothioneins (MTs) during pregnancy and embryonic development. This information, in turn, will provide clues as to the regulation and roles of MT in cancer cells. The MTs represent the best documented intracellular heavy metal (Zn, Cu, Cd) binding proteins, but they can also scavenge free hydroxyl radicals and dismutate superoxide anions. Therefore, MTs play pivotal roles in essential metal homeostasis and in protection from toxic metals and oxidative stresses. Zn or Cu deficiency leads to abnormal development and reproductive failure, whereas Cd is embryotoxic, teratogenic, and carcinogenic. Oxidative stress has been suggested as a central mechanism in aging and carcinogenesis. Our studies established that all four of the known MT genes are actively, but differentially expressed in specific cell-types that surround the developing mouse embryo from the time of implantation to near parturition. MT genes are also expressed in preimplantation blastocysts. These findings suggest that MTs have important functions during embryogenesis. The continuing broad Specific Aims of the proposed studies are to; 1) determine mechanisms regulating the cell-type specific expression of these genes in the reproductive tract and embryo, and 2) delineate the functions of these proteins during embryogenesis. These aims are being approached using molecular genetic and transgenic technologies. In Specific aim 1, we will; a) investigate the function and structure of E-boxes, found in the proximal regions of the mouse MT genes, in regulating patterns of expression in the maternal deciduum, visceral yolk sac endoderm, and placental spongiotrophoblasts; b) determine the roles of USF and USF-associated protein(s) in regulating cell-specific MT-I gene expression; c) identify and clone cell-specific bHLH transcription factors that interact with MT promoter E-boxes, and d) use transgenic mice we have created to monitor effects of maternal stresses on MT gene expression in the peri-implantation embryo. In Specific aim 2 we will delineate functions of metallothionein in Zn-homeostasis during embryogenesis using transgenic mice that over-express or have knockouts of the MT-I/II genes.
