Metal-responsive transcription factor 1 (MTF-1) is an essential protein required for mouse embryonic development. We report here the occurrence of sumoylation on MTF-1. Mutational studies demonstrated that sumoylation occurs on the lysine residue at position 627 (Lys(627)) of mouse MTF-1. Small ubiquitin-like modifier (SUMO)-1 was fused to the C terminus of MTF-1 to mimic the sumoylated form of the protein and it suppressed the transcriptional activity of MTF-1. The nuclear translocation activity, DNA-binding activity, and protein stability of SUMO-fused MTF-1 are similar to that of wild type MTF-1. The level of sumoylation was reduced by metal in a dose- and time-dependent manner. The fact that zinc reduces MTF-1 sumoylation makes the suppressive role of sumoylated MTF-1 in transcription physiologically less significant because the SUMO moiety of MTF-1 is removed when MTF-1 translocates into nucleus. We further identified a SUMO-interacting motif (SIM) on MTF-1. Remarkably, MTF-1 binds sumoylated MTF-1 and/or other cellular factors in a SIM-dependent manner. This interaction was disrupted by treating cells with zinc. Gel permeation chromatography demonstrated that MTF-1 forms SIM-dependent complexes. This cross-interaction transpires in the cytoplasm and markedly reduces upon nuclear translocation. It can therefore be concluded that SUMO conjugation and the SIM on MTF-1 do not play a critical role in suppressing transcriptional activity. Instead, MTF-1 forms complexes with cellular factors through SIM and SUMO moiety in the cytoplasm. The result explores a new understanding for the mode of MTF-1 assembly and regulation in cells.

Lizard embryos are nutritionally independent from their environment. During the early phases of oogenesis, the egg prepares for development by storing reserve organelles, proteins, and RNAs sufficient to allow the zygote to transform into a juvenile. This preparation also includes the storage of metallothionein (MT) transcripts. This study investigated the localization of these transcripts by in situ hybridization throughout Podarcis sicula developmental stages. Our data show that MT expression undergoes shifts in both regional and cellular localization. MT transcripts were detected early in the central nervous system, later in tissues implicated in metabolic processes. Results are discussed highlighting differences in lizard embryonic spatial and temporal MT expression compared with piscine, amphibian, and mammalian embryos. We hypothesize that, under natural conditions, the nutritionally closed system represented by the lizard egg protects the developing embryo from an unwanted excess of metals. This mechanism would make MT expression and accumulation in detoxifying organs in developing animals unnecessary until hatching and food intake begins. Conversely, the presence of MT transcripts during brain development may ensure the correct final architecture of this organ.

Hepcidin is a small acute phase peptide that regulates iron absorption. It is induced by inflammation and infection, but is repressed by anaemia and hypoxia. Here we further reveal that hepcidin transcription also involves interactions between functional metal response elements (MREs) in its promoter, and the MRE-binding transcription factor-1. Analysis of hepcidin mRNA and protein levels in hepatoma cells suggests that its expression may be regulated by divalent metal ions, with zinc inducing maximal effects on hepcidin levels. These data suggest that this peptide may be a pleiotropic sensor of divalent metals, some of which are xenobiotic environmental toxins.

ABSTRACT: BACKGROUND: Metal-responsive transcription factor 1 (MTF-1), which binds to metal response elements (MREs), plays a central role in transition metal detoxification and homeostasis. A Drosophila interactome analysis revealed two candidate dMTF-1 interactors, both of which are related to the small regulatory protein Dumpy-30 (Dpy-30) of the worm C. elegans. Dpy-30 is the founding member of a protein family involved in chromatin modifications, notably histone methylation. Mutants affect mating type in yeast and male mating in C. elegans. RESULTS: Constitutive expression of the stronger interactor, Dpy-30L1 (CG6444), in transgenic flies inhibits MTF-1 activity and results in elevated sensitivity to Cd(II) and Zn(II), an effect that could be rescued by co-overexpression of dMTF-1. Electrophoretic mobility shift assays (EMSA) suggest that Dpy-30L1 interferes with the binding of MTF-1 to its cognate MRE binding site. Dpy-30L1 is expressed in the larval brain, gonads, imaginal discs, salivary glands and in the brain, testes, ovaries and salivary glands of adult flies. Expression of the second interactor, Dpy-30L2 (CG11591), is restricted to larval male gonads, and to the testes of adult males. Consistent with these findings, dpy-30-like transcripts are also prominently expressed in mouse testes. Targeted gene disruption by homologous recombination revealed that dpy-30L1 knockout flies are viable and show no overt disruption of metal homeostasis. In contrast, the knockout of the male-specific dpy-30L2 gene results in male sterility, as does the double knockout of dpy-30L1 and dpy-30L2. A closer inspection showed that Dpy-30L2 is expressed in elongated spermatids but not in early or mature sperm. Mutant sperm had impaired motility and failed to accumulate in sperm storage organs of females. CONCLUSIONS: Our studies help to elucidate the physiological roles of the Dumpy-30 proteins, which are conserved from yeast to humans and typically act in concert with other nuclear proteins to modify chromatin structure and gene expression. The results from these studies reveal an inhibitory effect of Dpy-30L1 on MTF-1 and an essential role for Dpy-30L2 in male fertility.

ABSTRACT: BACKGROUND: Retinal degeneration is a main cause of blindness in humans. Neuroprotective therapies may be used to rescue retinal cells and preserve vision. Hypoxic preconditioning stabilizes the transcription factor HIF-1a in the retina and strongly protects photoreceptors in an animal model of light-induced retinal degeneration. To address the molecular mechanisms of the protection, we analyzed the transcriptome of the hypoxic retina using microarrays and real-time PCR. RESULTS: Hypoxic exposure induced a marked alteration in the retinal transcriptome with significantly different expression levels of 431 genes immediately after hypoxic exposure. The normal expression profile was restored within 16 hours of reoxygenation. Among the differentially regulated genes, several candidates for neuroprotection were identified like metallothionein-1 and -2, the HIF-1 target gene adrenomedullin and the gene encoding the antioxidative and cytoprotective enzyme paraoxonase 1 which was previously not known to be a hypoxia responsive gene in the retina. The strongly upregulated cyclin dependent kinase inhibitor p21 was excluded from being essential for neuroprotection. CONCLUSION: Our data suggest that neuroprotection after hypoxic preconditioning is the result of the differential expression of a multitude of genes which may act in concert to protect visual cells against a toxic insult.

Abstract Dietary zinc deficiency in mice is accompanied by enhanced expression of the zinc uptake transporter Slc39a4 (Zip4) and repressed expression of Slc39a5 (Zip5) in tissues which regulate zinc homeostasis (intestine, pancreas and visceral yolk sac). Herein, mechanisms controlling this differential expression were investigated. The induction of Zip4 mRNA during zinc deficiency, and its repression in response to zinc repletion were found to reflect changes in Zip4 mRNA stability and not changes in the relative rate of transcription of this gene. During zinc deficiency, ZIP4 protein levels are increased and this protein is localized on the apical membranes. Administration of an oral gavage of zinc caused ZIP4 internalization and degradation in enterocytes and visceral endoderm cells. Similarly, ZIP4 is induced by zinc deficiency in cultured mouse Hepa cells and is rapidly degraded in response to added zinc. Zip5 mRNA abundance does not change in response to zinc, but the translation of this mRNA was found to be zinc-responsive. During zinc deficiency, Zip5 mRNA remains associated with polysomes, while the protein is internalized and degraded in enterocytes, acinar cells and endoderm cells. After zinc-gavage, ZIP5 is rapidly resynthesized and targeted to the basolateral membranes of these cell types.

The insulator element at the 5' end of the chicken beta-globin locus acts as a barrier, protecting transgenes against silencing effects of adjacent heterochromatin. We showed earlier that the transcription factor USF1 binds within the insulator, and that this site is important for generating in adjacent nucleosomes histone modifications associated with active chromatin, and by inference, with barrier function. To understand the mechanism of USF1 action we have characterized USF1 containing complexes. USF1 interacts directly with the histone H4R3-specific methyltransferase, PRMT1. USF1, PRMT1, and the histone acetyltransferases (HATs) PCAF and SRC-1 form a complex with both H4R3 HMT and HAT activities. siRNA downregulation of USF1 results in localized loss of H4R3 methylation, and other histone modifications associated with euchromatin, at the insulator. A dominant negative peptide that interferes with USF1 binding to DNA causes silencing of an insulated reporter construct, indicating abolition of barrier function. These results show that USF1 plays a direct role in maintaining the barrier, supporting a model in which the insulator works as a barrier by maintaining a local environment of active chromatin.

There are indications that Cd-induced malformations in rodents are related to a disrupted flux of Zn to the developing embryo. The aim of the present study was to detect ZnT-1 (Slc30a1) and MT (Mt1) protein in structures within the decidua, yolk sac and embryo of mice and to determine whether Cd affects ZnT-1 or MT-1 gene expression in these tissues. ZnT-1 was detected in the placental labyrinth, in the ventral part around the floor plate, in the inner cell layers of the rhombencephalon and in the ventral area of the otic vesicle. MT protein was detected in the yolk sac and in the surface ectoderm of some embryonic areas, such as the pharyngeal arches. ZnT-1 and MT-1 transcripts were most abundant in the decidua and yolk sac, whereas the abundance of these genes was relatively low in the embryo. Cd exposure down-regulated ZnT-1 and up-regulated MT-1 gene expression in all structures investigated, indicating that maternal Cd exposure may alter Zn homeostasis in the conceptus.

The regulation of divalent zinc has been observed in a wide range of organisms. Since this metal is an essential nutrient, but also toxic in excess, zinc homeostasis is crucial for normal cellular functioning. The metal-responsive-element-binding transcription factor-1 (MTF-1) is a key regulator of zinc in higher eukaryotes ranging from insects to mammals. MTF-1 controls the expression of metallothioneins (MTs) and a number of other genes directly involved in the intracellular sequestration and transport of zinc. Although the diverse functions of MTF-1 extend well beyond zinc homeostasis to include stress-responses to heavy metal toxicity, oxidative stress, and selected chemical agents, in this review we focus on the recent advances in understanding the mechanisms whereby MTF-1 regulates MT gene expression to protect the cell from fluctuations in environmental zinc. Particular emphasis is devoted to recent studies involving the Cys(2)His(2) zinc finger DNA-binding domain of MTF-1, which is an important contributor to the zinc-sensing and metal-dependent transcriptional activation functions of this protein.

Mouse metal response element-binding transcription factor-1 (MTF-1) regulates the transcription of genes in response to a variety of stimuli, including exposure to zinc or cadmium, hypoxia, and oxidative stress. Each of these stresses may increase labile cellular zinc, leading to nuclear translocation, DNA binding, and transcriptional activation of metallothionein genes (MT genes) by MTF-1. Several lines of evidence suggest that the highly conserved six-zinc finger DNA-binding domain of MTF-1 also functions as a zinc-sensing domain. In this study, we investigated the potential role of the peptide linkers connecting the four N-terminal zinc fingers of MTF-1 in their zinc-sensing function. Each of these three linkers is unique, completely conserved among all known vertebrate MTF-1 orthologs, and different from the canonical Cys2His2 zinc finger TGEKP linker sequence. Replacing the RGEYT linker between zinc fingers 1 and 2 with TGEKP abolished the zinc-sensing function of MTF-1, resulting in constitutive DNA binding, nuclear translocation, and transcriptional activation of the MT-I gene. In contrast, swapping the TKEKP linker between fingers 2 and 3 with TGEKP had little effect on the metal-sensing functions of MTF-1, whereas swapping the canonical linker for the shorter TGKT linker between fingers 3 and 4 rendered MTF-1 less sensitive to zinc-dependent activation both in vivo and in vitro. These observations suggest a mechanism by which physiological concentrations of accessible cellular zinc affect MTF-1 activity. Zinc may modulate highly specific, linker-mediated zinc finger interactions in MTF-1, thus affecting its zinc- and DNA-binding activities, resulting in translocation to the nucleus and binding to the MT-I gene promoter.

Several ZIP genes (SLC39A family of metal transporters) play roles in zinc homeostasis. Herein, the temporal and spatial patterns of expression of the mouse ZIP1, 3, 4, and 5 genes in the developing intestine and the effects of maternal dietary zinc deficiency on these patterns of expression were examined. ZIP1 and ZIP3 genes, conserved members of the ZIP subfamily II, were found to be coexpressed during development. Expression of these genes was detected on day 14 of gestation in smooth muscle and the pseudostratified endoderm. By 5 days post-partum, prominent expression became restricted to muscle and connective stroma. In contrast, expression of ZIP4 and ZIP5 genes, members of the ZIP subfamily called LIV-1, coincided with epithelial morphogenesis. ZIP5 expression was detected on d16 of gestation and localized to the basolateral membranes of the single-layered epithelium. ZIP4 expression was detected on d18 of gestation and localized to the apical membrane of villus epithelial cells. When dams were fed a zinc-deficient diet beginning at parturition, ZIP4 expression in the nursing neonate was greatly induced. In contrast, neonatal ZIP5 expression remained unchanged, but this protein was removed from the basolateral membrane of the enterocyte. These responses to dietary zinc deficiency mimic those found in the adult intestine. These studies reveal cell-type-specific expression of ZIP genes during development of the intestine, and suggest that the mouse intestine can elicit an adaptive response to dietary zinc availability at birth.

Emerging evidence demonstrates that heart disease may originate during fetal development. This review will focus on the role of maternal nutrition in the development of the fetal cardiovascular system. Emphasis will be placed upon the concept that nutritional inadequacies during gestation may be major programming stimuli that alter fetal cardiac, as well as vascular, physiology and predispose an individual to cardiovascular abnormalities in postnatal life. It is hypothesized that this research area will yield new information, resulting in improved fetal nutrition, growth and development through efficient maternal nutrition before and during pregnancy and will form the basis for nutritional strategies for the primary prevention of cardiovascular disease.

An orthologue to the mammalian zinc transporter-1 (ZnT-1) gene was cloned from the intestine of the torafugu pufferfish (Takifugu rubripes), demonstrating that this gene predates the evolution of land-living vertebrates. TrZnT-1 shares overall topology with other members of the ZnT- family of zinc transporters, with six transmembrane domains (TM) including a large histidine-rich intracellular loop between TM IV and V and intracellular C- and N- termini. Expression of TrZnT-1 in a metallothionein (MT) acquiescent cell line suggested that this protein reduces intracellular Zn 2+ levels. Manipulation of the transporting media showed that several externally applied hydrominerals had no effect on TrZnT-1 activity. However addition of N-ethylmaleimide increased TrZnT-1 mediated transport possibly by increasing intracellular free Zn 2+ levels by Zn 2+-release from carrier proteins. Generation of a specific antibody and subsequent immunocytochemistry on fixed cells overexpressing TrZnT-1 indicated that the protein is localised to the plasma membrane in these cells. The genomic organisation of TrZnT-1 is the same as that in mammals with two exons. The upstream regulatory region of the TrZnT-1 gene contains several putative cis-acting elements including metal response elements (MREs) and a Sp1 site. Analysis of the DNA contigs surrounding the TrZnT-1 gene reveal limited synteny between corresponding regions in rat, mouse and human, however this was very low with only two syntenic genes, ZnT-1 and NEK2.

Most studies utilizing transgenic technology focus on the impact to traits of interest, rather than propagation of the transgene to offspring. In animals containing growth hormone constructs, transgene transmission to progeny follows a Mendelian pattern of inheritance in the first few generations following generation of a founder animal, but decreases in subsequent generations. In the present study, the ovine metallothionein 1a-ovine growth hormone (oMt1a-oGH) transgenic mouse was used to determine whether transgene transmission rate to progeny was affected by overexpression of ovine growth hormone in the transgenic parent. The oMt1a-oGH mouse is a useful model for assessing transgene transmission, as the construct is easily regulatable and transgene inactivation results in a return of plasma GH to basal levels. Male and female hemizygous oMt1a-oGH mice were assigned to 1 of 3 treatment groups:(1) mice never actively expressing the transgene,(2) mice actively expressing the transgene from 3 weeks of age, and (3) mice actively expressing the transgene from 3 to 11 (males) or 3 to 8 (females) weeks of age. Transgenic mice were mated to wild type animals and the resulting progeny were genotyped. Males never actively expressing the transgene passed on the transgene to progeny in a Mendelian fashion, while males actively expressing the transgene transmitted the transgene to a smaller than expected number of progeny. However, following inactivation of the oMt1a-oGH construct in transgenic males, subsequent offspring demonstrated Mendelian inheritance of the transgene. In contrast, females expressing the transgene from 3 to 8 weeks of age were able to pass on the oMt1a-oGH construct in a Mendelian fashion, but females from other treatment groups were not. In oMt1a-oGH males, reduced transgene transmission appears to be due to selection against transgenic gametes. In females, however, selection against the transgenic genotype likely occurs at the embryonic level.

Upstream stimulating factors (USF), USF-1 and USF-2, are members of the eucaryotic evolutionary conserved basic-Helix-Loop-Helix-Leucine Zipper transcription factor family. They interact with high affinity to cognate E-box regulatory elements (CANNTG), which are largely represented across the whole genome in eucaryotes. The ubiquitously expressed USF-transcription factors participate in distinct transcriptional processes, mediating recruitment of chromatin remodelling enzymes and interacting with co-activators and members of the transcription pre-initiation complex. Results obtained from both cell lines and knock-out mice indicates that USF factors are key regulators of a wide number of gene regulation networks, including the stress and immune responses, cell cycle and proliferation, lipid and glucid metabolism, and in melanocytes USF-1 has been implicated as a key UV-activated regulator of genes associated with pigmentation. This review will focus on general characteristics of the USF-transcription factors and their place in some regulatory networks.

BACKGROUND: In vivo electrotransfer is a physical method of gene delivery in various tissues and organs. It is a promising strategy for the systemic secretion of therapeutic proteins and for DNA vaccination. Nevertheless, for the success of gene therapy in clinics, it is essential to develop gene regulation systems. The existing systems described in the literature all rely on the creation of an artificial transcription factor and/or an inducer drug. New strategies based on endogenous regulatable elements are being developed. We have previously identified the murine metallothionein promoter as an endogenous promoter inducible by controlled electric stimuli applied for electrotransfer experiments. We report here a regulation strategy based on this murine metallothionein promoter in a plasmid context using electric pulses delivery as an inducer. METHODS: Plasmids containing different constructions of the murine metallothionein I (mMT-I) promoter were transfected in mice tibialis-cranalis muscles using the simple skeletal muscle electrotransfer method. The regulation system was studied with the murine secreted alkaline phosphatase (MUSEAP) reporter gene. RESULTS: The mMT-I promoter can be transiently induced in vivo by application of electric fields. Its inducibility was analyzed in a plasmid context. We demonstrated that the mechanism of this transcriptional induction is not mediated by the cellular entry of metal ions. The ARE (antioxidant-responsive element) sequence was identified as the element responsive to the electric field stimulation. CONCLUSIONS: This time-control of the expression of a therapeutic gene by physical stimuli could be of value in the context of gene regulation for gene therapy.

Genetic predisposition to cancers is significant to public health because a high proportion of cancers probably arise in a susceptible human subpopulation. Using a mouse model of gamma-ray-induced thymic lymphomas, we performed linkage analysis and haplotype mapping that suggested Mtf-1, metal-responsive transcription factor-1 (Mtf-1), as a candidate lymphoma susceptibility gene. Sequence analysis revealed a polymorphism of Mtf-1 that alters the corresponding amino acid at position 424 in the proline-rich domain from a serine in susceptibility strains to proline in resistant strains. The transcriptional activity of Mtf-1 encoding serine and proline was compared by transfecting the DNA to Mtf-1-null cells, and the change to proline conferred a higher metal responsiveness in transfections. Furthermore, the resistant congenic strains possessing the Mtf-1 allele of proline type exhibited higher radiation inducibility of target genes than susceptible background strains having the Mtf-1 allele of serine type. Since products of the targets such as metallothionein are able to suppress cellular stresses generated by irradiation, these results suggest that highly inducible strains having Mtf-1 of proline type are refractory to radiation effects and hence are resistant to lymphoma development.

The SLC30 family of cation diffusion transporters includes at least nine members in mammals, most of which have been documented to play a role in zinc transport. The founding member of this family, Znt1, was discovered by virtue of its ability to efflux zinc from cells and to protect them from zinc toxicity. However, its physiological functions remain unknown. To address this issue, mice with targeted knockout of the Znt1 gene were generated by homologous recombination in embryonic stem cells. Heterozygous Znt1 mice were viable. In contrast, homozygous Znt1 mice died in utero soon after implantation due to a catastrophic failure of embryonic development. Although extraembryonic membranes formed around these embryos, the embryo proper failed to undergo morphogenesis past the egg cylinder stage and was amorphous by d9 of pregnancy. Expression of the Znt1 gene was detected predominantly in trophoblasts and in the maternal deciduum during the postimplantation period (d5 to d8). The failure of homozygous Znt1 embryos to develop could not be rescued by manipulating maternal dietary zinc (either excess or deficiency) during pregnancy. However, embryos in Znt1 heterozygous females were approximately 3 times more likely to develop abnormally when exposed to maternal dietary zinc deficiency during later pregnancy than were those in wildtype females. These studies suggest that Znt1 serves an essential function of transporting maternal zinc into the embryonic environment during the egg cylinder stage of development, and further suggest that Znt1 plays a role in zinc homeostasis in adult mice.

The ZIP5 gene encodes a protein closely related to ZIP4, a zinc transporter mutated in the human genetic disorder acrodermatitis enteropathica. Herein, we demonstrate that mouse ZIP5 and ZIP4 genes are co-expressed in several tissues involved in zinc homeostasis (intestine, pancreas, embryonic yolk sac). However, unlike expression of the ZIP4 gene, which is induced during periods of zinc deficiency, ZIP5 gene expression is unaltered by dietary zinc. Immunohistochemistry localizes ZIP5 to the basolateral surfaces of enterocytes, acinar cells, and visceral endoderm cells in mice fed a zinc-adequate diet. However, this protein is removed from these cell surfaces and internalized during dietary zinc deficiency. In contrast, ZIP4 is induced and recruited to the apical surface of enterocytes and endoderm cells during zinc deficiency. In the pancreas, ZIP4 is expressed in beta-cells, whereas ZIP5 is expressed in acinar cells. These results suggest that the function of ZIP5 is antagonistic to that of ZIP4 in the control of zinc homeostasis; rather than functioning in the acquisition of dietary zinc, as does ZIP4, ZIP5 may function in the removal of zinc from the body. Thus, during periods when dietary zinc is replete, ZIP5 may function to remove zinc from the blood via the pancreas and intestine, the major sites of zinc excretion in mammals, whereas the acquisition of dietary zinc by intestinal ZIP4 would be minimal. In contrast, during periods of dietary zinc deficiency when secretion of zinc by the pancreas and intestine is minimized, ZIP5 is removed from the cell surface, and the intestinal uptake of zinc is augmented by induction of ZIP4.

Based on the effects of a selective experimental zinc deficiency in a rodent model we explore the use of transcriptome profiling for assessing nutrient-gene interactions in the liver at the molecular and cellular levels. Zinc deficiency caused pleiotropic alterations in mRNA/protein levels of hundreds of genes. In the context of observed metabolic alterations in hepatic metabolism, possible mechanisms are discussed for how a low zinc status may be sensed and transmitted into changes in various metabolic pathways. However, it also becomes obvious that analysis of such complex nutrient-gene interactions beyond the descriptional level is a real challenge for systems biology.