Tim Dalton

REVERSIBLE ACTIVATION OF MOUSE METAL RESPONSE ELEMENT-BINDING TRANSCRIPTION FACTOR 1 DNA BINDING INVOLVES ZINC INTERACTION WITH THE ZINC FINGER DOMAIN

The DNA-binding activity of the Zn finger protein metal response element-binding transcription factor 1 (MTF-1) was rapidly induced both in vivo in mouse Hepa cells, canine MDCK, and human HeLa cells after incubation in medium containing zinc and in vitro in whole-cell extracts to which zinc was added. Acquisition of DNA-binding capacity in the presence of free zinc was temperature and time dependent and did not occur at 4 degrees C. In contrast, activated MTF-1 binding to the metal response element occurred at 4 degrees C. After Zn activation, mouse MTF-1 binding activity was more sensitive to EDTA and was stabilized by DNA binding relative to the Zn finger transcription factor Sp1. After dilution of nuclear or whole-cell extracts from Zn-treated cells and incubation at 37 degrees C, mouse MTF-1 DNA-binding activity was no longer detected but could be completely reconstituted by the subsequent readdition of zinc. In vitro-synthesized, recombinant mouse MTF-1 displayed a similar, reversible temperature- and Zn-dependent activation of DNA-binding activity. Analysis of deletion mutants of recombinant MTF-1 suggests that the Zn finger domain is important for the Zn-dependent activation of DNA-binding capacity. Thus, mouse MTF-1 functions as a reversibly activated sensor of free zinc pools in the cell.

Activation of the complete mouse metallothionein gene locus in the maternal deciduum

The mouse metallothionein (MT) gene family consists of four known members (MT‐I through IV) clustered on chromosome 8. Studies reported herein examine the expression and regulation of the MT‐III and MT‐IV genes in specific cell types in the maternal reproductive tract, developing embryo, and fetus known to express the MT‐I and ‐II genes. MT‐III and MT‐IV mRNAs were absent from the visceral yolk sac, placenta, and fetal liver, tissues with high levels of MT‐I and MT‐II mRNAs. In contrast, MT‐III and MT‐IV mRNAs were both abundant in the maternal deciduum, and in experimentally induced deciduoma on 7 and 8 days postcoitum (1 dpc = vaginal plug), as are MT‐I and ‐II mRNAs. The abundance of each of these MT mRNAs increased coordinately during development of the deciduum (6–8 dpc), and in situ hybridization localized MT‐I, MT‐III, and MT‐IV mRNAs to the secondary decidual zone of the antimesometrial region on 8 dpc, where in some regions all of the cells were apparently positive. Thus, all of the known mouse MT genes are co‐expressed in at least some of the cells in the secondary decidual zone. Electrophoretic analysis of decidual MT suggested that the MT‐I, ‐II, and ‐III isoforms are abundant proteins in the secondary deciduum. Bacterial endotoxin‐lipopolysaccharide (LPS) and Zn are powerful inducers of MT‐I and MT‐II gene expression in many adult organs, whereas these agents apparently have little effect on MT‐III and MT‐IV gene expression. Neither of these agents significantly effected levels of decidual MT‐III or MT‐IV mRNAs in vivo or in primary cultures of decidual cells in vitro, and only modest effects of Zn on MT‐I mRNA levels were noted. During 2 days of in vitro culture, decidual cell MT‐I and MT‐III mRNA levels remained elevated while MT‐IV mRNA levels decreased. Thus, expression of the mouse MT gene locus in the deciduum appears to be developmentally regulated, and in this tissue, the MT genes are refractory to induction by Zn or inflammation.

Activation of the Chicken Metallothionein Promoter by Metals and Oxidative Stress in Cultured Cells and Transgenic Mice

Cis-acting elements in the chicken metallothionein promoter were tested for their ability to direct responses of reporter genes to metal ions and oxidative stress in transfected mouse cells and in transgenic mice. In addition, protein interactions with the promoter were analyzed by the electrophoretic mobility shift assay. In transient transfection assays and in transgenic mice, 107-bp of the chicken MT promoter was sufficient to direct responses to Zn. This promoter region also directed response to oxidative stress in transfected cells and transgenic mice, but in transgenic mice, maximal responsiveness to oxidative stress apparently involved other elements in the proximal promoter region (307-bp). The proximal 200-bp of the promoter contains sequences homologous to a metal response element (-47-bp), Sp1 binding sites (-70-bp and -161-bp), and an antioxidant response element (-189-bp). Electrophoretic mobility shift assay demonstrated that metal response element binding activity was low in control Hepa cell nuclear extracts, but was induced 6-fold after 45 min of H2O2 treatment. In contrast, Sp1 binding remained unchanged, and no evidence for specific binding to the core antioxidant response element consensus sequence was obtained. These studies demonstrate that cis-acting elements mediating induction of metallothionein gene expression by metals and oxidative stress are present in the chicken metallothionein promoter and suggest a role for increased binding of the transcription factor MTF-1 to the metal response element(s).

Transgenic mouse blastocysts that overexpress metallothionein-I resist cadmium toxicity in vitro

The role of metallothionein with regard to cadmium toxicity in vitro was investigated using preimplantation mouse blastocysts derived from a transgenic strain that constitutively overexpresses metallothionein‐I transgenes (MT‐I*). Northern blot and in situ hybridization revealed high levels of MT‐I mRNA in transgenic blastocysts when compared with control blastocysts, and reverse transcriptase‐polymerase chain reaction‐amplified MT‐I mRNA was almost exclusively MT‐I*. Moreover, pulse‐labeling experiments showed that the relative rate of synthesis of MT was 9‐fold higher in transgenic blastocysts. Cadmium (Cd2+) toxicity was assessed after incubating blastocysts for 4 hr in Whittens medium containing 50 μM Cd2+. Embryos that displayed abnormal morphology were judged “sensitive.” Transgenic blastocysts were more resistant to cadmium‐induced morphological changes than were control blastocysts. “Sensitive” and “resistant” blastocysts were individually genotyped by polymerase chain reaction, or they were transferred to foster mothers, and embryonic development to midterm was monitored. Of the blastocysts derived from mating heterozygous transgenic males with control females, 56% were transgenic before incubation with Cd2+, whereas 95% of the blastocysts that retained normal morphology after incubation were transgenic. Moreover, after Cd2+ exposure, transgenic blastocysts with normal morphology were nine times more likely to develop to midterm than were control blastocysts with normal morphology. Blastocysts with abnormal morphology failed to develop to midterm. These studies indicate that MT plays a central role in protection from Cd2+ toxicity within the physiological context of the developing mouse embryo.