Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Apr 11;21(7):1549–1554. doi: 10.1093/nar/21.7.1549

Purification of mouse MEP-1, a nuclear protein which binds to the metal regulatory elements of genes encoding metallothionein.

S Labbé 1, L Larouche 1, D Mailhot 1, C Séguin 1
PMCID: PMC309361  PMID: 8479904

Abstract

Metal regulatory elements (MREs) shared by metallothionein (MT) gene promoters are essential for metal induction of MT genes. MEP-1, a nuclear protein which binds to these elements has been purified from heavy metal-resistant mouse L cells using footprinting, Southwestern and UV cross-linking techniques to assay its binding activity. The purification scheme, starting from crude nuclear extracts, involved a combination of heparin-Sepharose and MRE-DNA affinity chromatography. The purified protein preparation showed a single polypeptide band of 108 kDa on polyacrylamide gel electrophoresis, and 2D-gel analyses revealed the presence of a protein species migrating as a single population of approximately 110 kDa. MEP-1 does not appear to be glycosylated since it eluted with the flow-through on a Wheat Germ Sepharose column. It was retained by a zinc-Chelating Sepharose column suggesting that amino acid residues (i.e., cysteine, histidine) which have an affinity for zinc ions are exposed on the protein surface. Binding studies with the purified protein indicated that it binds specifically to MRE sequences and that the binding can be abolished by a point mutation in the MRE core consensus sequence or by the addition of the chelating agent 1,10-phenanthroline. Binding activity can be restored by the addition of zinc ions to the chelated protein. These results suggest that MEP-1 is one of the major proteins interacting with MRE sequences.

Full text

PDF
1549

Images in this article

1550Image
on p.1550
1551Image
on p.1551
1551Image
on p.1551
1552Image
on p.1552
1552Image
on p.1552
1552Image
on p.1552

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersen R. D., Taplitz S. J., Oberbauer A. M., Calame K. L., Herschman H. R. Metal-dependent binding of a nuclear factor to the rat metallothionein-I promoter. Nucleic Acids Res. 1990 Oct 25;18(20):6049–6055. doi: 10.1093/nar/18.20.6049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersen R. D., Taplitz S. J., Wong S., Bristol G., Larkin B., Herschman H. R. Metal-dependent binding of a factor in vivo to the metal-responsive elements of the metallothionein 1 gene promoter. Mol Cell Biol. 1987 Oct;7(10):3574–3581. doi: 10.1128/mcb.7.10.3574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andrews G. K. Regulation of metallothionein gene expression. Prog Food Nutr Sci. 1990;14(2-3):193–258. [PubMed] [Google Scholar]
  4. Carter A. D., Felber B. K., Walling M. J., Jubier M. F., Schmidt C. J., Hamer D. H. Duplicated heavy metal control sequences of the mouse metallothionein-I gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7392–7396. doi: 10.1073/pnas.81.23.7392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carthew R. W., Chodosh L. A., Sharp P. A. The major late transcription factor binds to and activates the mouse metallothionein I promoter. Genes Dev. 1987 Nov;1(9):973–980. doi: 10.1101/gad.1.9.973. [DOI] [PubMed] [Google Scholar]
  6. Chodosh L. A., Carthew R. W., Sharp P. A. A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor. Mol Cell Biol. 1986 Dec;6(12):4723–4733. doi: 10.1128/mcb.6.12.4723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cousins R. J., Lee-Ambrose L. M. Nuclear zinc uptake and interactions and metallothionein gene expression are influenced by dietary zinc in rats. J Nutr. 1992 Jan;122(1):56–64. doi: 10.1093/jn/122.1.56. [DOI] [PubMed] [Google Scholar]
  8. Culotta V. C., Hamer D. H. Fine mapping of a mouse metallothionein gene metal response element. Mol Cell Biol. 1989 Mar;9(3):1376–1380. doi: 10.1128/mcb.9.3.1376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Czupryn M., Brown W. E., Vallee B. L. Zinc rapidly induces a metal response element-binding factor. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10395–10399. doi: 10.1073/pnas.89.21.10395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Foster R., Gedamu L. Functional analyses of promoter elements responsible for the differential expression of the human metallothionein (MT)-IG and MT-IF genes. J Biol Chem. 1991 May 25;266(15):9866–9875. [PubMed] [Google Scholar]
  11. Fürst P., Hu S., Hackett R., Hamer D. Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein. Cell. 1988 Nov 18;55(4):705–717. doi: 10.1016/0092-8674(88)90229-2. [DOI] [PubMed] [Google Scholar]
  12. Hamer D. H. Metallothionein. Annu Rev Biochem. 1986;55:913–951. doi: 10.1146/annurev.bi.55.070186.004405. [DOI] [PubMed] [Google Scholar]
  13. Imbert J., Culotta V., Fürst P., Gedamu L., Hamer D. Regulation of metallothionein gene transcription by metals. Adv Inorg Biochem. 1990;8:139–164. [PubMed] [Google Scholar]
  14. Imbert J., Zafarullah M., Culotta V. C., Gedamu L., Hamer D. Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes. Mol Cell Biol. 1989 Dec;9(12):5315–5323. doi: 10.1128/mcb.9.12.5315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jackson S. P., Tjian R. O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell. 1988 Oct 7;55(1):125–133. doi: 10.1016/0092-8674(88)90015-3. [DOI] [PubMed] [Google Scholar]
  16. Jackson S. P., Tjian R. Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1781–1785. doi: 10.1073/pnas.86.6.1781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Karin M., Haslinger A., Holtgreve H., Richards R. I., Krauter P., Westphal H. M., Beato M. Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene. Nature. 1984 Apr 5;308(5959):513–519. doi: 10.1038/308513a0. [DOI] [PubMed] [Google Scholar]
  18. Koizumi S., Suzuki K., Otsuka F. A nuclear factor that recognizes the metal-responsive elements of human metallothionein IIA gene. J Biol Chem. 1992 Sep 15;267(26):18659–18664. [PubMed] [Google Scholar]
  19. Labbé S., Prévost J., Remondelli P., Leone A., Séguin C. A nuclear factor binds to the metal regulatory elements of the mouse gene encoding metallothionein-I. Nucleic Acids Res. 1991 Aug 11;19(15):4225–4231. doi: 10.1093/nar/19.15.4225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lichtsteiner S., Schibler U. A glycosylated liver-specific transcription factor stimulates transcription of the albumin gene. Cell. 1989 Jun 30;57(7):1179–1187. doi: 10.1016/0092-8674(89)90055-x. [DOI] [PubMed] [Google Scholar]
  21. Mueller P. R., Salser S. J., Wold B. Constitutive and metal-inducible protein:DNA interactions at the mouse metallothionein I promoter examined by in vivo and in vitro footprinting. Genes Dev. 1988 Apr;2(4):412–427. doi: 10.1101/gad.2.4.412. [DOI] [PubMed] [Google Scholar]
  22. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  23. O'Halloran T. V., Frantz B., Shin M. K., Ralston D. M., Wright J. G. The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell. 1989 Jan 13;56(1):119–129. doi: 10.1016/0092-8674(89)90990-2. [DOI] [PubMed] [Google Scholar]
  24. Searle P. F. Zinc dependent binding of a liver nuclear factor to metal response element MRE-a of the mouse metallothionein-I gene and variant sequences. Nucleic Acids Res. 1990 Aug 25;18(16):4683–4690. doi: 10.1093/nar/18.16.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Seguin C., Hamer D. H. Regulation in vitro of metallothionein gene binding factors. Science. 1987 Mar 13;235(4794):1383–1387. doi: 10.1126/science.3103216. [DOI] [PubMed] [Google Scholar]
  26. Stuart G. W., Searle P. F., Chen H. Y., Brinster R. L., Palmiter R. D. A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7318–7322. doi: 10.1073/pnas.81.23.7318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stuart G. W., Searle P. F., Palmiter R. D. Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. 1985 Oct 31-Nov 6Nature. 317(6040):828–831. doi: 10.1038/317828a0. [DOI] [PubMed] [Google Scholar]
  28. Séguin C. A nuclear factor requires Zn2+ to bind a regulatory MRE element of the mouse gene encoding metallothionein-1. Gene. 1991 Jan 15;97(2):295–300. doi: 10.1016/0378-1119(91)90066-k. [DOI] [PubMed] [Google Scholar]
  29. Séguin C., Felber B. K., Carter A. D., Hamer D. H. Competition for cellular factors that activate metallothionein gene transcription. Nature. 1984 Dec 20;312(5996):781–785. doi: 10.1038/312781a0. [DOI] [PubMed] [Google Scholar]
  30. Séguin C., Prévost J. Detection of a nuclear protein that interacts with a metal regulatory element of the mouse metallothionein 1 gene. Nucleic Acids Res. 1988 Nov 25;16(22):10547–10560. doi: 10.1093/nar/16.22.10547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thiele D. J. ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene. Mol Cell Biol. 1988 Jul;8(7):2745–2752. doi: 10.1128/mcb.8.7.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thiele D. J. Metal-regulated transcription in eukaryotes. Nucleic Acids Res. 1992 Mar 25;20(6):1183–1191. doi: 10.1093/nar/20.6.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Thomassin H., Hamel D., Bernier D., Guertin M., Belanger L. Molecular cloning of two C/EBP-related proteins that bind to the promoter and the enhancer of the alpha 1-fetoprotein gene. Further analysis of C/EBP beta and C/EBP gamma. Nucleic Acids Res. 1992 Jun 25;20(12):3091–3098. doi: 10.1093/nar/20.12.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Welch J., Fogel S., Buchman C., Karin M. The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein. EMBO J. 1989 Jan;8(1):255–260. doi: 10.1002/j.1460-2075.1989.tb03371.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Westin G., Schaffner W. A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene. EMBO J. 1988 Dec 1;7(12):3763–3770. doi: 10.1002/j.1460-2075.1988.tb03260.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  37. Zhou P., Szczypka M. S., Sosinowski T., Thiele D. J. Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor. Mol Cell Biol. 1992 Sep;12(9):3766–3775. doi: 10.1128/mcb.12.9.3766. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES