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Greg Mayer

Regulation of squirrelfish metallothionein by zinc and estradiol: Implications in zinc homeostasis and sequestration. Greg Mayer. Overview. Introduction – heavy metals/Zn, metallothionein Activation of MT transcription by Zn in trout Squirrelfish MT Cloning / Sequencing / Analysis

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Greg Mayer

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  1. Regulation of squirrelfish metallothionein by zinc and estradiol: Implications in zinc homeostasis and sequestration Greg Mayer

  2. Overview • Introduction – heavy metals/Zn, metallothionein • Activation of MT transcription by Zn in trout • Squirrelfish MT Cloning / Sequencing / Analysis In vitro Zn influx in squirrelfish hepatocytes Transcriptional regulation of squirrelfish MT • Zebrafish transgenics

  3. Heavy Metals and the Environment Commonly found in environment Both natural and anthropogenic sources Non-degradable / persistent Both essential and toxic

  4. Zn as a Nutrient • Essential Element • All organisms need Zn to live and reproduce • More than 300 known proteins require Zn • Prosthetic group • active and co-active sites • Structural group (-S-Zn-S- bridges)

  5. Zn is a Toxicant…...at least to aquatic organisms • 96-h LC50 in fish: • Reported range: 0.070mg – 40mg l-1 • Toxicity increases with decreased hardness, decreased pH, increased AVS, etc. • Sublethal effects: • Impaired reproduction down to 0.050 mg l-1 • Avoidance reaction down to 0.006 mg l-1

  6. Metallothionein(MT) • Small metal-binding protein, 60 – 63 amino acid residues • Binds I-B and II-B metals with very high affinity: Kdfrom 10-11 M for Zn(II) to 10-19 M for Ag(I) • Synthesis is stimulated by increased intracellular concentrations of I-B and II-B metals • Metals organised in two clusters Courtesy of Dr. Peter Kille

  7. Metallothionein Biochemistry Fisher and Davie, 1998, PNAS

  8. GR Cortisol Jun/fos HO · MTF-1 Metallothionein Induction MT-Gene + Transcription AAA ZnT-1 Zn2+ THIONEIN Translation Zn2+ ZIP ? Zn2+ METALLOTHIONEIN

  9. Transcriptional activation machinery MRE Genes VI, Lewin, 1997

  10. MTF-1 Mediates MT Transcription by Metals and is Constitutively Active Acidic transactivation domain DNA-binding domain with 6 Zn-fingers Zn Zn Zn Zn Zn Zn MRE 5’ flanking region of MT gene

  11. NF-IL6 GRE API MRE Rainbow Trout MT-A Promoter TATA -1000 -800 -600 -400 -200 0 • 6 functional MRE (a-f) • 4 functional API sites • 1 complete putative GRE, 73% of consensus • 1 putative NF-IL6 site, 100% of consensus

  12. 5’-TGCPuCNCGG-3’ TATA Light MRE MRE MRE MRE MRE MRE Luciferase pGL-6MRE Transfection MT CHSE-214 RTG-2

  13. MT Promoter Activity in Zn-Exposed Cells Mayer, et al., 2003

  14. Conclusions: MT promoter activation by Zn • Zn activates the MT promoter in a dose dependant manner • Zn is approx 3 fold more toxic to CHSE-214 than to RTG-2

  15. * * Zn concentrations in squirrelfish Hogstrand, et al., 1996

  16. F M F F F F M F F M M F northern analysis of squirrelfish MT mRNA

  17. Hepatic MT mRNA vs. Liver Zn concentration in female squirrelfish 10 R2 = .80 8 6 Liver Zn (µmols/g) 4 2 0 0 1 2 3 MT mRNA (relative units) Mayer, unpublished

  18. Gonado Somatic Index vs. Liver Zn Concentration in female squirrelfish 10 R2 = .82 8 6 Liver Zn (µmols/g) 4 2 0 0 1 2 GSI Thompson, Mayer, 2002

  19. Gonado Somatic Index vs. Liver MT mRNA levels in female squirrelfish R2 = .85 2 1 MT mRNA (relative units) 0 0 1 2 GSI Mayer, unpublished

  20. Net 65Zn flux in female squirrelfish Hogstrand, et al., 1996

  21. What Regulates Zn / MT Homeostasis in FEMALE Squirrelfish? ?

  22. Sequence of the Squirrelfish MT locus 1 actatagggc acgcgtggtc gacggcccgg gctggtatcc tcatgctttg ctcccggctt actcaacccc acttcagagc 90 aggcgatgct ctcaataagt cagtgttaag gtctgcagtg ttaatcgagg ttagaacgaa tgtaattgaa tgtgtttgnt 180 gtctattcaa aactgtgcta caaacatttt gaagatataa acttagtgct ttctagtgag agaggttaat aagtgtgagt 270 gtggaatgag gctagcctaa gcgctgggtt tattgtagcc aactgcctta gattagccga gggctagaaa atgcaaacag 360 gaaaaggatc taaatatcca tatcattgtt ctcattagtt gctgacaggg cttccatgct catgcctgtc tgacttgggg 450 aacctgtgac tgacaactct cttttcagtc gctaccaaag ttacagacat gaaaacagta caaataacat cctatttgtc 540 agttttttta attactgtat acagtaaatc cattcgtttt gggctattca ttaaaacata tgtagatata tccagatatg 630 ttgtttgagt gttaaattga caagtttata taaaaaaggg caatacccga agcaactcaa ctcctctccc acccttattt 720 accgcttttg ttggtttgtt caactgcgag taattgtaat atttccgaag gcacttgaag gcagcttatt atcatcagcc 810 gtgtgtttac tctcagtatg ctcacggcgc tccagcggcc gagtgcagag cagagagaga ggggggggag ggaggagtgt 900 aaacagtgcg cttgtggtca gtgtgaccgt gtgcacagac tgcaggcggc tgagaaatat catccaatac aaacaggcca 990 aaattaatac aatgtcgtga cagtaaagga gccccccgtt ttgttattat actaattaat gagcttctga acgcactttg 1080 gctttgatct cattgcactt attcatgaca tcaacattga caataaacat tcagttgatt tttnaaagga caaaaatcaa 1170 ctcgattgtc agcagacttt taaaatagca tctaaacatt aacatcatcc aaaatacgtc aaacaaattt acaacggagg 1260 aatgtagaga ttaagtgtaa ctgaaaactt ttacactagg gacaattaac tctatctatc tatctatcta tctcacacac 1350 taagtgccca ctggaatatt ataggaaaaa tataaatata atttgttttt ttacataaac ataacacatt atttcctgta 1440 agacaaaatt actggatcat attgttatac tgtgtgttgt gctgtcttat ataaggttgt tttcctctgt ttttaatgaa 1530 tacatctgaa ctccggtgac cccttctttt catgtcattt gcataacaaa acgccaactt tcgtcctcgc ttgggaattt 1620 ctgtcctcaa gtcgtgtgca caaccaagcc ctcatttccc cctcattttt ttcaggtgat actgaccacg tgaaaatgtg 1710 acaggcgcat tttgcacccg gcccgttcct ctcggacgct ataaaatgcc gcacccctga ctgatactca caacactgag 1800 tctaccgaca cagaaacatc tttaatcaac cacagaagcg gaagactaag aaacttctcc agcatcgaca tggacccctg 1890 cgagtgctct aagagtgagt tctattttct ctcttcacct ctgttttcat ctcgcctctc attcacntaa tttgaccttg 1980 ttgtaggcta cataaaccag atgataatgt gatactgatc gacctgntgt gttgtagtgt tttctagctg gaacctgcag 2070 ctgcggtgga tcctgcacct gcacaaactg ctcctgcacc acctgcaaga agagtaagtt ctcttgattt ttttttctat 2060 gttactgata gatgtgtgct tccagtattt tgatttaaga tagaactttt tcacagcagc cattttgata tgcaatagta 2150 gaccgacacc aggtgttggc tactaatcac attagggggt ttgtcctgct cttcaattga tttgccttca gaatgttaaa 2240 atgtgcaagt gaccatgcaa attcaggttt tatcactttt ctttcccctt taaaaagagg tctggacatt gtctgagaaa 2330 tccagattct gggtcataat tggttaattg acaactgccc atgtgtttta atgttgctgc tcatgcaggg atgttgtgca 2420 gttaatccca gctcccaaca ctctctttag aggacaatga cttaacagat ttgtaactta aacctgcttg gaagcagact 2510 cttctgcctc aaatcgctga ctgaattgtg tcactaatcg cagcccaatc tgtctctcct ctttgcagct gctgcccctg 2600 ctgcccatcc ggctgcagca agtgcgcctc tggctgcgtg tgcaaaggga agacctgtga caccagctgc tgccagtgag 2690 aatccatctt gcagcagccc tgtcatcatg aaggcctatc atcatgaggc ctgtttctac tgtgaaatgt cttaactgca 2780 cttgcagtcg tctagagtag ttgtgtgttt tttgtacttt gatttcaatg ggataataaa

  23. NF-IL6 GRE API MRE Rainbow Trout MT-A Promoter TATA -1000 -800 -600 -400 -200 0 • 6 functional MRE (a-f) • 4 functional API sites • 1 complete putative GRE, 73% of consensus • 1 putative NF-IL6 site, 100% of consensus

  24. MRE B ERE B MRE C MRE A MRE D ARE AP-1 USF MRE E ERE A Squirrelfish MT Locus TATA Exon I Exon II Exon III -1750 -1500 -1250 -1000 -750 -500 -250 0 250 500 750 1000 Base pairs

  25. Squirrelfish MT Locus MRE B ERE B MRE C MRE A Exon I MRE D Exon II MRE E Exon III ERE A -1750 -1500 -1250 -1000 -750 -500 -250 0 250 500 750 1000 Base pairs

  26. Squirrelfish surgery and perfusion Squirrelfish hepatocyte culture for Zn/steroid testing Needed many replicates without sacrificing many fish

  27. Primary hepatocyte cell culture

  28. Analysis of qPCR 50 C E2-7 E2-7 C E2-7 E2-7 Zn200 Zn400 Zn200 Zn200 Zn200 ________________ Tmx

  29. Conclusions: mRNA regulation by Zn and E2 • Both Zn and E2 up-regulate MT transcription • Zn and E2 in combination act synergistically • E2 effects on transcription are blocked by tamoxifen

  30. Influence of Zn and E2 on 65Zn influx -------------------------------------------------------------------------------------------------------------------- Zn200 E-8 E-6 ____________ ____________ C Zn400 E-7 E2-8 E2-7 E2-6 E2-8 E2-7 E2-6 Zn200 Zn400

  31. Conclusions: 65Zn influx regulation byZn and E2 • Zn or E2 alone do not significantly increase 65Zn influx • Zn and E2 in combination increase 65Zn in a concentration dependant manner with regard to E2 • These findings are in agreement with MT mRNA up-regulation

  32. Hypothesis: - Female squirrelfish accumulate zinc to supply the larvae with zinc for retinal development?

  33. Seasonal Variations in Females Mayer, et al., submitted

  34. Zn and squirrelfish spawning

  35. Conclusions from squirrelfish • Squirrelfish MT promoter contains novel ERE / MRE pairs • Zn and estradiol act synergistically to up-regulate MT transcription • Up-regulation in MT mRNA is associated with increased Zn influx in cultured hepatocytes • An association between [Zn] and egg viability may indicate a physiological explanation for female-specific zinc accumulation

  36. Zebrafish Transgenics Transgenic Vector Design Ocean pout antifreeze protein Carp b-actin Mc-GFP "Insulator" terminator "Enhancer element " "Enhancer element " Sqfish MT promoter

  37. 1Day Control 50 µM Zn

  38. 2 Day Control 50 µM Zn

  39. 5 Day Control 50 µM Zn

  40. 20 Day No Fluor Control 50 µM Zn

  41. Acknowledgements Squirrelfish Dr. Christer Hogstrand Dr. Pat Walsh Dr. David Thompson Mr. Tom Capo Zebrafish Transgenics Dr. Pat Gibbs Dr. Mike Schmale NIEHS Marine and Freshwater Biomedical Sciences Center Grant -ES05705

  42. Heavy Metal in the Aquatic Environment

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