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Redox Regulation of Transcription Factors Governing Development Jenny Davis

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Redox Regulation of Transcription Factors Governing Development Jenny Davis

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    1. Redox Regulation of Transcription Factors Governing Development Jenny Davis Dr. Gary Merrill Dept. Biochemistry/Biophysics

    2. Presentation Outline I. Background II. Procedure III. Results IV. Discussion

    3. The Process of Expression 1) Replication 2) Transcription (DNA-RNA) 3) Translation (RNA-PROTEIN) 4) Protein Folding

    4. Eukaryotic Transcription Polymerase (Pol II) makes RNA from DNA. Transcription factors are essential for Pol II interaction with the promoter (TATA) and the start of transcription.

    5. Oct Proteins Earliest expressed homeodomain protein; inactivated at about the time of embryo implantation Hox Protein A family of over 20 protein that deal specifically with differentiation and identity of developing cells; first discovered in Drosophila; present in all higher eukaryotes. Pax Proteins Family of paired box proteins that facilitate segmentation in development.

    7. p53 p53 is a tumor suppressor protein that is activated by DNA damage and stimulates transcription of genes that arrest or delay the cell cycle. Dr. Gary Merrill has found that the ability of p53 to function as a transcription factor is thioredoxin reductase dependent.

    8. LexA-Gal4 LexA-Gal4 is a fusion of LexA, a binding protein, and Gal4, a transcriptional activator. It is thioredoxin reductase independent.

    10. The Thioredoxin System Thioredoxins are proteins that participates in redox reactions, via the reversible oxidation of an active site dithiol. Thioredoxin reductase reduces oxidized thioredoxin, using NADPH as electron donor.

    11. What’s the Big Deal About Redox? Oxidation or formation of disulfide bonds can inactivate redox sensitive transcription factors. Identification of oxidation-prone transcription factors may help explain why the expression of specific genes are sensitive to vascularization and oxygen levels.

    12. Hypothesis The transcription factors Oct, Hox, and Pax are thioredoxin reductase dependent.

    13. Procedure Grow yeast strains MY401(WT) and MY402 (Dtrr1) to .4 OD=107 cells/ml Transform yeast with effecter and reporter plasmid

    15.

    16.

    17. ?-galactosidase Assay

    20. Hox 1.1 & Hox 2.3 TRR1 Results

    26. Discussion All Hox strains studied appeared to be thioredoxin reductase independent because there were no significant changes in ?-galactosidase between TRR1 and ?trr1 strains. Oct3 may be thioredoxin reductase independent in yeast strains MY401 and MY402. Its activity was very low, however, so its redox regulation is inconclusive.

    27. Future Experiments Determine if transforming vectors sequentially instead of at the same time has any effect on redox nature of the yeast. Perform the same experiments on other ??trr1 yeast strains.

    28. Acknowledgments HHMI Kevin Ahern Gary Merrill & Lab Oregon State University

    29. Summary Slide Discussion

    31. Hox 1.1 & Hox 2.3 Conclusion: Hox activity is not dependent on the presence or absence of thioredoxin reductase.

    32. Hox Results

    33. Oct Results 1

    34. Oct Results 2 Oct 3 activity showed little activity in the WT (MY401) and thioredoxin reductase null strain (MY402).

    35. JD2 Results nmol ONP per 10^7 cells/min.

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