1. Redox Regulation of Transcription Factors Governing Development 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. Transcription Factors

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. Homeodomain Proteins - Transcription Factors with Important Roles in Development

6. Octamer Sequence Oct4 Oct4 is a member of the Oct family of transcription factors that binds the octamer consensus sequence ATGCAAAT Oct ATGCAAAT

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. p53 interaction with DNA

8. LexA-Gal4 LexA-Gal4 is a fusion of LexA, a binding protein, and Gal4, a transcriptional activator. It is thioredoxin reductase independent. Lex AGal4

9. C O N SH H C C O H2H2 H2H2 C O N S H C C O H2H2 H2H2 C N C H2H2 H2H2 CH OO S cysteinecystine 2 x The amino acid cysteine can undergo oxidation Redox Control of Transcription Factors SS S H S H protein dithiolprotein disulfide oxidation reduction

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. SS S H S H protein dithiol protein disulfide oxidation reduction Thioredoxin Reductase NADPH NADP

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. By using yeast lacking thioredoxin reductase, we can study whether this enzyme plays a role in activating transcription factors. S H S H Active oxidation reduction Thioredoxin Reductase S S Inactive

13. Procedure Grow yeast strains MY401(WT) and MY402 (  trr1) to.4 OD=10 7 cells/ml Grow yeast strains MY401(WT) and MY402 (  trr1) to.4 OD=10 7 cells/ml Transform yeast with effecter and reporter plasmid Transform yeast with effecter and reporter plasmid

14. Transformation Plasmids URA Lac Z Basal Promoter Oct URA LEU Basal Promoter Oct Effector Plasmid Reporter Plasmid B-gal The effector plasmid encodes for the transcription factor of interest The reporter plasmid carries a response element (Lac Z) that produces  -galactosidase in the presence of the specific transcription factor. Oct

15. Yeast DNA LEUURA3 MY401 MY402 Both plates lack supplements uracil and leucine which are required for yeast to grow. Therefore, only the yeast clones that take up both plasmids will be able to grow. v Yeast Transformants

16. Procedure Grow yeast strains MY401(WT) and MY402 (  trr1) to.4 OD=10 7 cells/ml Grow yeast strains MY401(WT) and MY402 (  trr1) to.4 OD=10 7 cells/ml Transform yeast with effecter and reporter plasmid Transform yeast with effecter and reporter plasmid Grow transformants on plates lacking uracil and leucine Grow transformants on plates lacking uracil and leucine Pick clones and grow transformants in selective medium Pick clones and grow transformants in selective medium Assay for ß-galactosidase to determine transcription factor transactivation. Assay for ß-galactosidase to determine transcription factor transactivation.

17.  -galactosidase Assay 1.Grow Cells to.4 absorbance (A600) 2. Freeze 10 min. in lq. Nitrogen B-Gal ONPG 4. Add ONPG which gets broken down in the presence of  -gal to produce yellow color. 3. Add Z-buffer with Sarkosyl and  -Mercaptoethanal 7. Compare color strength with pre-rxn absorbance 5. Measure absorbance (A420) of yellow color

18. Hox Effector Plasmids ß-galactosidase Activity

19. Hox Effector Plasmids ß-Galactosidase Activity with Basal Activity Subtracted Conclusion: Hox 1.3, Hox 3.1, and Hox 3.2 are not TRR1 dependent.

20. Hox 1.1 & Hox 2.3 TRR1 Results Effector Plasmid  -Gal Cells  -Gal/cell Hox1.1 0.142 (A 420 )(A 600 )(A 420 /A 600 ) 0.172 nmol ONP min/10 7 cells 0.202 0.239 0.432 0.389 0.594 0.398 0.508 Hox 2.3 YEP 181 0.050 0.134 0.026 0.374 0.388 0.356 0.134 0.345 0.073 Mean 0.000 0.084 0.004 0.280 0.398 0.314 0.000 0.211 0.013 Mean 5.060 3.390 4.320 4.260 ±.837 1.140 2.940 0.618 1.570 ± 1.22 0.000 0.179 0.108 0.632

21. 0.267 Hox 1.1 & Hox 2.3  trr1 Results nmol ONP min/10 7 cells Effector Plasmid  -Gal Cells  -Gal/cell Hox1.1 0.146 (A 420 )(A 600 )(A 420 /A 600 ) 2.990 1.360 4.700 0.4160.351 0.050 0.352 0.312 0.632 0.160 0.557 Hox 2.30.012 0.496 0.382 0.024 0.031 YEP 1810.0000.8320.000 0.316 0.3000.042 0.000 0.004 3.020 ±1.67 Mean 0.206 0.237 ±.04 Mean 0.000 1.190 0.397 Mean

22. Hox 1.1 & Hox 2.3 ß-Galactosidase Activity with Basal Activity Subtracted Conclusion: Hox 1.1 and Hox 2.3 are not TRR1 dependent. n=3

23. 10.30 Oct 3  trr1Results Oct 3  trr1 Results nmol ONP min/10 7 cells Effector Plasmid  -Gal Cells  -Gal/cell YEP Oct3 0.138 (A 420 )(A 600 )(A 420 /A 600 ) 3.100 2.980 ±.006 0.3000.460 0.291 0.175 0.306 0.230 0.951 0.761 p53 LexA-Gal4 2.9650.3727.970 2.7890.232 0.2341.643 12.02 7.021 0.7590.3022.513 1.7890.2038.813 1.2610.4183.017 1.876 3.960 Mean 36.50 12.32 Mean 19.72 ±.088 32.55 49.08 28.68 36.77 ±.066 Mean

24. Oct3 TRR1Results Oct3 TRR1 Results nmol ONP min/10 7 cells Effector Plasmid  -Gal Cells  -Gal/cell Oct 3 (A 420 )(A 600 )(A 420 /A 600 ) p53 0.0780.4620.169 0.074 0.069 0.688 0.558 0.108 0.124 0.690 0.438 0.504 0.544 ±.001 Mean 1.709 1.460 0.304 0.298 5.622 4.899 1.0590.2803.782 22.96 20.00 15.45 26.90 ±.023 Mean LexA-Gal4 2.789 0.354 0.314 0.3022.789 7.879 8.882 9.235 32.18 36.28 37.72 35.39 ±.018 Mean

25. Oct ß-galactosidase activity with Basal Activity Subtracted Conclusion: Oct 3 TRR1 dependence cannot be determined from these 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. 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. Perform the same experiments on other  trr1 yeast strains.

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

29. Summary Slide Discussion Discussion

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