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Redox Regulation of Redox Regulation of Transcription Factors Transcription Factors
Governing DevelopmentGoverning Development
Jenny Davis
Dr. Gary Merrill
Dept. Biochemistry/Biophysics
Presentation OutlinePresentation Outline
I. BackgroundI. Background
II. ProcedureII. Procedure
III. ResultsIII. Results
IV. DiscussionIV. Discussion
The Process of ExpressionThe Process of Expression
1) Replication1) Replication2) Transcription 2) Transcription (DNA-RNA)(DNA-RNA)
3) Translation 3) Translation (RNA-PROTEIN)(RNA-PROTEIN)
4) Protein Folding4) Protein Folding
Eukaryotic TranscriptionEukaryotic Transcription
Polymerase (Pol II) makes RNA from DNA. Transcription Polymerase (Pol II) makes RNA from DNA. Transcription factors are essential for Pol II interaction with the factors are essential for Pol II interaction with the promoter (TATA) and the start of transcription. promoter (TATA) and the start of transcription.
Transcription Factors
Oct ProteinsOct ProteinsEarliest expressed homeodomain protein; Earliest expressed homeodomain protein;
inactivated at about the time of embryo implantationinactivated at about the time of embryo implantation
Hox ProteinHox ProteinA family of over 20 protein that deal A family of over 20 protein that deal
specifically with differentiation and identity of specifically with differentiation and identity of developing cells; first discovered in Drosophila; developing cells; first discovered in Drosophila; present in all higher eukaryotes.present in all higher eukaryotes.
Pax ProteinsPax ProteinsFamily of paired box proteins that facilitate Family of paired box proteins that facilitate
segmentation in development.segmentation in development.
Homeodomain Proteins - Transcription Factors with Important Roles in Development
Octamer Sequence
Oct4
Oct4 is a member of the Oct family of transcription factors that binds the octamer consensus sequence
ATGCAAAT
Oct
ATGCAAAT
p53p53
p53 is a tumor suppressor p53 is a tumor suppressor protein that is activated by protein that is activated by DNA damage and DNA damage and stimulates transcription of stimulates transcription of genes that arrest or delay the genes that arrest or delay the cell cycle. cell cycle.
Dr. Gary Merrill has found Dr. Gary Merrill has found that the ability of p53 to that the ability of p53 to function as a transcription function as a transcription factor is factor is thioredoxin thioredoxin reductase dependent.reductase dependent.
p53 interaction with DNA
LexA-Gal4LexA-Gal4
LexA-Gal4 iLexA-Gal4 is a fusion of LexA, a binding protein, and s a fusion of LexA, a binding protein, and Gal4, a transcriptional activator. It is Gal4, a transcriptional activator. It is thioredoxin thioredoxin reductase independentreductase independent..
Lex A Gal4
C
O
N
SHH
C
C
O
H2
H2
C
O
N
SH
C
C
O
H2
H2
C
N
C
H2
H2C H
OO
S
cysteine cystine
2 x
The amino acid cysteine can undergo oxidation
Redox Control of Transcription FactorsRedox Control of Transcription Factors
S SSH
SH
protein dithiol protein disulfide
oxidation
reduction
The Thioredoxin SystemThe Thioredoxin SystemThioredoxins are proteins that Thioredoxins are proteins that participates in redox participates in redox reactions, via the reversible oxidation of an active site reactions, via the reversible oxidation of an active site dithiol.dithiol. Thioredoxin reductase reduces oxidized thioredoxin, Thioredoxin reductase reduces oxidized thioredoxin, using NADPH as electron donor.using NADPH as electron donor.
S SSH
SH
protein dithiol protein disulfide
oxidation
reductionThioredoxin Reductase
NADPH NADP
What’s the Big Deal About Redox?What’s the Big Deal About Redox?
Oxidation or formation of disulfide bonds can Oxidation or formation of disulfide bonds can inactivate redox sensitive transcription factors.inactivate redox sensitive transcription factors.
Identification of oxidation-prone transcription Identification of oxidation-prone transcription factors may help explain why the expression factors may help explain why the expression of specific genes are sensitive to of specific genes are sensitive to vascularization and oxygen levels. vascularization and oxygen levels.
HypothesisHypothesis
The transcription factors Oct, Hox, and Pax are The transcription factors Oct, Hox, and Pax are thioredoxin reductase dependent. thioredoxin reductase dependent.
By using yeast lacking thioredoxin reductase, we By using yeast lacking thioredoxin reductase, we can study whether this enzyme plays a role in can study whether this enzyme plays a role in activating transcription factors.activating transcription factors.
SH
SH
Active
oxidation
reduction
Thioredoxin Reductase
S S
Inactive
ProcedureProcedure
Grow yeast strains MY401(WT) and MY402 Grow yeast strains MY401(WT) and MY402 ((trr1trr1) to .4 OD=10) to .4 OD=1077 cells/ml cells/ml
Transform yeast with effecter and reporter Transform yeast with effecter and reporter plasmidplasmid
Transformation Plasmids
URAURA
Lac ZBasal Promoter
Oct
URALEU
Basal Promoter
Oct
Effector Plasmid
Reporter PlasmidB-gal
The effector plasmid encodes for the transcription factor of interest
The reporter plasmid carries a The reporter plasmid carries a response element (Lac Z) that response element (Lac Z) that produces produces -galactosidase in the -galactosidase in the presence of the specific presence of the specific transcription factor. transcription factor.
Oct
Yeast DNALEU URA3
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
ProcedureProcedureGrow yeast strains MY401(WT) and MY402 Grow yeast strains MY401(WT) and MY402 ((trr1trr1) to .4 OD=10) to .4 OD=1077 cells/ml cells/ml
Transform yeast with effecter and reporter plasmidTransform yeast with effecter and reporter plasmid
Grow transformants on plates lacking uracil and Grow transformants on plates lacking uracil and leucineleucine
Pick clones and grow transformants in selective Pick clones and grow transformants in selective mediummedium
Assay for Assay for ßß-galactosidase to determine -galactosidase to determine transcription factor transactivation.transcription factor transactivation.
-galactosidase Assay-galactosidase Assay
1. Grow Cells to.4 absorbance (A600)
2. Freeze 10 min. in lq. Nitrogen
B-Gal
ONPG
4. Add ONPG which 4. Add ONPG which gets broken down in the gets broken down in the presence of presence of -gal to -gal to produce yellow color.produce yellow color.
3. Add Z-buffer with Sarkosyl 3. Add Z-buffer with Sarkosyl and and -Mercaptoethanal-Mercaptoethanal
7. Compare color strength with
pre-rxn absorbance
5. Measure absorbance (A420) of yellow color
Hox Effector Plasmids ß-galactosidase Activity
n=3
n=2
n=3
n=3
n=3
n=3
n=1
n=3
n=3
n=3
n=3
n=3
0 1 2 3 4 5
ADH-Hox 1.1
cYEP-Hox 1.3
cYEP-Hox 3.1
cYEP-Hox 3.2
cYEP-Hox 2.3
YEP-181
nmol ONP/min/107 cells
trr1-
TRR1
n=3
n=3
n=3
n=3
n=3
n=3
n=3
n=3
n=3
n=3
0 1 2 3 4 5
ADH-Hox1.1
cYEP-Hox1.3
cYEP-Hox3.1
cYEP-Hox3.2
cYEP-Hox2.3
nmol ONP/min/107 cells
trr1-
TRR1
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.
Hox 1.1 & Hox 2.3 TRR1 ResultsHox 1.1 & Hox 2.3 TRR1 ResultsEffectorPlasmid -Gal Cells -Gal/cell
Hox1.1 0.142
(A420) (A600) (A420 /A600)
0.172
nmol ONP
min/107 cells
0.202
0.2390.4320.389
0.5940.3980.508
Hox 2.3
YEP 181
0.0500.1340.026
0.3740.3880.356
0.1340.3450.073
Mean
0.0000.0840.004
0.2800.3980.314
0.0000.2110.013
Mean
Mean
5.0603.3904.3204.260 ± .8371.1402.9400.6181.570 ± 1.220.0000.1790.1080.632
0.267
Hox 1.1 & Hox 2.3 Hox 1.1 & Hox 2.3 trr1trr1 Results Results
nmol ONPmin/107 cells
EffectorPlasmid -Gal Cells -Gal/cell
Hox1.10.146
(A420) (A600) (A420 /A600)
2.9901.3604.700
0.416 0.3510.0500.352
0.3120.632
0.1600.557
Hox 2.3 0.0120.012
0.4960.382
0.0240.031
YEP 181 0.000 0.832 0.0000.000 0.316
0.3000.0420.0000.004
3.020 ±1.67 Mean
0.206
0.237 ±.04Mean0.0000.0001.1900.397
Mean
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
n=2
n=3
0 2 4 6
ADH-Hox 1.1
cYEP-Hox 2.3
nmol ONP/min/10 7 cells
trr1-
TRR1
n=3
10.30
Oct 3 Oct 3 trr1trr1 ResultsResults
nmol ONPmin/107 cells
EffectorPlasmid -Gal Cells -Gal/cell
YEP Oct30.138
(A420) (A600) (A420 /A600)
3.1002.980 ±.006
0.300 0.4600.2910.175
0.3060.230
0.9510.761
p53
LexA-Gal4 2.965 0.372 7.9702.789 0.232
0.2341.64312.027.021
0.759 0.302 2.5131.789 0.203 8.8131.261 0.418 3.017
1.8763.960
Mean
36.5012.32
Mean 19.72 ±.08832.5549.0828.6836.77 ±.066Mean
Oct3 TRR1Oct3 TRR1 ResultsResults
nmol ONPmin/107 cells
EffectorPlasmid -Gal Cells -Gal/cell
Oct 3
(A420) (A600) (A420 /A600)
p53
0.078 0.462 0.1690.0740.069
0.6880.558
0.1080.124
0.6900.4380.5040.544 ±.001Mean
1.7091.460
0.3040.298
5.6224.899
1.059 0.280 3.782
22.9620.0015.4526.90 ±.023Mean
LexA-Gal4 2.7892.789
0.3540.3140.3022.789
7.8798.8829.235
32.1836.2837.7235.39 ±.018Mean
n=3
n=3
n=3
n=3
n=3
n=3
0 5 10 15 20 25 30 35 40 45 50
cYEPOct3;1A16W
PRS415GPDp53;PRS316p53RE-z
LexA-Gal4-LexOP-z
nmol ONP/min/107 cells
trr1-
TRR1
Oct ß-galactosidase activity with Basal Activity Subtracted
Conclusion: Oct 3 TRR1 dependence cannot be determined from these results.
DiscussionDiscussion
All Hox strains studied appeared to be All Hox strains studied appeared to be thioredoxin reductase independent because thioredoxin reductase independent because there were no significant changes in there were no significant changes in --galactosidase between TRR1 and galactosidase between TRR1 and trr1trr1 strains. strains.
Oct3 may be thioredoxin reductase Oct3 may be thioredoxin reductase independent in yeast strains MY401 and independent in yeast strains MY401 and MY402. Its activity was very low, however, MY402. Its activity was very low, however, so its redox regulation is inconclusive. so its redox regulation is inconclusive.
Future ExperimentsFuture Experiments
Determine if transforming vectors sequentially Determine if transforming vectors sequentially instead of at the same time has any effect on instead of at the same time has any effect on redox nature of the yeast.redox nature of the yeast.
Perform the same experiments on other Perform the same experiments on other trr1trr1 yeast strains.yeast strains.
AcknowledgmentsAcknowledgments
HHMIHHMI Kevin AhernKevin Ahern Gary Merrill & LabGary Merrill & Lab Oregon State UniversityOregon State University
Summary SlideSummary Slide
DiscussionDiscussion
Hox 1.1 & Hox 2.3Hox 1.1 & Hox 2.3Hox B-Gal Assay Results
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
ADH-Hox 1.1 cYEP-Hox 2.3 YEP 181
Effecter Plasmids
nmol ONP/10^7cells/min
TRR1
trr1-
TRR1-YEP181
trr1-YEP181
ConclusionConclusion: Hox activity is not dependent on the presence or : Hox activity is not dependent on the presence or absence of thioredoxin reductase. absence of thioredoxin reductase.
Hox ResultsHox Results
Oct Results 1Oct Results 1
MY401 & MY402
05
10
15202530
3540
cYEPOct3;1A16W
PRS415GPDp53;PRS3
16p53RE-z
LexA-Gal4-LexOP-z
YEP181;1A16W
YEP181;PRS316p53R
E-z
YEP 181;LexOP-z
Plasmids
nmol ONP/10^7cells/min
TRR1
trr1-
TRR1-YEP181
trr1-YEP181
Oct Results 2Oct Results 2MY401 & MY402 Oct
0
5
10
1520
25
30
35
cYEPOct3;1A16W
PRS415GPDp53;PRS3
16p53RE-zLexA-Gal4-LexOP-z
YEP181;1A16W
Plasmids
nmolONP/10^7cells/min
TRR1
trr1-
Oct 3 activity showed little activity in the WT Oct 3 activity showed little activity in the WT (MY401) and thioredoxin reductase null strain (MY401) and thioredoxin reductase null strain (MY402).(MY402).
JD2 ResultsJD2 Results
nmol ONP per 10^7 cells/min.nmol ONP per 10^7 cells/min.
0
10
20
30
40
50
60
70
80
90
100
Hox
Evx
Pax3
Pax1
Pax6
3-Oct
p53
Control