Redox Regulation of Transcription Factors Governing Development Jenny Davis

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Redox Regulation of Transcription Factors
Governing DevelopmentJenny Davis

• Dr. Gary Merrill
• Dept. Biochemistry/Biophysics

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Presentation Outline

• I. Background
• II. Procedure
• III. Results
• IV. Discussion

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The Process of Expression

• 1) Replication
• 2) Transcription (DNA-RNA)
• 3) Translation (RNA-PROTEIN)
• 4) Protein Folding

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Eukaryotic Transcription
Transcription Factors

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

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Homeodomain Proteins - Transcription Factors
with Important Roles in Development

• 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.

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Oct4
Oct4 is a member of the Oct family of
transcription factors that binds the octamer
consensus sequence
Octamer Sequence
ATGCAAAT
Oct
ATGCAAAT
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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
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LexA-Gal4

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

Lex A
Gal4
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Redox Control of Transcription Factors
N
H2
N
H2
2 x
C
S
H2
C
H2
C
H
S
C
H
O
C
O
C
O
O
cysteine
cystine
The amino acid cysteine can undergo oxidation
S
S
oxidation
reduction
protein dithiol
protein disulfide
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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.

S
S
oxidation
reduction
protein disulfide
Thioredoxin Reductase
NADP
NADPH
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Whats 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.

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Hypothesis

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

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

• Grow yeast strains MY401(WT) and MY402 (Dtrr1) to
  .4 OD107 cells/ml
• Transform yeast with effecter and reporter plasmid

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

Yeast Transformants
v
MY401
MY402
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Procedure

• Grow yeast strains MY401(WT) and MY402 (Dtrr1) to
  .4 OD107 cells/ml
• Transform yeast with effecter and reporter
  plasmid
• Grow transformants on plates lacking uracil and
  leucine
• Pick clones and grow transformants in selective
  medium
• Assay for ß-galactosidase to determine
  transcription factor transactivation.

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?-galactosidase Assay
2. Freeze 10 min. in lq. Nitrogen
3. Add Z-buffer with Sarkosyl and
?-Mercaptoethanal
ONPG

• Grow Cells to
• .4 absorbance (A600)

4. Add ONPG which gets broken down in the
presence of ?-gal to produce yellow color.
B-Gal
7. Compare color strength with pre-rxn absorbance
5. Measure absorbance (A420) of yellow color
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Hox Effector Plasmids ß-galactosidase Activity
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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.
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Hox 1.1 Hox 2.3 TRR1 Results
Effector Plasmid
b -Gal/cell
b -Gal
Cells
nmol ONP min/107 cells
(A420)
(A600)
(A420 /A600)
Hox1.1
0.142
0.239
0.594
5.060
0.172
0.432
0.398
3.390
0.202
0.389
0.508
4.320
4.260 .837
Mean
Hox 2.3
0.050
0.374
0.134
1.140
0.134
0.388
0.345
2.940
0.026
0.356
0.073
0.618
1.570 1.22
Mean
YEP 181
0.000
0.179
0.108
0.632
Mean
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Hox 1.1 Hox 2.3 Dtrr1 Results
Effector Plasmid
b -Gal/cell
b -Gal
Cells
nmol ONP min/107 cells
(A420)
(A600)
(A420 /A600)
Hox1.1
0.146
2.990
0.416
0.351
1.360
4.700
3.020 1.67
Mean
Hox 2.3
0.206
0.267
0.237 .04
Mean
YEP 181
0.000
0.000
0.004
1.190
0.397
Mean
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Hox 1.1 Hox 2.3 ß-Galactosidase Activity with
Basal Activity Subtracted
n3
Conclusion Hox 1.1 and Hox 2.3 are not TRR1
dependent.
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Oct 3 Dtrr1 Results
Effector Plasmid
b -Gal/cell
b -Gal
Cells
nmol ONP min/107 cells
(A420)
(A600)
(A420 /A600)
YEP Oct3
0.138
0.300
0.460
1.876
3.960
3.100
2.980 .006
Mean
p53
10.30
36.50
12.32
19.72 .088
Mean
LexA-Gal4
32.55
49.08
28.68
36.77 .066
Mean
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Oct3 TRR1 Results
Effector Plasmid
b -Gal/cell
b -Gal
Cells
nmol ONP min/107 cells
(A420)
(A600)
(A420 /A600)
Oct 3
0.544 .001
Mean
p53
LexA-Gal4
2.789
0.354
7.879
2.789
0.314
8.882
0.302
2.789
9.235
35.39 .018
Mean
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Oct ß-galactosidase activity with Basal Activity
Subtracted
Conclusion Oct 3 TRR1 dependence cannot be
determined from these results.
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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.

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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.

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Acknowledgments

• HHMI
• Kevin Ahern
• Gary Merrill Lab
• Oregon State University

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Summary Slide

• Discussion

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Hox 1.1 Hox 2.3

• Conclusion Hox activity is not dependent on
  the presence or absence of thioredoxin reductase.
  

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Hox Results
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Oct Results 1


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Oct Results 2

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

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JD2 Resultsnmol ONP per 107 cells/min.