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