junguk hur school of informatics & center for genomics and bioinformatics

Indiana UniversityBloomington, IN

Junguk HurSchool of Informatics &

Center for Genomics and

Bioinformatics

Characterization of Characterization of transcriptional responses to transcriptional responses to

environmental stress by environmental stress by differential location analysisdifferential location analysis

Capstone Presentation Junguk Hur05-26-2006 2

OVERVIEW

Background / Motivation

Location Analysis / Differential Binding

TF Response Classification

Comparison with Microarray

Conclusion / Future Work

Overview


Central Dogma

http://campus.queens.edu/faculty/jannr/bio103/tests/TEST2Help.htm

Overview

Background


Transcriptional Regulation

Albert et al., Molecular Cell Biology of the Cell

Overview

Background


Transcriptional RegulationOverview

Background

Transcription Factors


Response to EnvironmentOverview

Background

Broach et al. Curr Opin Microbiol 2004


Previous Studies - Expression

http://www.fao.org/DOCREP/003/X6884E/x6884e03.htm

High-throughput DNA Microarray – mRNA expression

Overview

Background


Previous Studies - Expression

Literature : Gene Expression Microarray Stress

• Exploring the metabolic and genetic control of gene expression on a genomic scale, DeRisi JL, et al. (1997) Science

• Genomic expression programs in the response of yeast cells to environmental changes, Gasch AP, et al. (2000) Mol Biol Cell

• Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source, Kuhn KM, et al. (2001) Mol Cell Biol

• Role of thioredoxin reductase in the Yap1p-dependent response to oxidative stress in Saccharomyces cerevisiae, Carmel-Harel O, et al. (2001) Mol Microbiol

• Transcriptional Remodeling in Response to Iron Deprivation in Saccharomyces cerevisiae, Shakoury-Elizeh M, et al. (2004). Mol Biol Cell

• Transcriptional response of steady-state yeast cultures to transient perturbations in carbon source, Ronen M and Botstein D (2005) Proc Natl Acad Sci

• About 900 publications

Overview

Background


Previous Studies – ChIP-Chip

Which genes are directly regulated by TFs?

Harbison et al. Nature 2004

Overview

Background

(Environment specific use of regulatory code)


Motivation and Goal

Limitation of Microarray data for understanding

regulatory system upon environmental change

Previous qualitative analysis of ChIP-Chip

Integration of heterogeneous data

ChIP-Chip (direct regulation) +

Microarray (direct/indirect regulation)

Quantitative analysis of TF binding

Better understanding of differential

responses of transcriptional regulatory

system via differential location analysis

Overview

Background


Location AnalysisGenome-wide Location Analysis : ChIP-on-Chip Exp.• In vivo assay based on

• ChIP (Chromatin Immuno-Precipitation)• High-throughput array experiment

Where and how strongly TF binds to

Overview

Background

Location Analysis


Differential Location Analysis

• Harbison et al. Nature 2004• Saccharomyces cerevisiae (budding yeast)• 204 TFs in 14 conditions (352 experiments)• Genome-wide location data (11,000 interactions)

Overview

Background

Location Analysis


ChIP-on-Chip 204 Yeast Transcription Factors (TFs)1

1 rich medium condition + 13 stress conditions 6540 genes & corresponding promoter sequences.

Microarray

Stress ConditionConcentratio

nTime point Array Ref.

H2O2 – oxidation 0.3 mM 20 mins cDNA 2

H2O2 – oxidation 0.32 mM 20 mins cDNA 3

H2O2 - oxidation 0.4 mM 20 mins oligo 4

SM (Sulfometuron methyl) – AA starvation

0.2 g/ml 15 mins cDNA 5

SM (Sulfometuron methyl) – AA starvation

0.2 g/ml 240 mins cDNA 5

Data Sets - IOverview

Background

Location Analysis


Data Sets - II

ChIP-on-Chip Data Preprocessing P-value and ratio data from Harbison et al. ‘NaN’ point removal Ratio value below 1 set to 1

Stress conditions 13 stress conditions 147 TF-cond pairs

Overview

Background

Location Analysis


Differential Binding Ratio

Rich medium Stress Condition

Increased binding in stress condition

Decreased binding in stress condition

How different the binding ratio btw different conditions???

Overview

Background

Location Analysis

Differential Binding


Differential Binding Measure

Ai, Bi: binding ratio of TF k to regulated region i

under stress and rich medium culture condition respectively

-1 Pik 1

Differential binding is represented as Pik by

using ChIP-chip binding ratio data between stress condition (A) and rich medium (B).

Overview

Background

Location Analysis


Rich medium Stress Condition

PiK > 0 UP

PiK < 0 DOWN


All ChIP-Chip Binding Data

Normal distribution

Distribution of Pik - I

Ex) FHL1 YPD (Rich Medium) vs SM (Amino acid starvation)

High-confidence Binding Data (p0.001)

Skewed to negative direction

Overview

Background

Location Analysis



HighUp vs HighDown

-10

10

30

50

70

90

110

130

-10 10 30 50 70 90 110 130

Num of Up regions

Num

of D

own r

egio

ns

Up vs Down

-30

0

30

60

90

120

150

180

210

-30 0 30 60 90 120 150 180 210

Num of Up regions

Num

of D

ow

n r

egio

ns

Distribution of Pik - II

• Number of UP/DOWN differentially bound regions (genes) for each TF-cond pair

• High-confidence data point only (p<0.001)

0.5 |Pik| 1

Overview

Background

Location Analysis


-1 Pik 1


Chi-Square Test - I

* Chi-Square tests for Pik of 147 TF-condition pairs

to check their distributions (Normal or skewed)* R-package & Perl scripts* P<0.05 Skewed distribution

Normal Dist. (p=0.97)

All ChIP-Chip Binding Data

Skewed Dist. (p=0.00003)

High-confidence Binding Data (p0.001)

Overview

Background

Location Analysis



Skewed DistributionChi-square test : p < 0.05105 TF-Cond pairs (out of 147) : 70%

Chi-Square Test - IIOverview

Background

Location Analysis



Classification of TFs - I

Depending on patterns of the skewed

pattern , 105 TF-condition pairs have been

grouped into three categories.

UP : More than 2/3 are in ‘+’

direction

DOWN : More than 2/3 are in ‘–’

direction

BOTH : Similar proportions of ‘+’

and ’-’

Overview

Background

Location Analysis


TF response classification


Classification of TFs - II

UP : YAP5_H2O2Hi BOTH : HPO4_Pi DOWN : SFP1_H2O2Lo

Overview

Background

Location Analysis




Differential Location Analysis - Summary

Comparison of TF binding

between rich medium and stress cond.

Represented by PiK

Distribution of PiK

Normal and Skewed

Chi-Square Tests

70% TF-cond pairs differential

distribution

Classification of TF responses

UP, DOWN, and BOTH

Overview

Background

Location Analysis




How differential binding?

1.1. Changes in the TF expressionChanges in the TF expression

2.2. Different TFBS signature for different Different TFBS signature for different

categorycategory

3.3. Interaction with other TFs expressionInteraction with other TFs expression

4.4. Modifications in TFs (protein level)Modifications in TFs (protein level)

5.5. Changes in physical structures Changes in physical structures

(epigenetic features) (epigenetic features)

6.6. Other unknown reasonsOther unknown reasons

Overview

Background

Location Analysis



Comp. with Microarray



Comparison with Microarray - I

ChIP-Chip vs

Microarray data

----------------------expression levels of

transcription factors (TFs).

----------------------H2O2 Low (oxidation)

----------------------TF-Cond pairs : 28Skewed : 23Normal : 5

Overview

Background

Location Analysis





Comparison with Microarray - II

SM (Sulfometuron

methyl)Amino acid starvation

--------------------------ChIP-Chip

0.2 g/ml final2 hours

--------------------------Microarray0.2 g/ml 15

minutes5.0 g/ml 4 hours

--------------------------

TF-Cond pairs : 34Skewed : 21Normal : 13

Overview

Background

Location Analysis





Comparison with Microarray - III

H2O2 Oxidation stress

SM (Sulfometuron methyl) Amino acid starvation

Overview

Background

Location Analysis





Comparison with Microarray - IV

50~60% of differentially binding TFs have NO significant

expression change

Changes of gene regulation (differential binding) cannot be

fully revealed by Microarray

Overview

Background

Location Analysis








categorycategory






Overview

Background

Location Analysis





Conclusion

Categorization of Transcription

Factors (TFs) depending on the

responses to stress

No significant expression changes in

about 50% of the tested TFs in response

to environmental changes.

Importance of integrating differential

binding of TFs with gene expression to

get the bigger picture

Overview

Background

Location Analysis






Future Work

Investigating the biological mechanism of

the differentially binding of TFs

1. Target gene of expression level

2. Post-translational modification

3. Protein-protein interaction

Comprehensive differential analysis

Integration

Diff. Expression

Diff. Location

Diff. Protein-Protein Int. (open)

Diff. Proteomics (open)

Overview

Background

Location Analysis




Diff. Binding Motif Anal.

TF-TF Corr.



References

1. Harbison, C.T., et al., Transcriptional regulatory code of a eukaryotic genome. Nature, 2004. 431(7004): p. 99-104. .

2. Shapira, M., E. Segal, and D. Botstein, Disruption of yeast forkhead-associated cell cycle transcription by oxidative stress. Mol Biol Cell, 2004. 15(12): p. 5659-69.

3. Gasch, A.P., et al., Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell, 2000. 11(12): p. 4241-57.

4. Causton, H.C., et al., Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell, 2001. 12(2): p. 323-37.

5. Jia, M.H., et al., Global expression profiling of yeast treated with an inhibitor of amino acid biosynthesis, sulfometuron methyl. Physiol Genomics, 2000. 3(2): p. 83-92.

Overview

Background

Location Analysis





TF-TF Corr.



Acknowledgements

Dr. Haixu Tang

Dr. Sun Kim

Dr. Mehmet Dalkilic

Dr. Predrag Radivojac

Seung-hee Bae

Sourav Roy

Capstone class 2006

School of informatics

Center for Genomics and Bioinformatics

Dr. Zhixiong Xue at DuPont

My Family





categorycategory







categorycategory

Overview

Background

Location Analysis






Different TFBS signatures?

Category “BOTH” has Category “BOTH” has

similarsimilar numbers of numbers of

Up-differentially Up-differentially

bound and Down-bound and Down-

differentially bound differentially bound

regions.regions.

Is there any difference Is there any difference

between these between these

TFBSs in different TFBSs in different

groups?groups?

Overview

Background

Location Analysis






Different TFBS signatures?

Step1. Collect putative TFBS A. Frankel’s motifs

(102TFs) B. Gary Stormo’s “Patser”

Step2. Create profiles from collected TFBS seqs

Step3. Profile comparison A. “MatCompare”

program by Michale Zhang

BOTH

Up seqs Down seqs

TFBSs TFBSs

Profile Profile

Step3

Step2

Step1

Overview

Background

Location Analysis






Different TFBS signatures?High-Threshold

Overview

Background

Location Analysis






Different TFBS signatures?Low-Threshold

Overview

Background

Location Analysis









categorycategory

3.3. Association with other TFsAssociation with other TFs





3.3. Association with other TFsAssociation with other TFs

Overview

Background

Location Analysis





TF-TF Corr.


TF-TF Correlation

TF

TF

Overview

Background

Location Analysis





TF-TF Corr.

junguk hur school of informatics & center for genomics and bioinformatics

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