Exploring the Exploring the Metabolic and Genetic Metabolic and Genetic

Control of Gene Control of Gene Expression on a Expression on a Genomic ScaleGenomic Scale

Exploring the Exploring the Metabolic and Genetic Metabolic and Genetic

Control of Gene Control of Gene Expression on a Expression on a Genomic ScaleGenomic Scale

Joseph L. DeRisi, Vishwanath R. Iyer, Patrick O. BrownJoseph L. DeRisi, Vishwanath R. Iyer, Patrick O. Brown

Science Vol. 278 October 24, 1997 p.680-6Science Vol. 278 October 24, 1997 p.680-6

What they asked…What they asked…What they asked…What they asked…• Question - Question - What changes occur at the What changes occur at the genetic level when yeast shifts from genetic level when yeast shifts from anaerobic to aerobic metabolism?anaerobic to aerobic metabolism?

Glucose

Ethanol + CO2

Ethanol + O2

““Diauxic Shift”Diauxic Shift”

CO2 and ATP

How can you approach How can you approach this question?this question?

How can you approach How can you approach this question?this question?

• Changes to proteinsChanges to proteins• Expression and post-translational Expression and post-translational modificationmodification

• Changes to cell structure / Changes to cell structure / morphologymorphology

• Changes in gene expression:Changes in gene expression:• Occurs relatively quicklyOccurs relatively quickly• Gives you a good idea which processes are Gives you a good idea which processes are being affectedbeing affected

• MICROARRAY technology now availableMICROARRAY technology now available

Measuring Gene Measuring Gene ExpressionExpression

Measuring Gene Measuring Gene ExpressionExpression

• How to measure changes in RNAHow to measure changes in RNA

SignalRegulation ofRegulation of

RNA transcriptionRNA transcription(( or or ))

Use a viral enzyme (Reverse Transcriptase) to make single-stranded DNA

from mRNA (cDNA).

Amplify the cDNA withPCR for detection on gel

Northern BlotNorthern Blot

Microarray TechnologyMicroarray TechnologyMicroarray TechnologyMicroarray Technology

YeastYeastcDNAcDNA

LibraryLibrary

Anaerobic Aerobic

Interpreting Interpreting MicroarraysMicroarraysInterpreting Interpreting MicroarraysMicroarrays

Increase

Decrease

NoChange

Control (Cy3) Treated (Cy5)

Why use a Microarray?Why use a Microarray?Why use a Microarray?Why use a Microarray?

• Quantitative Quantitative comparison of comparison of transcript levelstranscript levels

• Look at thousands Look at thousands of genes in one of genes in one experimentexperiment

• Can theoretically Can theoretically assess changes in assess changes in all genes of an all genes of an organismorganism

Experimental DesignExperimental DesignExperimental DesignExperimental Design• Took samples from a Took samples from a fermenting yeast culture fermenting yeast culture seven times over 2 hours.seven times over 2 hours.

• Isolated RNA from cells Isolated RNA from cells and prepared cDNA labelled and prepared cDNA labelled with Cy3 (with Cy3 (greengreen) for ) for initial measurement (time initial measurement (time zero) and Cy5 (zero) and Cy5 (redred) for ) for successive samples.successive samples.

• Equal amounts of cDNA were Equal amounts of cDNA were mixed from time zero and mixed from time zero and each time point.each time point.

What they found…What they found…What they found…What they found…

Organization Organization and and

AnalysisAnalysis

Organization Organization and and

AnalysisAnalysis• They set a They set a threshold of 2-threshold of 2-fold changefold change

• Not many genes Not many genes changes in first changes in first few hoursfew hours

• As yeast switched As yeast switched from fermentation from fermentation to respiration, to respiration, started to see started to see large changes in large changes in some genes.some genes.

Changes in gene Changes in gene expression during expression during diauxic shift:diauxic shift:

• ALD2 and ACS1 (fuel the ALD2 and ACS1 (fuel the TCA cycle)TCA cycle)

• Pyruvate carboxylase and Pyruvate carboxylase and Pyruvate decarboxylase Pyruvate decarboxylase (fuel TCA cycles and (fuel TCA cycles and gluconeogenesis)gluconeogenesis)

• PCK1 and PCK1 and FBP1 (pushing FBP1 (pushing reversal of glycolysis reversal of glycolysis pathway toward production of pathway toward production of glucose-6-phosphataseglucose-6-phosphatase

• Trehalose synthase and Trehalose synthase and glycogen synthase (promotes glycogen synthase (promotes storage of glucose)storage of glucose)

• genes associated protein genes associated protein synthesissynthesis

• On the contrary, On the contrary, in in mitochondrial ribosomal mitochondrial ribosomal proteins observed, proteins observed, emphasizing need for emphasizing need for mitochondrial biogenesis mitochondrial biogenesis (requirement for more (requirement for more energy?)energy?)

Gene families and temporal relationships

Gene families and temporal relationships

Other observations…Other observations…Other observations…Other observations…• Genes containing Genes containing a carbon source a carbon source response element response element (CSRE) in the (CSRE) in the promoter.promoter.

• As concentration As concentration of glucose went of glucose went down, repression down, repression was relieved on was relieved on gene gene transcription.transcription.

• Promoter analysis of effected genes revealed:Promoter analysis of effected genes revealed:• Stress response elements (STRE)Stress response elements (STRE)• HAP responsive genes (respiration)HAP responsive genes (respiration)• Rap1-regulated genes (ribosomal protein translation)Rap1-regulated genes (ribosomal protein translation)

• Also, increased expression of HAP4 and SIP4, transcription factors which control Also, increased expression of HAP4 and SIP4, transcription factors which control respirationrespiration

Identification of Gene Identification of Gene Families and Families and

PathwaysPathways

Limitations of Experimental Limitations of Experimental DesignDesign

Limitations of Experimental Limitations of Experimental DesignDesign

• Used primers for KNOWN / PREDICTED open Used primers for KNOWN / PREDICTED open reading frames in yeastreading frames in yeast

• Not useful if you want to identify new genesNot useful if you want to identify new genes• Can use primers which non-specifically amplify all Can use primers which non-specifically amplify all mRNAs.mRNAs.

• Threshold of 2-fold - may miss important Threshold of 2-fold - may miss important genes that are changing slightly, but have genes that are changing slightly, but have large effectslarge effects

• Multiple time points good (allows plotting Multiple time points good (allows plotting of changes over time and acts as an of changes over time and acts as an internal control) - but always hard to know internal control) - but always hard to know when the optimal times to choose are.when the optimal times to choose are.

• Adaptive changes in mutant strains.Adaptive changes in mutant strains.

Other applications of Other applications of microarrays: Effects of microarrays: Effects of individual mutations individual mutations

Other applications of Other applications of microarrays: Effects of microarrays: Effects of individual mutations individual mutations

• Create a strain of Create a strain of yeast with a mutation yeast with a mutation in one protein and in one protein and compare to wild-type compare to wild-type strain. (Gain or loss strain. (Gain or loss of function)of function)

• Gives information about Gives information about the genes regulated by the genes regulated by that protein.that protein.

• Mutated Tup1 (tup1Mutated Tup1 (tup1) ) and compared to WT.and compared to WT.

• Tup1 known to repress Tup1 known to repress gene transcription in gene transcription in response to glucose.response to glucose.

Identification of Identification of possible overlapping possible overlapping

functionsfunctions

Identification of Identification of possible overlapping possible overlapping

functionsfunctions

DiauxicDiauxicShiftShift tup1tup1

10%Shared

Therefore, tup1 might be integral in the repressing the Therefore, tup1 might be integral in the repressing the diauxic shift in the presence of glucose.diauxic shift in the presence of glucose.

Gain of Function: Gain of Function: Over-expression of Over-expression of

YAP1YAP1

Gain of Function: Gain of Function: Over-expression of Over-expression of

YAP1YAP1• Yap1 - transcription Yap1 - transcription factorfactor

• Identified genes Identified genes belonging to a class of belonging to a class of dehydrogenases/ dehydrogenases/ oxidoreductasesoxidoreductases

• Might play a protective Might play a protective role during oxidative role during oxidative stress.stress.

• 2/3 of genes induced 2/3 of genes induced had Yap1 binding sites had Yap1 binding sites in the promoter.in the promoter.

Conclusions and Conclusions and Directions:Directions:

Conclusions and Conclusions and Directions:Directions:

• Data agrees with what is already known Data agrees with what is already known about the pathways, therefore about the pathways, therefore demonstrates the robustness of this demonstrates the robustness of this technique.technique.

• Effected genes often grouped into Effected genes often grouped into distinct functional classes - distinct functional classes - specificity and accuracy.specificity and accuracy.

• Target genes often had predicted binding Target genes often had predicted binding sites for transcription factors.sites for transcription factors.

• Reproducible (0.87 correlation between Reproducible (0.87 correlation between duplicate experiments, 95% differed by a duplicate experiments, 95% differed by a factor of less than 2-fold)factor of less than 2-fold)

Microarray TechnologyMicroarray TechnologyMicroarray TechnologyMicroarray Technology• Strengths of this approachStrengths of this approach

• Lots of data!Lots of data!• Great starting point when screening for new targets or Great starting point when screening for new targets or pathwayspathways

• Screen many genes at one time with small amounts of RNAScreen many genes at one time with small amounts of RNA• QuantitativeQuantitative

• Weaknesses of the approachWeaknesses of the approach• Only investigate changes in RNA levels (transcription)Only investigate changes in RNA levels (transcription)• Poorly expressed genes (sensitivity)Poorly expressed genes (sensitivity)• Small changes can translate to large biological effects Small changes can translate to large biological effects (statistical error and elimination)(statistical error and elimination)

• Limited by the genes spotted on the chipLimited by the genes spotted on the chip• Cross-hybridization of cDNA with highly homologous Cross-hybridization of cDNA with highly homologous sequences (isoforms, families)sequences (isoforms, families)

• Statisitical analysis - false positives/negatives, Statisitical analysis - false positives/negatives, validation by other methodsvalidation by other methods

Applications for a Applications for a MicroarrayMicroarray

Applications for a Applications for a MicroarrayMicroarray

• Drug screens:Drug screens:• Search for desired patterns Search for desired patterns

of gene activationof gene activation• Identify potential toxic or Identify potential toxic or

beneficial effects of chemicalsbeneficial effects of chemicals• Tumor classification / prognosisTumor classification / prognosis• Assess effects of mutationsAssess effects of mutations• Identify the functions of Identify the functions of unclassified genesunclassified genes