S. Cerevisiae - Ocw.mit.edu

Lecture 22Eukaryotic Genes and Genomes IIIIn the last three lectures we have thought a lot about analyzing a regulatorysystem in S. cerevisiae, namely Gal regulation that involved a hand full of genes.These studies monitored the increased transcription of Gal genes in the presenceof galactose (and the absence of glucose); we saw that this regulation is achievedby particular proteins, or multiprotein complexes that bind to specific sequences inthe promoter region upstream from their target genes.What if I told you that it is now possible to do the following in S. cerevisiae: Monitor mRNA expression level for every gene in S. cerevisiae, in one singleexperiment.Monitor all the binding sites in the S. cerevisiae genome for eachtranscription factor in a single experiment.Determine all possible pair-wise interactions for every S. cerevisiae protein.Obviously I wouldn’t mention these possibilities if they weren’t already happening.What I want to do today is to introduce you to the idea of carrying out geneticanalyses on a global, genome-wide scale, and hopefully give you some examplesthat are relevant to what we have already learned along the way. So, this will bea technology oriented lecture, but with someapplication to what we have already learnedabout gene regulation in eukaryotes. It shouldS. cerevisiae5,800also be mentioned that what will be describedDrosophila14,000for S. cerevisiae, is theoretically possible forC. elegans19,000any organism whose genome has beenmouse22,500completely sequenced and the location of allhuman22,500the genes in that genome have beenFigure by MIT OCW.established. What we will learn today isalready being, or will be, applied to highereukaryotes and mammals.Monitor mRNA expression level for every gene in S. cerevisiae, in onesingle experiment: Global transcriptional profiling.Before we consider how it is possible to measure the levels of thousands of mRNAspecies, we will have to step back to consider how the levels of one or two mRNAspecies can be measured by Northern Blot analysis .and I know you must havelearned this in 7.01 if not in high school. Northern blot analysis is based upon thefact that DNA and RNA molecules that possess complementary base sequences willhybridize together to form a double stranded molecule. If the complementarity isperfect the duplex molecule is stable, if it is imperfect (with base pair mismatches)it is relatively less stable. This provides the specificity needed to identify perfectly

matched DNA:RNA duplexes (on Northern Blots) and DNA:DNA duplexes (onSouthern Blots). This specificity is needed to be sure we are measuring the levelof one particular transcript and that this is not contaminated with signal fromclosely related transcripts. RNA is isolated from cells, size fractionated on a gel;the thousands of mRNAs species form a smear on the gel which is punctuated bythe strong ribosomal RNA bands (28S and 18S) that do not interfere with theanalysis.Image removed due to copyright reasons.Please c.htmlFigure by MIT OCW.Northern BlotsImmobilized mRNA population hybridizedwith labeled DNA probe representing oneor two genesDNA MicroarraysImmobilized DNA probes representing allpossible genes hybridized with labeledmRNA populationDNA ClonesPCR amplificationpurificationroboticprintinghybridize targetto microarrayFigure by MIT OCW.The breakthrough in developing microarraysfor analyzing mRNA levels was to reverse thelogic – instead of immobilizing the mRNAs forhybridization with one or two labeledcomplementary DNA (cDNA) probes, allpossible cDNA probes are immobilized on asolid surface (usually glass slides). Thespotting of probes is achieved robotically; theDNA probes are designed to specificallyhybridize to only one nucleic acid sequencethat represents a single mRNA species. Thethousands of DNA probes are dispensed from96-well, or 384-well plates to an addressablesite on the solid surface. The mRNApopulation from each cell type purified andthen copied such that the copy is fluorescentlylabeled. This fluorescent population ishybridized to the immobilized probes, and theintensity of the fluorescence at each probespot is proportional to the number of copies ofthat specific mRNA species in the originalmRNA population.

So let’s look at how this would actually work in a real experiment. mRNA isisolated from yeast cells in state A (e.g., minus galactose) and from yeast cells instate B (e.g., plus galactose), and copies of each population is made such thatone fluoresces red and the other fluoresces green. After mixing, these fluorescentmolecules are hybridized to the slides containing 5,800 DNA probes, each onespecific for detecting hybridization of many copies of an individual mRNA species.Yeast in state AWhat’s happening at each spot?Yeast in state BIsolate mRNA ALabel copies of mRNAspecies with RED or TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTHybridize to themicroarraymRNA present much higher in State A than State BmRNA present much higher in State B than State AmRNA present at equal levels in States A and BHybridizationThe location and identity of each probe on themicroarray slide is known, and each probe isspecific for a single mRNA. The color andintensity of the fluorescence is measured byscanning the slide with lasers, and the relativeabundance of each mRNA in the cells of StateA vs State B can be calculated from theemitted fluorescence. i.e., the relative level of5,800 mRNAs can be compared between twopopulations of yeast cells.Presenting data for thousands of mRNA transcripts is clearly a challenge. Youcould present endless tables of data, but our brains are much more adept atrecognizing shapes, patterns and colors. Colored representations of up and downregulation of transcripts levels is the preferred way to present data.Northern Blot vs. MicroarrayEach colored vertical line in the horizontal lane displaysthe relative expression level of a single mRNAImages removed due to copyright reasons. Please see Lodish, Harvey, et. al. Molecular Cell Biology.5th ed. New York : W.H. Freeman and Company, 2004.

For our purposes here, let’s look at what genes are up-regulated when a glucosegrown culture of S. cerevisiae is shifted into galactose; what genes are upregulated under these conditions? Obviously transcripts for Gal1, Gal7 andGal10 genes will be up-regulated, as we have discussed in the last couple oflectures. In addition Gal2 (galactoseWhat transcripts have increased levelspermease) and Gal80 (the negativewhen shifted from glucose to galactose?regulator of the Gal4 transcriptionalactivator) are also induced; this waspreviously known, although we didn’tImages removed due to copyright reasons.discuss it directly in the previous lectures.Please see Ren, Bing., et.al. "Genome-wide LocationBut upon looking globally, it has becomeand Function of DNA Binding Proteins."clear that some other genes are also upScience 290, no. 5500 (Dec. 22, 2000): 2306-9.regulated. (This figure shows just a smallsnapshot of the response.) These additionalgenes are Fur4, Gcy1, Mth1, and Pcl10,and their co-regulation along with the Galgenes was previously unrealized. We willbe coming back to this later in the lecture.Monitor all the binding sites in the S. cerevisiae genome for eachtranscription factor in a single experiment.In the last lecture we talked about deletion analysis of cis-acting regulatorysequences identifying the location of UAS and URS sequences upstream of theGal1 gene. That the Gal4 transcriptional activator protein binds to the DNAsequence present at the URSGAL1 can be shown to happen in the test tube, butshowing that it is actually bound in a living cell is another matter. A method wasrecently developed for doing just that, and this method has been furtherdeveloped to determine transcription regulator binding across the whole genome.Chromatin Immuno Precipitation (ChIP)Living cellsH2COFormaldehydetreatment crosslinksproteins to DNADNA fragments thatthe transcriptionfactor was bound to inthe living yeast cellIsolate DNA withproteins crosslinked,shear into smallfragmentsReverse theformaldehydecrosslinks and get ridof proteinImmunoprecipitatespecific transcriptionfactor and its boundDNAImages removed due to copyright reasons. Please seeFigure 2 in Weinmann, Amy S. Novel ChIP-based Strategiesto Uncover Transcription Factor Target Genes in the Immune System.Nature Reviews Immunology 4 (2004): 381-386.This method takes advantage of the fact that formaldehyde crosslinks proteins toDNA in a way that can later be reversed.

For galactose grown yeast cells chromatin immunoprecipitation (ChIP) with anantibody that pulls down the Gal4 proteinA more complete view of galactoserevealed some surprises. In addition toinduced gene expression in S. cerevisiaeconfirming that Gal4 binds to the promotersregions upstream of the expected Gal genes,Images removed due to copyright reasons.the Gal4 protein also binds to the promoterPlease see Ren, Bing, et. al. "Genome-wideregions of 4 other genes, namely Fur4, Pcl10,Location and Function of DNA Binding Proteins."Mth1 (shown in the adjacent figure) and Gcy1Science 290, no. 5500 (Dec. 22, 2000): 2306-9.(not shown). Note that these genes wereshown to be induced by galactose in theprevious section. Just how the up-regulationof Fur4, Pcl10 and Mth1 might contribute tooptimizing the metabolism of galactose isshown in this figure, but the role Gcy1 plays isunclear. Clearly, taking a global look at what genes are up-regulated in thepresence of galactose, and taking a global look at what promoters are bound bythe Gal4 regulator, has clearly enriched our view of how S. cerevisiae adapts tothe presence of this sugar.The ChIP approach, followed by hybridization to DNA microarrays, was originallylimited to monitoringbinding of transcriptionalregulators for which thereImages removed due to copyright reasons.Please see Ren, Bing, et. al. "Genome-widewere good precipitatingLocation and Function of DNA Binding Proteins."antibodies. However, thisScience 290, no. 5500 (Dec. 22, 2000): 2306-9.limitation was recentlyeliminated by fusing anArrayed probeepitope TAG to eachsequences representthe upstream cisregulator gene. Thisacting regions of allepitope TAG is recognized5,800 genesby a strong antibody, andso a single antibody can “pull down”Regulatory ProteinRegulatory ProteinGene Promoter(immunoprecipitate) 100 differentbinds Gene Promoterregulatory proteins, each of which isexpressed in its own yeast strain.This has enabled a massive study toidentify all of the target genes foreach of 106 transcriptional regulatorsin S. cerevisiae growing in a definedmedium. A compilation of all the datahas revealed a number offundamentally different regulatorymotifs; these are shown in the