[PDF] What is a gene post-ENCODE? History and updated definition

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What isa gene,post-ENCODE ?

History andupdated definition

Mark B.Gerstein,

1,2,3,9

Can Bruce,

2,4

Joel S.Rozowsky,

2

Deyou Zheng,

2

Jiang Du,

3

Jan O.Korbel,

2,5

Olof Emanuelsson,

6

Zhengdong D.Zhang,

2

Sherman Weissman,

7 and MichaelSnyder 2,8 1 Program inComputational Biology& Bioinformatics,Yale University,New Haven,Connecticut 06511,USA; 2

Molecular

Biophysics &Biochemistry Department,Yale University,New Haven,Conne cticut06511, USA; 3

Computer ScienceDepartment,

Yale University,New Haven,Connecticut 06511,USA;

4 Center forMedical Informatics,Yale University,New Haven,Connecticut

06511, USA;

5 European MolecularBiology Laboratory,69117 Heidelberg,Germany; 6

Stockholm BioinformaticsCenter, Albanova

University Center,Stockholm University,SE-10691 Stockholm,Sweden; 7

Genetics Department,Yale University,New Haven,

Connecticut 06511,USA;

8 Molecular, Cellular,& DevelopmentalBiology Department,Yale University, NewHaven, Connecticut

06511, USA

While sequencingof thehuman genomesurprised uswith howmany protein-coding genesthere are,it didnot

fundamentally changeour perspectiveon whata geneis. Incontrast, theco mplexpatterns ofdispersed regulation

and pervasivetranscription uncoveredby theENCODE project,together with non-genicconservation andthe abundance ofnoncoding RNAgenes, havechallenged thenotion ofthe gene.T oillustrate this,we reviewthe

evolution ofoperational definitionsof agene overthe pastcentury - from theabstract elementsof heredityof

Mendel andMorgan tothe present-dayORFs enumeratedin thesequence databanks. Wethen summarizethe

current ENCODEfindings andprovide acomputational metaphorfor thecomp lexity.Finally, wepropose atentative

update tothe definitionof agene: Agene isa unionof genomicsequences encoding acoherent setof potentially

overlapping functionalproducts. Ourdefinition sidestepsthe complexities ofregulation andtranscription by

removing theformer altogetherfrom thedefinition andarguing thatfinal ,functional geneproducts (ratherthan

intermediate transcripts)should beused togroup togetherentities associated witha singlegene. Italso manifests

how integralthe conceptof biologicalfunction isin defininggenes.

Introduction

The classicalview ofa geneas adiscrete elementin the genome hasbeen shakenby ENCODE The ENCODEconsortium recentlycompleted itscharacterization of 1%of thehuman genomeby varioushigh-throughput experi- mental andcomputational techniquesdesigned tocharacterize functional elements(The ENCODEProject Consortium2007). This projectrepresents amajor milestonein thecharacterization of thehuman genome,and thecurrent findingsshow astriking picture ofcomplex molecularactivity. Whilethe landmarkhu- man genomesequencing surprisedmany withthe smallnumber (relative tosimpler organisms)of protein-codinggenes thatse- quence annotatorscould identify(

21,000, accordingto thelat-

est estimate[see www.ensembl.org]),ENCODE highlightedthe number andcomplexity ofthe RNAtranscripts thatthe genome produces. Inthis regard,ENCODE haschanged ourview of"what is agene" considerablymore thanthe sequencingof theHae- mophilus influenzaand humangenomes did(Fleisch- mann etal. 1995;Lander etal. 2001;Venter etal. 2001).The discrepancy betweenour previousprotein-centric viewof the gene andone thatis revealedby theextensive transcriptional activity ofthe genomeprompts usto reconsidernow whata gene

is. Here,we reviewhow theconcept ofthe genehas changedover the pastcentury, summarizethe currentthinking basedon thelatest ENCODEfindings, andpropose anew updatedgene defi-nition thattakes thesefindings intoaccount.

History ofthe gene,1860 tojust beforeENCODE

Definition 1860s-1900s:Gene asa discreteunit ofheredity The conceptof the"gene" hasevolved andbecome morecom- plex sinceit wasfirst proposed(see timelinein Fig.1, accom- panying poster).There arevarious definitionsof theterm, al- though commoninitial descriptionsinclude theability todeter- mine aparticular characteristicof anorganism andthe heritabil- ity ofthis characteristic.In particular,the wordgenewas firstused by WilhelmJohannsen in1909, basedon theconcept developed by GregorMendel in1866 (Mendel1866). Theword wasa de- rivative ofpangene,whichwas usedby HugoDe Vriesfor entities involved inpangenesis, Darwin'shypothetical mechanismof he- redity (Heimans1962). Johnannsencalled agene the"special conditions, foundationsand determinerswhich arepres- ent [inthe gametes]in unique,separate andthereby indepen- dent ways[by which]many characteristicsof theorganism are specified" (Johannsen1909, p.124). Theetymology ofthe term derives fromthe Greekgenesis("birth") orgenos("origin"). The related wordgeneticswas usedby thegeneticist WilliamBateson in 1905(http://www.jic.ac.uk/corporate/about/bateson.htm). Mendel showedthat whenbreeding plants,some traitssuch as heightor flowercolor donot appearblended intheir off- 9

Corresponding author.

E-mail Mark.Gerstein@yale.edu;fax (360)838-7861.

Article isonline athttp://www.genome.org/cgi/doi/10.1101/gr.63396 07. Freely availableonline throughthe Genome ResearchOpen Accessoption.

Perspective

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spring - that is,these traitsare passedon asdistinct, discrete entities (Mendel1866). Hiswork alsodemonstrated thatvaria- tions intraits werecaused byvariations ininheritable factors(or, in today s terminology,phenotype iscaused bygenotype). Itwas only afterMendel s workwas repeatedand rediscoveredby Carl Correns, Erichvon Tschermak-Seysenegg,and HugoDe Vriesin

1900 thatfurther workon thenature ofthe unitof inheritance

truly began(Tschermak 1900;Vries 1900;Rheinberger 1995).

Definition 1910s:Gene asa distinctlocus

In thenext majordevelopment, theAmerican geneticistThomas Hunt Morganand hisstudents werestudying thesegregation of mutations inDrosophila melanogaster.Theywere ableto explain their datawith amodel thatgenes arearranged linearly,and their ability tocross-over isproportional tothe distancethat separated them. Thefirst geneticmap wascreated in1913 (Sturtevant

1913), andMorgan andhis studentspublished The Mechanismof

Mendelian Inheritancein 1915(Morgan etal. 1915).To theearly geneticists, agene wasan abstractentity whoseexistence was reflected inthe wayphenotypes weretransmitted betweengen- erations. Themethodology usedby earlygeneticists involved mutations andrecombination, sothe genewas essentiallya locus whose sizewas determinedby mutationsthat inactivated(or activated) atrait ofinterest andby thesize ofthe recombining regions. Thefact thatgenetic linkagecorresponded tophysical locations onchromosomes wasshown later,in 1929,by Barbara McClintock, inher cytogeneticstudies onmaize (McClintock

1929).

Definition 1940s:Gene asa blueprintfor aprotein

Beadle andTatum (1941),who studiedNeurosporametabolism, discovered thatmutations ingenes couldcause defectsin stepsin metabolic pathways.This wasstated asthe "one gene,one en- zyme "view, whichlater became"one gene,one polypeptide."In this viewpoint,the geneis beingimplicitly consideredas the information behindthe individualmolecules ina biochemical pathway. Thisview becameprogressively moreexplicit and mechanistic inlater decades.

Definition 1950s:Gene asa physicalmolecule

The factthat heredityhas aphysical, molecularbasis wasdem- onstrated bythe observationthat Xrays couldcause mutations (Muller 1927).Griffith s (1928)demonstration thatsomething in virulent butdead Pneumococcusstrains couldbe takenup bylive nonvirulentPneumococcusand transformthem intovirulent bac- teria wasfurther evidencein thisdirection. Itwas latershown that thissubstance couldbe destroyedby theenzyme DNase (Avery etal. 1944).In 1955,Hershey andChase establishedthat the substanceactually transmittedby bacteriophageto their progeny isDNA andnot protein(Hershey andChase 1955).

Moreover, theidea thata gene

s productis adiffusible substance

underlies thecomplementation testthat wasused todefine genesin theearly yearsof bacteriology.A practicalview ofthe genewas that ofthe cistron,a regionof DNAdefined bymutations thatin transcould notgenetically complementeach other(Benzer

1955).

Definition 1960s:Gene astranscribed code

It wasthe solutionof thethree-dimensional structureof DNAby Watson andCrick in1953 (Watsonand Crick1953) thatex- plained howDNA couldfunction asthe moleculeof heredity. Base pairingexplained howgenetic informationcould becopied, and theexistence oftwo strandsexplained howoccasional errors in replicationcould leadto amutation inone ofthe daughter copies ofthe DNAmolecule. From the1960s on,molecular biologydeveloped ata rapid pace. TheRNA transcriptof theprotein-coding sequenceswas translated usingthe geneticcode (solvedin 1965by Nirenberget al. [1965]and S ll etal. [1965])into anamino acidsequence. Francis Crick(1958) summarizedthe flowof informationin gene expression asfrom nucleicacid toprotein (thebeginnings ofthe

Central Dogma

). However,there weresome immediateexcep- tions tothis: Itwas knownthat somegenes codenot forprotein but forfunctional RNAmolecules suchas rRNAand tRNA.In addition, inRNA virusesthe geneis madeof RNA.The molecular view ofthe genethat developedthrough the1960s canbe sum- marized ingeneral termsto bea coderesiding onnucleic acid that givesrise toa functionalproduct.

Definition 1970s

1980s: Geneas openreading frame(ORF)

sequence pattern The developmentof cloningand sequencingtechniques inthe

1970s, combinedwith knowledgeof thegenetic code,revolu-

tionized thefield ofmolecular biologyby providinga wealthof information onhow genesare organizedand expressed.The first gene tobe sequencedwas fromthe bacteriophageMS2, which was alsothe firstorganism tobe fullysequenced (Fierset al.1971,

1976). Theparallel developmentof computationaltools ledto

algorithms forthe identificationof genesbased ontheir se- quence characteristics(e.g., forreview, seeRogic etal. 2001).In many cases,a DNAsequence couldbe usedto inferstructure and function forthe geneand itsproducts. Thissituation createda new conceptof the"nominal gene,"which isdefined byits pre- dicted sequencerather thanas agenetic locusresponsible fora phenotype (Griffithsand Stotz2006). Theidentification ofmost genes insequenced genomesis basedeither ontheir similarityto other knowngenes, orthe statisticallysignificant signatureof a protein-coding sequence.In manycases, thegene effectivelybe- came identifiedas anannotated ORFin thegenome (Doolittle

1986).

Definition 1990s

2000s: Annotatedgenomic entity,

enumerated inthe databanks(current view,pre-ENCODE) The currentdefinition ofa geneused byscientific organizations that annotategenomes stillrelies onthe sequenceview. Thus,a gene wasdefined bythe HumanGenome NomenclatureOrgani- zation as"a DNAsegment thatcontributes tophenotype/ function. Inthe absenceof demonstratedfunction agene maybe characterized bysequence, transcriptionor homology "(Wain et al. 2002).Recently, theSequence OntologyConsortium report- edly calledthe genea "locatable regionof genomicsequence, corresponding toa unitof inheritance,which isassociated with Figure 1.(Enclosed poster)Timeline ofthe historyof theterm "gene." A terminvented almosta centuryago, "gene,"with itsbeguilingly simple orthography, hasbecome acentral conceptin biology.Given aspecific meaning atits coinage,this wordhas evolvedinto somethingcomplex and elusiveover theyears, reflectingour ever-expandingknowledge in genetics andin lifesciences atlarge. Thestunning discoveriesmade in the ENCODEProject - like manybefore thatsignificantly enrichedthe meaning ofthis term - areharbingers ofanother tideof changein our understanding ofwhat agene is.

Gerstein etal.

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regulatory regions,transcribed regionsand/or otherfunctional sequence regions "(Pearson 2006). The sequencingof firstthe Haemophilus influenzagenome and thenthe humangenome (Fleischmannet al.1995; Landeret al. 2001;Venter etal. 2001)led toan explosionin theamount of sequence thatdefinitions suchas theabove couldbe appliedto. In fact,there wasa hugepopular interestin countingthe number of genesin variousorganisms. Thisinterest wascrystallized origi- nally byGene Sweepstake s wageron thenumber ofgenes inthe human genome,which receivedextensive mediacoverage (Wade

2003).

It hasbeen pointedout thatthese enumerationsoverem- phasize traditional,protein-coding genes.In particular,when the number ofgenes presentin thehuman genomewas reportedin

2003, itwas acknowledgedthat toolittle wasknown aboutRNA-

coding genes,such thatthe givennumber wasthat ofprotein- coding genes.The Ensemblview ofthe genewas specificallysum- marized inthe rulesof theGene Sweepstakeas follows:"alterna- tively splicedtranscripts allbelong tothe samegene, evenif the proteins thatare producedare different. "(http://web.archive. A currentcomputational metaphor:Genes as"subroutines" in thegenomic operatingsystem Given thatcounting genesin thegenome issuch alarge-scale computational endeavorand thatgenes fundamentallydeal with information processing,the lexiconof computerscience natu- rally hasbeen increasinglyapplied todescribing them.In par- ticular, peoplein thecomputational biologycommunity have used thedescription ofa formallanguage todescribe thestruc- ture ofgenes invery muchthe sameway thatgrammars areused to describecomputer programs - withaprecise syntaxof up- stream regulation,exons, andintrons (Searls1997, 2001,2002). Moreover, onemetaphor thatis increasinglypopular fordescrib- ing genesis tothink ofthem interms ofsubroutines ina huge operating system(OS). Thatis, insofaras thenucleotides ofthe genome areput togetherinto acode thatis executedthrough the process oftranscription andtranslation, thegenome canbe thought ofas anoperating systemfor aliving being.Genes are then individualsubroutines inthis overallsystem thatare repeti- tively calledin theprocess oftranscription.

Problematic issueswith thecurrent definition

of agene There area numberof problematicaspects ofthe currentdefini- tion ofa gene,as appliedto thehuman genome,which wedis- cuss below.Several additionalcomplications aresummarized in

Table 1.

1. Generegulation

Jacob andMonod (1961),in theirstudy ofthe lacoperon of

Escherichia coli

,provideda paradigmfor themechanism ofregu- lation ofthe gene:It consistedof aregion ofDNA consistingof sequences codingfor oneor moreproteins, a"promoter"se- quence forthe bindingof RNApolymerase, andan "operator" sequence towhich regulatorygenes bind.Later, othersequences were foundto existthat couldaffect practicallyevery aspectof

gene regulationfrom transcriptionto mRNAdegradation andpost-translational modification.Such sequencescould residewithin thecoding sequenceas wellas inthe flankingregions, and inthe caseof enhancersand relatedelements, veryfar awayfrom thecoding sequence.Although functionallyrequired forthe expressionof thegene product,regulatory elements,espe- cially thedistant ones,made theconcept ofthe geneas acom- pact geneticlocus problematic.

Regulation isintegral tomany currentdefinitions ofthe gene. Inparticular, onecurrent textbookdefinition ofa genein molecular termsis theentire nucleicacid sequencethat isnec- essary forthe synthesisof afunctional polypeptide(or RNA) (Lodish etal. 2000).If thatimplies appropriatelyregulated syn- thesis, theDNA sequencesin agene wouldinclude notonly those codingfor thepre-mRNA andits flankingcontrol regions, but alsoenhancers. Moreover,many enhancersare distantalong the DNAsequence, althoughthey areactually quiteclose dueto three-dimensional chromatinstructure.

2. Overlappingand splicedgenes

Overlapping

As genes,mRNAs, andeventually completegenomes werese- quenced, thesimple operonmodel turnedout tobe applicable only togenes ofprokaryotes andtheir phages.Eukaryotes were different inmany respects,including geneticorganization and information flow.The modelof genesas hereditaryunits thatare nonoverlapping andcontinuous wasshown tobe incorrectby the precisemapping ofthe codingsequences ofgenes. Infact, some geneshave beenfound tooverlap oneanother, sharingthe same DNAsequence ina differentreading frameor onthe op- posite strand.The discontinuousstructure ofgenes potentially allows onegene tobe completelycontained insideanother one s intron, orone geneto overlapwith anotheron thesame strand without sharingany exonsor regulatoryelements.

Splicing

Splicing wasdiscovered in1977 (Bergetet al.1977; Chowet al.

1977; Gelinasand Roberts1977). Itsoon becameclear thatthe

gene wasnot asimple unitof heredityor function,but rathera series ofexons, codingfor, insome cases,discrete proteindo- mains, andseparated bylong noncodingstretches calledintrons. With alternativesplicing, onegenetic locuscould codefor mul- tiple differentmRNA transcripts.This discoverycomplicated the concept ofthe generadically. Forinstance, inthe sequencingof the genome,Celera defineda geneas "a locusof co-transcribed exons "(Venter etal. 2001),and Ensembl's GeneSweepstake Web page originallydefined agene as"a setof connectedtranscripts, " where"connected"meant sharingone exon(http://web.archive. org/web/20050428090317/www.ensembl.org/Genesweep). The latter definitionimplies thata groupof transcriptsmay sharea set ofexons, butno oneexon iscommon toall ofthem. Trans -splicing The phenomenonof trans-splicing(ligationof twoseparate mRNA molecules)further complicatedour understanding(Blu- menthal 2005).There areexamples oftranscripts fromthe same gene, orthe oppositeDNA strand,or evenanother chromosome, being joinedbefore beingspliced. Clearly,the classicalconcept of thegene as"a locus"no longerapplies forthese geneproducts whose DNAsequences arewidely separatedacross thegenome.

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Finally, anumber ofrecent studieshave highlighteda phenom- enon dubbedtandem chimerism,where twoconsecutive genes aretranscribed intoa singleRNA (Akivaet al.2006; Parra et al.2006). Thetranslation (aftersplicing) ofsuch RNAscan lead toa new,fused protein,having partsfrom bothoriginal proteins.3. Parasiticand mobilegenes A challengeto ourconcept ofthe genehas beenthat ofthe selfish or parasiticgene. Theidea firstproposed byRichard Dawkinsis that theunit ofevolution isnot theorganism butthe gene (Dawkins 1976).Organisms arejust toolsthat genesuse torep- Table 1.Phenomena complicatingthe conceptof thegene

PhenomenonDescriptionIssue

Gene locationand structure

Intronic genesA geneexists withinan intronof

another (Henikoffet al.1986) Two genesin thesame locus Genes withoverlapping readingframes ADNA regionmay codefor twodiffere nt protein productsin differentreading frames (Contreraset al.1977) No one-to-onecorrespondence betweenDNA and proteinsequence Enhancers, silencersDistant regulatoryelements (Spilianakiset al.

2005)DNA sequencesdetermining expressioncan be

widely separatedfrom oneanother in genome. Many-to-manyrelationship between genes andtheir enhancers.

Structural variation

Mobile elementsGenetic elementappears innew locationsover generations (McClintock1948) A geneticelement maybe notconstant inits location Gene rearrangements/structuralvariants DNArearrangement orsplicing insomatic cells results inmany alternativegene products (Earlyet al.1980) Gene structureis nothereditary, orstructure may differacross individualsor cells/tissues Copy-number variantsCopy numberof genes/regulatoryelements may differ betweenindividuals (Iafrateet al.2004; Sebat etal. 2004;Tuzun etal. 2005)Genetic elementsmay differin theirnumber

Epigenetics andchromosome structure

Epigenetic modifications,imprinting Inheritedinformation maynot beD NA-sequence based (e.g.,Dobrovic etal. 1988);a gene s expression dependson whetherit isof paternal ormaternal origin(Sager andKitchin

1975)Phenotype isnot determinedstrictly by

genotype Effect ofchromatin structureChromatin structure,which doesinfluence gene expression, onlyloosely associatedwith particular DNAsequences (Paul1972) Gene expressiondepends onpacking ofDNA.

DNA sequenceis notenough topredict gene

product.

Post-transcriptional events

Alternative splicingof RNAOne transcriptcan generatemultiple mRNAs, resulting indifferent proteinproducts (Berget et al.1977; Gelinasand Roberts1977) Multiple productsfrom onegenetic locus; information inDNA notlinearly relatedto that on proteinquotesdbs_dbs29.pdfusesText_35

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