Role of RCO 1 in the control of circadian gene expression and metabolic compensation of the Neurospora crassa circadian clock

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(1)!. ! ! PONTIFICA!UNIVERSIDAD!CATÓLICA!DE!CHILE! Facultad(de(Ciencias(Biológicas( Programa(de(Doctorado(en(Ciencias(Biológicas( Mención(Genética(Molecular(y(Microbiología( ! ! ! TESIS(DOCTORAL!. !. ! ! ! ! !. ROLE!OF!RCO31!IN!THE!CONTROL!OF!CIRCADIAN!GENE!EXPRESSION!AND!METABOLIC! COMPENSATION!OF!THE!NEUROSPORA)CRASSA)CIRCADIAN!CLOCK! ( ( ( ( Consuelo(Olivares(Yáñez( ( Agosto(2015(. !.

(2) !. 1!. ACKNOWLEDGEMENTS.. .. " " " I" want" to" thank" to" all" current" and" past" members" of" the" Larrondo" and" Vicuña" Laboratories"for"their"continuous"help"and"support:"it"was"a"pleasure"working"with"people" like" you." Particularly" Dr." Paulo" Canessa," I" will" always" be" grateful" for" all" the" support" and" guidance"that"you"gave"me"during"these"years."Also"I"would"like"to"mention"Dr."Alejandro" Montenegro,"Dr."Montserrat"Hevia,"Alejandra"Goity"and"Felipe"Muñoz"who"took"part,"not" only"intellectually,"but"in"many"other"ways"in"the"development"of"this"thesis."" " " I" would" like" to" deeply" thank" my" advisor," Dr." Luis" Larrondo," for" his" continuous" support"at"different"levels."It"was"a"pleasure"having"a"boss"as"intellectually"brilliant,"and,"at" the" same" time" able" to" understand" that" life" is" more" that" just" work." Thanks" again" for" the" support"given"both"professionally"and"personally."" " I"would"like"to"thank"the"DunlapKLoros"Labs."Dr."Jay"Dunlap"and"Dr."Jennifer"Loros:" I'm" really" grateful" for" how" you" welcome" me" into" your" lab," it" was" a" great" opportunity" working" with" you." I" would" also" like" to" thank" Dr." Jennifer" Hurley" and" Jillian" Emerson" for" important" technical" support," and" Dr." Arminja" Kettenbach," collaborator" of" some" of" the" experiments"performed"in"this"thesis." " Also"I"would"like"to"acknowledge"the"Comisión"Nacional"de"Investigación"Científica" y" Tecnológica" (CONICYT)" for" their" financial" support" in" the" form" of" a" doctoral" fellowship" (21100309),"internship"and"travels"grants." " And" especially" I" want" to" thank" my" family," my" parents," since" without" their" unconditional" support" I" would" not" have" arrived" to" this" moment:" I" love" you" with" all" my" heart."My"brothers"and"friends"for"staying"by"my"side"over"these"years,"for"understanding" the" long" hours" at" work," where" weekends" and" holidays" often" did" not" exist," thanks" for" listening"the"thousand"times"that"I"tried"to"explain"my"work,"I"think"that"by"now"almost"all" of"them"know"that"I"work"with"a"fungus!"Finally"in"would"like"to"that"my"thesis"committee" for"their"invaluable"input."" " The" work" developed" during" this" thesis" was" funded" by" the" Fondo" Nacional" de" Desarrollo"Científico"y"Tecnológico"(FONDECYT)"1090513"and"1131030"and"the"Millennium" Nucleus"for"Fungal"Integrative"and"Synthetic"Biology"(NC120043).".

(3) !. 2!. TABLE.OF.CONTENTS. " " " ACKNOWLEDGEMENTS......................................................................................................""1" " TABLE"OF"CONTENTS.........................................................................................................."2" " LIST"OF"FIGURES................................................................................................................."5" " LIST"OF"TABLES..................................................................................................................."7" " ABBREVIATIONS................................................................................................................."8" " ABSTRACT...........................................................................................................................""9" " INTRODUCTION" " " " " " " " " " 10" " 1.1"Circadian"Clocks"Mechanisms".....................................................................................""11" 1.2"The"Neurospora"Circadian"Oscillator"..........................................................................."14" " 1.3"Light"responses"and"photoadaptation"in"Neurospora"................................................."19" 1.4"Transcriptional"regulation"and"output"pathways........................................................."21" " 1.5"Metabolic"compensation"of"the"circadian"clock"""""......................................................"24" " MATERIALS"AND"METHODS" " " " " " " " " 27" " 2.1"Strains"and"growth"conditions"....................................................................................""27" 2.2"Yeast"recombinational"cloning....................................................................................""28" 2.3" Cloning" of" promoter" regions" for" luciferase" assays" at" the" his$3" locus" and" Neurospora" transformation..................................................................................................................""28" 2.4" Constructs" designed" for" integrative" transformation" at" loci" other" than" his$3" and" Neurospora"transformation..............................................................................................""31" ' 2.5"Search"of"regulatory"proteins"involved"in"the"control"of"circadian"expression…………""36" ".

(4) !. 3!. 2.6"Time"course"experiments"for"gene"expression"analysis……………………………………………""37" ' 2.7"Analysis"of"circadian"rhythmicity"through"the"race"tube"assay"……………………………….."40" " 2.8"In'vivo"bioluminescence"measurements......................................................................"42" " 2.9"Analysis"of"bioluminescence"data.……………………………………………………………………........"42" " 2.10"Protein"extraction,"CoKimmunoprecipitation"and"Western"blot.……………………………."43" " 2.11"Determination"of"FRQ"levels"through"the"Luciferase"assay"system..........................."43" " 2.12"MassKspectrometry"analysis.…………………………………………………………………………………."44" " 2.13"Protein"degradation"assay…………………………………………………………………………………….."45" " 2.14"Yeast"transformation"and"plasmid"retrieval……………………………………………………………"46"" " 2.15"Genomic"DNA"extraction………………………………………………………………………………………."46" " 2.16"RNA"extraction"and"reverse"transcription………………………………………………………………"47" " 2.17"RealKtime"PCR……………………………………………………………………………………………………....."50" . PRELIMINARY"RESULTS" " " " " " " " " 53" " HYPOTHESYS"" " " " " " " " " " " 55" " GENERAL"AIM"" " " " " " " " " " 55" " SPECIFIC"AIMS"" " " " " " " " " " 55" " RESULTS" " " " " " " " " " " 56" ' 3.1"Analysis"of"the"state"of"the"central"clock"in"the"absence"of"rco$1.……………………………" 56" 3.2"Evaluation"of"RCOK1"expression"pattern.…………………………………………………………………."68" 3.3"Search"of"genes"whose"expression"is"regulated"by"RCOK1."………………………………………"73" 3.4"RCOK1"is"required"for"proper"circadian"expression"of"some"of"its"target"genes…………"84" 3.5"Identification"of"proteins"that"interact"with"RCOK1..…………………………………………………"89".

(5) !. 4!. " 3.6"RCOK1"is"involved"in"the"control"of"metabolic"compensation"in"Neurospora'' crassa…………………………………………………………………………………………………………………………….104' ' 3.7"Reassesment"of"frq"expression"dynamics"and"the"molecular"bases"of"" period"determination"……………………………………………………………………………………………........108" " DISCUSSION"" " " " " " " " " " """"""""""""112" " ANNEXES" " " " " " " " " " """"""""""""128" " 1."RCOK1"putative"target"genes….......................................................................................128" " 2"Putative"target"genes"of"RCOK1"described"as"ccgs....................................................…….130" " 3."List"of"proteins"identified"through"Mass"Spectrometry………………………………………………133" " REFERENCES.......................................................................................................................134" . " " " " " " ".

(6) !. 5!. LIST.OF.FIGURES. . Figure"1.1."Common"elements"in"a"circadian"feedback"loop………………..…………………………"15"" Figure"2.1."Strategy"for"creating"knockKin"translational"constructs…………………………………"35" Figure"2.2."Time"course"experiments…………………………………………………………………………….."39" Figure"2.3."The"Race"Tube"assay"reveals"circadian"rhythms"in"conidiation"under"laboratory" conditions………………………………………………………………………………………………………………………"41" " Figure"3.1."Several"transcriptional"regulators"control"the"rhythmic"expression"of"the" output"reporter"con$10…………………………………………………………………….……………………………"58" " Figure"3.2."In"Δrco$1'overt"rhythms"in"conidiation"are"absent……………………………..…………"59" Figure"3.3."In"the"absence"of"RCOK1"the"circadian"clock"is"functional"although"period"" and"amplitude"are"affected…………………………………………………………….…………………………….."62" " Figure"3.4."Expression"of"destabilized"luciferase"at"the"cyclosporine"locus"reconfirms" that"RCOK1"is"not"an"essencial"coreKclock"component………………………...…………………………"63" " Figure"3.5."Rhythmic"frq/FRQ"expression"in"wt"and"Δrco$1"strains..………………………………."66" Figure"3.6."frq"displays"elevated"mRNA"and"protein"levels"in"the"absence"of"RCOK1..……."67" Figure"3.7."Analysis"of"RCOK1"circadian"expression"by"bioluminescent"reporters..…………."70" Figure"3.8."RCOK1"expression"at"the"mRNA"and"protein"levels…………………………….…………"71" Figure"3.9."RCOK1"exhibits"a"high"stability………………………………………………………………………"72" Figure"3.10."Functional"categories"overrepresented"using"FunCat"analysis……………………"76" Figure"3.11."Biological"processes"overrepresented"using"GO"classification……………………."77" Figure"3.12."RCOK1"regulates"the"expression"of"several"genes"involved"in"a"variety" "of"processes………….……………………………………………………………..……………………………………….""82" " Figure"3.13."Some"putative"RCOK1"targets"do"not"show"significant"misregulation"" in"its"absence.………………………………………………………………………………………………………………." 83".

(7) !. 6!. Figure"3.14."Some"RCOK1"target"genes"present"a"circadian"behavior……………………………..""85" Figure" 3.15." Schematic" representation" of" the" rco$1" (NCU06205)" gene" replacement" event" by"a"hygromicinKresistance"(hph)"cassette"through"homologous"recombination."………….""86" " Figure"3.16:"RCOK1"is"involved"in"proper"circadian"control"of"several"clock"control"" genes……………………………………………………………………………………………………………………………..""88" " Figure"3.17."Neurospora"genes"encoding"for"TF"homologous"to"Tup1"interactors"" exhibit"circadian"expression"………………………………………………………………………………………....""93" " Figure" 3.18." RCOK1" interacts" in' vivo" with" transcription" factor" involved" in" carbon" metabolism……………………………………………………………………………………………………………………."96" Figure"3.19."RCOK1"interacts"in'vivo"with"the"histone"deacetilase"HDA1…………………………""97" Figure"3.20."RCOK1"interacts"in'vivo"with"the"TF"HFSK2.…………………………………………………"102" Figure"3.21"State"of"the"central"oscillator"in"the"absence"of"selected"RCOK1"" interactors"……………………………………..………………………….………………………….……………………."103" " Figure"3.22."Alteration"of"asexual"development"in"the"absence"of"HSFK2"and"RCOK1..….."105" Figure"3.23."RCOK1"is"required"for"proper"metabolic"compensation"of"the"Neurospora"" clock"……………………………………………………………………………………………………………………………."107" " Figure"3.24."Discrepancy"between"the"oscillation"of"frq"expression"in"solid"and"" liquid"cultures"conditions"…………………………………………………………………………………………...."109" " Figure"3.25."Rhythmic"levels"and"higher"stability"of"FRQ"in"solid"media"cultures………….."111" " Figure"4.1."Diagram"of"RCOK1"role"in"the"circadian"clock"regulation"of"Neurospora………"124" " " " " ". ".

(8) !. 7!. LIST.OF.TABLES. " " " " Table"1."Primers"used"for"transcriptional"fusions"constructs…………………………………………."30" " Table"2."Primers"used"to"generate"the"translational"constructs…………………………………….."33" ! Table"3."Primer"used"to"check"the"mutants"strain………………………………………………………….""49" ! Table"4."Primers"and"conditions"used"for"qPCR"reactions………………………………………………."55" " Table"5."Putative"target"gene"of"RCOK1"with"functional"categories"of"interest……………….."78" " Table"6."Putative"recruiters"of"RCOK1"based"on"literature"available"for"S.'cerevisiae' and"S.'pombe"……………………………………………………………………………………………………….……….."91' " Table"7."MassKspectrometry"reveals"novels"TFs"and"chromatin"remodeling"proteins"that" interact"with"RCOK1…………………………………………………………………………………………..…………."100" " ! !.

(9) !. " bd" " c$box' ' CKterminus" CCD" " ccg' ' cDNA" " ChIPKseq" sequencing" CHX" " CK" " CoKIP" " DD" " DNA" " FCC" " FGSC" " FLO" " FRH" " FRQ" " FWDK1"" FWO" " GO" " kb" " KO" " LD" " LL" " LOV" " LUC" " min" " mRNA" " NKterminus" NF" " ORF" " PCR" " pLRE" " RCOK1" " SDS" " TF" " TTFL" " WC" " WCC" " WT" ". 8!. ABBREVIATIONS. band" clockKbox' carboxyKterminus" chargeKcoupled"device" clock"controlled"gene" complementary"DNA" chromatin"immunoprecipitation"followed"by"highKthroughput"DNA" cycloheximide" casein"kinase" coKimmunoprecipitation" constant"darkness" deoxyribonucleic"acid" FRQ/FRH"complex" Fungal'Genetic'Stock'Center" FRQKless"oscillator" FRQKINTERACTING"RNA"HELICASE" FREQUENCY" FKbox"WDK40"repeat"containing"proteinK1" FREQUENCY/WHITE"COLLAR"COMPLEX"oscillator" gene"ontology' kilobase" Knockout" light/dark" constant"light" light"oxygen"voltage" luciferase"from"Photinus'pyralis' minutes" messenger"ribonucleic"acid" amino"terminus" normalization"factor" open"reading"frame" polymerase"change"reaction" proximal"light"regulatory"element" regulator"of"conidiation"1" sodium"dodecyl"sulfate" transcription"factor" transcriptional/translational"feedback"loop" white"collar' WHITE"COLLAR"complex" Wild"type..

(10) !. 9!. ABSTRACT. ' Circadian" clocks" are" endogenous" molecular" timekeepers," which" organize" physiology"of"organisms"with"respect"to"the"external"world,"conferring"daily"rhythms"to"a" large" number" of" biological" processes" within" the" cell." These" clocks" are" present" in" various" organisms," impinging" close" to" 24" hours" rhythms" in" the" regulation" of" gene" expression," physiology"and"behavior."A"circadian"system"can"be"conceptualized"as"composed"of"three" parts:" input" mechanisms," a" central" oscillator" and" output" pathways." Although" a" detailed" molecular" description" of" the" core" oscillator" is" available" in" model" eukaryotes," there" is" limited"information"on"the"mechanisms"that"allows"it"to"regulate"rhythmic"processes."Such" “output"pathways”"are"the"least"characterized"aspect"of"circadian"systems."" The" filamentous" fungus" Neurospora' crassa' has" served" for" decades" as" a" model" organism"for"the"study"of"circadian"biology."In"an"effort"to"improve"current"knowledge"of" output" pathways" identifying" new" components" involved" in" this" process," a" genetic" screen" was" conducted" in" this" fungus," and" we" identified" potential" regulatory" candidates," among" which"we"characterized"the"coKrepressor"RCOK1"and"its"role"in"regulating"circadian"biology."" RCOK1" is" the" orthologue" of" the" Saccharomyces' cerevisiae" transcriptional" coK repressor"Tup1."Contrary"to"reports"that"emerged"while"developing"this"thesis,"we"provide" evidence" that" RCOK1" is" not" an" essencial" coreKclock" component" in" Neurospora." We" evaluated" the" status" of" the" central" clock" observing" that" expression" of" the" negative" element"of"the"core"oscillator,frequency'(frq),"remains"rhythmic"in"the"absence"of"RCOK1." Interestingly," in" this" mutant" both" amplitude" and" period" length" of" the" oscillations" were" affected." Importantly," we" observed" a" defect" in" metabolic" compensation" in" Δrco$1," such" that"highKglucose"concentrations"when"RCOK1"is"not"present"significantly"decrease"period." We" identified" several" proteins" involved" in" chromatin" remodeling" and" TFs" that" physically" interact" with" RCOK1" and," in" fact," the" absence" of" one" of" the" latter" group" also" leads"to"a"change"in"period."In"addition,"the"analysis"of"RCOK1"target"genes"revealed"that"it" plays"an"important"role"for"their"circadian"expression."In"summary,"these"results"indicate"a" dual"role"for"RCOK1:"although"it"is"not"essential"for"coreKclock"function,"it"regulates"proper" period"and"amplitude"of"frqKbased"expression,"whereas"it"is"also"required"in"output"for"the" rhythmic"regulation"of"several"clock$controlled'genes."" In"addition,"in"the"need"to"develop"sensitive"methods"to"follow"FRQ"expression"of" Δrco$1" under" solidKmedia" culture" conditions" this" thesis" also" contributed" to" resolve" key" aspects"of"the"clockworks."Thus,"with"this"same"methodology,"we"molecularly"confirmed" FRQ" data" that" was" inferred" from" bioluminescence" studies" of" a" Δfwd$1' strain." Thus," we" analyzed"FRQ"levels"in"a"KO"of"the"FKbox"protein"FWDK1"(a"ubiquitin"ligase"that"mediates" FRQ"proteasomal"degradation)"and"that"therefore"exhibits"decreased"FRQ"turnover."With" this,"we"provided"critical"evidence"showing"that"circadian"period"determination"does"not" relay"on"FRQ"stability,"but"instead"on"its"degree"of"posttranslational"modifications."" In'toto,"although"this"thesis"had"as"a"main"objective"to"contribute"defining"output" pathways" components," not" only" it" helped" advancing" the" latter," but" it" also" shed" light" on" basic" coreKclock" mechanisms" related" to" period" determination" and" also" to" metabolic" compensation."..

(11) !. 10!. . INTRODUCTION. " " " " Circadian" rhythms" are" part" of" the" history" of" life" in" this" planet," being" largely" involved"in"the"cyclic"control"of"a"myriad"of"biological"process"in"different"organisms."The" term" circadian" derives" from" the" latin' circa' diem,' or' “about" a" day”," and" as" it’s" name" indicates,"processes"under"circadian"regulation"have"a"duration"of"approximately"24"hours" or" a" day." These" rhythms" allow" organisms" to" anticipate" and" respond" to" daily" cyclic" transitions,"concentrating"activities"at"the"time"of"day"when"they"are"more"advantageous." Indeed," circadian" clocks," the" molecular" timing" machineries" underlying" these" oscillations," integrate" temporal" information" and" control" rhythms" in" gene" expression," biochemical" reactions," physiology" and" behavior" in" a" wide" range" of" organisms" and" it" is" generally" believed" that" they" confer" a" selective" advantage" (OUYANG' et' al.," 1998;" DODD' et' al.," 2005;" PARANJPE" and" SHARMA," 2005;" YERUSHALMI" and" GREEN," 2009)." Eclosion" in" flies," locomotory" activity" in" mammals" and" conidiation" in" fungi" are" some" of" the" several" examples" of" biological"process"that"exhibit"a"circadian"behavior"(SARGENT'et'al.,"1966;"BELLKPEDERSEN'et' al.,"2005;"MACKEY,"2007)." .. .. ..

(12) !. 1.1... 11!. Circadian.Clocks.Mechanisms.. " Circadian"rhythms"are"widespread"across"the"tree"of"life,"and"have"been"studied"in". a"broad"range"of"model"organisms"such"as"cyanobacteria,"fly,"fungi,"mammals"and"plants" (SARGENT' et' al.," 1966;" SWEENEY," 1987;" PITTENDRIGH," 1993;" DUNLAP," 2008)." In" most" of" these" organisms,"when"the"molecular"bases"of"the"clock"were"revealed,"it"was"observed"that"the" architecture" Kthe" molecular" designK" underlying" the" clockworks" in" eukaryotes" was" highly" conserved"(BELLKPEDERSEN'et'al.,"2005;"MACKEY,"2007)."This"came"as"a"surprise"as"the"genes" and"proteins"conforming"the"central"clock"machinery"are"genetically"unrelated,"consistent" with" the" fact" that" clocks" seem" to" have" evolved" at" least" three" independent" times" throughout"evolution"(TAUBER'et'al.,"2004;"ROSBASH,"2009)." " A" circadian" system" can" be" defined" as" a" threeKsystem" component," an" oscillator" or" pacemaker" and" two" signaling" pathways:" input" pathways" that" convey" external" signals" to" the"oscillator"(allowing"its"synchronization"with"the"environment),"and"output"pathways," which"temporally"regulate"diverse"cellular"processes." " The"current"view"of"circadian"oscillators"is"that"they"are"composed"of"positive"and" negative" elements," arranged" in" interlocked" positive" and" negative" feedback" loops." While" the" positive" feedback" loops" give" robustness" to" the" system," a" central" transcriptional/translational" negative" feedback" loop" (TTFL)" serves" as" the" main" timing" device"where"period"is"determined.""The"TTFL"is"conformed"of"positive"elements"that"drive".

(13) !. 12!. the"expression"of"negative"elements,"which"are"capable"to"negatively"regulate"their"own" expression." Importantly," the" positive" elements" transcriptionally" activate" two" types" of" genes:" clock" genes" (encoding" negative" elements)" and" firstKtier" clock" controlled" genes" (ccgs)."Thus"the"positive"elements"participate"in"the"main"TTFL"and"also"in"the"time"relay" that"leads"to"genome"wide"ccg"expression,"providing"control"of"the"rhythmic"activities"of" the"entire"organism.Figure.1.1." " " " " " ". ".

(14) !. 13!. " " " ". " Figure.1.1..Common.elements.in.a.circadian.feedback.loop... The" most" common" model" for" the" architecture" and" functioning" of" eukaryotic" circadian" pacemakers," involves" a" molecular" feedback" loop" in" which" positive" elements," working" usually"as"heterodimers"(not"shown),"activate"the"expression"of"negative"elements"(clock" genes)," which" ultimately" inhibit" the" activity" of" the" positive" elements," thus" negatively" regulating"their"own"expression."Positive"elements"also"regulate"the"rhythmic"expression" of"several"ccgs."" " ". ".

(15) !. 1.2.. ". 14!. The.Neurospora.Circadian.Oscillator.. The". circadian". oscillator". of". Neurospora'. crassa". is". based". on". a". transcriptional/translational"negative"feedback"loop"(TTFL)"where"the"positive"element"is"a" heterodimeric" transcriptional" complex," known" as" the" White" Collar" Complex" (WCC)" (CROSTHWAITE'et'al.,"1997),"composed"by"the"GATA"TFs"WCK1"and"WCK2,"and"the"negative" element" corresponds" the" protein" FREQUENCY" (FRQ)." Thus," the" positive" element" drives" frequency" (frq)" expression" and" the" resulting" protein" dimerizes," associates" with" an" RNA" helicase"named"FRH"(CHENG'et'al.,"2005)"forming"a"FFC"(FRQ,"FRH"complex)"that"acts"as"a" kinaseKrecruiting" platform," which" leads" to" the" inhibition" of" frq" own" expression" through" interaction"with"WCC"and"subsequent"WCCKinactivating"phosphorylation"events"(CHENG'et' al.," 2001;" DENAULT' et' al.," 2001;" FROEHLICH' et' al.," 2003;" SCHAFMEIER' et' al.," 2005;" HE' et' al.," 2006)"(Figure.1.2)."In"addition"to"their"role"in"the"Neurospora"central"oscillator,"WCK1"and" WCK2" are" also" essential" components" for" transcriptional" light" responses." Indeed," WCK1" (a" flavin" containing" protein)" can" also" acts" as" a" blue" light" photoreceptor," directly" activating" the" expression" of" over" a" hundred" genes" upon" light" exposure" (Approx." the" 5%" of" the" genome),"including"frq'itself'(FROEHLICH'et'al.,"2002;"HE'et'al.,"2002;"CHEN'et'al.,"2009;"SMITH' et' al.," 2010)" while" several" genes" that" exhibit" lightKinduction" also" possess" a" rhythmic" behavior,"indicating"that"these"processes"are"highly"related"(HURLEY'et'al.,"2014)." " The"cycle"begins"when"the"WCC"binds"to"a"specific"sequence"in"the"frq"promoter," known"as"the"ClockKbox"(C$box),"activating"frq"transcription."This"C$box"cisKelement"is"both".

(16) !. 15!. necessary"and"sufficient"for"frq"rhythmic"expression"(FROEHLICH'et'al.,"2003)."The"initiation" of"frq'transcription"leads"to"the"accumulation"of"frq"mRNA"and"therefore"of"FRQ"synthesis." After" its" translation," FRQ" starts" to" be" progressively! phosphorylated" and" homodimerizes" (CHENG'et'al.,"2001),"binding"with"FRH"forming"the"FFC"complex"(CHENG'et'al.,"2005)."The" homodimerization" is" important" for" the" circadian" function" of" FRQ," since" point" mutations" designed"to"abolish"the"selfKassociation"of"FRQ"result"in"an"arrhythmic"strain."Also"it"has" been" shown" that" the" interaction" of" FRQ" and" WCC" with" FRH" it" is" also" critical" for" the" circadian"rhythms"of"this"fungus,"regulating"FRQ"phosphorylation,"localization"and"stability" (GUO'et'al.,"2010;"SHI'et'al.,"2010;"CHA'et'al.,"2011)."" " ". FRH"is"a"homolog"of"Mtr4p,"which"is"a"wellKstudied"cofactor"of"the"Saccharomyces'. cerevisiae'exosome,"which"is"a"multiprotein"complex"that"regulates"RNA"metabolism"(GUO' et'al.,"2009)."Mtr4p"is"a"member"of"the"TRAMP"complex"and"plays"a"role"in"the"exosome"in" yeast," however" recent" studies" have" shown" that" the" role" of" FRH" in" the" clock" is" different" from"the"TRAMP/exosome"complex"function,"since"the"loss"of"its"helicase"activity"does"not" affect" the" clockworks" (HURLEY' et' al.," 2013)." Importantly," all" FRQ" in" the" cell" seems" to" be" associated"with"FRH"(CHENG'et'al.,"2005;"BAKER'et'al.,"2009)"as"part"of"the"FFC,"consistent" with"the"idea"of"FRH"being"a"Nanny"protein"that"helps"stabilizing"FRQ"intrinsically"disorder" (IDP)"nature"(HURLEY'et'al.,"2013)."This"protein"complex"acts"as"the"negative"element"of"the" circadian"clock:"when"it"enters"to"the"nucleus"it"is"able"to"interact"with"the"WCC"promoting" its" phosphorylation," which" leads" to" its" inactivation" and" clearance" from" the" nucleus" (SCHAFMEIER'et'al.,"2005;"HE'et'al.,"2006;"HONG'et'al.,"2008;"SCHAFMEIER'et'al.,"2008)."FRQ"is".

(17) !. 16!. also"subjected"to"extensive"and"sequential"phosphorylation"events,"which"are"essential"for" its"role"in"the"circadian"system"(BAKER'et'al.,"2009;"TANG'et'al.,"2009)."It"has"been"confirmed" that"at"least"85"serine/threonine"residues"of"FRQ"are"phosphorylated"in'vivo'(BAKER"et"al.," 2009)." When" FRQ" reaches" its" highest" levels" of" phosphorylation," its" interaction" with" the" WCC"is"impaired"and"FRQ"is"sent"to"degradation"by"the"ubiquitinKproteasome"pathway"(HE" and" LIU," 2005)" being" the" ubiquitin" E3" ligase" FWDK1" responsible" to" target" FRQ" to" degradation."However,"recent"findings"show"that"the"degradation"of"FRQ"is"not"necessary" for" restarting" a" new" circadian" cycle." Although" degradation" is" the" final" outcome" of" FRQ" posttranslational"modifications,"phosphorylation"may"be"the"key"mechanism"determining" clock"speed"and"the"point"at"which"the"next"cycle"of"frq"synthesis"is"initiated"(this"thesis" and" (LARRONDO' et' al.," 2015))." So," reaching" such" phosphorylation" levels" as" a" preKrequisite" for" the" next" stage" in" the" cycle" (which" is," release" of" FRQKmediate" repression" of" WCC" activity)"provides"the"necessary"delay"needed"for"the"feedback"to"last"approximately"24"h" (MERROW' et' al.," 1997;" SCHAFMEIER' et' al.," 2006)." By" the" late" subjective" night," and" in" the" absence"of"FRQ,"the"WCC"can"promote"frq'expression."As"a"result,"frq"mRNA"and"protein" levels" oscillate" daily," which" can" be" phenotypically" correlated" with" the" rhythmic" appearance" of" asexual" spores." Several" kinases" have" been" implicated" in" phosphorylating" FRQ," including" CKK1a," CAMKK1" and" CK2" (YANG' et' al.," 2001;" HE' et' al.," 2006)" and" some" of" these"kinases"play"relevant"roles"in"the"circadian"systems"of"other"organisms"(MIZOGUCHI'et' al.,"2006;"GALLEGO"and"VIRSHUP,"2007)."" " ". ".

(18) !. 17!. . Figure.1.2.Representation.of.the.circadian.clock.of.Neurospora." A)"The"WCC,"conformed"by"the"TFs"WCK1"and"WCK2,"acts"as"a"positive"element"promoting" frq"transcription."Once"FRQ"is"translated,"dimerizes"and"associated"with"FRQ,"forming"the" FFC"complex"(negative"element),"which"recruites"several"kinases"as"CK1"and"CK2." B)" The" FFC" complex," with" hypophosporylated" FRQ" is" able" to" interact" with" the" WCC" promoting" its" phosphorylation." Once" this" complex" is" phosphorylated" it" can" no" longer! promote"frq'synthesis,"and"existing"levels"of"frq'(mRNA)"decay."" C)" Hypophosphorylated" FRQ" is" target" of" consecutive" phosphorylations," and" finally" this" hyperphosphorylated" FRQ" is" recognized" by" the" ubiquitin" ligase" FWDK1" that" targets" it" for" degradation" through" the" ubiquitinKproteasome" pathway." Since" hyperphospohorylated" FRQ"fails"to"inhibit"the"WCC,"new"frq"mRNA"can"be"made,"and"new"(hypophosphoylated" FRQ)"can"be"synthesized"again."The"time"it"takes"to"complete"this"entire"cycle,"determines" the"period"of"the"circadian"clock"of"Neurospora.".

(19) !. ". 18!. FRQ"also"participates"in"a"positive"feedback"loop,"which"regulates"the"levels"of"the". WC"proteins"(LEE'et'al.,"2000;"CHENG'et'al.,"2001),"and"the"WC"proteins"regulate"their"own" expression"(CHENG'et'al.,"2001;"CHENG'et'al.,"2003)."Those"interlocked"loops"are"crucial"for" clock"robustness"and"stability." "" Thus,"under"circadian"conditions"the"WCC"binds"to"the'c$box,"driving"the"rhythmic" expression"of"frq,"while"upon"light"it"is"also"able"to"recognize"an"additional"sequence"in"the" frq'promoter"called"Proximal"Light"Regulatory"Element"(pLRE)"(FROEHLICH'et'al.,"2002)."This" sequence" has" been" identified" as" a" key" element" mediating" the" acute" transcriptional" response" of"frq' to" light," connecting" the" central" clock" with"environmental" time" (provided" by"new"photic"cues)"(FROEHLICH'et'al.,"2002)." " Among" the" phenotypic" traits" that" are" under" circadian" control" is" asexual" reproduction"(conidiation),"which"has"been"used"for"years"as"an"easyKtoKscore"readout"of" the"clock,"allowing"robust"evaluation"of"circadian"rhythmicity."Thus,"when"Neurospora"is" grown"under"constant"dark"conditions"(DD)"in"horizontal"hollowKglass"tubes"known"as"race" tubes,"the"generation"of"a"conidial"band"is"observed"every"22.5"hours"(PITTENDRIGH'et'al.," 1959;" SARGENT' et' al.," 1966)," revealing" the" period" dictated" by" the" circadian" oscillator." In" parallel,"a"new"system"for"the"evaluation"of"the"circadian"expression"has"been"adopted"in" different"organisms"(MILLAR'et'al.,"1992;"KONDO'et'al.,"1993;"BRANDES'et'al.,"1996;"GEUSZ'et' al.,"1997)."This"system,"which"is"based"on"the"use"of"luciferase"as"a"realKtime"reporter,"was" implemented"rather"recently"in"Neurospora,"compared"to"other"circadian"models"(MORGAN'.

(20) !. 19!. et' al.," 2003)." While" at" the" beginning" it" was" used" to" study" transcription" of" output" genes," over" the" years" Kto" improve" it’s" functioning" and" allow" the" study" of" genes" with" low" expression"such"as"frq"itselfK,"a"fully"codonKoptimized"version"of"luciferase"was"developed" (GOOCH' et' al.," 2008)." More" recently," a" new" version" of" luciferase" carrying" a" PEST" signal" (which" allows" a" faster" degradation" of" the" protein," and" therefore" a" shorter" halfKlife)" has" been"generated"(CESBRON'et'al.,"2013)."These"new"tools"allow"monitoring"the"state"of"clock" components"in"real"time"in"multiples"strains"at"a"time,"with"an"in$vivo'reporter"where"the" phenotypic"and"molecular"rhythms"can"be"studied"separately"(SHI'et'al.,"2007;"LARRONDO'et' al.,"2012;"MONTENEGROKMONTERO"and"LARRONDO,"2013;"LARRONDO'et'al.,"2015)." " " 1.3... Light.responses.and.photoadaptation.in.Neurospora... " ". As" previously" mentioned," besides" being" a" core" clock" component" WCK1" is" a" blue". light"photoreceptor"providing,"thus,"an"input"pathway"by"integrating"blue"light"information" into"the"circadian"system"and"guaranteeing"the"synchronization"of"the"endogenous"clock" to"the"24"h"dayKnight"cycle."" " ". The"protein"WCK1"contains"a"zincKfinger,"a"chromophoreKbinding"domain,"and"PAS". domains"for"protein–protein"interactions"(CROSTHWAITE'et'al.,"1997)."WCK1"senses"the"light" trough" the" specialized" PAS" domain" LOV" (LightKOxygenKVoltage)" (HE' et' al.," 2002)." When" cultures" grown" in" DD" conditions" are" exposed" to" light," the" LOV" domain" of" WCK1" binds" a" flavin" chromophore" (FAD)" promoting" the" dimerization" of" the" WCC," which" rapidly" binds,".

(21) !. 20!. and"induces"the"expression"of"lightKinduced"genes"(among"which"is"frq),"which"reach"peaks" of"expression"approximately"15"min"after"light"exposure;"then"RNA"levels"quickly"decrease" reaching" intermediate" levels" in" prolonged" constant" light" (LL)," a" process" called" photoadaptation"(SCHWERDTFEGER"and"LINDEN,"2001).""" ". "" VIVID" (VVD)," another" blueKlight" photoreceptor," plays" a" fundamental" role" in" the". photoadaptation"process"(CHEN'et'al.,"2010;"HUNT'et'al.,"2010;"MALZAHN'et'al.,"2010).'This" small"protein"also"bears"a"LOV"domain"(HEINTZEN'et'al.,"2001),"which"is"able"to"interact"with" the"LOVKPAS"domain"of"WCK1,"inhibiting"dimerization"of"the"WCC."This"causes"a"rapid"and" efficient" attenuation" of" lightKinduced" transcription" (MALZAHN' et' al.," 2010)." VVD' is" the" principal"protein"that"regulates"this"process,"however"other"proteins"also"play"a"role." " The" RCOK1/RCMK1" coKrepressor" complex" participates" in" the" photoadaptation" process"(OLMEDO'et'al.,"2010).'This"complex"is"homologous"to"the"Tup1/Ssn6"coKrepressor" complex" of" yeast," which" controls" the" expression" of" approximately" 7%" of" the" genome" (SMITH" and" JOHNSON," 2000;" GREEN" and" JOHNSON," 2004)." The" absence" of" the" RCOK1/RCMK1" repressor"complex"leads"to"several"alterations"affecting"differentially"transcription"of"lightK induced" genes;" lack" of" either" results" in" the" accumulation" of" some" transcripts" after" long" exposures"to"light"(OLMEDO'et'al.,"2010;"RUGERKHERREROS'et'al.,"2014),"similar"to"what"has" been"described"for"the"Δvvd'mutant"strain." " ..

(22) !. 1.4... 21!. Transcriptional.regulation.and.output.pathways.. " In" the" past" decades," coreKoscillator" composition" and" the" clockworks" have" been". exhaustively"studied"(CROSTHWAITE'et'al.,"1997;"GORL'et'al.,"2001;"YANG'et'al.,"2002;"CHENG'et' al.," 2005;" SCHAFMEIER' et' al.," 2005;" PREGUEIRO' et' al.," 2006;" GUO' et' al.," 2009;" BAKER' et' al.," 2011)." Nevertheless," it" has" not" been" trivial" to" identify" the" components" responsible" for" carrying" clock" information" from" the" central" oscillator" to" clockKcontrolled" biological" processes."" " Several"studies"show"that"about"20K40%"of"the"transcriptional"units"in"Neurospora" are"subjected"to"circadian"regulation"(ZHU'et'al.,"2001;"CORREA'et'al.,"2003;"NOWROUSIAN'et' al.,"2003;"DONG'et'al.,"2008;"HURLEY'et'al.,"2014)."But"even"when"an"important"part"of"the" genome" presents" a" circadian" regulation," only" few" biological" processes" have" been" described" as" circadian" regulated," and" even" a" smaller" amount" have" been" systematically" explored."Among"the"processes"that"have"received"more"attention"are"the"ones"related"to" osmotic"stress"and"carbon"metabolism."In"the"case"of"the"former,"the"signaling"from"the" clock"is"transmitted"trough"the"MAPK"pathway"(LAMB'et'al.,"2011;"LAMB'et'al.,"2012),"while" for"carbon"metabolism,"it"has"been"observed"that"the"TF"CSPK1"plays"an"important"role"in" regulating"the"circadian"expression"of"several"metabolicKrelated"rhythmic"genes"(SANCAR'et' al.," 2011)." This" data" indicated" that" circadian" gene" expression" can" be" regulated" through" several"mechanisms"(transcriptional,"postKtranscriptional,"postKtranslational,"etc.)." ".

(23) !. 22!. Based"on"the"fact"that"the"WCC,"central"component"of"the"clock,"is"formed"by"two" TFs"that"bind"directly"to"the"promoters"of"other"TFs"such"as"hsf$2,'adv$1,'sah$1,'csp$1,'cre$ 1,'etc."(SMITH'et'al.,"2010),"some"of"which"present"a"circadian"behavior"(LAMBREGHTS'et'al.," 2009;" SMITH' et' al.," 2010;" SANCAR' et' al.," 2011)," it" has" been" proposed" that" a" hierarchical" arrangement"of"transcriptional"regulators"may"be"the"main"mechanism"through"which"the" clock" regulates" rhythmic" gene" expression." However," and" despite" decades" of" using" Neurospora" as" research" model" organism," little" is" known" about" the" transcriptional" regulatory"networks"and"the"vast"majority"of"the"TFs"have"not"been"characterized."" " In"Neurospora,"the"most"studied"TFs"belong"to"the"GATA"family:"WCK1,"WCK2,"SRE," SUBK1," NITK2" and" ASDK4" (FU" and" MARZLUF," 1987;" CHIANG' et' al.," 1994;" FENG' et' al.," 2000;" HARRISON" and" MARZLUF," 2002;" CHEN' et' al.," 2009)." And" for" all" these" TFs" their" binding" site," biological" process" and" some" of" the" target" genes" have" been" determined" through" studies" based"on"Northern,"Western"blots,"EMSA,"etc."Each"TF"participated"in"the"regulation"of"a" specific" biological" process:" WCK1" and" WCK2" are" key" part" of" the" central" oscillator" and" of" light" responses" (CROSTHWAITE' et' al.," 1997;" HE' et' al.," 2002)," SUBK1" is" a" ccg" and" also" participates"in"photoresponses"(CHEN'et'al.,"2009;"SANCAR'et'al.,"2015)."NIT2"is"the"mayor" regulator"of"the"nitrogen"metabolism"(FU"and"MARZLUF,"1987),"while"SRE"participates"in"the" synthesis"of"siderophore,"whereas"ASDK4"has"an"important"role"in"ascus"formation"(FENG'et' al.," 2000;" HARRISON" and" MARZLUF," 2002)." Of" all" these," only" WCK1," WCK2" and" SUBK1" have" been"clearly"implicated"in"circadian"regulation." ".

(24) !. 23!. In" Neurospora" there" are" approximately" 272" predicted" TFs" (WEIRAUCH' et' al.," 2014)" (http://cisbp.ccbr.utoronto.ca/index.php)" and," beside" the" GATA" TFs" mentioned" before," only" a" few" more" TFs" have" been" characterized" in" detail." Just" recently," in" a" joint" effort" to" determine"the"DNA"sequence"specificity"of"TFs"belonging"to"different"eukaryotes"clades," through"a"proteinKbinding"microarrays"(PBMs)"strategy,"the"binding"specificity"for"146"TFs" (approx."50%"of"the"predicted"TFs"of"Neurospora)"was"identified"(WEIRAUCH'et'al.,"2014)." This"dataset"provides"a"unique"resource,"establishing"a"predictive"tool"to"inquire"about"the" possible" genes" regulated" by" each" TF." However," molecular" studies" are" necessary" to" corroborate"cohorts"of"target"genes"under"the"control"of"these"TFs"and"to"unravel"the"TFs" that"participate"in"the"circadian"output"pathways." " ! .. ..

(25) !. 1.5... 24!. Metabolic.compensation.of.the.circadian.clock... ! While" in" general" it" has" been" considered" that" outputs" from" the" clock" do" not" feed" back"to"influence"the"oscillator"itself"(BELLKPEDERSEN'et'al.,"1992),"lately"counter"examples" to" this" have" emerged" as" diverse" outputs" have" been" shown" to" influence" the" state" of" the" oscillator" (PREGUEIRO' et' al.," 2006;" O'NEILL' et' al.," 2008;" SANCAR' et' al.," 2012;" HONG' et' al.," 2013)."In"higher"organisms,"recent"findings"have"shown"that"the"regulation"of"metabolism" by" the" circadian" clock" and" its" components" is" reciprocal," and" that" coreKelements" of" the" circadian"clock"are"able"to"sense"the"alterations"in"cell"metabolism"(BREWER'et'al.,"2005;"LIU' et' al.," 2007;" KALSBEEK' et' al.," 2008;" NAKAHATA' et' al.," 2008;" NAKAHATA' et' al.," 2009;" BASS" and" TAKAHASHI,"2010;"GRIMALDI'et'al.,"2010;"CHO'et'al.,"2012;"JEYARAJ'et'al.,"2012)."" " In" Neurospora," the" relationship" between" metabolism" and" the" circadian" clock" has" been" only" recently" addressed" (SANCAR' et' al.," 2011;" HURLEY' et' al.," 2014)" For" instance," a" metabolic"compensation"mechanism"has"been"identified,"in"which"the"clock"maintains"its" periodicity" when" grown" under" different" glucose" concentrations." In" this" mechanism," the" repressor"CSPK1"plays"a"key"role"(SANCAR'et'al.,"2012),"as"the"absence"of"this"transcription" factor"leads"to"a"loss"of"metabolic"compensation"evidenced"as"period"shortening"at"highK glucose"levels."" " " ".

(26) !. 25!. As" it" was" mentioned" earlier," a" circadian" clock" can" be" defined" as" a" threeKsystem" component:"input"pathways,"central"core"an"output"pathway."For"several"years,"the"study" in"the"circadian"field"has"been"focused"into"the"two"first"areas"and"little"information"for" the"output"pathways"and"its"regulation"is"available."In"this"thesis,"by"employing"a"reverse" genetics" approach" and" luciferaseKbased" highKthroughput" screening," we" searched" for" transcriptional"regulators"involved"in"the"control"of"rhythmic"gene"expression"downstream" from"the"central"oscillator"and"the"biological"processes"that"are"controlled"by"them."This" screen"yielded"different"candidates"among"which,"it"was"the"coKrepressor"RCOK1."" " ". RCOK1"is"the"homolog"of"Tup1,"a"coKrepressor"that"regulates"approximately"7%"of". the"transcriptome"in"yeast"(SMITH"and"JOHNSON,"2000)."In"this"organism"this"protein"is"part" of"a"complex"with"Ssn6"and"it"is"able"to"control"gene"expression"of"a"variety"of"biological" processes"acting"mainly"as"a"repressor"(TREITEL"and"CARLSON,"1995)."This"complex"is"unable" to"bind"DNA"and"is"recruited"to"promoter"regions"through"the"interaction"with"specific"TFs" (KELEHER'et'al.,"1992)."Interaction"of"Tup1"with"at"least"10"TFs"has"been"described"(TREITEL" and" CARLSON," 1995;" ZITOMER' et' al.," 1997;" HUANG' et' al.," 1998;" PARK' et' al.," 1999;" PROFT" and" SERRANO,"1999;"ZHANG"and"REESE,"2004;"HANLON'et'al.,"2011),"which"can"explain"the"fact"that" Tup1"is"necessary"for"the"proper"regulation"of"multiple"and"diverse"processes,"associated" with"the"control"elicited"by"these"TFs."The"role"of"its"homolog"in"Neurospora,"RCOK1,"has" been" less" explored" although" as" mentioned" earlier" this" protein" is" now" known" to" be" important"for"photoadaptation"(OLMEDO'et'al.,"2010;"LEE'et'al.,"2011;"RUGERKHERREROS'et'al.," 2014)."".

(27) !. 26!. Interestingly," while" this" thesis" was" being" developed" the" ZnF_C2H2" TF" CSPK1" was" shown" to" regulate" the" expression" of" several" ccgs," which" indirectly" entails" RCOK1" in" this" process" through" interactions" with" CSPK1' (SANCAR" et" al.," 2011)." To" our" surprise," a" recent" publication"(ZHOU'et'al.,"2013)"that"appeared"as"this"thesis"was"under"course"claimed"an" essential"role"for"RCOK1"in"coreKclock"mechanisms,"such"that"in"the"absence"of"this"protein" circadian"expression"of"frq"ceased."Importantly,"the"results"obtained"in"this"thesis"do"not" endorse" such" claim" and" moreover," provide" compelling" results" contradicting" those" observations."" " ". Thus," based" on" the" results" obtained" in" a" genetic" screening" aimed" at" identifying". output"regulators,"this"thesis"set"into"studying"the"role"of"RCOK1"in"clock"output"in"order"to" improve" the" understanding" of" the" transcriptional" network" that" controls" the" circadian" expression"in"Neurospora."Not"only,"we"confirmed"the"importance"of"RCOK1"in"circadian" control,"but,"we"defined"that"RCOK1"is"not"an"essential"coreKclock"component,"although"we" identified" that" its" absence" lead" to" period" defects." Moreover," we" observed" that" in" the" absence" of" RCOK1" metabolic" compensation" is" broken," and" that" as" sugar" levels" are" increased" the" characteristic" long" period" observed" for" RCOK1" is" progressively" decreased," suggesting" a" different" mechanism" that" the" one" implied" for" CSPK1" in" metabolic" compensation." Finally," as" shown" at" the" end" of" the" results" section," some" of" the" experimental"protocols"developed"in"this"thesis"became"critical"in"reassessing"key"clocks" mechanisms," as" the" ones" associated" with" FRQ" stability" and" period" determination" (LARRONDO'et'al.,"2015).".