Genome-Wide Annotation and Expression Profiling of Cell Cycle Regulatory Genes in Chlamydomonas reinhardtii

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Genome-Wide Annotation and Expression Profiling of Cell Cycle Regulatory Genes in Chlamydomonas reinhardtii
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  Genome Analysis Genome-Wide Annotation and ExpressionProfiling of Cell Cycle Regulatory Genes in Chlamydomonas reinhardtii 1[w] Katerina Bisova, Dmitri M. Krylov, and James G. Umen* The Salk Institute for Biological Studies, La Jolla, California 92037 (K.B., J.G.U.); and National Center forBiotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda,Maryland 20894 (D.M.K.) Eukaryotic cell cycles are driven by a set of regulators that have undergone lineage-specific gene loss, duplication, ordivergence in different taxa. It is not known to what extent these genomic processes contribute to differences in cell cycleregulatory programs and cell division mechanisms among different taxonomic groups. We have undertaken a genome-widecharacterization of the cell cycle genes encoded by  Chlamydomonas reinhardtii , a unicellular eukaryote that is part of the greenalgal/land plant clade. Although Chlamydomonas cells divide by a noncanonical mechanism termed multiple fission, the cellcycle regulatory proteins from Chlamydomonas are remarkably similar to those found in higher plants and metazoans,including the proteins of the RB-E2F pathway that are absent in the fungal kingdom. Unlike in higher plants and vertebrateswhere cell cycle regulatory genes have undergone extensive duplication, most of the cell cycle regulators in Chlamydomonashave not. The relatively small number of cell cycle genes and growing molecular genetic toolkit position Chlamydomonas to become an important model for higher plant and metazoan cell cycles. It is well established that eukaryotic cell cycles arecontrolled by a conserved set of proteins. Centralamong these are cyclin-dependent kinases (CDKs;Nasmyth, 1996; Sherr, 1996; Johnson and Walker,1999; Mironov et al., 1999; Pavletich, 1999). CDKs aresubject to multiple regulatory mechanisms that causetheir activity and substrate specificity to oscillate andthereby drive cell cycle phase transitions. These regu-latory mechanisms include binding of cyclins that areobligatory activating subunits of CDKs, binding of inhibitory proteins, and activating or inhibitory phos-phorylation (Morgan, 1995). Research over the pasttwo decades has highlighted the remarkable conser-vation of these mechanisms, indicating that eukary-otes share a fundamentally conserved cell cycle(Morgan, 1997; Obaya and Sedivy, 2002; De Veylderet al., 2003; Dewitte and Murray, 2003).Despite the unity in underlying principles of cellcycle regulation, there are some important differencesthat remain to be elucidated. First, cell cycles thatdeviate in some way from the best characterizedG1-S-G2-M progression occur in some unicellulareukaryotes and are essential for the proper develop-ment of most multicellular eukaryotes, but the mech-anisms required to generate altered cell cycles are notfully understood (Joubes and Chevalier, 2000; Moserand Russell, 2000; Schwab et al., 2000; Edgar and Orr-Weaver,2001;Coffman,2004).Second,themechanicsof cell division in different taxa vary considerably, partic-ularly the mechanisms used for cytokinesis (Guertinet al., 2002). It is not known to what extent CDKs be-came adapted to their new substrates, or vice versa, asseparate division mechanisms evolved. Third, it is notclear why multicellular eukaryotes, such as plants andanimals, contain multiple CDKs, whereas in buddingyeast( Saccharomycescerevisiae )andfissionyeast( Schizo-saccharomyces pombe ),asingleCDKissufficienttodrivethe cell cycle. Until a broader spectrum of unicellulareukaryotes is examined, this question cannot be ad-dressed.The cell cycle regulators from the sequenced ge-nomes of animals, plants, and fungi show distinctpatterns of divergence, duplication, and gene loss.Higher plants are interesting because the CDK andcyclin family proteins have duplicated and divergedin them, thus giving rise to a novel CDK, CDKB(Mironov et al., 1999; Joubes et al., 2000; Vandepoeleet al., 2002), and several new cyclin families, includinga plant-specific D-type cyclin family that appears to befunctionally related to the animal D-cyclins (Meijerand Murray, 2000; Oakenfull et al., 2002). Unlike thefungi that lack the retinoblastoma (RB) pathway,homologs of RB and its binding partners E2F/DP arefound in higher plants and probably function ina similar manner to those of animals (Xie et al., 1996; 1 This work was supported by generous contributions to thelaboratory of J.G.U. from the H.N. and Frances C. Berger Founda-tion, the Joe W. and Dorothy Dorsett Brown Foundation, the Fritz B.Burns Foundation, The Arthur Vining Davis Foundations, TheLebensfeld Foundation, the John Stacy Lyons Memorial Foundation,The Gertrude E. Skelly Charitable Foundation, and the Irving A.Hansen Memorial Foundation.* Corresponding author; e-mail; fax 858–558–6379. [w] The online version of this article contains Web-only Plant Physiology,  February 2005, Vol. 137, pp. 475–491,    2005 American Society of Plant Biologists 475  www.plant.orgon September 30, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.  Huntley et al., 1998; Ramirez-Parra et al., 1999; Albaniet al., 2000; Boniotti and Gutierrez, 2001; Maricontiet al., 2002). The extensive duplications within higherplant cell cycle gene families have complicated thetask of reverse genetics. It is therefore of great interestto determine the extent to which cell cycle regulatorsin higher plants are conserved in simpler representa-tives of the plant kingdom. Chlamydomonas reinhardtii  is a chlorophyte alga thathas served as a model for plant cell biology and phys-iology (Goodenough, 1992; Rochaix, 1995; Gutmanand Niyogi, 2004), and whose genome has recently been sequenced ( Cell division in Chlamydomonasoccursby anoncanonical mechanism, termed multiplefission(Fig.1;SetlikandZachleder,1984;Donnanetal.,1985; John, 1987). The multiple fission cell cycle ischaracterized by a prolonged G1 period during whichcells may grow to many times their srcinal size. Thisgrowth phase is followed by a rapid series of ( n )alternating S (DNA synthesis) and M (mitotic) phases(Coleman, 1982), producing 2 n daughter cells of uni-form size. Size homeostasis is maintained by tworelated mechanisms. A mitotic sizer governs mothercell division number ( n ) so that daughter cell size issimilar regardless of mother cell size. A second sizeroperates during early G1 to control passage throughCommitment (Donnan and John, 1983, 1984), a cellcycle control point that is conceptually similar to Startin yeasts or the Restriction Point in animal cells (John,1984, 1987). Cells that have passed Commitment willdivide at least one time during the S/M phase. Amajor difference between Commitment and Start/Re-striction is that, after passage through Commitment,Chlamydomonas cells remaininG1for anadditional 5to 8 h, whether or not they continue to grow, and only begin the S/M phase after this delay. Under physio-logical conditions of alternating light and darkperiods, Chlamydomonas cells become highly syn-chronized so that growth occurs during the lightperiod and S/M occurs during a brief interval in thedark period (Fig. 2).Given its specialized cell cycle, its unicellular life-style, and its estimated divergence time from higherplants (Chlamydomonas and Arabidopsis [  Arabidopsisthaliana ] may have shared a common ancestor approx-imately 1.1 billion years ago [Hedges, 2002]), it might be expected that the cell cycle genes in Chlamydomo-nas would have diverged considerably from those inhigher plants and other eukaryotes. In this article, wehave identified and profiled the expression of the corecell cycle regulators from Chlamydomonas. Contraryto our initial expectation, we have found that Chla-mydomonas encodes orthologs of the major plantCDK and cyclin families. Chlamydomonas also en-codes two CDK subtypes and two cyclin subtypes thatare not found in higher plants, fungi, or animals.Besides cyclins and CDKs, we have found orthologs of wee1 kinase, a negative regulator of CDKs, CKS1/suc1, a CDK subunit, and potential CDC25 homologs.As expected from the presence of an RB-related gene,  MAT3  (Umen and Goodenough, 2001), we have alsoidentified Chlamydomonas E2F and DP orthologs.Unlike higher plants and animals, most of the corecell cycle regulatory genes in Chlamydomonas arepresent in single copy. We discuss these results in lightof their evolutionary implications and with respect toChlamydomonas as a model for higher plant andmetazoan cell cycles. RESULTSAnnotation Strategy The gene families for which we carried out compre-hensive searches were CDK, cyclin, RB-related, E2F/DP, CKS1/suc1, wee1, CDC25, and CDK inhibitors(CKIs). We first made use of automated annotations based on genome-wide BLASTsearches carried out aspartof the Chlamydomonas genome project and avail-able on the genome Web site ( This initial searchyielded a few high-scoring positives but mostly low-scoring or misannotated genes. A more productivestrategy involved the use of low-stringency reciprocalBLAST searches with representatives from each geneclass to query the conceptually translated Chlamydo-monas genome sequence: Typically, one or moreBLAST hits from Chlamydomonas could be found,and then the BLAST search was repeated with theChlamydomonascandidategenesagainsttheNationalCenter for Biotechnology Information (NCBI) nonre-dundant protein database and against the Chlamydo-monas genome to identify potential duplications oradditional family members. When possible, genemodel and expressed sequence tag (EST) evidencewere used to assemble predicted coding regions inorder to improve the search. Reverse transcription Figure 1.  Diagram of the multiple fission cell cycle. A clock-typediagram depicts the phases of the Chlamydomonas cell cycle. Most of the cycle is spent in G1, which is divided into two periods demarcatedbytheCommitmentpoint(seetextfor details).AttheendofG1,arapidseries of alternating S and M cycles generates 2 n  daughter cells of uniform size. The value of   n   is variable and is related to mother cellsize. Postmitotic mother cells with different values of   n   are depicted tothe right with 2, 4, 8, etc., daughters. Bisova et al.476 Plant Physiol. Vol. 137, 2005  www.plant.orgon September 30, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.  (RT)-PCR was used in some cases to confirm genemodels and to test for expression. Using these strate-gies, we found that a reciprocal orthology relationshipcouldbeestablishedformostofthecellcyclegenes.Forexample, the  CDKB  genes from Arabidopsis show thehighest similarity to a single gene model in Chlamy-domonas, and that gene model in Chlamydomonasshows the highest degree of similarity to the CDKBgenes from higher plants (Fig. 3; Table I). To comple-ment this approach,we also carried out BLASTsearch-esagainsttheconceptuallytranslatedChlamydomonasEST database that includes more than 100,000 se-quences. No cell cycle genes were identified in ourEST search that had not already been identified inthe sequenced genome, suggesting that our search isessentiallycomplete.Low-stringencySouthernblotsof severalgeneswerealsousedtoconfirmthatthekeycellcycle regulators are single copy (Supplemental Fig. 1).Phylogenetic analyses were carried out to more objec-tively place each family member within its properclade. The results from individual gene families arediscussed below, and the results of our annotation aresummarizedinTableI.Foramorecompletedescriptionof gene models and annotation evidence, see Supple-mental Table I. Synchronization and Expression Profiling A major advantage of using Chlamydomonas toinvestigate cell cycle regulation is the ease with whichcultures can be synchronized under physiological con-ditions. By growing cells phototrophically in alter-nating periods of light and dark, we were able tosynchronize our cultures so that they passed Commit-mentduringthemiddleofthelightperiod,enteredtheS/Mphaseofthecellcycleattheendofthelightperiod,andcompleteddivisionduringthedarkperiod(Fig.2).We used semiquantitative RT-PCR to examine theexpressionpatternofasubset ofthegenespredictedto becellcycleregulators.Expressionlevelsforeachgenewere determined in RNA samples prepared fromsynchronous cultures. The cultures from which RNAwas prepared were simultaneously monitored for cellsize (Fig. 2, A and B), passage through Commitment(Fig.2B),passagethroughmitosis(Fig.2,AandB),andhistone H1 kinase activity (Fig. 2C). Besides the anno-tated cell cycle regulatory genes, we also examined ex-pression of some genes encoding proteins required forS phase that were expected to be cell cycle regulated. Figure 2.  Synchronization of the cell cycle with alternating light anddark periods. A, Photomicrographs of cells at different time points witha scale bar in the bottom left corresponding to 10  m m. B, Graph of synchronized cultures. The top image depicts cell size (white squares).The bottom image depicts the fraction of cells that have passedCommitment (white circles), and progression through cell divisionwith mother cells that have undergone one (black circles), two (blacktriangles), and three (black squares) rounds of division. Division wascomplete by 17 h with approximately 60% of the cells dividing threetimes into eight daughters and approximately 40% dividing twice intofourdaughters.Thelight-darkphasingisindicatedbythewhiteorblackbars above the graphs. C, CKS1-purified histone H1 kinase activityassayed from extracts prepared at different time points. Each imageshows an autoradiograph indicating the extent of histone H1 phos-phorylation in the presence and absence of the CKI roscovitine. Chlamydomonas reinhardtii  Cell Cycle GenesPlant Physiol. Vol. 137, 2005 477  www.plant.orgon September 30, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.  CDK Family CDKs are Ser-Thr kinases that function in cell cycleregulation and in other processes such as transcrip-tion. The most widely conserved CDKs possess a ca-nonical PSTAIRE motif in the C-helix (De Bondt et al.,1993). In both fission and budding yeast, a singlePSTAIRE CDK is sufficient to regulate all the cell cyclephases (Mendenhall and Hodge, 1998; Moser andRussell, 2000). By contrast, metazoans and plantsencode several variant CDKs with functions in cellcycle regulation. In humans, there are three PSTAIRECDKs (CDK1/cdc2, CDK2, and CDK3) and a variantCDK4/6 subfamily with a P(I/L)ST(V/I)RE motif, allof which function in cell cycle regulation (Meyersonet al., 1992; Pines, 1995; Reed, 1997; Lee and Yang,2003). Higher plants encode only one PSTAIRE CDK,designated CDKA, that can functionally substitute forits yeast orthologs, cdc2/CDC28 (Ferreira et al., 1991;Hirt et al., 1991). Plant CDKs C, D, and E are variantsthat have metazoan counterparts CDK9, CDK7, andCDK8, respectively (Mironov et al., 1999; Joubes et al.,2000; Vandepoele et al., 2002). Plant-specific kinaseCDKB has been found in all plants and CDKF only inArabidopsis. B-type CDKs are expressed during theG2/M transition when they are thought to function(Mironov et al., 1999; Porceddu et al., 2001; Lee et al.,2003; Boudolf et al., 2004). D- and F-type CDKs areCDK-activating kinases (CAK) that serve to activateA-type CDKs (Umeda et al., 1998; Yamaguchi et al.,1998, 2000; Shimotohno et al., 2003). CDKD bindscyclin H to form a CAK complex (Yamaguchi et al.,2000), while CDKF, a distant relative of CDKD, is abletofunctionwithoutanybindingpartner.AnimalCDK8and CDK9, as well as plant C-type CDKs, have beenimplicated in transcriptional control (Oelgeschlager,2002;Barrocoetal.,2003),andnofunction hasyetbeendetermined for E-type CDKs in plants (Magyar et al.,1997).Chlamydomonas encodes a single ortholog for eachof the plant-type CDKs A, B, C, D, and E (genes weredesignated  CDKA1 ,  B1 ,  C1 ,  D1 , and  E1 , respectively) but does not encode an F-type CDK (Fig. 3; Table I;Supplemental Fig. 1). In addition, Chlamydomonasencodes four novel members of the CDK family thatare not orthologous to any known CDKs in plants orother CDKs in databases. Two of these novel CDKsencoded by genes designated  CDKG1  and  CDKG2  arerelated to each other, but are significantly diverged intheir predicted C-helices (SDSTIRE and AASTLRE, Figure 3.  Neighbor-joining tree of CDKs. Bootstrap values of 50% orhigher are shown for each clade. Am, Antirrhinum majus  ; At, Arabidopsis;Cr,  C. reinhardtii  ; Ce,  Caenorhabditis elegans  ; Dd,  Dictyostelium discoi- deum  ; Dt,  Dunaliella tertiolecta ; Hs, Homo sapiens  ; Os,  Oryza sativa ; Sc,budding yeast; Sp, fission yeast; Pp, Physcomitrella patens.  See Supple-mental Table II for GenBank accessionnumbers. Bisova et al.478 Plant Physiol. Vol. 137, 2005  www.plant.orgon September 30, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.  respectively). The third locus that we have designated CDKH1  encodes a protein with a PVSTIRE motif andforms a sister group with the CDKC family (Fig. 3).However,CDKH1ismoredistantlyrelatedtoChlamy-domonas CDKC1 than are the plant, metazoan, andslime mold CDKC orthologs, indicating that its dupli-cation preceded the divergence of these taxa. Thefourth CDK, encoded by the  CDKI1  gene, is the mostdiverged CDK that we identified and is very distantlyrelated to Arabidopsis CDKF, but it does not appear to beaCDKFortholog.Itdoesnotcontain theN-terminalinsertion that is characteristic of CDKF, and it does notshow a reciprocal best-hit relationship with CDKF inBLASTsearches.The expression profiles of Chlamydomonas CDKsare similar to those of their plant counterparts. mRNAfor  CDKA1  was present constitutively during the cellcycle with expression increasing as cells entered thegrowth phase at the beginning of the light period andincreasing further around the time of S/M phase(Fig. 4). Abundance of presumed ChlamydomonasCDKA protein (reacting with anti-PSTAIR antibody)was relatively constant during the cell cycle withphosphorylation-induced isoforms appearing duringS/M phase (John et al., 1989). Higher plant CDKAmessage and protein levels are also relatively constantduring the cell cycle (Martinez et al., 1992; Hemerlyet al., 1993; Magyar et al., 1997; Richard et al., 2001;Sorrell et al., 2001; Menges et al., 2003). mRNA for CDKB1  shows two peaks of expression, one cor-responding to passage through commitment and asecond, very strong peak during S/M phase (Fig. 4).Elevated expression at the time of mitosis wasdescribed for other members of the CDKB subfamilyin plants (Magyar et al., 1997; Richard et al., 2001;Sorrell et al., 2001; Menges et al., 2002; Menges andMurray,2002)andisconsistentwitharoleforCDKBinthe regulation of the G2/M transition (Porceddu et al.,2001; Lee et al., 2003; Boudolf et al., 2004). CDKC1  and  E1  are expressed constitutively as aretheir orthologs in higher plants and in animals (DeLuca et al., 1997; Magyar et al., 1997; Garriga et al.,1998). The Chlamydomonas-specific CDKs,  G1  and  H1 , are also expressed constitutively at a low level,with  CDKG1  message levels rising somewhat afterCommitment (Fig. 4). Although we were able to am-plify  CDKD1 , its abundance appears to be extremelylow, and the variability of amplification prevents us Table I.  Summary of annotated cell cycle genes in Chlamydomonas  Gene model names are according to Chlamydomonas genome version 2 ( na, Not applicable;Yes, successful amplification and subcloning of RT-PCR fragment; No, amplification was tried but was not successful; nd, RT-PCR was not done. Chlamydomonas Gene Model Gene Name Protein Family Protein Subfamily Signature Motif Putative Function(s) EST RT-PCR 2 C_1630009  CDKA1  CDK CDKA PSTAIRE G1/S, G2/M Yes YesC_1340024  CDKB1  CDK CDKB PSTTLRE G2/M Yes YesC_1730005  CDKC1  CDK CDKC/CDK9 PITAIRE Unknown Yes YesC_700049  CDKD1  CDK CDKD/CDK7 DPTLARE CAK Yes YesC_1700010/11  CDKE1  CDK CDKE/CDK8 SPTAIRE Unknown No YesC_270112  CDKG1  CDK Novel SDSTIRE Unknown Yes YesC_1020028  CDKG2  CDK Novel AASTLRE Unknown Yes NoC_980026  CDKH1  CDK Novel PVTSIRE Unknown No YesC_740039  CDKI1  CDK Novel PDVVVRE Unknown Yes ndC_120118  CYCA1  Cyclin A LVEVSEEY S/G2/M Yes YesC_1420018  CYCB1  Cyclin B HLKF G2/M Yes YesC_460084  CYCC1  Cyclin C Transcription No NoC_140186  CYCD1  Cyclin D G1/S No NoC_290120  CYCD2  Cyclin D LQCDE G1/S Yes YesC_1460039  CYCD3  Cyclin D LFCGE G1/S Yes YesC_570097  CYCL1  Cyclin L Unknown Yes ndC_320095  CYCM1  Cyclin Novel Unknown Yes ndC_660038  CYCU1  Cyclin U Unknown Yes ndC_1740016  CYCT1  Cyclin T Unknown No ndC_1630021  CYCAB1  Cyclin Novel A/B related Unknown No YesC_1840020  MAT3  RBR na G1/S, S/M Yes YesC_180138  E2F1  E2F E2F G1/S, S/M No YesC_570078  DP1  E2F DP G1/S, S/M Yes YesC_210071  E2FR1  E2F E2F? Unknown No NoC_980002  WEE1  WEE1 na G2/M Yes YesNone a CKS1  CKS1 na CDK subunit Yes YesC_980016  RDP1  cdc25-like na HCHGSKVRGP G2/M No YesC_410005  RDP2  cdc25-like na HCMFSQQRGP G2/M No YesC_100077  RDP3  cdc25-like na HCHFSKVRGP G2/M Yes nd a Gene is located between gene models C_860084 and C_860085. Chlamydomonas reinhardtii  Cell Cycle GenesPlant Physiol. Vol. 137, 2005 479  www.plant.orgon September 30, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.
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