Phylogenetic analysis reveals the evolution and diversification of cyclins in eukaryotes

Molecular Phylogenetics and Evolution 66 (2013) 1002–1010

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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Phylogenetic analysis reveals the evolution and diversification of cyclins in eukaryotes Zhaowu Ma a,1, Yuliang Wu a,1, Jialu Jin b, Jun Yan a, Shuzhen Kuang a, Mi Zhou a, Yuexuan Zhang a,⇑, An-Yuan Guo a,⇑ a Hubei Bioinformatics & Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, PR China b School of Medicine, Hebi Vocational and Technical College, Hebi, Henan 458000, PR China

a r t i c l e

i n f o

Article history: Received 19 July 2012 Revised 5 December 2012 Accepted 10 December 2012 Available online 20 December 2012 Keywords: Cyclins Reclassification Evolution Phylogenetic tree

a b s t r a c t Cyclins are a family of diverse proteins that play fundamental roles in regulating cell cycle progression in Eukaryotes. Cyclins have been identified from protists to higher Eukaryotes, while its evolution remains vague and the findings turn out controversial. Current classification of cyclins is mainly based on their functions, which may not be appropriate for the systematic evolutionary analysis. In this work, we performed comparative and phylogenetic analysis of cyclins to investigate their classification, origin and evolution. Cyclins originated in early Eukaryotes and evolved from protists to plants, fungi and animals. Based on the phylogenetic tree, cyclins can be divided into three major groups designated as the group I, II and III with different functions and features. Group I plays key roles in cell cycle, group II varied in actions are kingdom (plant, fungi and animal) specific, and group III functions in transcription regulation. Our results showed that the dominating cyclins (group I) diverged from protists to plants, fungi and animals, while divergence of the other cyclins (groups II and III) has occurred in protists. We also discussed the evolutionary relationships between cyclins and cyclin-dependent kinases (CDKs) and found that the cyclins have undergone divergence in protists before the divergence of animal CDKs. This reclassification and evolutionary analysis of cyclins might facilitate understanding eukaryotic cell cycle control. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Cell cycle is a series of events that take place in a cell leading to its genome replication and chromosome division to maintain genome integrity. Cyclins are key components of the core cell cycle engine driving cell cycle progression. They comprise a diverse family of proteins that were first discovered in sea urchin eggs and later the homologs were identified in protists, animals and plants (Evans et al., 1983; Murray, 2004). Cyclins share a conserved motif named the cyclin box which is necessary for the binding and activation of cyclin-dependent kinase (CDK) (Murray, 2004). The progression of the cell cycle is controlled by the alternating activity of different groups of CDK/cyclin complexes (Galderisi et al., 2003). Cyclins accumulate and degrade periodically during the cell cycle and determine the timing of the CDK activity (Morgan, 1997). Each cyclin can interact with several CDKs in mammalian cells, and each CDK interacts with a specific subset of cyclins (Dhavan and Tsai, 2001). Depending on their structural similarity and periodic accu⇑ Corresponding authors. Fax: +86 27 8779 3177. E-mail addresses: [email protected] (Y. Zhang), [email protected] (A.-Y. Guo). 1 These authors contributed equally to this work. 1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.12.007

mulation patterns, cyclins are grouped into G1-specific (C-, D-, and E-types) and mitotic (A- and B-types) cyclins (Fung and Poon, 2005; Pines, 1993). Although cyclins are key proteins in the cell division and organism development, their origin and evolutionary process was rarely studied and remains vague. There are at least 28 cyclin genes in human genome and they have been designated into more than 10 types based on their functional relevance and sequence analysis (Malumbres and Barbacid, 2005). At least 43 cyclins were identified in Arabidopsis with putative roles in cell cycle progression (Wang et al., 2004). The plant cyclin nomenclature is based on the functional similarity with the mammalian counterparts (Inze and De Veylder, 2006). A typical animal or plant cyclin contains a conserved region called the cyclin core, which is approximately 250 amino acids and consists of two domains: Cyclin_N and Cyclin_C (Nugent et al., 1991). The Cyclin_N domain in cyclins is about 100 amino acids in length and highly conserved with a CDK-binding site. However, the Cyclin_C domain is less conserved and may be absent in some cyclins (Horne et al., 1996). Previous research proposed that two major groups of cyclins had distinct functions. Cyclins A, B, D and E work with their CDK partners and play pivotal roles in the regulation of cell cycle progression, while another group including cyclins C, H, K, L and T tends to play roles in the transcriptional regulation aided

Z. Ma et al. / Molecular Phylogenetics and Evolution 66 (2013) 1002–1010

by RNA polymerase II (Bregman et al., 2000). Interestingly, TFIIB is a general transcription factor linking the TATA-binding protein (TBP) and RNA polymerase II (Weinmann, 1992). A previous study indicated that TFIIB in proto-Eukaryotes or prokaryotes was close to the common ancestor of cyclins (Gibson et al., 1994). Taken together, cyclins may be derived from relevant components of transcriptional regulation. It would lead to fundamental understanding to explore the origin, evolution, classification, divergence of cyclins and their interrelated proteins (CDKs) in cell cycle. Cyclins are highly conserved proteins identified in plants, animals, fungi and protists (Morgan, 1997; Nurse, 1990). In recent years, as the genome was being sequenced, cyclins in some species were identified and characterized on a genome-wide scale. Briefly, 49 cyclins were identified and categorized into 10 families in the Arabidopsis genome (Wang et al., 2004) and 49 cyclins forming nine families were detected in the rice genome (La et al., 2006). A recent genome-wide study reported that 59 cyclin genes were identified in maize and grouped into six types according to protein domains (Hu et al., 2010). The phylogeny of cyclins has only been reported for a few types of cyclins (e.g., D-Type) (Menges et al., 2007) or in some species (e.g., Arabidopsis or rice) (La et al., 2006; Wang et al., 2004). A recent phylogenomic analysis showed that the four major cyclin families (A-, B-, D- and E-type) in animals and fungi evolving with the amino acid changes on the surface of its binding motif with CDK (Gunbin et al., 2011). However, the evolutionary history about the cyclins evolving from the primitive protists to higher Eukaryotes has not been systematically studied. The current classification (A, B, C and D types etc.) of cyclins is based on the functional relevancy. However the functions of many cyclins have not been characterized. Here we presented a comprehensive classification together with sequence and phylogenetic analysis of cyclins. This analysis was performed to examine their categories across species and explore the evolution of cyclins in Eukaryotes. Furthermore, we proposed a model to show the evolutionary history of cyclins and their evolutionary relationships with CDKs. This reclassification and evolutionary analysis of cyclins might facilitate the cell cycle studies across species.

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JGI and other resources (Davidson et al., 2009). The non-cyclin proteins which also contain a similar Cyclin_N domain, such as CABLES2, CNTD1, and CNTD2, were removed using Blast. 2.2. Phylogenetic analyses We chose the amino acid sequences of the most conserved Cyclin_N domain for phylogenetic analysis. ClustalX (v2.0) (Larkin et al., 2007) were used for the multiple sequence alignment of the Cyclin_N domain and the alignment results were manually refined. Several cyclin proteins were excluded from the phylogenetic analysis because of their incomplete Cyclin_N domain. Maximum likelihood (ML) and Bayesian methods were used to construct phylogenetic trees. Bayesian analysis were performed using MrBayes 3.1.2 for 1,600,000 generations with Markov chain sampling every 100 generations (Ronquist and Huelsenbeck, 2003). The ML analysis was performed using PhyML3.0 (Guindon and Gascuel, 2003) with 100 bootstrap replicates and the best substitution models were determined with the Akaike Information Criterion (AIC) using ProtTest 2.4 (Abascal et al., 2005). The confidence degree of nodes in the phylogenetic trees was performed by bootstrap method with 100 replicates. 2.3. Identification of cyclin motifs Based on the complete protein sequence alignment conducted by ClustalX, we retrieved several conserved motifs manually. The sequence WebLogos of cyclin box motif and signature motif were produced by online server (http://weblogo.berkeley.edu/logo.cgi) based on the alignment results produced by ClustalW. Phosphorylation site was predicted by the GPS (http://gps.biocuckoo.org/1.1/) (Xue et al., 2005), while the PEST region (a hydrophilic domain related to proteolysis) was identified from epestfind website online (http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind). 3. Results 3.1. The distribution of cyclins in Eukaryotes

2. Materials and methods 2.1. Data retrieval The complete proteome data of Caenorhabditis elegans, Ciona intestinalis, Danio rerio, Drosophila melanogaster, Gallus gallus, Homo sapiens and Saccharomyces cerevisiae were downloaded from the Ensembl database (http://www.ensembl.org/). Data of Physcomitrella patens, Chlamydomonas reinhardti, Dictyostelium purpureum, Bigelowiella natans and Leishmania major were downloaded from JGI (http://www.jgi.doe.gov/), while data of Oryza Sativa and Arabidopsis thaliana were downloaded from MSU Rice Genome database (http://www.plantbiology.msu.edu/) and TAIR (www.arabidopsis.org/). HMMER (http://hmmer.wustl.edu/) search using the Pfam profile PF00134 (Cyclin_N domain) and PF02984 (Cyclin_C domain) against the proteome sequences above were performed (Evalue < 1e 4) and the results were manually refined to obtain the cyclin-like proteins. Pfam HMMER model has been used to identify the cyclin-like proteins which serve as the candidate cyclin proteins. Each candidate protein has one or more isoforms because of the alternative splicing. For the Ensembl data, we consider the longest isoform as the representative one and eliminate the redundant protein sequences of a uniform Ensembl gene ID, while CD-HIT (http://weizhong-lab.ucsd.edu/cdhit_suite/cgi-bin/index.cgi) was used to remove the redundancy for those genome data obtained from the

To investigate the evolutionary history of cyclins, we identified the cyclins by characterizing the Cyclin_N domain among the proteomes of representative species in protists, plants, fungi and animals (Table 1, Supplementary Tables S1 and S2). Some cyclins were discovered in primitive eukaryotic species (Bigelowiella natans, Dictyostelium purpureum and Leishmania major). Searching for the Cyclin_N domain was also performed by BLAST against JGI genome databases, NCBI NR database and NCBI microbial genomes (http:// www.ncbi.nlm.nih.gov/sutils/genom_table.cgi), no cyclin sequence was found in prokaryotic species. Our data displayed that plants and animals shared many types of cyclins including A-, B-, C-, D-, F-, H-, I-, L- and T-type, whereas E-, G-, J-, K-, O-, and Y-type cyclins were specific to animals, and Pand SDS type cyclins were unique to plants. Four major classes of animal cyclins (A-, B-, D- and E-type) are involved in cell cycle regulation, and only B- and D-type cyclins are present in protists. In addition, other types, such as C-, H-, L- and T-protist cyclins, are also present in protists (Table 1). It is surprising that the total number of cyclins in the B. natans genome is 23 (nine cyclin B). In plants, the distribution of cyclins in Chlorophyta (Chlamydomonas reinhardtii), a lower unicellular plant, is similar to that in protists. Many types of cyclins in plants emerged from moss (Physcomitrella patens). However, the types and numbers of cyclin genes in rice and Arabidopsis genomes are larger (over 40) than those in lower plants (below 10). In animals, many cyclins were identified from C. elegans to vertebrates. There are 11 cyclins in the C. elegans gen-

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Table 1 Distribution of cyclins among protists, plants, fungi and animals. Kingdom

Species

Animals

Homo sapiens Gallus gallus Danio rerio Ciona intestinalis Drosophila melanogaster Caenorhabditis elegans Saccharomyces cerevisiae Arabidopsis thaliana Oryza sativa Physcomitrella patens Chlamydomonas reinhardtii Bigelowiella natans Dictyostelium purpureum Leishmania major

Fungi Plants

Protists

Group I

Group II

Group III

Total

A

B

D

E

F

G

I

J

O

SDS

CLB

CLN

P

Y

PCL

C

H

L

K

T

SSN

CCL1

2 2 1 3 1 1 – 10 6 8 – – – –

3 1 4 1 2 4 – 9 7 1 2 9 2 3

3 3 5 1 1 1 – 10 13 3 – 2 – 1

2 2 2 2 1 1 – – – – – – – –

1 1 1 – – – – – 6 – – – – –

2 2 2 – 1 – – – – – – – – –

2 2 2 1 – 1 – – – – 4 – – –

2 2 2 – 1 – – – – – – – – –

1 – – 1 – – – – – – – – – –

– – – – – – – 1 1 – – – – –

– – – – – – 6 – – – – – – –

– – – – – – 3 – – – – – – –

– – – – – – – 8 3 3 –

3 2 2 – 1 1 – – – – – 5 6 2

– – – – – – 3 – – – –

1 1 1 – 1 – – 2 1 1 1 2 1 –

1 1 1 1 1 – 1 1 1 2 – – 3 –

2 1 2 1 1 1 – 1 1 1 1 2 1 –

1 1 1 2 1 1 – – – – – 3 1 –

2 1 3 1 1 – – 5 3 3 –

– – – – – – 1 – – – – – – –

– – – – – – 1 – – – – – – –

ome and at least 28 cyclins in human. The number of cyclins in vertebrates is about twice of that in invertebrates. Compared with human and animals, plants possess less cyclin types and more cyclins in each type. In humans, any cyclin types contain no more than three members; however, more than half of cyclin types in Arabidopsis and rice possess at least four cyclins. For example, there are 10 and 13 members of D-type cyclins in Arabidopsis and rice, respectively. On the contrary, only 3 and 5 D-type cyclins are present in human and zebrafish individually. 3.2. Reclassification and phylogenetic analysis of cyclins To carry out a comprehensive evolutionary study on the cyclins, we constructed phylogenetic trees based on Cyclin_N domain alignment in cyclins by maximum likelihood (ML) and Bayesian methods (Figs. 1 and 2). To simplify the tree and show it clearly, we chose some representative species for phylogenetic analysis in Fig. 1: human, zebrafish, fly, yeast, Arabidopsis, moss and protists. Results showed that both phylogenetic trees by ML and Bayesian methods had similar topology. The current classification of cyclins is based on functional similarity but not on sequence similarity and may not reflect the evolutionary process of cyclins. Here, the cyclins can be divided into three major groups based on the phylogenetic tree and were designated as the group I, II and III. As shown in Fig. 1, each group of cyclins formed a relatively separate clade, and the relationship among these clades was also consistent with that of the phylogeny of plants and animals (Fig. 2). In plants, fungi and animals, the group I includes the A-, B-, D-, E-, F-, G-, I-, -J, O-, SDS, CLB and CLN type cyclins. Group II includes the P-, Y- and PCL type cyclins. Group III includes the C-, H-, K-, L-, T-, SSN and CCL1 type cyclins. As shown in Fig. 2, plants contained more cyclins genes than animals, especially for the A-, B- and D-type cyclins, which may indicate that they are more important in plants. Group I contains the majority (60%) of cyclins in plants and animals. Some cyclin genes, such as SDS from Arabidopsis and rice, are distantly related to all other cyclins. In group II, P-type cyclins were present only in plants, and Y-type cyclins were specific to animals. In addition, the group III includes C-, H-, L- and T-type cyclins in plants and animals. Cyclins in group III are more conserved than cyclins in groups I and II. And the cyclins gene numbers of group III are similar in plants (24) and animals (26). K-type cyclins are unique to animals and they are closely related to T-type cyclins in animals. Our analysis indicated that most types of cyclins are present in both plants and animals. As mentioned above, our reclassification of group I, II and III cyclins facilitates evolutionary analysis on diverse cyclins. Since plants, fungi and animals may evolve from different groups of pro-

28 22 29 14 13 11 15 47 42 22 8 23 14 6

tists, how about the groups of cyclins in protists? We performed multi-sequence alignment and constructed a phylogenetic tree for protist cyclins (B. natans, D. purpureum and L. major) (Fig. 3). Similar to Fig. 1 in plants, fungi and animals, these cyclin-like genes are also divided into three groups in protists. Based on tree in Fig. 3, cyclin-like genes in protists were designated as B/D-protist, II-protist and C/H/L/T-protist. The B/D-protist was less divergence in protists, while the II-protist and C/H/L/T-protist groups were expanded in protists. The C/H/L/T type cyclins in plants and animals are involved in the regulation of RNA polymerase II transcription (Oelgeschlager, 2002), which may indicate that the primary function of C/H/L/T-protist is related to transcription regulation. The group I cyclins in protists only contained B and D-types, while group I cyclins in plants, fungi and animals have diverged into more than 10 types. On the contrary, the types of group II and group III cyclins in plants, fungi and animals are similar to those in protists. 3.3. Conserved cyclin domains and motifs The Cyclin_N domain contains about 100 amino acids and spans the CDK-binding region with a conserved cyclin signature of eight amino acids (Wang et al., 2004). To further analyze the functional motifs of cyclins, we characterized several domains and motifs in cyclins and showed them in Table 2, including Cyclin_C domain, cyclin box motif and cyclin signature (Supplementary Tables S1 and S2). Cyclin box is required for cyclins to associate with specific CDKs and regulate their kinase activity during the cell cycle (Murray, 2004). The Cyclin_N domain is the defining domain for cyclins, and present in most of the known cyclins. The Cyclin_C domain is less conserved and absent in some cyclins. H- and P-type cyclins in plants lack Cyclin_C domains. Our phylogenetic trees showed that H- and P-type cyclins are more closely related to each other than related to other types (Fig. 2). In animals, T- and Y-type cyclins also lack Cyclin_C domains. In fungi, exclusive of CLB cyclins, most of cyclins (SSH, PCL, SSN, CLN) lack Cyclin_C domains. Several specific cyclins, such as the human cyclin G1 and G2, Arabidopsis C-, D-, H-, T-, L- and P-type cyclins, have the Cyclin_N domain but not the Cyclin_C domain, suggesting that the Cyclin_C domain may not be critical for their functions. In order to analyze conserved sites in cyclins, we used the WebLogo tool based on the multiple alignments of cyclin box motifs and signature sequences (Supplementary Fig. S1). Both of motifs contain the well-conserved eight amino acids. Almost all of group I cyclins have a conserved Arg (R) residue and a conserved Trp (W) residue in the cyclin box motif, while almost all of the cyclins have a conserved Val (V) residue in the fourth amino acid residue of the signature sequence. The well-conserved cyclin box motif and sig-

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Fig. 1. Phylogram for cyclins investigated in protists, plants, fungi and animals. Numbers associated with branches are the bootstrap support values calculated by maximum likelihood (ML) and the Bayesian methods. The unrooted phylogenetic tree is based on the Cyclin_N domain sequences. ML phylogenetic tree was performed with PhyML 3.0 using the m LG + G model of amino acid substitution with 100 bootstrap replicates. Three major groups of cyclins are separated with black lines. Dicpu: Dictyostelium purpureum; Leima: Leishmania major; Bigna: Bigelowiella natans; moss: Physcomitrella patens; AT: Arabidopsis thaliana; yeast: Saccharomyces cerevisiae; fly: Drosophila melanogaster; zebrafish: Danio rerio; human: Homo sapiens.

nature sequence of cyclins are closely related to bind and activate CDKs. Some other conserved motifs also regulate cyclin functions, such as degradation and phosphorylation. We provided an overview of protein structure and putative CDK phosphorylation sites in plant cyclins (Supplementary Fig. S2). The PEST sequence enriches in Pro [P], Gln [E], Ser [S], and Thr [T] and acts as a signal peptide for cyclin degradation (De Luca et al., 2003). Our results showed that PEST sequence was mainly present in plant B- and D-type cyclins. The majority of the potential phosphorylation sites were identified at the C terminal of D-type cyclins. Strikingly, we also noted that animal CYCL sequences contained many possible CDK phosphorylation sites at the terminus of Cyclin_C domain. Cyclin L associates with a kinase to phosphorylate histone H1, the CTD, and SR protein SC35 (Dickinson et al., 2002). In addition,

CYCB3 and CYCT sequences contain many possible CDK phosphorylation sites, but the potential roles remain unknown.

4. Discussion Cyclins are a family of diverse proteins that play fundamental roles in regulating cell cycle progression in Eukaryotes. Since the discovery of cyclins, many studies have showed that cyclins were highly complex and diverse in different organisms (BuendiaMonreal et al., 2011; La et al., 2006; Nieduszynski et al., 2002; Obaya and Sedivy, 2002). The machinery regulating cell cycle was highly conserved in eukaryotic evolution, while the numbers of cyclins and CDKs were expanded with the species evolution (Murray and Marks, 2001). However, the evolution of cyclins has

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A

B

Fig. 2. The cyclin family in plants and animals. Phylogenetic tree for cyclins in plants (A) and animals (B). Numbers associated with branches are the bootstrap support values calculated by maximum likelihood (ML) method and the Bayesian methods. Two ML phylogenetic trees were performed with PhyML 3.0 using the m JTT + I + G + F (plants)/m LG + G (animals) model of amino acid substitution with 100 bootstrap replicates. AT: Arabidopsis thaliana; rice: Oryza Sativa; moss: Physcomitrella patens; alga: Chlamydomonas reinhardti; human: Homo sapiens; chick: Gallus gallus; zebrafish: Danio rerio; sea squirt: Ciona intestinalis; fly: Drosophila melanogaster; C. elegans: Caenorhabditis elegans.

not been systematically surveyed in Eukaryotes. Here, we undertook a comparative and phylogenetic analysis of eukaryotic cyclins to explore their origin and evolution. We also conducted the evolutionary analysis between cyclins and CDKs to elaborate their evolutionary relationships. 4.1. An evolutionary model of cyclins in Eukaryotes Based on our phylogenetic results, we proposed a scenario to infer the evolutionary history of the cyclins (Fig. 4). Previous study has implied an origin of the cyclins after the archaebacterial/

eukaryotic divergence (Gibson et al., 1994). Here, our phylogenetic analysis indicated that the diversified cyclins can be categorized into three groups in protists, whereas they may emerge with a variety of functions in eukaryotic evolution and become more complex in plants, fungi and animals with increased types and quantities. This model did not include the evolution of some specific cyclins, such as SDS type and F-type in rice, I-type in Chlorophyta. Our reclassification of cyclins indicated that the group I cyclins diverged from protists to plants, fungi and animals. In animals, A-, B-, E-, F-, -J, O-type cyclins derived from the B-protist cyclins of protists. Likewise, plant A-, B- type cyclins and yeast CLB-, CLN-

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Fig. 3. Phylogenetic tree for cyclin-like genes in protists. Cyclin-like genes in protists were divided into three groups, group I formed two relatively clades, group II and III divergence have occurred in protists. ML phylogenetic tree was performed with PhyML 3.0 using the m LG + G + F model of amino acid substitution with 100 bootstrap replicates. The numbers at the branches represent bootstrap values. The IDs of cyclin-like genes for slime molds Dictyostelium purpureum (Dicpu) in JGI and Ensembl database were annotated in the corner, while the Leishmania major (Leima) proteome shows the same ID in JGI and Ensembl database and the Bigelowiella natans (Bigna) proteome is only available in JGI database but not in Ensembl database.

type cyclins derived from the B-protist cyclins. The clade of D-protist cyclins evolved into D-, G- and I- types in animals. Similarly, a large number of D cyclins were expanded in plants. As shown in Table 1 and Fig. 3, B- and D-protist cyclins in protists can be categorized into group I. Our results indicated that cyclin D, G and I also formed a large clade in group I (Figs. 1 and 4). In group II and III, it is obvious that group II-protist and C/H/L/T-protist cyclins have occurred large divergence in early protists. During subsequent evolution, most of cyclins descended from protists to higher Eukaryotes. 4.2. The diversification of cyclins in eukaryotic evolution Cyclins show a great diversification and complexity from protists to plants, fungi and animals. We reclassified all cyclins into three groups with distinct functional characteristics. The group I cyclins in plants and animals play pivotal roles in cell cycle. The group II cyclins contained three types of cyclins and their divergence might be posterior to the eukaryotic speciation. Y-type cyclins were specific to animals, P-type cyclins were unique to plants, and PCLs were exclusively present in fungi (Liu and Finley, 2010; Measday et al., 1997; Torres Acosta et al., 2004). The group III cyclins including cyclin C, H, K, L and T, share common feature that they are involved in the regulation of RNA polymerase II transcription (Bregman et al., 2000). Hence, we reclassified the cyclins based

on the phylogenetic analyses and suggested that the functional evolution of cyclins was coupled with the eukaryotic evolution. 4.2.1. Group I cyclins are extensively expanded from protists to higher Eukaryotes The group I cyclins derived from the B- and D-protist have more members than group II and III because of the large numbers of A-, B-, D- and E-type cyclins in group I. Most of group I cyclins perform crucial functions in cell cycle in higher eukaryotic organisms including plants and animals. The cyclin A/B with corresponding CDKs regulate the S phase progression and the G2/M transition; cyclin D controls the G1/S transition by interacting with CDK4 and CDK6, allowing cells to progress from G1 into the S phase, while cyclin E in animals interacts with CDK2 to promote G1/S transition (Coudreuse and Nurse, 2010; Vandepoele et al., 2002). It has been suggested that some of the A-, B- and D-type cyclins in plants may play similar roles to those in animals (La et al., 2006). Moreover, detailed analysis of plant-specific types of cyclins may potentially uncover plant-specific functions of cyclins, perhaps in the regulation of unique aspects of the plant cell cycle (Wang et al., 2004). In particular, plant cells are not mobile, which may need more diverse cyclins to fulfill the plant-specific regulatory pathways during growth and development (Meijer and Murray, 2001). Our data retrieval showed that B-type cyclins were present from the

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Table 2 List of cyclin conserved motifs. Group

Types

Species

Group I

A-type B1/2-type B3-type D-type E-type F-type G-type I-type J-type O-type SDS-type CLB CLN P-type Y-type PCL C-type H-type K-type L-type T-type SSN CCL1

Plant & Plant & Animal Plant & Animal Plant & Animal Plant & Animal Animal Plant Yeast Yeast Plant Animal Yeast Plant & Plant & Animal Plant & Plant & Yeast Yeast

Group II

Group III

a b

animal animal animal animal animal

animal animal animal animal

C_domaina

Cyclin box motifb

Cyclin signature

Y Y Y Y Y Y Y Y Y Y Y Y N N N N Y Plant: N animal:Y Y Y Plant: Y animal: N N N

MRxILxDW MRxILxDW MRxxLVDW MRxxVAxW MRxILLDW xRYILxDW – xRDExxxW SRxxLxxS – – xRxxLxDW – – xRxxAxLS – KRFYARYS KRFxLxxS HRFYMFHS xRDVINVF HRFYxxxS – –

LxEVx[D/E]EY Lx[E/Q]VHxKF [L/M]VEVQxxF M[L/M][E/Q]VCEEQ L[L/M]EVCEVY LVExxTxK – – – – – [L/R]VxxH[E/N]KF LxxLSVxT – – – LKxIDPxL VMxxHPxx – – – – –

‘‘Y’’: Contain Cyclin C_domain, N: no Cyclin C_domain. ‘‘x’’: In the motif and signature indicates non-conserved amino acid.

lated to A-, B- and D-type cyclins in plants and animals, which suggested that the functions of F-type cyclins might be similar to that of these types (La et al., 2006). Unlike the loci of other types of cyclin genes spreading over the whole genome, most of the F-type cyclins were located on chromosome 2, forming two compact blocks in rice (La et al., 2006). So it is suggested that the F-type genes might originate later than the other types of cyclin genes and perform specific functions in monocotyledons.

Fig. 4. A model for the evolutionary process of cyclins from protists to plants, animals and fungi. The evolutionary diversification of cyclins occurred from protists to higher Eukaryotes. The group I occurred large divergence in eukaryotic evolution, while group II and III were conserved in eukaryotic evolution. Wavy line: large divergence in the evolution, straight line: less divergence or conserved in the evolution.

primordial protists to higher Eukaryotes. The functional specificity of B-type cyclins is determined by their intrinsic activities of targeting CDK1 toward different replication factors (Hu and Aparicio, 2005). Some cyclin types only exist in specific lineages, for example Etype cyclins were present only in animals. E-type cyclins are known as very important regulators in G1/S-checkpoint control in animals. However, E-type cyclins or homologs have not been found in plant kingdom so far, thus, the analogous functions may be substituted by the other cyclins. It was reported that the tobacco A-type cyclin Nicta;CycA3;2 acted as an early G1/S-activated gene, which was analogous to E-type cyclins in animals (Yu et al., 2003). In addition, our results showed that cyclin F shares the high similarity on amino acid sequences with cyclin A (Fig. 1). It has been reported that F-type cyclins were closely re-

4.2.2. Group II cyclins: specificity in eukaryotic speciation The group II cyclins include P-, Y- and PCL type cyclins. P-type cyclins are specific to plants, Y-type cyclins are specific to animals (Table 1), and PCL cyclins are specific to fungi. Cyclin P is a type of ‘CDK-orphan’ cyclin, which does not form cyclin/CDK complex during the cell cycle (Malumbres and Barbacid, 2005). Cyclin P is involved in cell division, cell differentiation, and the nutritional status of the cell (Torres Acosta et al., 2004). The large number of P-type cyclins in plants suggests their essential function in plants. By genome-wide analysis of cyclin family in rice, a numbers of Ptype cyclins were identified and formed a separate clade (La et al., 2006). In Arabidopsis, P-type cyclins were enriched in proliferating cells and mildly expressed in differentiating and mature tissues. Previous researches indicated that cyclin Y orthologs are identified based on the genomes that are fully sequenced in some metazoans, including bilaterians, cnidarians, and the placozoan (Trichoplax adhaerens) (Liu and Finley, 2010). Cyclin Y is also found in the choanoflagellate, Monosiga brevicollis, which is the closest known unicellular relative of metazoans, suggesting that the Y-type cyclins originated prior to occurrence of the multicellular species (Liu and Finley, 2010). Cyclin Y proteins from all of these species share substantial sequence identity and appear to be conserved throughout the metazoans. This suggests that cyclin Y has an important and conserved function in metazoans. In addition, cyclin Y is a membrane-associated cyclin that acts as a cell-cycle regulator in Wnt signaling pathway during G2/M phase (Davidson et al., 2009; Jiang et al., 2009). There is no Wnt signaling pathway in plants, so Y-type cyclins may carry out specific functions in animal kingdoms. We speculated that these prototypical cyclins

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had various roles in gene expression or metabolism in respective species, while they evolved to control cell division in complex eukaryotic cells. Then, as the cyclin family diversified during eukaryotic evolution, many of new members became specialized, performing some specific roles in regulating particular aspects of cell cycle progression in different species. 4.2.3. Group III cyclins: regulate RNA polymerase II transcription Cyclin C, H, K, L and T were classified into the group III cyclins. It is surprising that these cyclins have undergone great duplications in protists and much less divergence in plants and animals. These cyclins were proved to be involved in the regulation of RNA polymerase II-mediated transcription (Bregman et al., 2000). Cyclin K, as a CDK9 regulatory subunit, participates in RNA polymerase IImediated transcription (Fu et al., 1999). The CDK9/cyclin T complex is able to activate gene expression in a catalytic-dependent manner, phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II (De Luca et al., 2003). In the evolution of animal cyclins, cyclin K and the closely related cyclin T originated from T-protist. They interact with CDK9 to form multiple nuclear complexes collectively referred as positive transcription elongation factor b (P-TEFb) (Baek et al., 2007). These functions of cyclin K and T in group III were conserved across different species. 4.3. The evolutionary relationships between cyclins and CDKs CDKs are a family of serine/threonine kinases and are cell cycle engines for the eukaryotic cell cycle progression. All CDK proteins share similar sequences and are activated by relevant cyclins (Morgan, 1995). At early stages of evolution, the eukaryotic cell cycle may be controlled by a subset of extant kinases. Then, a series of duplications of kinase genes occurred during subsequent evolution and the number of kinases including CDKs gradually increases (Krylov et al., 2003). So far, it is unclear that the evolutionary relationship between cyclins and CDKs in eukaryotic evolution. Recently, Malumbres and Barbacid summarized the evolutionary history of CDKs from yeast to human in a comprehensive review (Malumbres and Barbacid, 2009). Here, we supplemented a representative species of protist (Dictyostelium purpureum) and further gained insight into the connection between CDK and cyclins to explore the evolutionary history of CDKs and cyclins in Eukaryotes (Fig. 5). Our results indicated that the divergence of dominating cyclins (group I) has occurred in plants, fungi and animals, while the group II and III cyclins have diverged in protists. On the contrary, most of animal CDKs emerged from invertebrates (C. elegans), and they may be evolved from protists. The number of CDKs and cyclins has increased considerably during the evolution (Malumbres and Barbacid, 2009). The kinases of Cdc28 and Pho85 are activated by

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a large number of cyclins, whereas the other kinases (Ctk1, Sgv1, Kin28 and Srb10) associate with a single specific cyclin. Why have Eukaryotes evolved an increasing number of CDKs? The most plausible explanation is that multi-cellular organisms require additional levels of regulation to maintain genome integrity of a wide repertoire of cell types (Malumbres and Barbacid, 2009). The CDK1–3 originated from CDK1 in protists and mainly interacted with the group I cyclins. The CDK5 plays a simple role in our evolutionary model and interacts with cyclin I in group I and cyclin Y in group II. The CDK7–13, and CDK20 share a common feature interacting with group III cyclins, although they originated from several ancestors individually. Since the CDKs can be divided into three sections according to their binding cyclins, it provides additional evidence to support our reclassification of all cyclins. It has been suggested that multiple autonomous biochemical oscillators entrained each other to an approximate aggregate rhythm in primordial eukaryotes, then cyclin-CDK oscillation could have evolved and yielded a stable PL structure (Lu and Cross, 2010). So it is suggested that diverse cyclins and CDKs may co-evolve in the eukaryotic speciation. The evolutionary processes in cyclins and CDKs might form some specific partnerships to control cell cycle in a coordinated manner. CDK1/2/3 interact with A-, B- and E-type cyclins individually to function throughout G1/S, S and M phase (Morgan, 2008). Remarkably, the CDK4/6 which play crucial roles in G1/S transition cannot be identified in fungi and protists, thus their ancestors were unknown. Their functions can be carried out in yeast by a simple and universal CDK (Cdc28) partnering with various members of CLNs and CLBs. The CDK5 may be evolved from the same kinase in protists and interact with group II cyclins. Pho85 plays a distinctive role in sensing mitogenic signals for G1 progression (Malumbres and Barbacid, 2005). In addition, these CDKs associating with group III cyclins, have close ties with the transcriptional machinery and probably control gene expression through regulation of the RNA polymerase II holoenzyme and transcription factors (Malumbres and Barbacid, 2009). 4.4. Concluding remarks In summary, cyclins may be evolutionarily derived from a common ancestor. Our current results suggested that the ancestral cyclins emerged prior to the divergence of protists. Roberts proposed that cyclins may have first been used to facilitate substrate targeting by the ancestral CDKs, functionally separating them from the signal transduction enzymes and ultimately from each other (Roberts, 1999). The number of CDKs and cyclins has increased considerably during the evolution. They are involved in processes including cell cycle, transcription, and differentiation (Malumbres and Barbacid, 2009). This work indicated that the cyclins, prior to

Fig. 5. The co-evolution between cyclins and CDKs. Malumbres and Barbacid have reviewed an evolutionary model of CDKs from yeast to human in 2009 (Malumbres and Barbacid, 2009). We supplemented a representative species of protist (Dictyostelium purpureum) and designated the cyclins based on CDK partners. D. purpureum, S. cervisiea, C. elegans, D. melanogaster and H. sapiens were chosen as the representative species of protists, fungi and animals.

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