Convergent evolution of RFX transcription factors and ciliary genes predated the origin of metazoans
© Chu et al; licensee BioMed Central Ltd. 2010
Received: 9 December 2009
Accepted: 4 May 2010
Published: 4 May 2010
Intraflagellar transport (IFT) genes, which are critical for the development and function of cilia and flagella in metazoans, are tightly regulated by the Regulatory Factor X (RFX) transcription factors (TFs). However, how and when their evolutionary relationship was established remains unknown.
We have identified evidence suggesting that RFX TFs and IFT genes evolved independently and their evolution converged before the first appearance of metazoans. Both ciliary genes and RFX TFs exist in all metazoans as well as some unicellular eukaryotes. However, while RFX TFs and IFT genes are found simultaneously in all sequenced metazoan genomes, RFX TFs do not co-exist with IFT genes in most pre-metazoans and thus do not regulate them in these organisms. For example, neither the budding yeast nor the fission yeast possesses cilia although both have well-defined RFX TFs. Conversely, most unicellular eukaryotes, including the green alga Chlamydomonas reinhardtii, have typical cilia and well conserved IFT genes but lack RFX TFs. Outside of metazoans, RFX TFs and IFT genes co-exist only in choanoflagellates including M. brevicollis, and only one fungus Allomyces macrogynus of the 51 sequenced fungus genomes. M. brevicollis has two putative RFX genes and a full complement of ciliary genes.
The evolution of RFX TFs and IFT genes were independent in pre-metazoans. We propose that their convergence in evolution, or the acquired transcriptional regulation of IFT genes by RFX TFs, played a pivotal role in the establishment of metazoan.
All metazoans and many unicellular eukaryotes have functional cilia (also known as flagella) . Both motile and immotile cilia (also known as sensory or primary cilia) hold many receptors for sensing environmental signals. Cilia may offer competitive advantages to ciliated organisms by allowing them to avoid predation and also to track nutritionally rich resources . It is thus not surprising that cilia and most ciliary genes are deeply conserved, both in structure and function, in the "tree of life". Such high levels of conservation suggest a common evolutionary origin . Ciliary defects have been associated with defective development in the nematode Caenorhabditis elegans  as well as a growing list of devastating human genetic disease conditions collectively called ciliopathies, including polycystic kidney disease (PKD), Bardet-Biedl syndrome (BBS), Alstrome syndrome, Joubert syndrome, Meckel-Gruber syndrome, and primary ciliary dyskinesia [4, 5]. In mammals, cilia are found on essentially all cell types, highlighting the critical role cilia play . One essential cellular process in cilia is the intraflagellar transport (IFT) that is responsible for the assembly and maintenance of eukaryotic cilia. The IFT machinery consists of four basic molecular modules: (a) motors, (b) Complex A, (c) Complex B, and (d) BBS complex [7, 8].
How IFT genes are regulated at the transcriptional level remained largely unknown until this century when Swoboda and colleagues discovered in C. elegans that many IFT genes are regulated by DAF-19, a RFX type transcription factor . Mutations in daf-19 resulted in defects in cilia development and constitutive dauer formation . DAF-19 binds to X-box motif, which is a highly conserved cis-regulatory element first discovered in mammals [3, 9]. Ciliary genes in C. elegans often contain one or more putative X-box motifs 100 bp - 250 bp upstream of the coding sequences [3, 4, 10, 11]. In addition, ciliary genes and cilia development in the fruit fly Drosophila melanogaster were also suggested to be regulated by RFX TFs . Two RFX genes dRFX and dRFX2  have been identified in D. melanogaster. dRFX was identified through a homology search for the RFX DNA binding domain (DBD) and dRFX2 was identified through yeast-one-hybrid (Y1H) screening for transcription factors that bind to a putative promoter sequence [13, 14]. Notably, dRFX2 has not been found in the D. melanogaster genome sequences, suggesting that it is likely located within the heterochromatin regions (William Gelbart, personal communication).
RFX TFs were first identified in mammals as binding proteins of the X-box motif . Through bioinformatics searches and molecular characterization, seven RFX genes--RFX1-7 have been found in mammals [16, 17]. Different mammalian RFX genes show differential but overlapping expression patterns , suggesting that they have complementary and cooperative roles in regulating genes in many different biological pathways. Indeed, mammalian RFX TFs have been shown to interact with each other and with many additional co-factors . Accumulating evidence confirms that RFX genes regulate development and function of cilia in mammals as well. For instance, RFX3 knockout in mice led to abnormal cilia development in both brain  and pancreas .
Outside of metazoans, however, there is no evidence suggesting that IFT genes are regulated by RFX TFs. No RFX TFs have been reported in the green alga Chlamydomonas reinhardtii, a popular model organism for studying cilia biology. Conversely, RFX TFs exist in organisms including the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe that do not have cilia , suggesting that RFX TFs do not regulate ciliary genes in these organisms. Based on these observations, we hypothesize that IFT genes and RFX TFs evolved independently and that their evolution converged at some point. To test this hypothesis, we have identified and examined IFT genes and RFX TFs in hundreds of fully sequence genomes that have become available recently.
Molecular evolution of ciliary genes
Molecular evolution of RFX TFs
We found candidate RFX TFs in all sequenced metazoan genomes (Figure 1). In addition to the RFX TFs that have been reported previously, including seven RFX TFs found in mammals , DAF-19 in C. elegans , and dRFX , we found many RFX genes that have not been described previously. We have identified seven RFX genes (RFX1-7) in all vertebrate genomes except fish genomes, which have nine putative RFX genes (RFX1-9). We have also identified four RFX genes in Ciona intesttinalis, six in the purple sea urchin (Strongylocentrotus purpuratus), and five in the sea anemone (Nematastella vectensis). In D. melanogaster, in addition to the two RFX genes reported previously--dRFX and dRFX2, we have identified a novel RFX TF, which we named dRFX1. Interestingly, among all metazoans examined, nematodes including C. elegans are the only organisms that possess just one RFX gene.
RFX TFs are also found in some non-metazoans. Of the 51 fungus species examined, we identified single RFX TFs in 44 species, including the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe, as previously reported , as well as a ciliated fungus Allomyces macrogynus, whose genome was recently sequenced by the Fungal Genome Initiative of the Broad Institute http://www.broadinstitute.org/annotation/fungi/fgi/. All unicellular organisms we have examined possess either one RFX gene (fungi) or none except for the choanoflagellates. For example, M. brevicollis, which was recently sequenced , contain two genes (Mbre_cRFX1 and Mbre_cRFX2) with well-defined RFX DBDs.
RFX DBD sequences are the defining features of all known RFXs and show high similarity (>40% PID) to each other. However, there are a small number of additional proteins that contain domains that show weaker similarity (<30% PID) to known RFX DBDs. In particular, a gene (ARID2) in the human genome contains a RFX-like domain that shows 29% PID to the human RFX1 DBD. Among the nine residues that have direct contact with DNA sequences, five can be found in the RFX-like domain found in ARID2. ARID2, whose function as a transcription factor has not been well studied, has orthologs in all mammals as well as other vertebrates (data not shown). Additionally, a gene in M. brevicollis also shows weak similarity (27%) to known RFX DBDs (five of the nine residues that have direct contact with DNA are conserved). We name this novel gene Mbre_cRFX3. Because of their low similarity to known RFX DBDs, these RFX like genes--ARID2 and Mbre_cRFX3--are not regarded as RFX TFs in this project and thus are not examined further. No RFX genes have been found in any bacteria, ancient bacteria, or plants (Figure 1).
In the inferred phylogenetic tree, the nematodes are the only metazoans that have only one RFX TF--DAF-19, which groups together with the mammalian RFX1-3 group (Figure 5). It was proposed previously that prior to the complete sequencing of the C. elegans genome, more RFX TFs should exist in C. elegans . However, exhaustive searches of the completed C. elegans genome revealed no traces of additional RFX genes, suggesting that RFX genes corresponding to other RFX groups (RFX4-6 and RFX5-7) were lost in the last common ancestor of the nematode species. In fact, none of the seven sequenced nematode genomes have more than one RFX TF (Additional file 1).
Evolutionary relationship between ciliary genes and RFX TFs
The above comprehensive identification of IFT genes and RFX TFs shows clearly that all metazoans have both ciliary genes and RFX genes. Since IFT genes have been demonstrated to be regulated by RFX TFs in C. elegans, D. melanogaster, and humans, IFT genes in all metazoans are likely regulated by RFX TFs. Our analysis strongly suggests that IFT genes and RFX TFs evolved independently. In addition to the budding yeast (S. cerevisiae) and fission yeast (S. pombe), we have identified 41 fungus species that have single RFX genes but no IFT genes, thus RFX genes in these species do not regulate ciliary genes expression. Indeed, Crt1/RFX in the budding yeast plays a role in DNA damage response . Outside of metazoans, only two sequenced genomes have both IFT and RFX genes, the choanoflagellate M. brevicollis and the fungus A. macrogynus. Outside of metazoans, choanoflagellates, and fungi, none of the sequenced genomes possess a single RFX gene, regardless of the possession of IFT genes.
This is the first project to comprehensively identify and compare RFX TFs in the entire "tree of life" since Emery and colleagues described RFXs in humans (RFX1-5), mice (RFX1-3 and RFX5), C. elegans, and the budding and fission yeasts domains more than a decade ago . In this paper, we identified for the first time (1) nine RFX genes in all sequenced fish genomes; (2) two RFX genes in the choanoflagellate M. brevicollis genome; (3) single RFX genes in many fungus genomes. Additionally, we have identified RFX genes in many vertebrates. Furthermore, we have identified a third RFX (dRFX1) in the fruit fly D. melanogaster. Based on our phylogenetic analysis of all RFX TFs identified in the "tree of life", we have confirmed the hypothesis proposed by Emery and colleagues that C. elegans has lost RFX genes as it evolved .
The evolution of multicellular metazoans from a unicellular protozoan ancestor represents a major and what we consider to be the most spectacular transition in the "history of life". This transition is demonstrated by the abrupt appearance of a huge variety of metazoans in the fossil record approximately 560 million years ago during the Cambrian explosion . Many environmental, ecological, and other evolutionary factors have been proposed to have contributed to this transition [36, 37]. Great efforts have been made to understand this transition by studying protein-coding regions of numerous genes and gene families that are ubiquitous in and limited to metazoans. Findings obtained in these studies showed that many genes and gene families previously found to be expressed only in metazoans are also found in choanoflagellates giving evidence that metazoans arose from choanoflagellates. For example, work by King and colleagues clearly demonstrated that choanoflagellates have a receptor tyrosine kinase that is found in metazoans but not in other eukaryotes . Manning and colleagues searched the sequenced choanoflagellates M. brevicollis genome , and identified a highly elaborate tyrosine kinase signaling network . Many additional genes are shared by M. brevicollis and metazoans, including cadherin, which are essential for metazoan development , and transcription factors such as P53 and Myc . These findings encouraged additional large scale searches, including the UNICORN (unicellular opisthokont research initiative) project , for genes and gene families critical for the transition from unicellularity to multicellularity. However, accumulating evidence is showing that these genes predated the origin of metazoans and played different roles from their counterparts in metazoans. Thus these genes, even though some have been co-opted to perform novel functions in metazoans, are probably not be the main driving force underlying the transition from unicellular protozoans to multicelluar metazoans.
What then was the main factor driving this transition? In contrast to coding sequences of genes, which are usually under strong purifying selection, regulatory sequences show much more rapid evolution. Compelling evidence suggests that changes in cis-regulatory sequences and transcriptional regulation in general play a pivotal role in evolution [37, 40]. Kingsley and colleagues recently identified changes in cis-regulatory modules that dictate dramatic changes in pigmentation in sticklebacks and humans . Thus the transition from unicellular flagellates to multicellular metazoans may have been driven by innovations at the transcriptional level. The convergent evolution of RFX TFs and ciliary genes (IFT genes in particular) in the common ancestor of metazoans and choanoflagellates prompt us to propose that the acquired tight control of ciliary genes at the transcription level by RFX TFs served as one of the critical driving forces in the establishment of multicellularity and the rise of metazoans.
RFX TFs and IFT genes evolved independently in pre-metazoans and their convergence, or the acquired transcriptional regulation of IFT genes by RFX TFs, may have played a pivotal role in the establishment of metazoan.
All sequence data (both genomic DNA sequences and gene annotation data including cDNA and protein sequences) were downloaded from public databases. The list of genomes and the data source are described in Additional file 2. The initial set of DNA binding domains that were used as queries for BLAST searches were taken from Human RFX1-7 , C. elegans DAF-19 , D. melanogaster dRFX , and yeast RFX1 .
Identification of RFX TFs
We carried out similarity searches using WU-BLAST (version 2.2.6; http://blast.wustl.edu) with e-value 0.01 and without sequence filter (option -F). The initial set of DBDs was used as query to search against all the mammalian proteomes (entire collection of protein peptides). The resulting DBDs were added to the query list and used to search against arthropods. The iteration of adding DBD and blasting continues until all species have been searched. A hit is accepted as a candidate DBD if the corrected percent identity over the entire domain length is >= 40%. The corrected percent identity was calculated as the number of identical positions divided by total length of the query. We also searched for candidate RFX TFs in genome sequences (DNA sequences) to ensure that no RFX TFs have been missed in the gene annotations.
Identification of ciliary genes
We carried out similarity searches using WU-BLAST (version 2.2.6; http://blast.wustl.edu) with e-value 0.01 and without sequence filter (without -F). Human protein sequences were taken from NCBI and used as queries (See accession number in Additional file 3). PID was calculated as the number of identical amino acids reported by WU-BLAST over the entire length of the query.
Phylogenetic analysis was done using MEGA4 . Multiple sequence alignment was done using CLUSTALW (included in META4) with default settings. Phylogenetic trees were inferred using the Neighbor-Joining method.
Functional domain identification and analysis
Sequences for activation, B, C, and D domains were taken from previous publications. The multiple sequence alignment was performed for each domain and used as input for hmmbuild to generate a HMM profile for each domain. hmmsearch was used to scan the proteome of selected species to find regions of similar profile. Both hmmbuild and hmmsearch are part of the HMMER suite http://hmmer.janelia.org.
We thank Syed Aftab and Lucie Semenec who participated in the early phase of this project. We thank B. Brandhorst, L. Quarmby, R. Johnsen, M. Tarailo-Graovac, C. Frech, and I. Vergara for critical reading of the manuscript and for their suggestions. This project is supported by a Discovery Grants (to D.L.B. and N.C.) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. J.S.C.C. holds an NSERC doctoral fellowship. D.L.B. is a Canada Research Chair in Genomics. N.C. is a Michael Smith Foundation for Health Research (MSFHR) Scholar and a Canadian Institutes of Health Research (CIHR) New Investigator.
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