Convergent evolution of RFX transcription factors and ciliary genes predated the origin of metazoans

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

The conservation of RFX TFs and ciliary IFT components in selected species. These species were selected to provide a wide sampling of the "tree of life". The phylogenetic relationship between each species was derived from the "Tree of Life Web Project" [44]. Species indicated with "*" have ciliated cells based on published evidence. The 'RFX #' column shows the number of putative RFX TFs identified in this project or reported previously. The grey scale table shows the sequence conservation of individual ciliary components in each species. Darker shade represents higher sequence similarity and conservation. The numbers in each box indicate the percent identity revealed by the alignments between IFT genes and their corresponding human orthologs.

Figure 2

DBDs of RFX TFs are highly conserved. Representative DBDs from humans (hRFX1-7), C. elegans (DAF-19), D. melanogaster (dRFX), S. serevisiae (sRFX1), and M. brevicollis (Mbre_cRFX1 and Mbre_cRFX2). DBDs from different species show high similarity at the peptide level. Nine residues of DBD that directly contact DNA (indicated by arrows) are essentially identical for all RFX TFs.

Figure 3

Predicted protein domains of RFX TFs in representative species. The defining domain of all RFX TFs--DBD--is shown in red. Other domains including the activation domain (green), the B domain (purple), C domain (blue), and D domain (orange) are not present in all RFX TFs. In the left column, Y stands for the budding yeast S. cerevisiae, M for Monosiga brevicollis, C for C. elegans, and D for D. melanogaster.

Figure 4

Sequence alignment between M. brevicollis Mbre_cRFX1 and Human RFX1-3 with all functional domains highlighted. Amino acid residues are color coded with darker color representing higher conservation. Putative functional domains are encircled and labeled.

Figure 5

The phylogenetic tree of all RFX DBDs found in this study. Each distinct group of RFX is labeled. Labels for each putative RFX refer to records in Additional file 1. The colored branches indicate three major groups of RFX TFs: RFX1-3 in red, RFX4-6 in green, and RFX5-7 in blue. The fish RFX8 TFs cluster with the RFX1-3 group, while the fish RFX9 TFs cluster with the RFX5-7 group. All nematodes are grouped together (labeled DAF-19) with the RFX1-3 group. Some insects RFX TFs (labeled as dRFX) group with RFX1-3, while others (labeled dRFX1) with RFX5-7. M. brevicollis (cRFX1 and cRFX2) are shown in open squares (□). Drosophila dRFX2 [14] is shown in the tree but it is not found in the sequenced D. melanogaster genome nor any sequenced Drosophila genome. It is likely located in the heterochromatic region (William Gelbart, personal communication). The phylogenetic tree was inferred using the Neighbor-Joining method [45]. Phylogenetic analysis was performed using MEGA4 [46].

Figure 6

RFX TF-mediated transcription and the origin of metazoans. The common ancestor of metazoans, choanoflagellates, and fungi was likely a ciliated unicellular eukaryote with a single RFX TF. Over the course of evolution, some fungi lost RFX TFs while preserving cilia, some lost cilia but kept RFX, and some lost both. Only a few fungi kept both cilia and RFX. The last common ancestor (LCA) of Monosiga and metazoans preserved both cilia and RFX.