Regulatory Factor X (RFX)-mediated transcriptional rewiring of ciliary genes in animals

Abstract

Cilia were present in the last eukaryotic common ancestor (LECA) and were retained by most organisms spanning all extant eukaryotic lineages, including organisms in the Unikonta (Amoebozoa, fungi, choanoflagellates, and animals), Archaeplastida, Excavata, Chromalveolata, and Rhizaria. In certain animals, including humans, ciliary gene regulation is mediated by Regulatory Factor X (RFX) transcription factors (TFs). RFX TFs bind X-box promoter motifs and thereby positively regulate >50 ciliary genes. Though RFX-mediated ciliary gene regulation has been studied in several bilaterian animals, little is known about the evolutionary conservation of ciliary gene regulation. Here, we explore the evolutionary relationships between RFX TFs and cilia. By sampling the genome sequences of >120 eukaryotic organisms, we show that RFX TFs are exclusively found in unikont organisms (whether ciliated or not), but are completely absent from the genome sequences of all nonunikont organisms (again, whether ciliated or not). Sampling the promoter sequences of 12 highly conserved ciliary genes from 23 diverse unikont and nonunikont organisms further revealed that phylogenetic footprints of X-box promoter motif sequences are found exclusively in ciliary genes of certain animals. Thus, there is no correlation between cilia/ciliary genes and the presence or absence of RFX TFs and X-box promoter motifs in nonanimal unikont and in nonunikont organisms. These data suggest that RFX TFs originated early in the unikont lineage, distinctly after cilia evolved. The evolutionary model that best explains these observations indicates that the transcriptional rewiring of many ciliary genes by RFX TFs occurred early in the animal lineage.

Fig. 1.

Schematic diagram depicting the evolutionary conservation of cilia, RFX TFs, and the control of ciliary genes by RFX TFs. Representative organisms from all major eukaryotic phylogenetic supergroups are included. Dashed lines depict supergroups with an uncertain rooting. For each organism, the presence (+) or absence (−) of cilia, RFX TFs, and of ciliary genes that contain X-box DNA footprints or proven X-box promoter motifs, the binding site for RFX TFs, are indicated. The evolutionary loss of cilia is specified only for unikont organisms. All nonunikont organisms listed contain cilia.

Fig. 2.

Multiple-sequence alignment of the 76-residue DNA-binding domain (DBD) of RFX TFs from select unikont organisms. For each organism the presence (+) or absence (−) of cilia and of ciliary genes that contain X-box DNA footprints or proven X-box promoter motifs, the binding site for RFX TFs, are indicated. Structural data were derived from a multiwavelength anomalous dispersion model of human RFX1 (30).

Fig. 3.

Identification of X-box DNA footprints or proven X-box motifs in 1-kb promoter regions of evolutionarily conserved ciliary genes from select unikont and nonunikont organisms. (A) The annotated gene ortholog of every sampled ciliary gene is listed from humans and other ciliated model organisms. Dashes represent genes that are believed to have been lost from the genome of the respective organism. (B) Percent of sampled organisms that contain X-box DNA footprints or proven X-box motifs in ciliary and nonciliary gene promoters from organisms that contain and do not contain RFX TFs. “Expected” ciliary genes contain an experimentally proven X-box promoter motif in C. elegans. “Unknown” ciliary genes either do not contain an X-box promoter motif (KAP3 and IFT74) or are absent from the genome (SPAG6 and CCDC147) in C. elegans. “Not expected” genes are nonciliary and nontarget genes of RFX TFs. The false-positive rate (dashed line) was calculated by averaging the cumulative number of random X-box hits identified in a complete set of all sampled promoters after sequence scrambling. (C) Percent of all “expected” and “unknown” ciliary gene promoters that contain X-box DNA footprints or proven X-box promoter motifs in representative unikont organisms. No RFX TF ortholog was identified in B. dendrobatidis (negative control).