Gene prediction and RFX transcriptional regulation analysis using comparative genomics

Regulatory Factor X (RFX) is a family of transcription factors (TF) that is conserved in all metazoans, in some fungi, and in only a few single-cellular organisms. Seven members are found in mammals, nine in fishes, three in fruit flies, and a single member in nematodes and fungi. RFX is involved in many different roles in humans, but a particular function that is conserved in many metazoans is its regulation of ciliogenesis. Probing over 150 genomes for the presence of RFX and ciliary genes led to the understanding of how RFX-cilia regulatory interaction occurred in evolution. Molecular phylogenetic analysis revealed that RFX is only found in metazoans, in some fungi, and in only one unicellular organism, Monosiga brevicollis. However, ciliary genes did not co-exist with RFX genes except in Allomyces macrogynus and Monosiga brevicollis. The data showed that RFX and cilia evolved independently until the time just before the establishment of metazoans. These results suggest that RFX TFs acquired the role of transcriptional regulation on ciliary genes before metazoans arose and such gain-in-function could be a driving force for metazoan evolution. RFX regulate genes via a regulatory motif called the X-box motif. My laboratory, as well as others, has identified novel RFX target genes in C. elegans. However, accumulating evidence suggest more RFX genes could be uncovered and some of these genes could be regulated by divergent X-box motifs. Additional RFX target genes with divergent X-box motifs were identified in C. elegans by first revising the gene set in C. briggsae, C. remanei, and C. brenneri using a novel homology-based gene finder, genBlastG. Comparing the four genomes with the revised gene set revealed promoter regions with conserved X-box motif in all species except in C. elegans. Detailed examination revealed divergent X-box motifs in these regions. Mutagenesis experiments in the region upstream of F25B4.2 showed that divergent X-box motifs could drive gene expression and may repress gene expression as well. This study provides a deeper understanding regarding the evolution and mechanism of a conserved and important transcription factor.