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Evolutionary History of GLIS Genes Illuminates Their Roles in Cell Reprograming and Ciliogenesis

Abstract The GLIS family transcription factors, GLIS1 and GLIS3, potentiate generation of induced pluripotent stem cells (iPSCs). In contrast, another GLIS family member, GLIS2, suppresses cell reprograming. To understand how these disparate roles arose, we examined evolutionary origins and genomic organization of GLIS genes. Comprehensive phylogenetic analysis shows that GLIS1 and GLIS3 originated during vertebrate whole genome duplication, whereas GLIS2 is a sister group to the GLIS1/3 and GLI families. This result is consistent with their opposing functions in cell reprograming. Glis1 evolved faster than Glis3, losing many protein-interacting motifs. This suggests that Glis1 acquired new functions under weakened evolutionary constraints. In fact, GLIS1 induces induced pluripotent stem cells more strongly. Transcriptomic data from various animal embryos demonstrate that glis1 is maternally expressed in some tetrapods, whereas vertebrate glis3 and invertebrate glis1/3 genes are rarely expressed in oocytes, suggesting that vertebrate (or tetrapod) Glis1 acquired a new expression domain and function as a maternal factor. Furthermore, comparative genomic analysis reveals that glis1/3 is part of a bilaterian-specific gene cluster, together with rfx3, ndc1, hspb11, and lrrc42. Because known functions of these genes are related to cilia formation and function, the last common ancestor of bilaterians may have acquired this cluster by shuffling gene order to establish more sophisticated epithelial tissues involving cilia. This evolutionary study highlights the significance of GLIS1/3 for cell reprograming, development, and diseases in ciliated organs such as lung, kidney, and pancreas.

**Ndc1 was separated into two gene models in L. anatina.

Fig. S2 The original NJ tree before rearrangement with ORTHOSCOPE analysis
In ORTHOSCOPE analysis (Fig. S1), an NJ tree was constructed using nucleotide sequences, excluding 3 rd codon positions. A scale bar of branch length indicates substitutions per site.

Fig. S4 An ML tree of GLIS genes using nucleotide sequences.
Using nucleotide sequences of genes selected in ORTHOSCOPE analysis (Fig. S1), an ML tree was constructed. Bootstrap support values for nodes are indicated (n=100). Monophylies of GLIS1/3, GLIS2, GLI, GLI/GLIS1/3, and GLI/GLIS1/3/GLIS2 were strongly supported (>93) as highlighted with magenta. Longer branch length of Glis1 genes in both trees indicates that more substitutions have accumulated in Glis1 after the whole-genome duplication in vertebrates.

Fig. S6 Sequence alignment of Glis1/3 genes demonstrates loss of protein interacting motifs in Glis1
Alignment of amino acid sequences of Glis1 (NP_152629) is also indicated by a red arrow. The dashed green box indicates a SUFU binding motif of Glis1 conserved only in mammals, and located different position from strongly conserved motif in Glis3 (green box). We assume that this motif is not functional. Expression levels of glis1/3 genes are shown in the same format as in Fig. 3.
(A) Early human embryos (Xue, et al. 2013). GLIS1 was filtered from the analysis because its expression levels are very low (average RPKM < 0.5) in all stages. GLIS3 expression is also low throughout early embryogenesis in humans.
(B) Early mouse embryos (Tang, et al. 2011). These data showed results similar to those in Fig. 3A.