The evolutionary diversification of LSF and Grainyhead transcription factors preceded the radiation of basal animal lineages

Abstract

Background: The transcription factors of the LSF/Grainyhead (GRH) family are characterized by the possession of a distinctive DNA-binding domain that bears no clear relationship to other known DNA-binding domains, with the possible exception of the p53 core domain. In triploblastic animals, the LSF and GRH subfamilies have diverged extensively with respect to their biological roles, general expression patterns, and mechanism of DNA binding. For example, Grainyhead (GRH) homologs are expressed primarily in the epidermis, and they appear to play an ancient role in maintaining the epidermal barrier. By contrast, LSF homologs are more widely expressed, and they regulate general cellular functions such as cell cycle progression and survival in addition to cell-lineage specific gene expression.

Results: To illuminate the early evolution of this family and reconstruct the functional divergence of LSF and GRH, we compared homologs from 18 phylogenetically diverse taxa, including four basal animals (Nematostella vectensis, Vallicula multiformis, Trichoplax adhaerens, and Amphimedon queenslandica), a choanoflagellate (Monosiga brevicollis) and several fungi. Phylogenetic and bioinformatic analyses of these sequences indicate that (1) the LSF/GRH gene family originated prior to the animal-fungal divergence, and (2) the functional diversification of the LSF and GRH subfamilies occurred prior to the divergence between sponges and eumetazoans. Aspects of the domain architecture of LSF/GRH proteins are well conserved between fungi, choanoflagellates, and metazoans, though within the Metazoa, the LSF and GRH families are clearly distinct. We failed to identify a convincing LSF/GRH homolog in the sequenced genomes of the algae Volvox carteri and Chlamydomonas reinhardtii or the amoebozoan Dictyostelium purpureum. Interestingly, the ancestral GRH locus has become split into two separate loci in the sea anemone Nematostella, with one locus encoding a DNA binding domain and the other locus encoding the dimerization domain.

Conclusions: In metazoans, LSF and GRH proteins play a number of roles that are essential to achieving and maintaining multicellularity. It is now clear that this protein family already existed in the unicellular ancestor of animals, choanoflagellates, and fungi. However, the diversification of distinct LSF and GRH subfamilies appears to be a metazoan invention. Given the conserved role of GRH in maintaining epithelial integrity in vertebrates, insects, and nematodes, it is noteworthy that the evolutionary origin of Grh appears roughly coincident with the evolutionary origin of the epithelium.

Figure 1

Motif architecture of LSF and GRH proteins from 10 metazoan taxa, a choanoflagellate, and two fungi. Conserved motifs were identified using MEME, as described in the methods. Motifs (colored boxes) and inter-motif regions (thick black lines) were drawn to scale except for certain lengthy inter-motif regions, which were truncated by 50% (0.5×). Thin colored lines highlight motif conservation between proteins. The relative relationships among taxa depicted here reflect a general consensus among molecular phylogenetic analyses [62-68], although there continues to be controversy surrounding key elements of the phylogeny including the placement of ctenophores [69] and the monophyly of the triploblasts [70-72].

Figure 2

Top-scoring motif sequences and consensus motif architecture. Metazoan LSF proteins, metazoan GRH proteins, and fungal proteins can be distinguished by their consensus motif architectures (top). The consensus diagrams include all motifs that were found in at least one member of the respective group (Fig. 1). The best matches for each sequence motif identified by MEME are shown below the diagrams. The correspondence between these conserved motifs and known functional domains are indicated by boxes.

Figure 3

Phylogeny of LSF and GRH proteins. The tree shown is based on a neighbor-joining analysis of the amino acids in the gap free alignment. Numbers at nodes represent bootstrap support. The tree is drawn as though rooted between the metazoan sequences and the fungal sequences. Branch length is shown in terms of expected number of substitutions per residue (bar at lower right).

Figure 4

Mapping of Nev-Grh1 and Nev-Grh2 ESTs to separate loci. The NevGrh1 and NevGrh2 loci (enclosed in boxes) are flanked by distinct genes and are found on distinct, non-overlapping genomic scaffolds. Exons are indicated by black boxes, and introns are represented by solid black lines. Dotted lines represent the intergenic sequence leading to the nearest flanking genes. Flanking genes are named by species and NCBI number. The EST contigs for each locus are represented as thick blue lines beneath the exons that encode them. Figure is not to scale.