Common ancestry of heterodimerizing TALE homeobox transcription factors across Metazoa and Archaeplastida

Abstract

Background: Complex multicellularity requires elaborate developmental mechanisms, often based on the versatility of heterodimeric transcription factor (TF) interactions. Homeobox TFs in the TALE superclass are deeply embedded in the gene regulatory networks that orchestrate embryogenesis. Knotted-like homeobox (KNOX) TFs, homologous to animal MEIS, have been found to drive the haploid-to-diploid transition in both unicellular green algae and land plants via heterodimerization with other TALE superclass TFs, demonstrating remarkable functional conservation of a developmental TF across lineages that diverged one billion years ago. Here, we sought to delineate whether TALE-TALE heterodimerization is ancestral to eukaryotes.

Results: We analyzed TALE endowment in the algal radiations of Archaeplastida, ancestral to land plants. Homeodomain phylogeny and bioinformatics analysis partitioned TALEs into two broad groups, KNOX and non-KNOX. Each group shares previously defined heterodimerization domains, plant KNOX-homology in the KNOX group and animal PBC-homology in the non-KNOX group, indicating their deep ancestry. Protein-protein interaction experiments showed that the TALEs in the two groups all participated in heterodimerization.

Conclusions: Our study indicates that the TF dyads consisting of KNOX/MEIS and PBC-containing TALEs must have evolved early in eukaryotic evolution. Based on our results, we hypothesize that in early eukaryotes, the TALE heterodimeric configuration provided transcription-on switches via dimerization-dependent subcellular localization, ensuring execution of the haploid-to-diploid transition only when the gamete fusion is correctly executed between appropriate partner gametes. The TALE switch then diversified in the several lineages that engage in a complex multicellular organization.

Keywords: Archaeplastida evolution; Developmental mechanism; KNOX transcription factor; PBC-homology; TALE-class homeobox; Transcription factor heterodimerization.

Fig. 1

Common origin of heterodimerizing TALE homeobox TFs. Hypothesized homodimerizing proto-TALE protein (top) duplicated before the eukaryotic radiations into animals/fungi/amoebae vs. algae/plants. Lineage-specific diversification soon followed, generating heterodimeric configurations distinct at the phylum-level. (Left) Each lineage possesses one or two classes of potential heterodimeric TALEs, which are summarized onto the eukaryotic phylogeny. A representative species name is given for each analyzed lineage. (Right) Summary of TALE configurations, coupling members of the PBC/PBX/GLX group that shares PBC-homology domains and of the MEIS/KNOX group that shows homology in the KN-A/B domains N-terminal to the homeodomain. Lightly shaded boxes depict homology domains, whose names are provided above. Open areas in the domain boxes indicate the absence of MEINOX-motif for PBX-Red, KN-A for KNOX-Red1 and ELK for KNOX-Red2. Colored vertical lines in the HD indicate two shared introns at 44/45 (orange over “H” in HD) and 48(2/3) (blue over “D” in HD), whose alternating existence between the two groups suggests independent diversification of TALE heterodimerization. HD: Homeodomain; PBL-C: PBL-Chloro; PBL-R: PBL-Red

Fig. 2

Maximum likelihood (ML) phylogeny of the TALE superclass homeodomain in Archaeplastida supports ancient division between KNOX- and non-KNOX TALE groups. The ML trees were generated from the homeodomain alignment with 70 amino acid positions. The consensus tree out of 1000 bootstrap trees is shown. The three numbers at critical nodes show %bootstrap, %SH, and Bayesian posterior probability in support of clades. The tree contains two outgroup clades marked by black squares at nodes, and two Archaeplastida clades, one combining most KNOX sequences marked by the red square and the other combining all non-KNOX sequences marked by the blue square. Vertical bars on the right depict the distribution of outgroup in black, KNOX in red, and non-KNOX sequences in blue. Red dots by the sequence names indicate the presence of KN-A or KN-B domains, and blue dots indicate the presence of a PBC-homology domain. Truncated sequences not available for homology domain analysis are marked with open black boxes. Filled black boxes indicate the absence of a KN-A/B or PBC-homology domain. Proposed classification is indicated by the vertical lines. Dotted vertical lines indicate suggested class members placed outside the main clade for the class in the phylogeny. PBX-Red sequences are found in four separate clades, marked by purple shades on the blue section of the vertical bars. Two PBX-Red sequences marked by the purple square are exceptionally found in the KNOX-Red1 clade, having divergent amino acids at highly conserved positions at Trp19, His23, and Lys31 in their homeodomain, suggesting their false association with the KNOX-Red1. Colors of the sequence names indicate their phylogenetic group: Blue for Glaucophyta, purple for Rhodophyta, green for prasinophytes, light blue for the chlorophytes, orange for Streptophyta, and black for outgroups. The ruler shows genetic distance. Details of the sequences analyzed by this phylogeny are provided in Additional file 1: Table S2. *Gloeochaete_wittrockiana_014496 is considered as a sequence from a bannelid-type amoeba that contaminated the original culture (SAG46.84) for the MMETSP1089 transcriptome. **Association of KNOX-Red2 class sequences to Amorphea PBC sequences is attributed to a shared WFGN motif determining DNA-binding specificity of the homeodomain via convergent evolution

Fig. 3

Archaeplastida non-KNOX group TALEs possess a PBC-like domain (PBL) consisting of N-terminal MEINOX homology and C-terminal PBC-B homology. Amino acid letters in black with gray shades, in white with light shades, and in white with black shades show more than 60%, 80%, or 100% similarity in each column. Inverse red triangles indicate the discarded sequences in un-aligned insertions. a PBL-Glauco domain alignment, including two Glaucophyta sequences sharing homology in both MEINOX homology and C-terminal half of the PBC-B domain with non-Archaeplastida TALE sequences. Red box indicates the ELK domain. b PBL-Red domain alignment. All Rhodophyta non-KNOX sequences possess a PBL domain with poor MEINOX homology. c PBL-Chloro domain alignment. Cyanophora_paradox_20927.63 is included for comparison. Picocystis_salinarum_02499 is a founding member of GLX class with a PBL-Chloro domain. d Comparison among PBL domains. The top row shows the consensus made from the alignment of (a), (b), and (c) combined and the lower consensus sequences are collected from the individual alignments presented in (a), (b), and (c)

Fig. 4

TALE TFs engage in heterodimerization networks between KNOX and non-KNOX groups. The bait constructs conjugated to the GAL4 DNA-binding domain (DBD) and the prey constructs conjugated to the GAL4 transcriptional activation domain (AD) are listed in the table. Construct combinations, numbered 1–8, are arranged in wedges clock-wise, starting at 9 o’clock as labeled in the -LT panels. Confirmed interacting pairs are shown in bold faces in the table. The laminin and T-Antigen (T-Ag) pair, known to be interacting partners, was plated in the 8th sector as a positive control. a Assays using M. commoda TALEs. b Assays using O. tauri TALEs. c Assays using P. salinarum TALEs. KNOX-tr refers to the N-terminal truncated KNOX construct for preventing self-activation. d Detailed construct information is provided in Additional file 1: Table S5