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

<jats:title>Abstract</jats:title><jats:p>Homeobox transcription factors (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, representing remarkable functional conservation of a developmental TF across lineages that diverged one billion years ago. To delineate the ancestry of TALE-TALE heterodimerization, 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. These results indicate that the TF dyads consisting of KNOX/MEIS and PBC-containing TALEs must have evolved early in eukaryotic evolution, a likely function being to accurately execute the haploid-to-diploid transitions during sexual development.</jats:p><jats:sec><jats:title>Author summary</jats:title><jats:p>Complex multicellularity requires elaborate developmental mechanisms, often based on the versatility of heterodimeric transcription factor (TF) interactions. Highly conserved TALE-superclass homeobox TF networks in major eukaryotic lineages suggest deep ancestry of developmental mechanisms. Our results support the hypothesis 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, a system that then diversified in the several lineages that engage in complex multicellular organization.</jats:p></jats:sec>

section_content: The homeobox transcription factors (TFs) are ubiquitous in eukaryotes, carrying a DNAbinding homeodomain typically 60 amino acids, that folds into three -helices [1]. The atypical or TALE (Three Amino acid Length Extension) superclass of homeobox TFs shares a three-amino-acid insertion between helix 1 and 2 and plays essential roles during embryonic development by participating in interactive TF networks. In animals, MEIS-and PBC-class TALE proteins, such as Meis/Hth and Pbx/Exd, form heterodimers that in turn form ternary complexes with HOX-class homeobox TFs, determining cellular fates along the anterior-posterior axis of the developing embryo [2, 3]. In plants, the interacting KNOX-and BELL-class TFs in the TALE group play critical roles during organ formation and the vegetative-to-reproductive transition in the undifferentiated cell mass known as the shoot apical meristem [4, 5]. 

The heterodimerization of TALE proteins serves as a trigger for precise execution of developmental programs. Prior to heterodimerization, animal PBX proteins are localized in the cytosol, and upon binding to MEIS, they translocate to the nucleus [6, 7]. Similar heterodimerization-dependent translocation is also observed for KNOX-BELL pairs in the plant Arabidopsis, implying that this mechanism is a conserved regulatory feature of TALE proteins [8]. In addition, TALE proteins differ in their DNA-binding specificity [9, 10], which is primarily determined by the homeodomain residues at positions 47, 50, and 54 [11], and heterodimerization increases target affinity by bringing two such DNA-binding domains together. TALE-heterodimerization is mediated by class-specific homology domains located on the Nterminal side adjacent to the homeodomain [12, 13]. Animal MEIS and plant KNOX class proteins share readily identifiable homology in their heterodimerization domain, leading to the proposal of an ancestral TALE class named MEINOX [12]. In contrast, their partner classes --PBC and BELL --exhibit no apparent homology in their heterodimerization domains. Short shared sequence motifs and common secondary structures have been found within the heterodimerization domains between MEINOX and PBC or BELL [14, 15], but their extent of the homology requires adequate taxon sampling to recover ancestral relationships. 

An ancestral functions of TALE-TALE heterodimerization was revealed in studies of the unicellular green alga Chlamydomonas reinhardtii: the KNOX ortholog GSM1 and a second TALE protein GSP1 form heterodimers immediately after the fusion of sexual gametes, and these drive the haploid-to-diploid transition by activating >200 diploid-specific genes and inactivating >100 haploid-specific genes [10, 16, 17]. In subsequent studies, plant-type TALE-TALE heterodimers between KNOX and BELL were shown to be required for the haploid-todiploid transition of the moss Physcomitrella patens [18, 19]. Given the conserved role of TALE heterodimerization as a developmental switch in the sexual life cycle of the plant lineage, understanding its origins and diversification promises to shed light on the evolution of developmental mechanisms during eukaryotic radiation and the emergence of land plants. 

To delineate the ancestry of plant-type TALE heterodimerization, we performed a phylogenetic and bioinformatics analysis of TALE TFs in the three algal radiations of the Archaeplastida supergroup, the descendants of a single endosymbiosis event > one billion years ago [20, 21]. Our analysis showed that the TALEs were already diversified into two groups at the origin of Archaeplastida, one sharing KNOX-homology and the other sharing PBC-homology. Together with our protein-protein interaction data, we propose that all TALE classes participate in heterodimerization networks via the KNOX-and PBC-homology domains between the two ancestral groups.
section_name: Introduction

section_name: Results

section_content: The Archaeplastida consists of three monophyletic phyla [22, 23] (Fig 1 ). 1) Viridiplantae include two divisions, Chlorophyta --chlorophytes and prasinophytes (a paraphyletic group of seven lineages [24] ) --and Streptophyta --charophyte algae and land plants [25]. 2) Rhodophyta (red algae) include diverse unicellular and multicellular organisms that diverge into four major lineages [26] (S1 Spreadsheet). 3) Glaucophyta members include only four cultured genera and possess plastids that carry ancestral features of the cyanobacterial symbiont that gave rise to photosynthetic organelles in eukaryotes [27]. 

To collect all the available homeobox protein sequences, we performed BLAST and Pfammotif searches against non-plant genomes and transcriptome assemblies throughout the Archaeplastida (S1 Spreadsheet), identifying 327 proteins from 55 species as the Archaeplastida homeobox collection (29 genomes and 18 transcriptomes; S2 Spreadsheet). 

Of these, 102 possessed the defining feature of TALE proteins, a three-amino-acid insertion between aa positions 23-24 in the homeodomain [28] 2) The non-KNOX group, including the BELL and GSP1 homologs, contained clades of mixed taxonomic affiliations. These analyses showed that the TALE proteins had already diverged into two groups before the evolution of the Archaeplastida and that the KNOX-group is highly conserved throughout Archaeplastida.
section_name: TALEs in Archaeplastida are divided into two groups, KNOX and non-KNOX

section_name: KNOX group sequences share the same heterodimerization domains throughout

section_content: The next question was whether the plant KNOX class originated prior to the Viridiplantae phylum. The plant KNOX proteins and the Chlorophyta GSM1 possess KNOX-homology sequences, consisting of KN-A, KN-B and ELK domains, required for their heterodimerization with other TALE proteins [10] ; therefore, the presence of the KNOX homology sequences suggest functional homology to the plant KNOX class. To collect homology domains without prior information, we performed ad-hoc homology domain searches among the KNOX group sequences. Using the identified homology domains as anchors, we carefully curated an alignment of the KNOX-group sequences combined with any other TALE sequences with a KNOX-homology, (S2 Fig). From this KNOX alignment, we defined KNOX-homologs as having amino acid similarity scores >50% for at least two of the three domains comprising the KNOX-homology region (S3 Spreadsheet for calculated domain homology). Using this criterion, all KNOX group sequences (excluding partial sequences) possessed the KNOX homology (
section_name: Archaeplastida

section_content: In Viridiplantae, we found a single KNOX homolog in most Chlorophyta species, whereas Available TALE sequences were limited for the Glaucophyta. We found a single KNOX homolog in two species, which possessed KN-A and KN-B domains but lacked an ELK domain. We termed these KNOX-Glauco.
section_name: KNOX classes diverged independently among the algal phyla

section_content: Following the identification of KNOX homologs, the non-KNOX group in the Archaeplastida was redefined as lacking KN-A and KN-B domains. Further classification of the non-KNOX group was challenging due to its highly divergent homeodomain sequences. However, we noticed that the number of non-KNOX genes per species was largely invariable: one in most Rhodophyta and Glaucophyta genomes and two in the majority of Chlorophyta genomes, suggesting their conservation within each radiation. 

Our ad-hoc homology search provided critical information for non-KNOX classification, identifying a homology domain shared among all Glaucophyta and Rhodophyta non-KNOX sequences (Fig 3A and 3B ). Since this domain showed a similarity to the second half of the animal PBC-B domain (Pfam ID: PF03792) known as heterodimerization domain [12], we named this domain PBL (PBC-B Like). Accordingly, we classified all the non-KNOX TALEs Two divergent non-KNOX classes in Chlorophyta led to a critical question about their dyadic networks. Previously studies had shown that TALE heterodimers required interaction between MEIS and PBC domains in animals and between KNOX and PBL domains in Chlamydomonas [6, 10]. It was, therefore, predicted that all Glaucophyta and Rhodophyta TALEs form heterodimers via their KNOX-and PBL-homology domains. On the other hand, it remained to be tested whether the Chlorophyta TALEs lacking a PBL-domain can form heterodimers with other TALEs. 

To characterize interaction network of TALE class proteins in Chlorophyta, we selected three prasinophyte species for protein-protein interaction assays: two species containing Mam-A and Mam-B genes (Micromonas commoda and Ostreococcus tauri), and another species (Picocystis salinarum), whose transcriptome contained one GLX and one Class-B sequence. 

In all three species, we found that KNOX homologs interacted with all examined non-KNOX proteins in Mam-A, Mam-B, Class-B, and GLX class (Fig 4A -4C ). No interaction was observed between the two non-KNOX proteins in any of the three species (Fig 4A -4C ). 

Similar to the GLX-KNOX heterodimerization, Mam-A and Mam-B also required additional domains outside the homeodomain for their heterodimerization with the KNOX homologs (S8 Fig). These results showed that the all divergent non-KNOX TALEs maintained their original activity to form heterodimers with the KNOX homologs. Observed interacting network among the TALE sequences is summarized in S9A Fig.
section_name: Non-KNOX group TALEs possess animal type PBC-homology domain, suggesting a shared ancestry between Archaeplastida and Metazoa

section_content: Our discovery of the PBC-homology in Archaeplastida suggests common ancestry of the heterodimerizing TALES between Metazoa and Archaeplastida. It also predicted that other eukaryotic lineages might possess TALEs with the PBC-homology. Outside animals, the Pfam database contains only two PBC-B domain-harboring sequences, one from a Cryptophyta species (Guillardia theta, ID_137502) and the other from an Amoebozoa species (Acanthamoeba castillian, ID:XP_004342337) [31]. We further examined the Excavata group, near to the posited root of eukaryotic phylogeny [22]. A search of two genomes (Naegleria gruberi and Bodo saltans) collected 12 TALE homeobox sequences in N. gruberi, and none in B. saltans, of which we found one with a PBC-homology domain
section_name: TALE heterodimerization evolved early in eukaryotic history

section_content: The ubiquitous presence of dyadic TALEs raised next question: Are all the dyadic TALEs reported in this study the descendants of a single ancestral dyad, or do they result from lineage-specific evolution from a single prototypical TALE (proto-TALE) that does not engage in heterodimerization. To probe deep ancestry, we examined intron-retention, this being regarded as a long-preserved character and less prone to occur by homoplasy (a character displayed by a set of species but not present in their common ancestor) [32]. Five
section_name: Intron-retention supports the parallel evolution of the heterodimeric TALE classes during eukaryotic radiations

section_content: . CC-BY 4. 0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (which was this version posted August 10, 2018. ; https://doi. org/10. 1101/389700 doi: bioRxiv preprint 10
section_name: Discussion

section_content: Our study shows that all three Archaeplastida phyla possess TALEs, diverged into two groups with distinct heterodimerization domains, the KNOX group with KN-A/KN-B domains and the PBX (or GLX) group with PBL domains. The similarity between the KNOX/PBX and the animal MEIS/PBC dyads led us to identify homologous heterodimerization domains in the TALEs of other eukaryotic lineages including Excavata. Based on our findings, we hypothesize that the TALE heterodimerization arose very early in eukaryotic evolution. 

During > 1 BY of Archaeplastida history, TALE TF networks have undergone three duplication events compared to the simple dyadic TALEs in Glaucophyta. In Viridiplantae, the KNOX class persists as a single member throughout the mostly unicellular Chlorophyta, whereas it duplicated into KNOX1 and KNOX2 in the multicellular Streptophyta [33]. In
section_name: TALE endowment in Archaeplastida

section_content: The BELL class is the only non-KNOX class in land plants, sharing a POX (Pre-homeobox) domain (PF07526) [13] and lacking an identifiable PBL domain. The K. flaccidum genome, the only genome available in the charophyte lineage from which land plant emerged, contained three non-KNOX sequences, all possessing a PBL domain ( Therefore, the lack of PBL-homology in the plant BELL class appears to be due to divergence or domain loss from an old charophyte class that had PBL-homology. We found an intron at the 24[2/3] homeodomain position of a K. flaccidum GLX homolog, which was previously identified as being specific to the plant BELL class (S8A Fig) [12], suggesting that the plant BELL class evolved from an ancestral GLX gene. More taxon sampling in charophytes is needed to confirm this inference.
section_name: Is the plant BELL class homologous to the Chlorophyta GLX class?

section_content: We found two conserved intron positions and shared sequence motifs between the KNOXand PBX-groups, generating our hypothesis that a proto-TALE protein initially engaged in homodimerization and then duplicated and diversified into two heterodimerizing classes (Fig [10, 34], coupled with numerous combinations of distinct DNA-binding modules that fine-tune target specificity, then generated customized transcription-on switches.
section_name: What would have been the critical drivers of TALE heterodimerization networks emerging from ancestral homodimers?

section_content: During sexual development, it is critical to accurately detect the fusion of two cells before initiating diploid development and to make sure that the mating combines correct partner gametes. TF heterodimerization can implement both steps if one TF partner is contributed by each gamete. In fact, TALE heterodimerization plays a central role as a developmental switch for the haploid-to-diploid transition in green algae and land plants [10, 19]. A similar haploid-to-diploid transition triggered by TF heterodimerization has recently been documented in Dictyostelium [35] and is well described in Basidiomycete fungi that utilize non-TALE homeobox proteins such as bW and bE [36, 37]. 

Discovery of new prokaryotic life forms, especially in the Archaea domain, suggests that multiple symbiotic mergers of different life forms evolved into the proto-eukaryotes, possibly first as a symbiotic community, which then evolved into the last eukaryotic common ancestors (LECA) that rapidly diverged into the eukaryotic supergroups [38] [39] [40]. This eukaryogenesis model predicts that the proto-eukaryotes  LECA transition required the faithful transmission of traits between progenitor cells and their progeny to evolve as individual lineages by Darwinian selection. Under this hypothesis, we anticipate that the generation of the LECA may have been driven by the sexual mechanisms that distinguish a cellular merger between the common descendants from a merger between unrelated community members. Our proposal for the evolution of heterodimeric TALEs from the homodimeric proto-TALE may provide one of the necessary mechanisms for the first sexual mode of reproduction that might have driven the generation of the LECA from its protoeukaryotic ancestors.
section_name: 1, S9A Fig). Heterodimerization-dependent subcellular localization

section_content: Plant studies have shown that the duplicated KNOX classes serve distinct functions: the plant KNOX1 class regulates the differentiation of an undifferentiated cell mass into spores in mosses or leafy organs in vascular plants, and the plant KNOX2 class regulates the transition from haploid gametophytes to diploid sporophytes in mosses and controls secondary cell wall development in vascular plants [18, [41] [42] [43]. We propose that the duplicated TALE heterodimers in the Streptophyta allowed independent regulation of cellular differentiation and life cycle transitions, priming the emergence of land plants by expanding the diploid phase of their life cycle from a dormant zygospore to a multicellular individual bearing many meiotic spores. The repertoire of TALE heterodimers continued to expand during land plant evolution, serving all the major organ differentiation programs in the diploid phase of their life cycle. 

Can a similar expansion of TALE heterodimers be found during Metazoan evolution? Our search for TALE TFs in unicellular relatives of the Metazoa --Spingoeca and Monosigarevealed a simple configuration with one MEIS-and one PBC-like TALE (S12, S13 Fig), whereas at the Metazoan base one finds at least three MEIS-related classes and two PBCrelated classes [44]. These findings suggest the occurrence of a similar expansion of a founding dyad during Metazoan evolution. Therefore, in both plants and animals, the TALE TF network seems to be redeployed for complex multicellularity, departing from its posited original function in sexual development. 

Our results suggest that TALE TF networks represent early-evolving developmental mechanisms. That said, the emergence of complex multicellularity doubtless required more than TF networks. TF-based developmental cues need to be propagated via chromatin-level regulatory mechanisms that establish the cellular memory during embryo development. The extent to which chromatin-level regulatory mechanisms are involved in the development of unicellular organisms is a critical question in elucidating the origins of complex multicellularity.
section_name: Does expansion of heterodimerizing TALE TFs relate to the emergence of multicellular complexity?

section_name: Materials and methods

section_content: Axenic Micromonas commoda (RCC299) and Ostreococcus tauri (OTH95) were maintained in Keller medium [45] in artificial seawater at room temperature. One hundred mL of a 14day-old culture was harvested for genomic DNA extraction. Picocystis salinarum (CCMP1897) was obtained from the National Center for Marine Algae and Microbiota (NCMA), maintained in L1 medium [46] in artificial sea water, and plated on 1. 5% Bactoagarcontaining media for single-colony isolation. Genomic DNA of P. salinarum was then obtained from a culture derived from one colony.
section_name: Strains and culture conditions

section_content: Archaeplastida algal TALE homeodomains were collected from the available genomes and transcriptomes listed in S1 Spreadsheet. Details of how TALE sequence was collected is provided in S1A Methods. After excluding nearly identical sequences, a total of 96 sequences together with 18 reference TALE sequences were made into the final homeodomain alignment with 70 unambiguously aligned positions with eight gapped and one constant sites. Details of phylogenetic reconstruction is provided in S1B Methods.
section_name: Phylogenetic analysis and classification of homeobox genes

section_content: The entire TALE collection was divided into multiple groups representing major clades in the homeodomain tree. Each group was individually analyzed by running MEME4. 12 in the motif-discovery mode with default option collecting up to 10 motifs at http://meme-suite. org/ [47]. The search provided multiple non-overlapping motifs, many of which were combined according to previously identified domains such as bipartite KN-A/KN-B, ELK, and HD [14] and independent domain searches against the INTERPRO database (http://www. ebi. ac. uk/interpro/) [48]. All the collected TALE-associated homology domains were aligned to generate HMM motifs, which we used to test if these homology domains are specific to the TALE sequences. All the homology domain information was used to locate any error in gene predictions, and gene models were updated if necessary (Details of the gene model curation is provided in S1C Methods).
section_name: Bioinformatics analysis

section_content: Introns within the homeodomain were collected and labeled as site numbers of the homeodomain. If an intron is between two codons it is denoted N/N+1, where N is the last amino acid site number of the preceding exon; introns within a codon are denoted N[n/n+1], where n is one or two for the codon nucleotide position relative to the splice-sites. Authors: Sunjoo Joo, Ming Hsiu Wang, Gary Lui, Jenny Lee, Andrew Barnas, Eunsoo Kim, Sebastian Sudek, Alexandra Z. Worden, and Jae-Hyeok Lee.
section_name: Intron comparison

section_name: Yeast

section_name: Acknowledgements

section_content: This work was supported by Discovery Grant 418471-12 from the Natural Sciences and Engineering Research Council (NSERC) (to J. -H. L. ), by the Korea CCS R&D Center (KCRC), Korean Ministry of Science, grant no. 2016M1A8A1925345 (to J. -H. L. ), NSF-IOS0843119 (to A. Z. W. ), GBMF 3788 (to A. Z. W. ), and NSF CAREER-1453639

(to E. K. ). Postdoctoral support of S. J. was from NSF -IOS0843506 (awarded to Ursula W. Goodenough ). We thank Mary Berbee and her laboratory members for valuable comments on the manuscript.
section_name: Acknowledgements