https://orcid.org/0000-0001-8194-5214
Figures
Abstract
The C2H2 zinc finger gene cucoid establishes anterior-posterior (AP) polarity in the early embryo of culicine mosquitoes. This gene is unrelated to genes that establish embryo polarity in other fly species (Diptera), such as the homeobox gene bicoid, which serves this function in the traditional model organism Drosophila melanogaster. The cucoid gene is a conserved single copy gene across lower dipterans but nothing is known about its function in other species, and its evolution in higher dipterans, including Drosophila, is unresolved. We found that cucoid is a member of the ZAD-containing C2H2 zinc finger (ZAD-ZNF) gene family and is orthologous to 27 of the 91 members of this family in D. melanogaster, including M1BP, ranshi, ouib, nom, zaf1, odj, Nnk, trem, Zif, and eighteen uncharacterized genes. Available knowledge of the functions of cucoid orthologs in Drosophila melanogaster suggest that the progenitor of this lineage specific expansion may have played a role in regulating chromatin. We also describe many aspects of the gene duplication history of cucoid in the brachyceran lineage of D. melanogaster, thereby providing a framework for predicting potential redundancies among these genes in D. melanogaster.
Citation: Li M, Kasan K, Saha Z, Yoon Y, Schmidt-Ott U (2023) Twenty-seven ZAD-ZNF genes of Drosophila melanogaster are orthologous to the embryo polarity determining mosquito gene cucoid. PLoS ONE 18(1): e0274716. https://doi.org/10.1371/journal.pone.0274716
Editor: René Massimiliano Marsano, University of Bari: Universita degli Studi di Bari Aldo Moro, ITALY
Received: September 1, 2022; Accepted: December 16, 2022; Published: January 3, 2023
Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: ML was the recipient of a fellowship from the Top Student Training Program of Peking University, China, for an International Summer Research Experience for Undergraduates (REU) program of the Biological Sciences Division at the University of Chicago. ZS was the recipient of a Research Foundations in Genetics and Genomics summer fellowship of the Biological Sciences Collegiate Division at the University of Chicago. YY was the recipient of an award of University of Chicago Henry Hinds Funds for Graduate Student Research in Evolutionary Biology. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM127366. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Dipteran insects (true flies) begin embryogenesis with 12 or 13 synchronous nuclear division cycles [1–4]. During this syncytial phase of embryonic development, a uniform blastoderm forms in the cortical layer of the egg, activates the zygotic genome [5–7], and establishes axial polarity [8,9]. Anterior determinants (ADs) establish the embryo’s head-to-tail polarity via transcription factor gradients. In the fruit fly Drosophila melanogaster (D. melanogaster), the AD is encoded by the homeobox gene bicoid [10], which has been studied extensively [11–17]. However, genes unrelated to bicoid are being used in species of different dipteran lineages for the same developmental task [18,19]. This evolutionary plasticity, along with the simple anatomy of early dipteran embryos and their amenability to experimental perturbation in non-traditional model organisms, set the stage for an attractive experimental system to study the molecular and evolutionary basis of transcriptional network stability and co-option of new central players in embryo development.
What are the molecular mechanisms that guide the co-option of new ADs? Bicoid has many target genes [15,20–22], but it remains unclear how it adopted them. The bicoid gene evolved from a gene duplication of the Hox3 ortholog of flies (also known as zerknüllt or zen), more than 145 million years ago [23–25]. The diverged DNA-binding specificity of Bicoid, compared to its closest paralogs, prompted detailed studies on the evolution of its DNA-binding homeodomain using ancestral sequence reconstruction, quantitative in vitro DNA binding assays, and in vivo rescue experiments in Drosophila embryos [26–29]. These studies emphasized the importance of mutations that altered DNA-binding specificity of the Bicoid protein. It was also shown that a feed-forward relay integrates certain regulatory activities of Bicoid and Orthodenticle via shared DNA binding sites [28]. These homeodomain proteins have qualitatively similar DNA affinity and Orthodenticle has a conserved zygotic function in head development, which raised the question of whether Bicoid took over functions of Orthodenticle [29]. However, comparative studies revealed ADs with distinct DNA binding domains and DNA affinities and suggest that the AD of the last common ancestor of dipterans was encoded by pangolin (Tcf) [18]. Therefore, the co-option of new ADs in different fly lineages may not require shared target sites between the old and new ADs.
Why do specific genes adopt the AD function in addition to their other roles? The identification of AD gene orthologs in Drosophila melanogaster provides a useful starting point because many of its gene functions have been analyzed. For example, odd-paired, the only zic (zinc finger of the cerebellum) gene family member of flies [30], opens specific chromatin regions to advance the temporal progression of zygotic pattern formation in Drosophila embryos [31,32]. This function appears to be conserved in moth flies, where odd-paired additionally adopted the AD function by acquiring a maternal transcription variant [18]. The ability of Odd-paired protein to drive the accessibility of specific chromatin regions, which is also a property of Bicoid [22,33], could have facilitated their convergent co-option as ADs.
In culicine mosquitoes (e.g., Aedes and Culex), a previously uncharacterized C2H2 zinc finger gene, named cucoid, adopted the AD function. In these species, three cucoid transcript isoforms with alternative 3’ ends have been identified in embryos. The shortest isoform is expressed maternally and is localized at the anterior pole of the egg. In culicine mosquitoes, knockdown of cucoid by RNAi results in ectopic expression of posterior genes at the anterior and the double abdomen phenotype [18]. However, the function of cucoid orthologs in other species is unknown and obscured by a complex evolution of this gene in higher flies, including Drosophila melanogaster [18]. Here we show that cucoid is a member of the ZAD-ZNF gene family and is orthologous to at least 27 of D. melanogaster’s 91 ZAD-ZNF genes [34]. ZAD-ZNF gene family members encode C2H2 zinc finger proteins with an N-terminal Zinc-finger-associated domain (ZAD) [34–39]. Most cucoid orthologs of D. melanogaster have not yet been characterized but those that have been named and studied predominantly function in early development and oogenesis and may affect chromatin states.
2. Materials and methods
2.1 Cucoid structure prediction and identification of cucoid orthologs
Protein structure was predicted using AlphaFold2_advanced with default settings [40]. Cucoid orthologs were identified by reciprocal protein BLAST using default E value cut-off threshold of 0.05 while setting maximum target sequences to 5000 to detect all potential orthologs in the target species (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [41]. As queries we used Cucoid sequences from Culex quinquefasciatus (C. quinquefasciatus; GenBank identifier QFQ59547.1) and Aedes aegypti (A. aegypti; GenBank identifier XP_021704552.1). The Cucoid sequence of C. quinquefasciatus is referred to as C-isoform in GenBank but corresponds to the non-truncated A-isoform in [18]. The Cucoid sequence of A. aegypti is referred to as myoneurin in GenBank and correspond to the non-truncated A-isoform in [18]. The name myoneurin for cucoid in A. aegypti appears to be a misnomer due to spurious similarity with non-orthologous myoneurin genes in other species. Therefore, cucoid and myoneurin are not synonyms and the name myoneurin should not be used to designate cucoid orthologs. Candidate orthologs were searched for conserved domains using NCBI’s Conserved Domain Database (CDD) with the server’s default E value cut-off of ≤ 0.01 [42]. C2H2 zinc finger proteins with ZAD (also known as zf-AD, smart00868, or pfam07776) were retained for reciprocal BLAST in A. aegypti and C. quinquefasciatus, using server default E value cut-offs of ≤ 0.05.
Conservation of Cucoid clade genes of Drosophila melanogaster within the Brachycera was assessed by reciprocal protein BLAST in Drosophila virilis, Lucilia cuprina, Bactrocera dorsalis, and Hermetia illucens. Syntenies of Cucoid orthologs in these species were examined in GenBank and illustrated using the IBS server [43]. Accession numbers are provided as supplementary material (S1 Table).
Since the assembly of the robber fly Proctacanthus coquilletti in GenBank (GenBank identifier GCA_001932985.1) is not annotated, we identified candidate exons of P. coquilletti cucoid orthologs (S2 Table), using tblastn with default settings [41] and Hermetia illucens (H. illucens) Cucoid orthologs and D. melanogaster CG9215, CG4424, and CG14711 as queries. Protein sequences of four candidate Cucoid orthologs from the robber fly were assembled manually and used for reciprocal protein BLAST in H. illucens and D. melanogaster.
2.2 Protein alignment and phylogenetic analysis
The list of D. melanogaster ZAD-ZNF genes has been reported elsewhere [34]. The respective protein sequences were downloaded from GenBank. MAFFT alignments were generated by MAFFT v7.471with the L-INS-i strategy (https://mafft.cbrc.jp/alignment/software/) [44]. Protein alignments were visualized using Geneious Prime 2021.2.2 (https://www.geneious.com/). For the protein tree with 91 ZAD-ZNF sequences, the raw alignments were trimmed using TrimAl v1.3 (http://trimal.cgenomics.org/) [45] with a conservation threshold of 20, a gap threshold of 0.8, and a similarity threshold of 0.05 to remove highly variable positions (columns). The trimmed alignments were further divided into two partitions corresponding to ZAD and ZNF regions. The best molecular substitution model for each partition was selected by partition merging strategy (MFP+MERGE) using ModelFinder [46] implemented in IQ-TREE v2.1.3 [47], based on Bayesian Information Criterion (BIC). Maximum likelihood trees were then built based on the selected substitution models, with branch support values generated by the implemented ultrafast bootstrap approximation [48], setting replicates to 3000. A majority rule consensus tree was generated form bootstrap trees and visualized by FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). Trees are unrooted unless otherwise stated. Accession numbers of all sequences used in protein trees (S1 Table) and full-length alignments of D. melanogaster ZAD-ZNF proteins (S1 File) and the Cucoid orthologs from D. melanogaster and D. virilis (S2 File) are provided as supporting information.
3. Results and discussion
3.1 Cucoid is a ZAD-ZNF protein with 27 orthologs in Drosophila melanogaster
Reciprocal protein BLAST identified single copy cucoid orthologs in all major branches of the lower (non-brachyceran) Diptera, including Tipulomorpha, Culicomorpha, Psychodomorpha [18], and Bibionomorpha (this study), as well as in the insect orders Siphonaptera (fleas) and Lepidoptera (butterflies and moths) (S1 Fig). No cucoid orthologs were found in other insect orders, suggesting that cucoid evolved during the radiation of holometabolous insects.
The genome of Drosophila melanogaster encodes around 300 C2H2 zinc finger proteins [37,39], including multiple candidate orthologs of cucoid. To aid in the identification of cucoid orthologs in D. melanogaster, we searched for diagnostic domains and motifs of Cucoid using protein alignments and protein folding software [49] (Figs 1 and S1). The alignment was constructed with previously reported single-copy Cucoid orthologs from the mosquitoes Culex quinquefasciatus, Aedes aegypti, and Anopheles gambiae (Culicidae), the harlequin fly Chironomus riparius (Chironomidae), the moth fly Clogmia albipunctata (Psychodidae), and the crane fly Nephrotoma suturalis (Tipulidae) [18], as well as newly identified single-copy Cucoid orthologs from the gall midge Contarinia nasturtii (Cecidomyiidae), the cat flea Ctenocephalides felis (Siphonaptera), and the silk moth Bombyx mori (Lepidoptera) that we retrieved from sequences deposited in GenBank (S1 Fig and S1 Table). The gall midge belongs to the Bibionomorpha, the putative sister taxon of the Brachycera [50], while the cat flea and the silk moth represent close outgroups of the Diptera [51]. We focused on these lineage representatives because of the advanced state of genome resources for these species and because they yielded best matches in reciprocal protein BLAST with our query sequences.
Structure of a Cucoid homodimer (maternal isoform from C. quinquefasciatus, GenBank: QFQ59549.1) as predicted by AlphaFold2 (AlphaFold identifier for Cucoid structure: A0A5P8HWN4) is shown with the two chains colored in red and blue above a simplified sketch of full-length Cucoid protein with the N-terminal end to the left and the C-terminal end to the right, and ZAD and ZNFs marked by colored rectangles.
Cucoid proteins typically contain five C2H2 zinc finger domains. However, the Cucoid ortholog of Chironomus lacks zinc fingers 4 and 5, and culicine mosquitoes also express shorter isoforms without the 5th (Culex) or 5th, 4th, and C-terminal half of the 3rd zinc finger domains (Aedes). We found that all these Cucoid orthologs also contain a conserved N-terminal domain, known as Zinc-finger-associated domain (ZAD; Figs 1 and S1) [35,38]. The ZAD is stabilized by zinc coordination via four invariant cysteine residues and can drive dimerization [37,52] and nuclear localization [53].
Holometabolous insects evolved many ZAD-ZNF genes through lineage-specific gene duplications [35,36,39], especially in dipterans. For example, 147 ZAD-ZNF proteins have been found in Anopheles gambiae [36] and 91 in Drosophila melanogaster [34]. To identify the ZAD-ZNF proteins in D. melanogaster most similar to Cucoid, we conducted protein BLAST with all 91 ZAD-ZNF proteins of D. melanogaster in A. aegypti and C. quinquefasciatus. The same seventeen Drosophila sequences retrieved cucoid in Aedes and Culex (54.1% sequence conservation). The corresponding genes were therefore considered candidate orthologs of cucoid (Table 1).
Next, we generated a Maximum Likelihood protein tree with all 91 ZAD-ZNF proteins of D. melanogaster and examined the distribution of the candidate Cucoid orthologs on this protein tree (Fig 2). All seventeen candidate orthologs (marked by red triangles in Fig 2) mapped to a monophyletic clade of 27 ZAD-ZNF proteins (henceforth Cucoid clade). The Cucoid clade can be subdivided into two subclades with 9 members (subclade A) and 18 members (subclade B), respectively. All 9 members of subclade A were included in the original list of seventeen candidate Cucoid orthologs (Table 1). The top hit of that list, CG9215, also belongs to subclade A. The 18 members of subclade B experienced on average an elevated substitution rate. This subclade includes 10 genes that we did not recover using reciprocal protein BLAST. These 10 genes do not form a monophyletic clade but are nested within the Cucoid clade and are therefore probably true Cucoid orthologs. We therefore conclude that at least 27 of D. melanogaster’s 91 ZAD-ZNF genes are orthologous to cucoid.
3.2 Genomic organization and relationship of genes of the Cucoid clade
All proteins of the Cucoid clade, except CG9215, are encoded by genes on the right arm of chromosome 3 and are organized in five gene complexes of 4–5 genes and three isolated singletons (Fig 3). These 26 genes share intron positions among each other and with the cucoid orthologs from lower dipterans (Fig 4), consistent with an evolutionary origin by DNA-based tandem gene duplications. CG9215 is an intron-less gene on the X-chromosome that may have evolved by retro-transposition from the singleton zif, its most likely parent gene (Fig 2). We denoted each gene cluster in D. melanogaster by the member that gave the smallest E value in reciprocal BLAST with Cucoid (Table 1), that is: M1BP cluster (orange), CG4424 cluster (blue), CG14711 cluster (green), and CG31441 cluster (purple). One cluster (light blue) did not include any of the genes that we identified by reciprocal BLAST and was named after a previously described gene, oddjob (odj) [54].
Genes of the Cucoid clade are distributed in clusters on the right arm of chromosome 3, except CG9215 (not shown) which is located on the X chromosome. M1BP cluster (orange), CG14711 cluster (green), CG4424 cluster (blue), Odj cluster (light blue), CG31441 cluster (purple), Zif (black), other dispersed genes (outlined).
Multiple sequence alignment of Cucoid orthologs from lower dipterans and Cucoid clade members of D. melanogaster. Conserved intron positions are boxed, and the red zigzags represent the splicing points. Similarity of aligned amino acids was assessed using the Blosum62 matrix with black representing 100% similarity, dark grey 80–100% similarity, light grey 60–80% similarity, and white less than 60% similarity.
The M1BP cluster genes form a monophyletic clade that can be traced to a single M1BP-like precursor gene, preserved in other schizophoran flies such as blow flies and tephritid fruit flies (see below). The first duplication of the M1BP-like precursor gave birth to M1BP/ranshi and nom/ouib/CG8159. The precursor of nom/ouib/CG8159 duplicated twice, first generating nom/ouib and CG8159 and then generating nom and ouib (Fig 2). These duplications occurred before the split of the D. virilis and D. melanogaster lineages [55]. M1PB/ranshi duplicated after the split of D. melanogaster and Drosophila ananassae [55].
The other cucoid-related gene clusters of D. melanogaster do not form monophyletic clades. These incongruences between our protein tree and clustering in the D. melanogaster genome could have resulted from limitations of the phylogenetic inference methods that we used to build the protein tree, such as model choice and long-branch attraction [56], or non-allelic recombination (gene conversion) within the ZAD-ZNF family [57,58]. However, in D. virilis, genes related to members of the CG14711 cluster (green), the CG31441 cluster (purple), and the Odj cluster (light blue) form a single gene complex with a different gene order, and this gene order is consistent with the inferred close relationship of neighboring Cucoid clade genes (Fig 5A). Based on synteny in D. virilis and phylogenetic inference with D. virilis and D. melanogaster orthologs (Fig 5B), CG18764 of the CG14711 cluster (green) is paralogous to all genes of the Odj cluster (light blue) as well as CG6689 of the CG31441 cluster (purple). Additionally, we infer that CG6689 is a paralog of CG17803 of the Odj cluster, even though a gene-specific N-terminal THAP (Thanatos Associated Proteins) domain [59–61] that CG6689 inherited from its precursor, CG6689/CG17803, is not preserved in CG17803 (S2 Fig). Finally, we infer that D. virilis lost the Nnk/CG17806 precursor because a CG17806-like precursor gene existed before the split of D. melanogaster and D. virilis but was not found in D. virilis. Duplication of the Nnk/CG17806 precursor occurred after the split of the D. willistoni lineage from D. melanogaster lineage [55].
(A) Synteny of cucoid orthologs in D. virilis. Genes are color coded to indicate their relationship to gene clusters in D. melanogaster (see Fig 3). (B) Manually rooted maximum likelihood protein tree of Cucoid orthologs from D. melanogaster and D. virilis. Note that D. virilis has two CG17801 orthologs.
3.3 The Cucoid clade in Drosophila outgroups
Lineage-specific gene family expansions may reflect innovations or adaptations [38], but it is unknown why the number of ZAD-ZNF genes independently increased so much in multiple lineages of the Holometabola. To better understand when and how the Cucoid clade expanded, we searched for orthologs of the Cucoid clade members in representatives of other schizophoran fly species, including a blow fly (Lucilia cuprina) and a tephritid fruit fly (Bactrocera dorsalis), and in two representatives of the lower Brachycera, including a soldier fly (Hermetia illucens) and a robber fly (Proctacanthus coquilleti). While non-brachyceran dipterans have single cucoid orthologs (see section 3.1), we identified multiple cucoid orthologs in all the brachyceran species, albeit in lower numbers than in Drosophila.
In Lucilia and Bactrocera, we identified a single M1BP-like gene (orange), and several genes related to the CG4424 cluster (dark blue) and the CG14711 cluster (green), respectively, as well as putative orthologs of zif and CG9215 (Fig 6A and 6B). The presence of a M1BP-like gene in these unrelated species (they represent paraphyletic/parallel lineages of the Schizophora [62]) suggests that the M1BP cluster expanded during, rather than before the radiation of the Schizophora in the Tertiary epoch [63]. Whether the expansion of the M1BP cluster within the Schizophora resulted in subfunctionalization or the acquisition of new gene functions or a mix of both remains unknown, due to the lack of functional comparisons of the M1BP-like gene in lower Schizophora with their multiple orthologs in Drosophila.
Genes are color coded to indicate their relationship to gene clusters in D. melanogaster. (A) Synteny of cucoid orthologs in Lucilia cuprina. (B) Synteny of cucoid orthologs in Bactrocera dorsalis. (C) Synteny of cucoid orthologs in Hermetia illucens.
Two additional features of the genomic organization of Cucoid clade genes in Bactrocera dorsalis deserve attention. First, the M1BP-like gene of this species is in the immediate vicinity of the CG14711 cluster (green, Fig 6A). This finding may suggest that the founder gene of the M1BP cluster originated as an offshoot of the CG14711 cluster, even though this is not apparent in the protein tree (Fig 2). Second, CG14711 and CG14710 of B. dorsalis have merged; the predicted protein has two linear ZAD-ZNFs structures that correspond to CG14710 and CG14711, respectively. Whether these genes resulted from the same duplication is unclear. Phylogenetic analysis suggests that CG14711 is more closely related to CG4424 than to CG14710. However, since CG14711 and CG4424 are more similar to Cucoid than CG14710 and other members of the CG14711 and CG4424 clusters, the inferred close relationship of CG14711 and CG4424 might reflect their less diverged status rather than their duplication history.
In the genomes of lower Brachycera, we identified five cucoid loci on chromosome 6 of the soldier fly Hermetia illucens (Hil_cucoid_1–5, GenBank accession numbers: XP_037925088.1, XP_037925165.1, XP_037924715.1, CAD7093451.1, XP_037922166.1) [64] (Fig 6C) and four cucoid loci in the robber fly Proctacanthus coquilleti (Pco_cucoid_1–4) [65] (S2 Table), which seem to be orthologous to Hermetia cucoid orthologs 1, 2, 3, and 4/5, respectively (S3 Fig). The lower brachyceran Cucoid proteins 1 and 2 are closely related to CG9215 judged by protein BLAST E value but retain conserved introns and are therefore potentially orthologous to CG9215/Zif, whereas the lower brachyceran Cucoid orthologs 3 and 4 are closely related to CG4424 and CG14711, respectively. Therefore, the last common ancestor of soldier flies, robber flies, and Drosophila may have had at least three cucoid orthologs, including a CG4424-like member, CG14711-like member, and a CG9215-like ortholog of the Zif/CG9215 precursor. No Hil_5 ortholog was found in D. melanogaster and the robber fly Proctacanthus coquilletti. Its location within an intron of Hil_4 suggests that it was born by lineage-specific duplication of Hil_4. Thus, Hil_5 may also be orthologous to CG14711.
4. Conclusions
D. melanogaster contains at least 27 cucoid orthologs, that is, almost one third of the 91 ZAD ZNF genes of this species. Reciprocal BLAST, phylogenetic inference, and genomic organization suggest that the Cucoid clade of D. melanogaster expanded gradually in the brachyceran lineage (Fig 7), while its founder gene was already present in the last common ancestor of butterflies, fleas, and flies. The last common ancestor of the brachyceran species that we analyzed may have had three cucoid orthologs that encoded proteins similar to CG9215/zif, CG4424, and CG14711. We infer this because in protein BLAST against Drosophila proteins, H. illucens and P. coquilletti Cucoid proteins 1 and 2 recovered CG9215 as the best hit, and their Cucoid orthologs 3 and 4 recovered CG4424 and CG14711 as the best hits, respectively. The founder of the monophyletic M1BP-cluster originated before the radiation of the Schizophora. All other clusters of D. melanogaster may not have monophyletic origins.
Inferred gene duplications in the Cucoid clade based on data in this study and D. melanogaster gene ages reported elsewhere [55]. For details see text. Gene clusters (hexagons) and gene loci (triangles) are indicated. For color code see Fig 2.
Our study was motivated by the question of what is known about cucoid orthologs in Drosophila melanogaster. Most of the 27 cucoid orthologs of D. melanogaster that we identified in this study did not affect viability when downregulated in previous large-scale screens (Table 2) [34,66–68]. However, several orthologs have been characterized in greater depth and display diverse, essential functions. For example, M1BP binds core promoters of thousands of genes and functions during transcription activation and polymerase Ⅱ pausing while promoting chromatin accessibility surrounding the transcription start sites [69,70]. Other genes in the M1BP cluster show more specialized functions: ranshi regulates oocyte differentiation [71], nom functions in muscle development, and ouib is necessary during ecdysteroid synthesis by regulating spookier [72,73]. The closely related genes odj and Nnk have essential functions in heterochromatin regulation [34], zaf1 is a chromosome architecture protein that serves as insulator in Drosophila melanogaster [74], trem is required for binding Mei-P22 on meiotic chromosomes to initiate double strand breaks for homologous recombination [75], and Zif is required for the expression and asymmetric localization of aPKC in neuroblast cells to regulate their polarity and self-renewal [76,77]. All other genes of the Cucoid clade remain uncharacterized. Taken together, our study suggests that many cucoid orthologs of D. melanogaster function in oogenesis and embryogenesis and several of them modify chromatin states. It will be interesting to find out whether single copy Cucoid orthologs from lower dipterans function in similar ways to some D. melanogaster orthologs and what structural and/or regulatory features enable Cucoid in culicine mosquitoes to regulate early zygotic segmentation genes.
Supporting information
S2 Table. Locations of cucoid orthologs in P. coquiletti inferred from tblastn.
https://doi.org/10.1371/journal.pone.0274716.s002
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S1 File. Full length alignment of 91 D. melanogaster ZAD-ZNF protiens.
https://doi.org/10.1371/journal.pone.0274716.s003
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S2 File. Full length alignment of D. melanogaster and D. virilis Cucoid orthologs.
https://doi.org/10.1371/journal.pone.0274716.s004
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S1 Fig. Cucoid protein alignment and prediction of Cucoid homodimer.
(A) Multiple sequence alignment of Cucoid orthologs from Aedes aegypti (Aae), Anopheles gambiae (Aga), Bombyx mori (Bmo), Chironomus riparius (Cri), Clogmia albipunctata (Cal), Contarinia nasturtii (Cna), Ctenocephalides felis (Cfe), Culex quinquefasciatus (Cqu), and Nephrotoma suturalis (Nsu). (B) A plot of the predicted alignment error of the best model acquired from AlphaFold2 output which estimates the distance error for every pair of residues. Both axes represent the positions on the dimer of Cucoid maternal isoform (499 aa) from C. quinquefasciatus. The color key is measured in angstrom. Very low position errors are found for the overlapping of residues in the ZAD dimer as well as between zinc fingers on the same strand, indicating true packing of these domains. (C) The plot of predicted local distance difference test (pLDDT) per position gives a confidence level between 0–100 for each residue. All models predict ZAD and ZNF domain with very high confidence, whereas the highly variable linker regions get deficient support.
https://doi.org/10.1371/journal.pone.0274716.s005
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S2 Fig. CG6689 acquired a DNA-binding THAP domain.
The THAP domains from 9 THAP-containing proteins in D. melanogaster and a THAP-like fragment from CG17803 are shown in alignment here. The color code for each column is based on similarity of aligned amino acids, with black representing high similarity and white representing no similarity. The N-terminal THAP domain of CG6689 is absent in all other Cucoid orthologs including its most recent paralog, CG17803, which has incomplete THAP features. THAP is a zinc-coordinating DNA binding domain with a conserved C2CH structure and shares features with the DNA binding domain of the P element transposase [59,60]. THAP-domain-containing proteins have been found in human, D. melanogaster, and C. elegans [59]. In the nine D. melanogaster proteins that have this domain, only CG6689 and CG10431 belong to the ZAD-ZNF family. CG10431 is only distantly related to CG6689 and located on a different chromosome (2L), suggesting that even within the ZAD-ZNF family THAP domains evolved de novo. The THAP domain of CG6689 is encoded by the first two exons of this gene, which are only conserved in CG17803 (Fig 4).
https://doi.org/10.1371/journal.pone.0274716.s006
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S3 Fig. Phylogeny of Cucoid orthologs in H. illucnes and P. coquilletti.
A phylogenetic tree with Cucoid orthologs in H. illucnes, P. coquilletti and lower flies was constructed based on an untrimmed alignment using 3 partitions inlucing ZAD, ZNF, and the other regions. Regions outside the ZAD and ZNF domains include diagnostic features useful for inferring orthology. This tree suggests that Hil_cucoid_1 to Hil_cucoid_4 are orthologous to Pco_cucoid_1 to Pco_cucoid_4, respectively.
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Acknowledgments
We thank our colleagues Dr. Phoebe Rice and Dr. Manyuan Long for helpful discussions, and Dr. Shengqian Xia and Dylan Sosa in the Long lab for technical assistance.
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