CoBra is the Brachyury-class T-box transcription factor of the amoeboid filasterean Capsaspora owczarzaki, a unicellular relative of animals whose life cycle has filopodial amoeba, aggregative and cystic stages. It is the only Brachyury known outside animals. Its single T-box DNA-binding domain keeps most of the DNA-contact and dimerization residues of animal T-box proteins. Protein-binding microarrays show that it recognizes the same T-box motif as mouse Brachyury (T). In Capsaspora, an antibody against CoBra enriches regulatory sites carrying Brachyury motifs. These sites lie mostly in first introns and 5' UTRs and are active in the amoeboid and aggregative stages. The predicted targets include genes for cell polarity, phagocytosis, metabolism and GPCR signalling, and the targets shared with mouse Brachyury are enriched for actin-cytoskeleton and cell-motility functions. When expressed in Xenopus embryos, CoBra partly rescues a dominant-negative Brachyury phenotype, but unlike animal Brachyury it activates every mesendodermal gene tested. This lack of target selectivity maps to its N- and C-terminal regions, not to the T-box, and the authors attribute it to missing cofactor interactions. No genetic loss-of-function data exist in Capsaspora. The identification of UniProt entry A0A0D2VUC6 (CAOG_005512, automatically named "TBX19 protein") as the CoBra of the published studies is inferred, not stated by those papers. It rests on sequence similarity to the T subfamily and on an arginine at the position of Xenopus Brachyury Lys149 that the 2013 study reports for CoBra.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000785 chromatin | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer from the deep T-box family node PTN000137774. Supported in Capsaspora itself: ChIP-qPCR with an anti-CoBra antibody enriches ATAC-defined regulatory sites carrying Brachyury motifs over random motif sites [PMID:27114036]. Reason: A sequence-specific transcription factor shown by ChIP to occupy regulatory sites in its own organism is located at chromatin. The ChIP-qPCR covers 20 sites, and the antibody target is the "CoBra" of that study, which we map to A0A0D2VUC6 by inference [file:projects/ORIGINS_OF_MULTICELLULARITY/capsaspora-tbox/RESULTS.md]. The term is a cellular location, not an animal-specific process, so there is no propagation problem. Supporting Evidence: PMID:27114036 we developed an anti-Capsaspora-Bra (CoBra) antibody (Figures 6E and 6F) and performed ChIP-qPCR experiments PMID:27114036 The ATAC-defined Bra regulatory sites were strongly enriched in CoBra compared with random motifs (Figure 6G), validating our Bra target prediction approach. |
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IEA GO_REF:0000120 | ACCEPT | Summary: Combined InterPro and TreeGrafter inference from the T-box domain. Supported by two independent experiments: in vitro, universal protein-binding microarrays give CoBra a motif highly similar to that of mouse Brachyury [PMID:24043797]; in Capsaspora cells, CoBra occupies Bra-motif regulatory sites near transcription start sites, mostly in first introns and 5' UTRs [PMID:27114036]. Reason: The T-box domain is intact and its sequence-specific DNA binding has been shown directly. The bound sites lie in cis-regulatory regions of protein-coding genes and carry active promoter and enhancer marks (H3K4me3, H3K27ac), so the RNA polymerase II cis-regulatory term is appropriate. The PBM used recombinant protein; occupancy in vivo was tested by ChIP-qPCR at 20 selected sites, not genome-wide. A NEW row cannot duplicate a GOA term, so the Capsaspora ChIP-qPCR result (which would support an IDA for this term from PMID:27114036) is recorded here; the in vitro PBM result is added as a NEW sequence-specific DNA binding row. Supporting Evidence: PMID:24043797 Our results indicate that CoBra has a highly similar motif to that determined in the mouse Bra-homolog, called T PMID:27114036 these inferred Bra sites are preferentially located at the first intron and 5′ UTR and are predominantly associated with the filopodial amoeba and aggregative stages |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer from the T-box family node. CoBra binds the conserved T-box motif [PMID:24043797], occupies regulatory sites in Capsaspora [PMID:27114036], and when expressed in Xenopus embryos it activates mesendodermal target genes and partly rescues a dominant-negative Brachyury phenotype [PMID:24043797]. Reason: This is the core molecular function, consistent with the core function chosen for human TBXT. Binding comes from Capsaspora protein and Capsaspora chromatin. Transcriptional activation has been shown only heterologously, in Xenopus embryos and animal caps, where CoBra acts as a nonselective "pan-T-box" activator. A more specific activator term (GO:0001228) is plausible but was not demonstrated in Capsaspora, so the general term is kept. The node lies at the T-box family root and a sequence-specific DNA-binding transcription factor activity fits every member, so the transfer is sound. Supporting Evidence: PMID:24043797 Our data show that C. owczarzaki Brachyury ( CoBra ) can partially rescue Xenopus laevis embryos injected with a dominant negative XBra construct. PMID:24043797 The conserved binding motif also helps to explain the ability of CoBra to rescue endogenous XBra and to activate several downstream T-box targets in Xenopus , but without the specificity of XBra PMID:27114036 The ATAC-defined Bra regulatory sites were strongly enriched in CoBra compared with random motifs (Figure 6G), validating our Bra target prediction approach. |
| GO:0001708 cell fate specification | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter transfer from PTN000137774, the deep T-box family node that also gives human TBXT its IBA for this term. The IBD donors are animal T-box genes (fly, worm, zebrafish, mouse), whose fate-specifying roles are embryonic. For CoBra, the only fate-related data are heterologous: in Xenopus embryos it partly rescues mesoderm and muscle gene expression after dominant-negative XBra, which the authors interpret cautiously [PMID:24043797]. Reason: GO defines cell fate specification as designating a cell to follow a developmental path. Capsaspora is unicellular, and no study links CoBra to the choice between its temporal life stages; Bra-motif sites are active in both the amoeboid and aggregative stages [PMID:27114036]. The Xenopus result shows only that the protein can partly substitute for a frog transcription factor in a frog embryo. Even there CoBra activated every mesendodermal gene tested, so it lacks the target selectivity that lets animal Brachyury specify particular fates [PMID:24043797]. The term is formally allowed for all cellular organisms (QuickGO only_in_taxon NCBITaxon:131567), so this is not a taxon-constraint violation. It is an animal developmental outcome placed at a node that also covers the unicellular filasterean and fungal T-box proteins. Human TBXT keeps this term (non-core). The propagation is valid for the animal donors but not for this target. Propagation Review Root cause: PROPAGATION BAD Failure modes: LINEAGE OR TAXON MISMATCH Sources checked: PANTHER:PTN000137774 SUPPORTS SOURCE BUT NOT TARGET Deep T-box family node whose cell fate specification IBD is seeded by animal T-box genes (fly, worm, zebrafish, mouse) with embryonic roles. The node also spans unicellular filasterean T-box proteins such as CoBra, for which no fate-specifying role has been shown in their own organism. Supporting Evidence: PMID:24043797 Thus, these results should be interpreted with caution; we can only conclude that both CoBra and CoTbx3 can roughly mimic endogenous XBra function. PMID:24043797 both CoBra and CoTbx3 (a member of the Tbx7 class) behave as what we call “pan-Tbox” genes, activating all potential targets |
| GO:0003700 DNA-binding transcription factor activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the T-box and Brachyury domain signatures. Correct, but less specific than GO:0000981, which this protein also carries. Reason: The T-box domain is present and functional (sequence-specific binding in vitro and occupancy in Capsaspora), and the protein activates transcription in a heterologous embryo. The term is a correct parent of the core function. Supporting Evidence: PMID:24043797 Experimental determination of the binding preferences of the C. owczarzaki Brachyury results in a similar motif to that of metazoan Brachyury and other T-box classes. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Combined InterPro, subcellular-location and TreeGrafter inference. CoBra has not been imaged in Capsaspora, but it is recovered from Capsaspora chromatin by ChIP [PMID:27114036], which places it in the nucleus. Reason: A transcription factor that occupies genomic regulatory sites must reside in the nucleus. The inference is sound, though no direct localization (imaging or fractionation) has been reported. Supporting Evidence: PMID:27114036 ChIP-seq and ChIP-qPCR were performed at three different life stages using antibodies against H3K4me3, H3K4me1, H3K27ac, H3K36me3, RNApolII, and CoBra |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the T-box domain. Correct but general; GO:0006357 and GO:0045893 are also present. Reason: A DNA-binding transcription factor regulates transcription. The term is correct and is a parent of the more specific process rows. Supporting Evidence: PMID:24043797 The conserved binding motif also helps to explain the ability of CoBra to rescue endogenous XBra and to activate several downstream T-box targets in Xenopus , but without the specificity of XBra |
| GO:0006357 regulation of transcription by RNA polymerase II | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer from the T-box family node. CoBra-bound sites in Capsaspora lie at the 5' ends of protein-coding genes and carry active promoter marks [PMID:27114036]. In Xenopus, CoBra activates RNA polymerase II-transcribed mesendodermal genes [PMID:24043797]. Reason: Follows from the DNA-binding transcription factor activity. The process is generic and holds for every T-box family member, so the transfer from the deep node is appropriate here, unlike the developmental row. The genes CoBra regulates directly in Capsaspora are inferred from motifs and ChIP-qPCR, not from loss-of-function expression data. Supporting Evidence: PMID:27114036 Accordingly, these Capsaspora-Bra sites are also more strongly correlated with the activating marks H3K4me3 and H3K27ac in these two stages |
| GO:0045893 positive regulation of DNA-templated transcription | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the T-box DNA-binding domain signature. In Xenopus animal caps and embryos, CoBra strongly activates all mesendodermal genes tested [PMID:24043797]. In Capsaspora, Bra-motif sites are associated with the activating histone marks H3K4me3 and H3K27ac [PMID:27114036]. Reason: Brachyury-class proteins are transcriptional activators. Activation by CoBra has been shown directly only in the heterologous Xenopus system. The Capsaspora data are correlative: bound sites carry activating marks, but no CoBra knockout or knockdown expression study exists. Accepted, with activation in Capsaspora itself still to be tested. Supporting Evidence: PMID:24043797 a different molecular phenotype was observed in embryos injected with C. owczarzaki Brachyury ( CoBra ) and Tbx7 ( CoTbx3 ) mRNAs, which strongly activated all mesendodermal genes PMID:27114036 Accordingly, these Capsaspora-Bra sites are also more strongly correlated with the activating marks H3K4me3 and H3K27ac in these two stages |
| GO:0043565 sequence-specific DNA binding | IDA PMID:24043797 Early evolution of the T-box transcription factor family. | NEW | Summary: Universal protein-binding microarrays (PBM) with CoBra protein give a DNA motif highly similar to that of mouse Brachyury (T) and to other T-box classes [PMID:24043797]. Reason: Direct in vitro assay of the Capsaspora protein's DNA-binding specificity (not a heterologous functional assay), so IDA. GOA has no experimental row for this protein. The term records the PBM result. The in vivo, cis-regulatory binding from ChIP in Capsaspora is recorded on the existing GO:0000978 row. Supporting Evidence: PMID:24043797 To further investigate the function of C. owczarzaki Brachyury, we determined its binding preferences using universal PBM PMID:24043797 Our results indicate that CoBra has a highly similar motif to that determined in the mouse Bra-homolog, called T |
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Download this section (compressed HTML)Q: Is UniProt A0A0D2VUC6 (CAOG_005512) the CoBra cDNA used in the 2013 Xenopus and PBM experiments and the antigen of the 2016 anti-CoBra antibody? The papers give no locus ID, and the assignment here rests on T-subfamily similarity and the arginine at the Xenopus Bra K149 position.
Q: PANTHER places A0A0D2VUC6 in subfamily PTHR11267:SF181 (OPTOMOTOR-BLIND PROTEIN, a Tbx2-class name), while the 2013 phylogeny places CoBra at the base of the Brachyury class. Is the subfamily assignment an error?
Q: Does CoBra activate or repress its predicted targets in Capsaspora, and does it regulate the transitions between the amoeboid, aggregative and cystic stages?
Q: Which cofactors, if any, does CoBra bind in Capsaspora, given that the cofactor Smad1 implicated in restricting animal Brachyury targets is absent?
Experiment: Generate a CoBra knockout or inducible knockdown with the Capsaspora genome-editing methods now available, and combine RNA-seq with genome-wide CoBra ChIP-seq or CUT&RUN to define direct targets. Score cell motility, filopodia and aggregation.
Hypothesis: CoBra directly activates a cell-polarity and motility gene network in Capsaspora.
Type: genetic loss of function / chromatin profiling
Experiment: Immunoprecipitate with the anti-CoBra antibody followed by mass spectrometry, or epitope-tag CAOG_005512 and repeat ChIP-qPCR at the published sites, to confirm the locus assignment.
Hypothesis: The anti-CoBra antibody recognizes the CAOG_005512 product.
Type: protein identification
Experiment: Immunostain CoBra or image a tagged transgene in filopodial, aggregative and cystic cells to test stage-dependent nuclear localization.
Hypothesis: CoBra is nuclear in all three life stages.
Type: localization / imaging
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