id: A0A0D2VUC6 gene_symbol: CoBra aliases: - CAOG_005512 - Capsaspora Brachyury - Co-Bra product_type: PROTEIN status: DRAFT taxon: id: NCBITaxon:595528 label: Capsaspora owczarzaki ATCC 30864 description: >- 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. existing_annotations: - term: id: GO:0000785 label: chromatin evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: located_in supporting_entities: - PANTHER:PTN000137774 review: 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]. action: ACCEPT 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. supported_by: - reference_id: PMID:27114036 supporting_text: >- we developed an anti-Capsaspora-Bra (CoBra) antibody (Figures 6E and 6F) and performed ChIP-qPCR experiments - reference_id: PMID:27114036 supporting_text: >- The ATAC-defined Bra regulatory sites were strongly enriched in CoBra compared with random motifs (Figure 6G), validating our Bra target prediction approach. - term: id: GO:0000978 label: RNA polymerase II cis-regulatory region sequence-specific DNA binding evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables supporting_entities: - InterPro:IPR001699 - PANTHER:PTN000137774 review: 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]. action: ACCEPT 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. supported_by: - reference_id: PMID:24043797 supporting_text: >- Our results indicate that CoBra has a highly similar motif to that determined in the mouse Bra-homolog, called T - reference_id: PMID:27114036 supporting_text: >- 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 - term: id: GO:0000981 label: DNA-binding transcription factor activity, RNA polymerase II-specific evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: enables supporting_entities: - PANTHER:PTN000137774 review: 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]. action: ACCEPT 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. supported_by: - reference_id: PMID:24043797 supporting_text: >- Our data show that C. owczarzaki Brachyury ( CoBra ) can partially rescue Xenopus laevis embryos injected with a dominant negative XBra construct. - reference_id: PMID:24043797 supporting_text: >- 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 - reference_id: PMID:27114036 supporting_text: >- The ATAC-defined Bra regulatory sites were strongly enriched in CoBra compared with random motifs (Figure 6G), validating our Bra target prediction approach. - term: id: GO:0001708 label: cell fate specification evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in supporting_entities: - PANTHER:PTN000137774 review: 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]. action: REMOVE 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. supported_by: - reference_id: PMID:24043797 supporting_text: >- Thus, these results should be interpreted with caution; we can only conclude that both CoBra and CoTbx3 can roughly mimic endogenous XBra function. - reference_id: PMID:24043797 supporting_text: >- both CoBra and CoTbx3 (a member of the Tbx7 class) behave as what we call “pan-Tbox” genes, activating all potential targets propagation_review: root_cause: PROPAGATION_BAD failure_modes: - LINEAGE_OR_TAXON_MISMATCH source_entities: - source_id: PANTHER:PTN000137774 source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET comment: >- 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. - term: id: GO:0003700 label: DNA-binding transcription factor activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR001699 - InterPro:IPR002070 - InterPro:IPR008967 - InterPro:IPR018186 - InterPro:IPR036960 - InterPro:IPR046360 review: summary: >- InterPro2GO mapping from the T-box and Brachyury domain signatures. Correct, but less specific than GO:0000981, which this protein also carries. action: ACCEPT 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. supported_by: - reference_id: PMID:24043797 supporting_text: >- 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. - term: id: GO:0005634 label: nucleus evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: located_in supporting_entities: - InterPro:IPR002070 - InterPro:IPR018186 - InterPro:IPR036960 - UniProtKB-SubCell:SL-0191 - PANTHER:PTN000137774 review: 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. action: ACCEPT 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. supported_by: - reference_id: PMID:27114036 supporting_text: >- ChIP-seq and ChIP-qPCR were performed at three different life stages using antibodies against H3K4me3, H3K4me1, H3K27ac, H3K36me3, RNApolII, and CoBra - term: id: GO:0006355 label: regulation of DNA-templated transcription evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in supporting_entities: - InterPro:IPR001699 - InterPro:IPR002070 - InterPro:IPR008967 - InterPro:IPR018186 - InterPro:IPR036960 review: summary: >- InterPro2GO mapping from the T-box domain. Correct but general; GO:0006357 and GO:0045893 are also present. action: ACCEPT reason: >- A DNA-binding transcription factor regulates transcription. The term is correct and is a parent of the more specific process rows. supported_by: - reference_id: PMID:24043797 supporting_text: >- 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 - term: id: GO:0006357 label: regulation of transcription by RNA polymerase II evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in supporting_entities: - PANTHER:PTN000137774 review: 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]. action: ACCEPT 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. supported_by: - reference_id: PMID:27114036 supporting_text: >- Accordingly, these Capsaspora-Bra sites are also more strongly correlated with the activating marks H3K4me3 and H3K27ac in these two stages - term: id: GO:0045893 label: positive regulation of DNA-templated transcription evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in supporting_entities: - InterPro:IPR046360 review: 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]. action: ACCEPT 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. supported_by: - reference_id: PMID:24043797 supporting_text: >- a different molecular phenotype was observed in embryos injected with C. owczarzaki Brachyury ( CoBra ) and Tbx7 ( CoTbx3 ) mRNAs, which strongly activated all mesendodermal genes - reference_id: PMID:27114036 supporting_text: >- Accordingly, these Capsaspora-Bra sites are also more strongly correlated with the activating marks H3K4me3 and H3K27ac in these two stages - term: id: GO:0043565 label: sequence-specific DNA binding evidence_type: IDA original_reference_id: PMID:24043797 review: 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]. action: NEW 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. supported_by: - reference_id: PMID:24043797 supporting_text: >- To further investigate the function of C. owczarzaki Brachyury, we determined its binding preferences using universal PBM - reference_id: PMID:24043797 supporting_text: >- Our results indicate that CoBra has a highly similar motif to that determined in the mouse Bra-homolog, called T core_functions: - description: >- Brachyury-class T-box transcription factor that binds the conserved T-box DNA motif and occupies cis-regulatory regions of genes in Capsaspora, mainly at first introns and 5' UTRs that are active in the amoeboid and aggregative stages. Predicted targets include cell polarity, phagocytosis and motility genes, some shared with mouse Brachyury. When expressed in Xenopus embryos it acts as a nonselective activator of T-box target genes and lacks the target specificity of animal Brachyury. Its role in Capsaspora biology has not been tested genetically. molecular_function: id: GO:0000981 label: DNA-binding transcription factor activity, RNA polymerase II-specific directly_involved_in: - id: GO:0006357 label: regulation of transcription by RNA polymerase II locations: - id: GO:0005634 label: nucleus - id: GO:0000785 label: chromatin supported_by: - reference_id: PMID:24043797 supporting_text: >- Our results indicate that CoBra has a highly similar motif to that determined in the mouse Bra-homolog, called T - reference_id: PMID:27114036 supporting_text: >- The ATAC-defined Bra regulatory sites were strongly enriched in CoBra compared with random motifs (Figure 6G), validating our Bra target prediction approach. - reference_id: PMID:24043797 supporting_text: >- 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 suggested_questions: - question: >- 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. - question: >- 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? - question: >- Does CoBra activate or repress its predicted targets in Capsaspora, and does it regulate the transitions between the amoeboid, aggregative and cystic stages? - question: >- Which cofactors, if any, does CoBra bind in Capsaspora, given that the cofactor Smad1 implicated in restricting animal Brachyury targets is absent? suggested_experiments: - hypothesis: >- CoBra directly activates a cell-polarity and motility gene network in Capsaspora. description: >- 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. experiment_type: genetic loss of function / chromatin profiling - hypothesis: The anti-CoBra antibody recognizes the CAOG_005512 product. description: >- 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. experiment_type: protein identification - hypothesis: CoBra is nuclear in all three life stages. description: >- Immunostain CoBra or image a tagged transgene in filopodial, aggregative and cystic cells to test stage-dependent nuclear localization. experiment_type: localization / imaging references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000118 title: TreeGrafter-generated GO annotations findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:24043797 title: Early evolution of the T-box transcription factor family. reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Full text cached (PMC3791752). The primary CoBra paper: phylogeny, Xenopus rescue and overexpression, XBra/CoBra chimeras and universal PBM. All functional assays except the PBM are heterologous (Xenopus). No locus ID is given for CoBra; the mapping to A0A0D2VUC6 is our inference. findings: - statement: CoBra partially rescues dominant-negative XBra Xenopus embryos. supporting_text: >- Our data show that C. owczarzaki Brachyury ( CoBra ) can partially rescue Xenopus laevis embryos injected with a dominant negative XBra construct. - statement: >- Unlike animal Brachyury, CoBra activates all mesendodermal genes tested in Xenopus. supporting_text: >- which strongly activated all mesendodermal genes - statement: >- Chimeras map the specificity difference to the N- and C-terminal regions, not the T-box. supporting_text: >- N-terminal and C-terminal domains, even though they do not contain any recognizable conserved amino acidic motifs, could largely account for the metazoan Brachyury homologs specificity - statement: CoBra binds a DNA motif highly similar to that of mouse Brachyury (PBM). supporting_text: >- Our results indicate that CoBra has a highly similar motif to that determined in the mouse Bra-homolog, called T - statement: CoBra has Arg at the position of Xenopus Bra Lys149. supporting_text: >- despite the presence of an Arginine (R) instead of a Lysine (K) in the CoBra protein - id: PMID:27114036 title: The Dynamic Regulatory Genome of Capsaspora and the Origin of Animal Multicellularity. reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Full text cached. Provides the only in vivo evidence for CoBra: Bra-motif regulatory sites from ATAC-seq, their stage association and histone marks, and ChIP-qPCR with an anti-CoBra antibody at 20 sites. The target network is inferred from motifs, not from loss of function. findings: - statement: >- Bra-motif regulatory sites are mainly in first introns and 5' UTRs and are associated with the amoeboid and aggregative stages. supporting_text: >- 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 - statement: CoBra ChIP-qPCR validates the predicted sites. supporting_text: >- The ATAC-defined Bra regulatory sites were strongly enriched in CoBra compared with random motifs (Figure 6G), validating our Bra target prediction approach. - statement: >- Targets shared with mouse Brachyury are enriched in actin-cytoskeleton and motility functions. supporting_text: >- those shared orthologs are enriched in actin cytoskeleton and amoeboidal cell-motility functions - id: file:projects/ORIGINS_OF_MULTICELLULARITY/capsaspora-tbox/RESULTS.md title: Which Capsaspora T-box protein is Brachyury (CoBra)? reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Our own reproducible analysis (tbox_assign.py). Assigns A0A0D2VUC6 as CoBra from T-box domain similarity and the arginine at the Xenopus Bra K149 position. Not a phylogeny, so the assignment is an inference. findings: - statement: A0A0D2VUC6 (CAOG_005512) is assigned as CoBra. supporting_text: >- A0A0D2VUC6 (CAOG_005512) is CoBra.