Sp-FoxB (originally cloned as Spfkh1) is the purple sea urchin forkhead box B class transcription factor: a 360-residue, basic (pI 9.96) protein with an N-terminal winged-helix Fork-head DNA-binding domain, a nuclear localization signal and a transactivation region. It is a regulatory gene of the skeletogenic micromere / primary mesenchyme cell lineage, activated in the late tier of the skeletogenic gene regulatory network by inputs from the double-negative-gate outputs Tbr and Alx1 (in an Ets1-Alx1 feed-forward arrangement), alongside dri and foxo. Together with Alx1, Ets1, Tbr, Tel, Erg, Hex and Dri its product is required as a driver of the skeletogenic differentiation gene batteries that build the larval biomineral skeleton, and in Lytechinus the orthologue is required for the apical-constriction step of primary mesenchyme cell epithelial-mesenchymal transition. Additional, later expression domains outside the skeletogenic lineage have been reported, and its upstream cis-regulatory region shows signatures of directional selection in natural populations.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of RNA polymerase II cis-regulatory sequence-specific DNA binding at the forkhead family node PTN000930906 (mouse/rat Foxa, human FOXA/FOXB-type proteins, worm, fly, yeast forkheads). Sp-FoxB carries an intact Fork-head winged-helix DNA-binding domain (residues 13-107) and sits in the FoxB subclass, so inheritance of family-wide sequence-specific DNA binding is expected. Reason: The family-level judgment is sound and the cloning paper confirms the DNA-binding domain; no S. purpuratus binding-site or EMSA data exist for FoxB itself, so this remains inferred rather than demonstrated. Supporting Evidence: PMID:10433970 Spfkh1 is a Strongylocentrotus purpuratus transcription factor that contains a winged helix DNA binding domain. PMID:10433970 this gene falls into Class II of the winged helix transcription factors. |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of sequence-specific DNA-binding transcription factor activity from the forkhead family node. Consistent with the cloning paper (DNA-binding domain, NLS and transactivation domain in one ORF) and with the gene regulatory network role, in which the FoxB product is one of the drivers required for transcription of the skeletogenic differentiation gene batteries. Reason: Core molecular function of the gene product; the IBA is corroborated by the domain structure and by morpholino-based network perturbation in S. purpuratus. Supporting Evidence: PMID:10433970 Spfkh1 is transcribed in one open reading frame that contains the DNA binding domain, nuclear localization signal and transactivation domain. PMID:18413610 these differentiation genes require as drivers products of all of the now familiar components of the skeletogenic regulatory state ( alx1 , ets1 , tbr , tel , erg , hex , foxb , dri ) and in addition a factor that is at this stage ubiquitously present, Hnf6 ( 42 ). |
| GO:0003677 DNA binding | IEA GO_REF:0000104 | MODIFY | Summary: Generic UniRule DNA binding from the Fork-head domain signature. Correct but uninformative; the IBA row already states the specific activity (RNA polymerase II cis-regulatory region sequence-specific DNA binding). Reason: Too general; the forkhead domain binds sequence-specifically at RNA polymerase II cis-regulatory regions, which is captured by the accepted IBA GO:0000978. Proposed replacements: RNA polymerase II cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:10433970 Spfkh1 is a Strongylocentrotus purpuratus transcription factor that contains a winged helix DNA binding domain. |
| GO:0003700 DNA-binding transcription factor activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the Fork-head domain signatures to DNA-binding transcription factor activity. Correct, but the RNA polymerase II-specific child (GO:0000981) is already asserted by IBA and better describes a metazoan forkhead factor. Reason: Replace with the more specific accepted term GO:0000981; the parent adds nothing. Proposed replacements: DNA-binding transcription factor activity, RNA polymerase II-specific Supporting Evidence: PMID:10433970 Spfkh1 is transcribed in one open reading frame that contains the DNA binding domain, nuclear localization signal and transactivation domain. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference that the forkhead family factor is active in the nucleus. The cloning paper identifies a nuclear localization signal in the Spfkh1 ORF, and a DNA-binding transcription factor must act in the nucleus. Reason: Consistent with the predicted NLS and with transcription factor function; no direct immunolocalization is available for Sp-FoxB. Supporting Evidence: PMID:10433970 Spfkh1 is transcribed in one open reading frame that contains the DNA binding domain, nuclear localization signal and transactivation domain. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: UniProt subcellular-location (UniRule/PROSITE Fork-head rule) inference of nuclear localization; redundant with the IBA row but correct. Reason: Correct location for a forkhead transcription factor with a predicted NLS. Supporting Evidence: PMID:10433970 Spfkh1 is transcribed in one open reading frame that contains the DNA binding domain, nuclear localization signal and transactivation domain. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping to generic regulation of DNA-templated transcription. The network perturbation data show that the FoxB product is required for (i.e. positively drives) transcription of RNA polymerase II skeletogenic differentiation genes. Reason: Too general; the gene already carries the IBA GO:0006357 and the literature supports the positive, RNA polymerase II-specific child GO:0045944 proposed below. Proposed replacements: positive regulation of transcription by RNA polymerase II Supporting Evidence: PMID:18413610 these differentiation genes require as drivers products of all of the now familiar components of the skeletogenic regulatory state ( alx1 , ets1 , tbr , tel , erg , hex , foxb , dri ) and in addition a factor that is at this stage ubiquitously present, Hnf6 ( 42 ). |
| GO:0006357 regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of regulation of RNA polymerase II transcription from the forkhead family node. Supported by the skeletogenic GRN perturbation analysis in which foxb is one of the regulatory genes whose products drive the differentiation gene batteries. Reason: Correct and corroborated by S. purpuratus perturbation data; the sign (positive) is captured by the NEW GO:0045944 row. Supporting Evidence: PMID:18413610 these differentiation genes require as drivers products of all of the now familiar components of the skeletogenic regulatory state ( alx1 , ets1 , tbr , tel , erg , hex , foxb , dri ) and in addition a factor that is at this stage ubiquitously present, Hnf6 ( 42 ). PMID:22238426 The final tier of regulators of skeletogenesis, foxO , foxB , and deadringer , are activated by the inputs that have become available. |
| GO:0009653 anatomical structure morphogenesis | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Very broad PAINT inference (anatomical structure morphogenesis) from vertebrate, fly and worm forkhead genes with morphogenetic phenotypes. For Sp-FoxB the relevant morphogenetic context is larval skeleton formation and primary mesenchyme cell ingression, but the S. purpuratus evidence is transcriptional (drives biomineral differentiation genes), and the ingression/EMT requirement is shown only for the Lytechinus variegatus orthologue. Reason: Not wrong (the gene acts within a morphogenetic programme) but too general and downstream to be a core function; the specific contribution is better captured by GO:0070169 and GO:0045944. Supporting Evidence: PMID:24598159 Three TFs highest in the GRN specified and activated EMT (alx1, ets1, tbr) and the 10 TFs downstream of those (tel, erg, hex, tgif, snail, twist, foxn2/3, dri, foxb, foxo) were also required for EMT. PMID:24598159 To observe regulatory control of EMT directly, we used the sea urchin Lytechinus variegatus |
| GO:0030154 cell differentiation | IBA GO_REF:0000033 | MODIFY | Summary: Broad PAINT inference of cell differentiation from forkhead family members. In S. purpuratus FoxB is a component of the skeletogenic regulatory state that drives the biomineral differentiation gene batteries of the primary mesenchyme cells. Reason: Replace the generic term with the specific process the gene product drives, positive regulation of biomineral tissue development (GO:0070169), following the convention used for the other skeletogenic regulators alx1 and ets1. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000930906 SUPPORTS TRANSFER Forkhead family node; the generic cell-differentiation inference holds for the family and for sea urchin FoxB (a driver of the skeletogenic differentiation gene batteries), but the root-level term is uninformative and the target evidence supports the specific process, positive regulation of biomineral tissue development. Proposed replacements: positive regulation of biomineral tissue development Supporting Evidence: PMID:18413610 these differentiation genes require as drivers products of all of the now familiar components of the skeletogenic regulatory state ( alx1 , ets1 , tbr , tel , erg , hex , foxb , dri ) and in addition a factor that is at this stage ubiquitously present, Hnf6 ( 42 ). PMID:18413610 the activation of the sets of genes ( 22 ) that actually constitute the skeletal biomineral and cause the cells to execute the many cell biology functions required for skeletal deposition. |
| GO:0043565 sequence-specific DNA binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping of the Fork-head domain to sequence-specific DNA binding; correct for a winged-helix forkhead factor, though less specific than the IBA GO:0000978. Reason: Correct; retained as a non-contradictory generic parent of the accepted specific binding term. Supporting Evidence: PMID:10433970 Spfkh1 is a Strongylocentrotus purpuratus transcription factor that contains a winged helix DNA binding domain. |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IMP PMID:18413610 Global regulatory logic for specification of an embryonic ce... | NEW | Summary: In the S. purpuratus skeletogenic GRN, foxb is activated by Tbr and Alx1 and its product is one of the regulatory-state components whose morpholino knockdown reduces transcription of the skeletogenic differentiation genes (Oliveri et al. 2008, Fig. 6A and Table S1), i.e. FoxB positively regulates RNA polymerase II transcription of its target battery. Reason: Specific, signed child of the accepted IBA GO:0006357 and the informative replacement for the IEA GO:0006355. The quantitative perturbation data sit in the supplementary table, so the row rests on the main-text statement that the differentiation genes require FoxB as a driver; direct binding to target cis-regulatory modules has not been shown. Supporting Evidence: PMID:18413610 these differentiation genes require as drivers products of all of the now familiar components of the skeletogenic regulatory state ( alx1 , ets1 , tbr , tel , erg , hex , foxb , dri ) and in addition a factor that is at this stage ubiquitously present, Hnf6 ( 42 ). PMID:18413610 The tbr and alx1 genes also provide inputs into the additional skeletogenic differentiation genes dri and foxb. PMID:22238426 The final tier of regulators of skeletogenesis, foxO , foxB , and deadringer , are activated by the inputs that have become available. PMID:29558892 a feedforward mechanism originally proposed by Oliveri and co-workers to account for the regulation of Sp-msp130, Sp- msp103L, and Sp-foxb [11] may control a large fraction of the effector genes in the PMC GRN |
| GO:0070169 positive regulation of biomineral tissue development | IMP PMID:18413610 Global regulatory logic for specification of an embryonic ce... | NEW | Summary: The differentiation genes that FoxB helps drive are the biomineralization genes that constitute the larval skeletal biomineral (msp130-type and spicule matrix genes); FoxB is listed with Alx1, Ets1, Tbr, Tel, Erg, Hex and Dri as a required driver of these batteries. Reason: Participation test: the FoxB product itself performs a regulatory step (transcriptional activation of biomineral genes) within the primary mesenchyme cells, rather than being merely required upstream. Comparator check: the same term is proposed for the co-acting skeletogenic regulators alx1 and ets1 in this repository, and vertebrate transcription factors driving mineralized-tissue genes carry it. Kept minimal (one lineage/process term) because the only evidence is GRN perturbation; the EMT requirement shown in Lytechinus is recorded in notes rather than annotated. Supporting Evidence: PMID:18413610 these differentiation genes require as drivers products of all of the now familiar components of the skeletogenic regulatory state ( alx1 , ets1 , tbr , tel , erg , hex , foxb , dri ) and in addition a factor that is at this stage ubiquitously present, Hnf6 ( 42 ). PMID:18413610 the activation of the sets of genes ( 22 ) that actually constitute the skeletal biomineral and cause the cells to execute the many cell biology functions required for skeletal deposition. PMID:18413610 The tbr and alx1 genes also provide inputs into the additional skeletogenic differentiation genes dri and foxb. |
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Download this section (compressed HTML)Q: Which skeletogenic differentiation genes are direct FoxB targets? Oliveri et al. 2008 list foxb among the required drivers, but no FoxB binding site has been validated by cis-regulatory mutagenesis, and the PMC CRM atlases (Shashikant et al. 2018) do not report a FoxB motif.
Q: Is the Lytechinus variegatus finding that foxb is required for the apical-constriction step of primary mesenchyme cell EMT reproducible in S. purpuratus? If so, GO:0010718 (positive regulation of epithelial to mesenchymal transition), already proposed for alx1 and ets1, would also apply to Sp-FoxB.
Q: What are the later, non-skeletogenic expression domains of Sp-FoxB (Minokawa et al. 2004; Tu et al. 2006) and do they reflect separate regulatory functions, e.g. in the oral ectoderm or gut, as the Wnt/hedgehog-responsive promoter sites proposed in 1999 might suggest?
Experiment: Morpholino knockdown of Sp-foxb followed by NanoString/QPCR of the biomineralization battery (msp130, sm50, sm30, p16, p19, cyp1) and whole-embryo skeletal phenotyping at 48 h, to quantify which differentiation genes depend on FoxB in S. purpuratus and whether a skeleton-minus or ingression-defective phenotype results.
Experiment: EMSA or CUT&RUN with tagged Sp-FoxB on the msp130 and msp103L cis-regulatory modules, to test whether the feed-forward Ets1/Alx1/FoxB wiring involves direct FoxB occupancy of effector-gene CRMs.
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