Six1/2 is the single sea urchin member of the SIX1/SIX2 subgroup of SIX-class homeodomain transcription factors (sine oculis family), with an N-terminal Six domain that mediates cofactor binding, a central homeodomain that recognises the GGGTATCA-type Six consensus, and a His-rich C-terminus. In the purple sea urchin embryo it is a late-tier regulatory gene of the aboral non-skeletogenic mesoderm (pigment cell precursor) regulatory state, switched on in the mesenchyme blastula after gataE, downstream of Delta/Notch signalling, Gcm and GataE, together with its cofactor Eya. Acting in the nucleus as a sequence-specific activator, Six1/2 provides a positive input through a Six site in the late cis-regulatory module of gcm, forming with Gcm a positive intergenic feedback loop that sustains gcm expression in specified pigment cell precursors after the initiating Delta signal has ceased; its knockdown lowers gcm output about three-fold but does not prevent pigment cell formation. Later it is expressed with pax6, eya, dach and soxE in the aboral veg2-derived cells at the archenteron tip and in the left coelomic pouch, where its expression depends on BMP signalling and is excluded from the right side by Nodal; in the related sea urchin Lytechinus variegatus this coelomic pouch Pax6/Six3/Six1/2/Eya/Dach1 subcircuit directs the homing of the small micromeres.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference from the SIX family node PTN000044272, seeded by mouse and human SIX factors with cis-regulatory binding data. The sea urchin protein has an intact homeodomain (residues 120-180), and cis-regulatory work in this species identifies a Six1 consensus match (GGGTATCA) as one of the two critical elements of the gcm late module, whose mutation reduces output in parallel with Six1 knockdown. Experimental support merged from the primary literature: site deletion in the gcm late module and six1 morpholino give concordant reductions of module output, which the authors interpret as a direct interaction of Six1 with the GGGTAT element. Reason: The IBA is consistent with cis/trans perturbation evidence for a functional Six site in an RNA polymerase II cis-regulatory module of gcm; this is the correct specific DNA-binding term. The sea urchin evidence is inferential (no in vitro binding or ChIP), so it is recorded here rather than as a separate experimental row. Supporting Evidence: PMID:22509525 Cis-perturbation analyses reveal that the two critical elements within this late module are consensus matches to Gcm and Six1 binding sites. PMID:22509525 The similarity of outcomes from these cis- and trans-perturbations is consistent with a direct interaction of Six1 with this GGGTAT element in the late module. We conclude Six1 is an important regulator of gcm expression in the second phase that begins after PMC ingression. |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of sequence-specific transcription factor activity from Drosophila sine oculis, mouse and human SIX genes and zebrafish six1. Sea urchin Six1/2 behaves in vivo as a transcription factor whose depletion lowers endogenous gcm and gcm late-module reporter output, and whose consensus site in that module is required, so inheritance of the family-level function is confirmed for this target. Reason: Core molecular function; confirmed in this species by the concordant cis- and trans-perturbation of the gcm late module. The sign of the activity (activator) is proposed as a NEW row below. Supporting Evidence: PMID:22509525 Trans-perturbations with anti-sense morpholinos reveal a co-dependency between six1 and gcm. Six1 mRNA levels fall sharply after Gcm is depleted, while depleting Six1 leads to significant reductions in output of endogenous gcm or modular-reporters. |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the homeobox conserved-site signature. Correct for a SIX homeodomain transcription factor and redundant with the IBA row above, which is confirmed experimentally for this gene. Reason: Correct electronic assignment of the core molecular function; experimental support is carried on the IBA row for the same term. Supporting Evidence: PMID:22509525 We conclude Six1 is an important regulator of gcm expression in the second phase that begins after PMC ingression. |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | MODIFY | Summary: Generic UniRule/InterPro assignment from the homeodomain signatures. Not wrong, but the experimentally supported binding for this gene is to a Six consensus element in the RNA polymerase II cis-regulatory late module of gcm, which is captured by the more specific term already in GOA. Reason: Replace the generic parent with RNA polymerase II cis-regulatory region sequence-specific DNA binding, which is supported by the cis-perturbation of the Six site in the gcm late module and is already present as an IBA row. Proposed replacements: RNA polymerase II cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:22509525 Cis-perturbation analyses reveal that the two critical elements within this late module are consensus matches to Gcm and Six1 binding sites. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of nuclear activity from the SIX family node, broadly seeded across fly, worm, mouse and human. Appropriate for a homeodomain transcription factor that acts on the gcm cis-regulatory module; no direct localisation study exists in sea urchin, but the function demonstrated (regulation of a target gene via a cis-regulatory site) is nuclear by definition. Reason: Correct cellular component for a DNA-binding transcription factor; consistent with the UniRule nucleus row and with the in vivo transcriptional role. Supporting Evidence: PMID:22509525 We conclude Six1 is an important regulator of gcm expression in the second phase that begins after PMC ingression. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule subcellular-location assignment from the homeobox PROSITE rule. Correct for a homeodomain transcription factor and redundant with the IBA nucleus row. Reason: Correct electronic assignment of the site of action of this transcription factor. |
| GO:0005667 transcription regulator complex | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference, seeded by a single mouse SIX gene, that SIX factors act within a transcription regulator complex, which for this family is the Six-Eya (and Dach) complex. In sea urchins the Eya cofactor is co-expressed with six1/2 in the aboral non-skeletogenic mesoderm and in the coelomic pouch, and Eya and Six1/2 knockdowns give matching regulatory effects in Lytechinus variegatus, consistent with a Six1/2-Eya complex, although no biochemical complex has been isolated from sea urchin. Reason: A single well-characterised donor is adequate support for a PAINT node placement, and the Six-Eya cofactor relationship is documented in sea urchin by co-expression and synergistic perturbation outcomes. Kept as non-core in the sense that the complex itself has not been demonstrated biochemically in this species. Supporting Evidence: PMID:23261933 The six1/2 cofactor eya is also expressed in the aboral NSM (not shown). PMID:26402456 Knowing that Eya is a transcriptional co-activator of Six1/2, Eya must positively regulate itself, six3, and six1/2 by co-acting with Six1/2. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | MODIFY | Summary: Generic InterPro2GO process term from the homeodomain signatures. The gene product demonstrably regulates transcription of gcm from an RNA polymerase II cis-regulatory module, and the direction is positive, so the specific RNA polymerase II terms are better supported. Reason: Too general; replace with positive regulation of transcription by RNA polymerase II, which is supported by the three-fold loss of endogenous gcm and reporter output after Six1 depletion and by the requirement for the Six site in the gcm late module. Proposed replacements: positive regulation of transcription by RNA polymerase II Supporting Evidence: PMID:22509525 injection of 250µM six1-MO resulted in an average three-fold lower peak output of endogenous gcm in real time PCR assays (Table 1). |
| GO:0006357 regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of regulation of RNA polymerase II transcription from the SIX family node. Confirmed in sea urchin by the cis- and trans-perturbation of the gcm late module, where Six1 is a positive input that sustains gcm expression in pigment cell precursors after the Delta signal has ceased; the positive sign is proposed as a NEW, more specific row. Reason: Correct process term, grounded in this species by morpholino and cis-regulatory data on the gcm late module. Supporting Evidence: PMID:22509525 These results support the conclusion gcm and six1 comprise a positive intergenic feedback loop in the mesodermal GRN. PMID:23261933 This work demonstrated the institution of a feedback relation with six1/2, which serves to stabilize the aboral NSM regulatory state in addition to positive gcm auto-regulation. |
| GO:0001228 DNA-binding transcription activator activity, RNA polymerase II-specific | IMP PMID:22509525 Cis-regulatory logic driving glial cells missing: self-susta... | NEW | Summary: In S. purpuratus the Six1 consensus element GGGTAT is one of the two critical elements of the gcm late cis-regulatory module; its deletion reduces module output, and translation-blocking six1 morpholino reduces endogenous gcm and late-module reporter output three- to four-fold at peak, with the gataE morpholino (which specifically depletes six1 mRNA) giving an independent confirmation. The concordance of cis and trans perturbations establishes Six1/2 as a positive, sequence-specific input into an RNA polymerase II-transcribed target. Reason: The sign of the transcription factor activity is established by combined cis-regulatory site mutagenesis and knockdown on a defined target; the parent GO:0000981 is already in GOA, so the activator child is the only new information. Evidence is IMP (morpholino) corroborated by reporter cis-perturbation. Supporting Evidence: PMID:22509525 The similarity of outcomes from these cis- and trans-perturbations is consistent with a direct interaction of Six1 with this GGGTAT element in the late module. We conclude Six1 is an important regulator of gcm expression in the second phase that begins after PMC ingression. PMID:22509525 injection of 250µM six1-MO resulted in an average three-fold lower peak output of endogenous gcm in real time PCR assays (Table 1). PMID:22509525 a Six1 consensus sequence, GGGTATCA (UniPROBE, (Newburger and Bulyk, 2009) |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IMP PMID:22509525 Cis-regulatory logic driving glial cells missing: self-susta... | NEW | Summary: Six1/2 depletion lowers peak endogenous gcm mRNA from about 3600 to about 1200 copies per embryo and reduces gcm late-module reporter output three- to four-fold, and the Six site in that module is required for its output, so Six1/2 positively regulates RNA polymerase II transcription of gcm; with Gcm autoregulation this constitutes the positive intergenic feedback loop that locks down gcm expression in pigment cell precursors. The same positive sign is seen in the coelomic pouch subcircuit of Lytechinus variegatus, where Six1/2 is upstream of six3, eya and itself. Reason: Six1/2 itself performs the regulatory step (binding the gcm late module and driving its output), so this is participation rather than mere necessity; the term is the signed child of GO:0006357, which GOA already carries by IBA. Downstream consequences for the pigment cell state are not annotated, because six1 knockdown does not alter pigment cell formation. Supporting Evidence: PMID:22509525 These results support the conclusion gcm and six1 comprise a positive intergenic feedback loop in the mesodermal GRN. PMID:22509525 injection of 250µM six1-MO resulted in an average three-fold lower peak output of endogenous gcm in real time PCR assays (Table 1). PMID:22238426 A small subnetwork downstream of gcm includes the gatae gene and the six1/2 gene, which feeds back on gcm , as well as on an auto-regulatory feed from gcm onto itself PMID:26402456 Six1/2 is seen to be upstream of six3, eya, and six1/2. |
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Download this section (compressed HTML)Q: Does sea urchin Six1/2 require Eya as a co-activator at the gcm late module, and do Six1/2 and Eya physically interact as in vertebrates and Drosophila?
Q: What does Six1/2 do in the left coelomic pouch (hydrocoel) downstream of BMP signalling, and does the Lytechinus small-micromere homing function hold in S. purpuratus?
Q: Which GataE input into six1/2 is direct, and does Six1/2 feed back on gataE as well as on gcm?
Experiment: Mutagenesis of the GGGTATCA Six site in the gcm late module reporter combined with six1 morpholino and with co-injection of six1 mRNA, to test epistasis and confirm that the site mediates the Six1/2 input.
Type: cis-regulatory reporter analysis
Experiment: DNA-affinity capture or ChIP with tagged Six1/2 at mesenchyme blastula and early pluteus to identify direct targets in pigment cell precursors and in the left coelomic pouch.
Type: ChIP-seq
Experiment: Mesoderm-targeted six1/2 knockdown in S. purpuratus followed by scoring of small micromere homing and left coelomic pouch marker expression, to test whether the Lytechinus coelomic pouch function is conserved.
Type: morpholino knockdown
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Direct binding of Six1/2 to the GGGTAT element of the gcm late module is inferred from the concordance of site deletion and morpholino effects; no in vitro binding, ChIP or Six1/2 expression construct data exist for sea urchin.
OPEN BIOLOGY
Provenance (the field's own admissions):
Gap: Apart from gcm, the direct targets of Six1/2 in pigment cell precursors and in the left coelomic pouch are unknown, and its function in the S. purpuratus coelomic pouch has not been tested by perturbation.
OPEN BIOLOGY
Provenance (the field's own admissions):
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