Gcm is the single sea urchin orthologue of the Drosophila glial cells missing transcription factor, carrying an N-terminal GCM DNA-binding domain that recognises the ATGCGGRY consensus and C-terminal trans-activation domains. In the purple sea urchin embryo it is the immediate transcriptional target of Delta/Notch signalling from the skeletogenic micromeres, switched on in the adjacent veg2 cells at late cleavage through Su(H) sites in its middle cis-regulatory module, and it is the top-level, pioneering regulator of the aboral non-skeletogenic mesoderm (pigment cell) regulatory state. Acting in the nucleus as a sequence-specific activator, Gcm directly drives the GATA factor gene gataE and the pigment differentiation gene polyketide synthase (pks), and, through a late module bearing Gcm and Six1 sites, sustains its own expression in a gcm-gataE-six1 feedback loop that locks down pigment cell fate. Morpholino knockdown produces larvae with no pigment cells and abolishes the pigment differentiation gene battery, while forced expression in skeletogenic cells converts them to pigment cells. Its expression is confined by Alx1 repression in the skeletogenic lineage, FoxA repression in the endoderm and Not-dependent clearance from the oral mesoderm.
| 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 GCM family node seeded by human GCM2. The sea urchin protein has the GCM DNA-binding domain (residues 50-208), and cis-regulatory work in this species shows that consensus Gcm sites (ATGCGGRY) in the pks -1.5 kb region and in the gcm late module are required for output of those modules, i.e. Gcm binds cis-regulatory DNA of RNA polymerase II transcribed genes. Experimental support merged from the primary literature: Predicted Gcm consensus sites in the pks cis-regulatory region and in the gcm late cis-regulatory module are functionally required in reporter assays, demonstrating sequence-specific binding of Gcm to RNA polymerase II cis-regulatory DNA in vivo. Reason: The IBA is consistent with direct cis-regulatory evidence for Gcm sites in pks and gcm regulatory modules; the term is the correct specific DNA-binding term. A separate experimental NEW row would duplicate this GOA term, so its evidence is carried here: Site-directed mutagenesis of Gcm consensus sites in two separate cis-regulatory modules gives loss of output; this is the standard evidence for cis-regulatory-region sequence-specific DNA binding by a transcription factor. Supporting Evidence: PMID:20122918 Predicted DNA-binding sites for SpGcm, SpGataE and SpKrl are located within this region. The mutagenesis of these DNA-binding sites indicated that SpGcm, SpGataE and SpKrl are direct positive regulators of SpPks. 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: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 gcm and human GCM1/GCM2. Sea urchin Gcm behaves in vivo as a DNA-binding transcription factor that directly activates gataE and pks, so the inheritance of the family-level function is confirmed for this target. Reason: Core molecular function; confirmed in this species by cis-regulatory mutagenesis of Gcm sites in target genes. Supporting Evidence: PMID:23261933 A linchpin of this network is gataE which as we show is a direct Gcm target and part of a feedback loop locking down the aboral regulatory state. |
| GO:0001228 DNA-binding transcription activator activity, RNA polymerase II-specific | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the GCM family entry. The sign of the activity is established experimentally for sea urchin Gcm, which is a direct positive regulator of pks (site mutagenesis) and of gataE, and autoregulates its own late module positively. Experimental support merged from the primary literature: Mutagenesis of predicted Gcm binding sites in the -1.5 kb pks cis-regulatory region, which suffices to recapitulate pks expression in pigment cells, reduces reporter output, establishing Gcm as a direct positive regulator of an RNA polymerase II target. Independently, Gcm sites in the gcm late module are required for peak output (autoregulation), gataE is a direct Gcm target, and forced gcm expression in skeletogenic cells sharply up-regulates pks. Reason: The electronic activator assignment matches direct cis-regulatory evidence in this species; an experimental row for the same term is added below. A separate experimental NEW row would duplicate this GOA term, so its evidence is carried here: The sign (activator) and directness (site-dependent cis-regulatory output) are both demonstrated in this species, so the activator-specific MF is warranted beyond the electronic assignment. Supporting Evidence: PMID:20122918 The mutagenesis of these DNA-binding sites indicated that SpGcm, SpGataE and SpKrl are direct positive regulators of SpPks. PMID:22509525 These different assays consistently show Gcm sites are required for peak output from the late module. PMID:23261933 A linchpin of this network is gataE which as we show is a direct Gcm target and part of a feedback loop locking down the aboral regulatory state. |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | MODIFY | Summary: Domain-level electronic inference from the GCM DNA-binding domain. Correct but generic; the informative binding term (GO:0000978) is already present and supported by cis-regulatory evidence. Reason: The GCM domain binds RNA polymerase II cis-regulatory DNA in a sequence-specific manner (Gcm consensus sites in pks and gcm modules), so the more specific term GO:0000978 should replace the bare DNA binding term. Proposed replacements: RNA polymerase II cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:22509525 This candidate Gcm site matched the published consensus ATGCGGRY |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of nuclear site of action from fly and mammalian GCM proteins. Sea urchin Gcm is a transcription factor whose only characterised activity is cis-regulatory activation of nuclear target genes, so the nucleus is the location in which it is active; no immunolocalisation exists in this species. Reason: Consistent with the transcription factor function demonstrated in vivo. Supporting Evidence: PMID:22509525 output from this recombinant BAC has the potential to enter nuclei and bind to consensus Gcm sites. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic subcellular-location inference from the GCM domain rule; redundant with the IBA row but correct. Reason: A DNA-binding transcriptional activator of gataE and pks must be nuclear. Supporting Evidence: PMID:23261933 A linchpin of this network is gataE which as we show is a direct Gcm target and part of a feedback loop locking down the aboral regulatory state. |
| GO:0006351 DNA-templated transcription | IEA GO_REF:0000104 | MODIFY | Summary: UniRule transfer of a very general transcription term. Gcm is a sequence-specific regulator of RNA polymerase II transcription, not a component of the transcription machinery; the appropriate process is regulation of transcription by RNA polymerase II (already present as an IBA row). Reason: Too general and mis-framed; replace with the RNA polymerase II regulatory term that describes what Gcm does. Proposed replacements: regulation of transcription by RNA polymerase II Supporting Evidence: PMID:22306924 Gcm itself is an activator required, in a feed forward relationship with respect to the D/N input, for gataE expression to occur in the NSM. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Correct but general parent of GO:0006357, which is present with IBA support. Kept as a non-core general term. Reason: Redundant with the more specific RNA polymerase II term; not wrong. |
| GO:0006357 regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that is fully borne out in the sea urchin, where Gcm regulates transcription of gataE, six1, pks and the pigment differentiation battery. Reason: Core process; the positive-regulation child is supported experimentally below. Supporting Evidence: PMID:23261933 As expected, Gcm MASO causes severe decreases in transcript levels of the differentiation genes bpnt, dopt, fmo, papss, pks, and sult. |
| GO:0042063 gliogenesis | IBA GO_REF:0000033 | REMOVE | Summary: The PAINT node carrying gliogenesis is seeded by Drosophila gcm and gcm2 and by mouse Gcm1. The glial function is a Drosophila-specific deployment of the family; mammalian Gcm1/Gcm2 act in placenta and parathyroid, and sea urchin gcm is expressed exclusively in the mesodermal pigment cell lineage from late cleavage onward, never in neural or glial tissue, and its knockdown phenotype is loss of pigment cells. There is therefore target-specific evidence that this lineage does not use gcm for gliogenesis, so the propagation to this node over-reaches. Reason: Over-propagated phylogenetic inference contradicted by the expression and loss-of-function phenotype of the sea urchin gene (pigment cell specification, not glial development). Propagation Review Root cause: PROPAGATION BAD Failure modes: LINEAGE OR TAXON MISMATCH CONTEXT OR TISSUE MISMATCH Sources checked: PANTHER:PTN000275807 SUPPORTS SOURCE BUT NOT TARGET GCM-family node whose gliogenesis inference is seeded by Drosophila gcm/gcm2; the sea urchin gene is expressed only in the mesodermal pigment-cell lineage and its knockdown phenotype is loss of pigment cells, so the glial deployment is lineage-specific and does not transfer. Supporting Evidence: PMID:12027439 Spgcm, an orthologue of the fruit fly gene glial cells missing, which is first expressed specifically and exclusively in part of the prospective secondary mesenchyme (mesodermal) domain at late-cleavage blastula stage PMID:16925988 Here, we show that microinjection of a spgcm antisense morpholino oligonucleotide results in larvae without pigment cells. |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IEA GO_REF:0000108 | ACCEPT | Summary: Inter-ontology inference from the activator MF, and independently supported by experiment: Gcm morpholino knockdown severely reduces transcripts of gataE, six1/2, z166 and the pigment differentiation genes bpnt, dopt, fmo, papss, pks and sult; gataE and pks are direct cis-regulatory targets, and Gcm positively autoregulates its own late module. Reason: The electronic row is retained as the carrier of this term (a separate experimental NEW row would duplicate it). Participation test passed for gataE, pks and gcm itself, where the Gcm binding sites have been mapped; the knockdown data extend this to the immediate aboral NSM battery. Supporting Evidence: PMID:20122918 The mutagenesis of these DNA-binding sites indicated that SpGcm, SpGataE and SpKrl are direct positive regulators of SpPks. PMID:23261933 As expected, Gcm MASO causes severe decreases in transcript levels of the differentiation genes bpnt, dopt, fmo, papss, pks, and sult. PMID:22306924 Gcm itself is an activator required, in a feed forward relationship with respect to the D/N input, for gataE expression to occur in the NSM. PMID:22509525 The self-sustaining expression of gcm that the late module promotes effectively locks down the pigment cell fate. |
| GO:0007501 mesodermal cell fate specification | IMP PMID:16925988 cis-regulatory processing of Notch signaling input to the se... | NEW | Summary: Gcm is the direct genomic target of Delta/Notch signalling in the veg2 cells and the first regulatory gene of the aboral non-skeletogenic mesoderm (pigment cell precursor) state. Morpholino knockdown gives larvae without pigment cells, and rewiring gcm under tbr control converts skeletogenic mesoderm cells into pigment cells in the absence of Notch signalling, so gcm expression is necessary and sufficient to specify this mesodermal sub-fate. Gcm itself performs the first regulatory step (activation of gataE and the downstream aboral NSM circuit). Reason: Gcm is the transcription factor that executes the specification step downstream of the Delta signal (participation, not merely necessity); the sibling GRN regulators Alx1 and Pmar1 carry the same term for the skeletogenic mesoderm lineage. Downstream repression of skeletogenic and oral-NSM genes is indirect and not annotated. Supporting Evidence: PMID:16925988 These results confirm that this gene is required for pigment cell specification. PMID:22238426 The result of reengineering the regulatory circuitry in this way was to divert the developmental program of these cells from skeletogenesis to pigment cell formation, confirming a direct prediction of the GRN. PMID:22509525 Gcm is required in veg2 progeny during late cleavages for the early phase of pigment cell precursor specification. PMID:23261933 First the gcm gene acts as the primary, novel, regulatory state pioneer activated by the D/N signal |
| GO:0050942 positive regulation of pigment cell differentiation | IMP PMID:23261933 Diversification of oral and aboral mesodermal regulatory sta... | NEW | Summary: Gcm directly activates the pigment differentiation gene pks (echinochrome polyketide synthase) via mapped Gcm sites, and Gcm knockdown abolishes the pigment cell differentiation battery (pks, fmo, sult, dopt, papss, bpnt) and pigment cells themselves; its self-sustaining late-module expression persists in differentiated, dispersed pigment cells. The same role is confirmed in other euechinoids. Reason: Gcm provides the direct transcriptional input into the differentiation gene battery, so it positively regulates pigment cell differentiation rather than merely being upstream of it; the sibling regulator GataE carries this term for the same battery. Supporting Evidence: PMID:23261933 Gcm provides a direct input into pigment cell differentiation genes, which begin to be expressed even before gastrulation PMID:20122918 The mutagenesis of these DNA-binding sites indicated that SpGcm, SpGataE and SpKrl are direct positive regulators of SpPks. PMID:16925988 Here, we show that microinjection of a spgcm antisense morpholino oligonucleotide results in larvae without pigment cells. PMID:27046223 We confirmed previous results by demonstrating that gcm is involved in pigment cell differentiation. |
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Download this section (compressed HTML)Q: Which Gcm site(s) in the gataE cis-regulatory module mediate the direct Gcm input, and does Gcm bind them cooperatively with Su(H)?
Q: Does sea urchin Gcm have any function outside the pigment cell lineage (e.g. in adult or larval immune cells), given its persistent expression in dispersed pigment cells?
Q: Through which intermediate repressor does forced gcm expression shut down ets1 and the skeletogenic regulatory state?
Experiment: ChIP-seq or DNA-affinity capture with a tagged Gcm in mesenchyme blastula embryos to map genome-wide Gcm occupancy and confirm direct binding to gataE, six1 and the pigment differentiation battery.
Type: ChIP-seq
Experiment: Site-directed mutagenesis of predicted Gcm consensus sites in the gataE early NSM module in reporter assays, with and without Gcm MASO, to establish the gataE linkage at cis-regulatory resolution.
Type: cis-regulatory reporter analysis
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The repression of skeletogenic regulatory genes (ets1, alx1, tbr) and of oral NSM genes after gcm expression is indirect; the aboral repressor(s) acting downstream of Gcm/GataE have not been identified.
OPEN BIOLOGY
Provenance (the field's own admissions):
Gap: Gcm binding to the gataE cis-regulatory module is inferred from kinetics, knockdown and Notch-independence rather than from mapped Gcm sites, and no Gcm ChIP or in vitro binding data exist for this species.
OPEN BIOLOGY
Provenance (the field's own admissions):
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