Sp-HesC is a Hairy/Enhancer-of-split (HES) family basic helix-loop-helix transcriptional repressor of the purple sea urchin, with an N-terminal bHLH DNA-binding domain, an Orange domain and a C-terminal WRPW motif that recruits the Groucho/TLE corepressor. In the early embryo hesC is transcribed zygotically from about 8 h after fertilization in every cell except the large micromere lineage, where the paired-homeodomain repressor Pmar1 shuts it off. HesC is the second repressor of the pmar1-hesC double-negative gate that initiates skeletogenic mesoderm specification: it directly represses the micromere regulatory genes alx1, ets1, tbr and the Notch ligand gene delta through cis-regulatory target sites (a class C E-box in the delta proximal module, proximal and distal sites at alx1, a proximal site at tbr), so that these genes are transcribed only where HesC is absent. Blocking hesC translation phenocopies global Pmar1 expression, converting all cells of the embryo into ingressing skeletogenic mesenchyme. Later, hesC is repressed by Blimp1 in the non-skeletogenic mesoderm and activated by Delta-Notch signaling through a Su(H) site, and its withdrawal permits delta expression to sweep from mesoderm into endoderm. This micromere-gating role of HesC is an evolutionary novelty of euechinoid sea urchins; cidaroid and starfish hesC do not occupy the same position, although the family's ancestral role as a Notch-responsive repressor in ectodermal lateral inhibition is retained in echinoids.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000122 negative regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the HES/HEY PAINT family for negative regulation of Pol II transcription. This is precisely the experimentally demonstrated function of Sp-HesC: it is the second repressor of the pmar1-hesC double-negative gate and directly represses delta, alx1, ets1 and tbr outside the micromere lineage. Reason: The phylogenetic inference is confirmed for this species by knockdown (hesC morpholino derepresses delta/alx1/ets1/tbr in every cell and phenocopies global pmar1 overexpression) and by cis-regulatory analysis (mutating HesC sites in the delta proximal CRM and in the alx1 BAC gives ubiquitous reporter expression). Core function. Supporting Evidence: PMID:17636127 Thus, HesC functions to repress micromere lineage specification in all cells other than the micromere lineage PMID:17636127 As logically required, blockade of hesC mRNA translation and global overexpression of pmar1 mRNA have the same effect, which is to cause all of the cells of the embryo to express micromere-specific genes. PMID:19104065 These results indicate that the expression of delta in the NSM results from activation by Runx and repression by HesC through sites in the proximal CRM. PMID:21723273 In the complete context of the alx1 GFP BAC, when either the proximal or distal HesC binding site is mutated, striking ectopic expression results |
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: IBA for sequence-specific binding to Pol II cis-regulatory regions. Functional HesC target sites have been mapped and mutated in the delta proximal CRM (a class C E-box palindrome), in the alx1 locus (proximal and distal sites) and in a proximal tbr module; in each case loss of the site releases ectopic expression. Reason: Site-directed mutagenesis in reporter/BAC constructs in the sea urchin embryo defines the cis-regulatory sequences through which HesC acts, and the protein carries a canonical bHLH DNA-binding domain. Direct in vitro binding (EMSA/ChIP) has not been reported for the sea urchin protein, so the mapped sites are functionally rather than biochemically defined; the IBA nevertheless states the correct activity. Supporting Evidence: PMID:19104065 These results indicate that the expression of delta in the NSM results from activation by Runx and repression by HesC through sites in the proximal CRM. PMID:21723273 Thus we have experimentally identified, and by mutation functionally characterized the genomic target sites responsible for direct spatial repression by the hesc gene product, and for activation by Ets1. PMID:19679118 early skeletogenic expression is controlled by an intron enhancer plus a proximal region containing a HesC site as predicted from network analysis |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IBA GO_REF:0000033 | ACCEPT | Summary: IBA for Pol II-specific DNA-binding transcription factor activity. Correct and core; the sea urchin data further fix the sign as repression, so the child term GO:0001227 is proposed as a NEW row rather than replacing this one. Reason: HesC is a bHLH-Orange-WRPW repressor whose transcription-factor activity on defined target genes (delta, alx1, tbr) has been shown by cis-regulatory dissection and knockdown in S. purpuratus. Supporting Evidence: PMID:17636127 Because HesC has a WRPW sequence, like all of its orthologues, it must act as a repressor, and we have shown that it does not interfere with pmar1 expression. PMID:18413610 The primary regulatory genes of the skeletogenic micromere specification GRN are subject to direct HesC repression. |
| GO:0003677 DNA binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the Orange domain (IPR003650) to generic DNA binding. HesC does bind DNA, but through its bHLH domain, and much more specific terms are supported by the cis-regulatory evidence. Reason: The term is true but uninformative, and the mapping source is odd: the Orange domain is a helical protein-interaction/specificity domain, not the DNA-contacting element. The specific activity supported by site mutagenesis is RNA polymerase II cis-regulatory region sequence-specific DNA binding (GO:0000978), already present as an accepted IBA. Proposed replacements: RNA polymerase II cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:21723273 Thus we have experimentally identified, and by mutation functionally characterized the genomic target sites responsible for direct spatial repression by the hesc gene product, and for activation by Ets1. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: IBA nucleus (is_active_in) from HES-family PAINT node. Consistent with the protein being a sequence-specific transcriptional repressor acting on target cis-regulatory modules. Reason: No antibody or fusion-protein localization exists for the sea urchin protein, but every demonstrated function (repression through defined genomic target sites, recruitment of Groucho/TLE via WRPW) is nuclear; the inference is sound. Supporting Evidence: PMID:19104065 encoding group E bHLH and Orange domains and a C-terminal WRPW motif thought to recruit the Groucho/TLE corepressor |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule/SubCell-keyword mapping to nucleus. Redundant with the IBA row but correct. Reason: Sequence-based nuclear localization inference for a bHLH transcriptional repressor; consistent with all functional data. |
| GO:0006351 DNA-templated transcription | IEA GO_REF:0000104 | MODIFY | Summary: UniRule keyword-derived 'DNA-templated transcription'. A sequence-specific transcription factor regulates transcription of specific genes; it is not itself a participant in the transcription process, which is reserved for the polymerase and general machinery. Reason: Per GO convention, DNA-binding transcription factors are annotated to regulation terms, not to GO:0006351 itself. The specific regulatory process demonstrated for HesC is negative regulation of transcription by RNA polymerase II (GO:0000122). Proposed replacements: negative regulation of transcription by RNA polymerase II Supporting Evidence: PMID:18413610 The primary regulatory genes of the skeletogenic micromere specification GRN are subject to direct HesC repression. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000120 | MODIFY | Summary: InterPro/UniRule mapping to the general 'regulation of DNA-templated transcription'. Correct but too general: the direction (repression) and polymerase (Pol II) are established. Reason: All experimental evidence shows repression of Pol II-transcribed regulatory genes; the accepted IBA GO:0000122 already captures the specific process and supersedes this parent. Proposed replacements: negative regulation of transcription by RNA polymerase II Supporting Evidence: PMID:17636127 Thus, HesC functions to repress micromere lineage specification in all cells other than the micromere lineage |
| GO:0009952 anterior/posterior pattern specification | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from PAINT node PTN000105428, seeded by vertebrate Hes/Her/Hey genes of the somite segmentation clock and by C. elegans lin-22 seam-cell patterning. No anterior/posterior patterning function has been reported for sea urchin HesC; its characterized function is to restrict the skeletogenic (micromere) regulatory state along the animal-vegetal axis, a euechinoid-specific gate. Reason: The IBD node placement bundles lineage-specific vertebrate/nematode A/P roles onto a bilaterian HES node. In S. purpuratus, hesC perturbation converts the whole embryo to skeletogenic mesenchyme with no reported axial patterning defect, and the double-negative gate in which HesC acts is an evolutionary novelty of euechinoids (cidaroids and starfish do not use hesC this way). The term is not contradicted, but nothing in the target supports it and the family evidence points to independently evolved patterning roles, so it should not be treated as a function of this gene. Propagation Review Root cause: PROPAGATION BAD Failure modes: LINEAGE OR TAXON MISMATCH CONTEXT OR TISSUE MISMATCH Sources checked: PANTHER:PTN000105428 SUPPORTS SOURCE BUT NOT TARGET Bilaterian-level HES node whose A/P inference is seeded by vertebrate segmentation-clock Hes/Her/Hey genes and C. elegans lin-22; nothing in the target supports an axial patterning role and the documented Sp-HesC function (micromere gating) is a euechinoid novelty. MGI:MGI:2135679 SUPPORTS SOURCE BUT NOT TARGET Mouse Hes7 somite segmentation-clock evidence; a vertebrate-specific deployment of the family, not shown in echinoderms. WB:WBGene00003008 SUPPORTS SOURCE BUT NOT TARGET C. elegans lin-22 seam-cell A/P patterning; lineage-specific context with no echinoderm counterpart reported. Supporting Evidence: PMID:17636127 Thus, HesC functions to repress micromere lineage specification in all cells other than the micromere lineage PMID:24924196 the expression pattern and function of hesC in P. baculosa were inconsistent with the DNG model, suggesting that the euechinoid-type DNG is not utilized during cidaroid mesenchyme specification |
| GO:0046983 protein dimerization activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro mapping of the bHLH domain (IPR011598/IPR036638) to protein dimerization activity. HES proteins bind DNA as bHLH dimers, so the term is plausible, but no dimerization partner or assay has been reported for Sp-HesC. Reason: Structural inference from the bHLH fold; a generic binding term that does not describe the gene's function in the GRN. Retained as a non-core, structurally implied property. Supporting Evidence: PMID:19104065 encoding group E bHLH and Orange domains and a C-terminal WRPW motif thought to recruit the Groucho/TLE corepressor |
| GO:0050767 regulation of neurogenesis | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA for regulation of neurogenesis from the classic Notch-target role of Hes genes in lateral inhibition. In S. purpuratus hesC is Notch-responsive (a Su(H) site at +2) and expressed in the apical plate, and in the cidaroid Eucidaris tribuloides hesc perturbation mirrors delta perturbation in ectodermal lateral inhibition; a neurogenic role for Sp-HesC itself has not been tested directly. Reason: Plausible ancestral function of the family, supported in echinoids by expression, cis-regulatory Notch input and cidaroid perturbation data, but the core documented function of Sp-HesC is mesodermal (micromere) fate restriction. Kept as non-core pending direct evidence in S. purpuratus. Supporting Evidence: PMID:19104065 the hesC cis -regulatory system responds negatively to Blimp1 repression and positively to Delta-Notch signaling PMID:29249002 Similarly, hesc perturbation mirrored the effects of delta perturbation. |
| GO:0001227 DNA-binding transcription repressor activity, RNA polymerase II-specific | IDA PMID:19104065 Gene regulatory network subcircuit controlling a dynamic spa... | NEW | Summary: Proposed. GOA carries only the direction-neutral parent GO:0000981; the sea urchin data establish that HesC is a dedicated repressor acting through defined cis-regulatory sites of Pol II-transcribed regulatory genes (delta, alx1, tbr). Reason: Mutating the HesC class C E-box in the delta proximal CRM (PMID:19104065) or either HesC site in the alx1 BAC (PMID:21723273) causes ubiquitous/ectopic reporter expression, i.e. loss of repression at the level of the target cis-regulatory module; hesC knockdown derepresses the same genes embryo-wide (PMID:17636127); the protein carries the Groucho/TLE-recruiting WRPW motif. This is the definition of GO:0001227. Evidence IDA on the cis-regulatory reporter assays; additive to the accepted parent. Supporting Evidence: PMID:19104065 These results indicate that the expression of delta in the NSM results from activation by Runx and repression by HesC through sites in the proximal CRM. PMID:21723273 Thus we have experimentally identified, and by mutation functionally characterized the genomic target sites responsible for direct spatial repression by the hesc gene product, and for activation by Ets1. PMID:19104065 Because HesC is the dominant repressor of delta , this demonstrates a causal chain linking Blimp1 with delta PMID:17636127 Because HesC has a WRPW sequence, like all of its orthologues, it must act as a repressor, and we have shown that it does not interfere with pmar1 expression. |
| GO:0042662 negative regulation of mesodermal cell fate specification | IMP PMID:17636127 A missing link in the sea urchin embryo gene regulatory netw... | NEW | Summary: Proposed. HesC is the repressor that keeps the skeletogenic mesoderm (large micromere lineage) regulatory state off in every other cell of the embryo; removing it converts all cells to skeletogenic mesenchyme. Reason: Participation test: HesC itself performs the regulatory step, direct transcriptional repression of the mesodermal specification genes alx1, ets1, tbr and delta, and the cis-regulatory sites through which it does so are mapped. hesC morpholino causes all cells to express the micromere regulatory state, ingress and adopt mesenchymal form (PMID:17636127); in other euechinoids hesC knockdown likewise increases mesenchyme cell number (PMID:27046223). The term is used for transcriptional regulators that restrict mesodermal fate (e.g. Mesp1, Dkk1 lineages in QuickGO), so it is the conventional term for this role. It is not an ancestor/descendant of any term already on the gene. Downstream skeletogenesis/biomineralization terms are deliberately not proposed (consequences of the fate decision, executed by other genes). Supporting Evidence: PMID:17636127 Thus, HesC functions to repress micromere lineage specification in all cells other than the micromere lineage PMID:17636127 As logically required, blockade of hesC mRNA translation and global overexpression of pmar1 mRNA have the same effect, which is to cause all of the cells of the embryo to express micromere-specific genes. PMID:17636127 the amount of transcript of delta and alx1 had increased 4- to 7-fold above normal in the two experiments performed PMID:18413610 The primary regulatory genes of the skeletogenic micromere specification GRN are subject to direct HesC repression. PMID:27046223 The number of larval mesenchyme cells increased when the translation of hesC was inhibited |
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Download this section (compressed HTML)Q: Should the HesC-delta relationship be represented as HesC 'has input' delta (negative regulation of delta transcription) rather than as a Notch-pathway regulation term? HesC controls where the ligand gene is transcribed, which is one transcriptional step removed from the pathway.
Q: Does Sp-HesC bind the mapped class C E-box (CACGCG) sites directly? No EMSA or ChIP has been reported for the sea urchin protein; binding-site identity rests on mutagenesis and the HES family consensus.
Q: Is the IBA anterior/posterior pattern specification row from PTN000105428 appropriate for non-vertebrate members of this node, given that the seeding evidence is the vertebrate segmentation clock and C. elegans lin-22?
Experiment: ChIP-qPCR or EMSA with recombinant Sp-HesC on the delta proximal CRM (+359 E-box), the alx1 proximal/distal sites and the tbr proximal site to convert the functional site assignments into direct binding evidence.
Experiment: Immunolocalization or GFP-tagged HesC BAC to document nuclear localization and protein clearance kinetics in the micromere lineage relative to alx1/delta activation, addressing the timing discrepancy raised by quantitative FISH.
Experiment: Test whether Sp-HesC is required for serotonergic neuron restriction in the apical plate (hesC morpholino with neural markers), which would ground the family-level regulation-of-neurogenesis inference in this species.
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