Snai2 (Slug) is a Snail-family C2H2 zinc-finger transcriptional repressor of Xenopus laevis (this entry is the L homeolog). An N-terminal SNAG domain recruits corepressors, including Ajuba-family LIM proteins and the PRC2 histone methyltransferase EZH2, while five C-terminal zinc fingers bind E-box (CAGGTG/CACCTG) elements in RNA polymerase II promoters. snai2 is switched on at late gastrula in the prospective neural crest, after snai1 and downstream of the neural plate border specifiers (Pax3, Zic1) and of Wnt, FGF and BMP-attenuation signals, and is maintained in premigratory and migrating cranial and trunk crest and in lateral plate mesoderm. It acts as a neural crest specifier: early inhibition prevents formation of crest precursors and Snai2 cooperates with Wnt to establish crest identity, although it cannot induce crest alone. Later it is required for crest epithelial-to-mesenchymal transition and migration, during which it occupies the E-cadherin promoter and recruits PRC2 to deposit H3K27me3, dampening (though not abolishing) E-cadherin expression. It also supports early mesoderm and cell survival through a regulatory loop with NF-kappaB. Snail border expression is ancestral in chordates (amphioxus Snail is transiently expressed at the neural plate border), whereas the separate Snai1/Snai2 paralogs and the specifier-type expression dynamics of snai2 are vertebrate features; lamprey has a single snail gene.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: IBA (PAINT node PTN002810938, Snail/Slug/Scratch clade) assigning RNA polymerase II cis-regulatory sequence-specific DNA binding. Xenopus Snai2 occupies E-box elements of the E-cadherin promoter in neural crest. Reason: Snail-family zinc fingers bind E-box (CAGGTG/CACCTG) elements in Pol II promoters; this is the conserved activity of the clade and is confirmed for frog Snai2 by ChIP of tagged Snai2 at E-boxes near the E-cadherin TSS and 2.2 kb upstream in late-neurula embryos. Correct and core. Supporting Evidence: PMID:25617436 Snail2 bound preferentially to the E-boxes located around the TSS |
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter IEA for the same Pol II cis-regulatory sequence-specific DNA binding. Reason: Same assertion as the IBA, supported by frog ChIP evidence at E-cadherin E-boxes. Correct. Supporting Evidence: PMID:25617436 Snail2 bound preferentially to the E-boxes located around the TSS |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IBA GO_REF:0000033 | ACCEPT | Summary: IBA for DNA-binding transcription factor activity, RNA polymerase II-specific. Reason: Correct for the Snail clade and for frog Snai2. The activity is more precisely repression: an Engrailed-repressor fusion of the XSlug DNA-binding domain mimics wild-type XSlug while an activator fusion inhibits it, and Snai2 represses E-cadherin by recruiting PRC2. The more specific repressor term (GO:0001227) is added as NEW rather than modifying the phylogenetic annotation, as done for foxd3-a and sox9-a in this project. Supporting Evidence: PMID:10772801 The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. PMID:25617436 The data thus demonstrate that Snail2 helps to recruit PRC2 to the E-cad promoter to set up repressive histone modification marks that result in inhibition of E-cad expression during neural crest EMT. |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter IEA for DNA-binding transcription factor activity, RNA polymerase II-specific. Reason: Correct, if general; repressor activity is captured by the NEW GO:0001227 row. Supporting Evidence: PMID:10772801 The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. |
| GO:0001755 neural crest cell migration | IDA PMID:12490555 Snail precedes slug in the genetic cascade required for the ... | ACCEPT | Summary: Neural crest cell migration from the Aybar et al. 2003 conditional gain/loss-of-function study (activated Xslug-GR increased migrating Slug-positive crest). Reason: Several independent Xenopus lines of evidence agree that Snai2 acts during crest migration, not only specification: hormone-inducible inhibition of XSlug at later stages blocks migration without blocking precursor formation; antisense slug RNA inhibits migration and loss of cartilage derivatives, rescued by Slug or Snail; Snai2 directly represses E-cadherin through PRC2 at the stage of crest EMT. Snai2 does work in this process (transcriptional repression of epithelial adhesion genes) and the role is conserved (chick Slug). Core. The cached Aybar abstract foregrounds Snail, but the curator had full text and the role is independently supported. Supporting Evidence: PMID:12490555 Snail lies upstream of Slug in the genetic cascade leading to neural crest formation PMID:10772801 inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration PMID:10452849 These studies indicate that XSlug is required for neural crest migration PMID:25617436 The data thus demonstrate that Snail2 helps to recruit PRC2 to the E-cad promoter to set up repressive histone modification marks that result in inhibition of E-cad expression during neural crest EMT. |
| GO:0001755 neural crest cell migration | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning IEA for neural crest cell migration. Reason: Consistent with the experimental evidence for frog Snai2 (see IDA row). Supporting Evidence: PMID:10772801 inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration PMID:10452849 These studies indicate that XSlug is required for neural crest migration |
| GO:0005515 protein binding | IPI PMID:27189455 Elongator Protein 3 (Elp3) stabilizes Snail1 and regulates n... | REMOVE | Summary: IPI with Elp3 (Q5HZM6): mouse Elp3 co-immunoprecipitated Xenopus Slug, and xSlug rescued Elp3 morphant migration defects. Reason: Generic protein binding is uninformative. In the paper, Elp3 binds the zinc-finger region of Snail1 and stabilises it against ubiquitin-mediated degradation; the Slug co-IP is a supplementary extension. Elp3 is an upstream regulator of Snail-protein stability rather than a corepressor or a partner through which Snai2 executes its function, so no more specific Snai2 molecular function follows from it. Removal does not dispute the interaction. Informative Snai2 partner binding (SNAG-dependent Ajuba LIM corepressors, EZH2/PRC2) is captured instead as NEW transcription corepressor binding. Supporting Evidence: PMID:27189455 Indeed, mElp3 pulled down efficiently xSlug in a co-IP experiment PMID:27189455 Thus, we favor the model that Elp3 binds and stabilizes Snail1, which is required for the transactivation of mesenchymal genes. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: nucleus (is_active_in). Reason: Snai2 is a chromatin-bound transcriptional repressor; its activity (E-box occupancy, PRC2 recruitment and H3K27me3 deposition at the E-cadherin promoter) is nuclear. Endogenous frog protein localisation has not been imaged, but the IBA is well founded. Supporting Evidence: PMID:25617436 The data thus demonstrate that Snail2 helps to recruit PRC2 to the E-cad promoter to set up repressive histone modification marks that result in inhibition of E-cad expression during neural crest EMT. file:XENLA/snai2/snai2-uniprot.txt SUBCELLULAR LOCATION: Nucleus |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location IEA: nucleus. Reason: Consistent with nuclear transcriptional repressor activity. Supporting Evidence: file:XENLA/snai2/snai2-uniprot.txt SUBCELLULAR LOCATION: Nucleus |
| GO:0006355 regulation of DNA-templated transcription | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: regulation of DNA-templated transcription. Reason: Correct but general; Snai2 negatively regulates Pol II transcription (E-cadherin). The specific process GO:0000122 is added as NEW; the broad IBA is retained. Supporting Evidence: PMID:10772801 The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter IEA: regulation of DNA-templated transcription. Reason: Correct, general; see IBA row. Supporting Evidence: PMID:10772801 The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. |
| GO:0006357 regulation of transcription by RNA polymerase II | IEA GO_REF:0000108 | ACCEPT | Summary: Inter-ontology IEA (from GO:0000981): regulation of transcription by RNA polymerase II. Reason: Logically entailed by Pol II-specific DNA-binding TF activity; correct. The direction (negative) is captured by NEW GO:0000122. Supporting Evidence: PMID:25617436 The data thus demonstrate that Snail2 helps to recruit PRC2 to the E-cad promoter to set up repressive histone modification marks that result in inhibition of E-cad expression during neural crest EMT. |
| GO:0014029 neural crest formation | TAS PMID:15242799 Cardiac neural crest ablation alters Id2 gene expression in ... | MODIFY | Summary: TAS (AgBase) to neural crest formation from Martinsen et al. 2004, a chick/Xenopus cardiac neural crest ablation study of Id2 expression. Reason: The cited paper (abstract-only in cache) concerns Id2 expression after cardiac crest ablation and does not appear to study Slug function; Slug was probably cited or used as a crest marker. The biological claim that Snai2 acts in crest formation is nevertheless sound. Following the project convention adopted for sox10 and sox9-a, genes acting on the border region go to the proposed neural plate border formation term (GO:0014029 is proposed for obsoletion), while crest specifiers are placed at GO:0014036 neural crest cell fate specification (part_of GO:0014034 part_of GO:0014029). Snai2 is clearly a specifier, not a border gene: it comes on after snai1 inside the prospective crest, and its knockdown leaves the border genes zic1 and pax3 intact. The weak TAS reference should ideally be replaced by the Xenopus experimental papers. Proposed replacements: neural crest cell fate specification Supporting Evidence: PMID:25617436 By contrast, knockdown of Snail2 did not appreciably diminish either Zic1 or Pax3 levels PMID:7720581 A related zinc-finger gene called Slug (Xslu) is expressed specifically in this tissue (i.e. the prospective crest) when the convergence extension movements are completed. PMID:10772801 inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration |
| GO:0014036 neural crest cell fate specification | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA IEA: neural crest cell fate specification. Reason: Matches the experimental evidence; this is the right layer for Snai2. Supporting Evidence: PMID:10772801 inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration PMID:25617436 By contrast, knockdown of Snail2 did not appreciably diminish either Zic1 or Pax3 levels |
| GO:0014036 neural crest cell fate specification | IGI PMID:12490555 Snail precedes slug in the genetic cascade required for the ... | ACCEPT | Summary: IGI with snai1 (P19382) from Aybar et al. 2003: dominant-negative/rescue epistasis placing Snail upstream of Slug in crest specification. Reason: Synthesised picture: snai2 is expressed in the prospective crest after the border is established; early inhibition of XSlug prevents crest precursor formation; Slug MO abolishes Sox9 crest expression; Snai2 overexpression expands crest markers (in an EZH2-dependent way) and, together with Wnt, is sufficient to establish a crest state, though alone it is insufficient to induce crest in naive ectoderm. Its loss does not affect the border genes zic1/pax3. This places Snai2 in the neural crest specifier layer, downstream of the border specifiers and of snai1, and GO:0014036 is the right term. Core. Supporting Evidence: PMID:12490555 Snail lies upstream of Slug in the genetic cascade leading to neural crest formation PMID:12490555 Slug alone is unable to induce other neural crest markers in animal cap assays PMID:10772801 inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration PMID:17205110 Injection of the Slug MO into one cell of a two cell embryo blocks the expression of Sox9 on the injected side PMID:25617436 By contrast, knockdown of Snail2 did not appreciably diminish either Zic1 or Pax3 levels PMID:25931449 Snail2 together with Wnt signaling is sufficient to establish a neural crest state PMID:9609823 Overexpression of the zinc finger transcription factor Slug, one of the earliest markers of neural crest formation, is insufficient for neural crest induction. |
| GO:0001227 DNA-binding transcription repressor activity, RNA polymerase II-specific | IDA PMID:25617436 Snail2/Slug cooperates with Polycomb repressive complex 2 (P... | NEW | Summary: Snai2 is a sequence-specific Pol II transcriptional repressor. Reason: Engrailed-repressor fusion of the XSlug DNA-binding domain phenocopies wild-type XSlug whereas a Gal4-activator fusion is inhibitory (LaBonne and Bronner-Fraser 2000); tagged Snai2 occupies E-boxes of the E-cadherin promoter and is required for EZH2 occupancy and H3K27me3 there, with E-cadherin RNA rising in Snai2 morphant crest at EMT stages (Tien 2015). The SNAG repression domain is conserved. Consistent with the repressor terms added for foxd3-a in this project. Supporting Evidence: PMID:10772801 The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. PMID:25617436 Snail2 bound preferentially to the E-boxes located around the TSS PMID:25617436 The data thus demonstrate that Snail2 helps to recruit PRC2 to the E-cad promoter to set up repressive histone modification marks that result in inhibition of E-cad expression during neural crest EMT. file:XENLA/snai2/snai2-uniprot.txt Probable transcriptional repressor. |
| GO:0000122 negative regulation of transcription by RNA polymerase II | IDA PMID:25617436 Snail2/Slug cooperates with Polycomb repressive complex 2 (P... | NEW | Summary: Snai2 represses Pol II target genes, directly including E-cadherin (cdh1) in the neural crest. Reason: Direct promoter occupancy plus Snai2-dependent PRC2 recruitment and H3K27me3 at the E-cadherin promoter during crest EMT; repressor-fusion mimicry in vivo. Supporting Evidence: PMID:25617436 The data thus demonstrate that Snail2 helps to recruit PRC2 to the E-cad promoter to set up repressive histone modification marks that result in inhibition of E-cad expression during neural crest EMT. PMID:10772801 The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. |
| GO:0001222 transcription corepressor binding | IPI PMID:25617436 Snail2/Slug cooperates with Polycomb repressive complex 2 (P... | NEW | Summary: Snai2 binds corepressors: EZH2 (catalytic subunit of PRC2) directly, and the Ajuba-family LIM proteins (Ajuba, LIMD1, WTIP) through its SNAG domain. Reason: Replaces the uninformative protein-binding class for Snai2 partners with the functionally meaningful one. EZH2 binds Snai2 directly and is recruited by it to repress E-cadherin; Ajuba LIM proteins act as SNAG-dependent Snail/Slug corepressors required for crest development in Xenopus. Supporting Evidence: PMID:25617436 EZH2 interacts directly with Snail2 PMID:25617436 Snail2 helps to anchor EZH2 to its target genes to deposit repressive chromatin marks. PMID:18331720 Ajuba LIM proteins contribute to neural crest development as Snail/Slug corepressors and are required for in vivo Snail/Slug function |
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Download this section (compressed HTML)Q: GO has no neural plate border specification/formation term. Snai2 is a crest specifier, not a border gene (zic1/pax3 are unaffected by its loss), so it should sit at GO:0014036 regardless; but should the AgBase TAS to GO:0014029 neural crest formation be withdrawn or re-supported by Xenopus experimental papers before GO:0014029 itself is obsoleted?
Q: Which crest functions are snai2-specific rather than shared with snai1? Snail and Slug rescue each other in overexpression and maintain each other's expression; snai1 (Tier 3) is also a blastula competence factor whereas snai2 behaves as a definitive crest factor in Xenopus.
Q: Do snai2.L (Q91924) and snai2.S (Q90VW5) differ in expression or activity, and should the experimental annotations on Q91924 also apply to the S homeolog?
Q: Should the Slug-NF-kappaB survival loop and early mesoderm role (Zhang et al. 2006) be annotated (e.g. negative regulation of apoptotic process), or is it a non-core consequence of crest/mesoderm loss?
Q: Should Snai2 carry GO:0036032 neural crest cell delamination (defined as negative regulation of cell adhesion)? Snai2 recruits PRC2 to the E-cadherin promoter and its knockdown raises E-cadherin RNA ~1.5-fold at EMT stages, but migrating Xenopus cranial crest retain E-cadherin and need it to migrate (Huang et al. 2016), and no Snai2-specific delamination phenotype has been scored in frog. Withheld pending a direct delamination assay.
Q: Lamprey has a single snail gene with affinities to both snai1 and snai2, and amphioxus Snail is the only crest specifier expressed (transiently) at the neural plate border: was snai2's crest-specifier deployment acquired after the gnathostome Snai1/Snai2 duplication by subfunctionalisation of an ancestral border-expressed Snail?
Experiment: Homeolog-resolved tagged-Snai2 CUT&RUN in dissected Xenopus neural folds at stages 14 and 19, combined with RNA-seq of snai2 F0 CRISPR crest, to define direct targets at specification versus EMT stages.
Hypothesis: Snai2 directly represses a set of epithelial and alternative-fate genes beyond E-cadherin in premigratory crest.
Type: ChIP-seq
Experiment: Single and combined F0 CRISPR knockouts of snai1 and snai2 (both homeologs) scoring border, specifier and migration markers and cdh1 derepression over time.
Hypothesis: snai1 and snai2 are partially redundant in crest specification but snai2 is specifically required at EMT.
Type: CRISPR knockout
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