Alx1 is a paired-class (Q50) homeodomain transcription factor of the Cart1/Alx3/Alx4 subfamily, with a C-terminal OAR motif and a lineage-specific 41-residue "Domain 2" (D2) motif that distinguishes it from its paralogue Alx4. In the sea urchin embryo it is transcribed exclusively in the large micromere lineage, the founder cells of the skeletogenic primary mesenchyme cells (PMCs), from the first interphase after the large micromeres are born. Its expression is cell-autonomous, is driven by Ets1 and by Alx1 itself, and is confined to the micromere lineage by the Pmar1/HesC double-negative gate: HesC represses alx1 everywhere else in the embryo through defined HesC sites in the alx1 cis-regulatory system. Alx1 binds palindromic and TAAT half-site sequences as a dimer and acts as a direct activator of PMC regulatory genes (including its own gene) and of a large battery of terminal effector genes, including those required for epithelial-mesenchymal transition (ingression) and for calcium carbonate biomineralization. Morpholino knockdown blocks PMC specification and produces skeleton-less embryos in which the micromere domain instead expresses the non-skeletogenic mesoderm regulator gcm, while ectopic alx1 activates the skeletogenic program in other lineages. Alx1 is a conserved regulator of skeletal cell identity across echinoderms, and vertebrate Alx3/Alx4/Cart1 proteins play analogous roles in craniofacial and limb skeletogenesis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the homeobox signature. Fully consistent with the experimental record: Alx1 is a sequence-specific homeodomain factor whose direct transcriptional inputs into PMC regulatory and effector genes have been shown by cis-regulatory analysis, ChIP-seq and EMSA in S. purpuratus. Reason: Core molecular function of the gene product, supported by direct evidence (a more specific activator child term is proposed as NEW below). Supporting Evidence: PMID:21723273 we demonstrate that Alx1 protein is both an immediate, direct, auto-activator, and at higher concentrations an auto-repressor PMID:31331943 we used genome-wide ChIP-seq to identify ... Alx1-binding sites and direct gene targets |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | MODIFY | Summary: Generic homeodomain-derived DNA binding. True but uninformative; the experimentally demonstrated binding is sequence-specific (palindromic and TAAT half sites in cis-regulatory modules). Reason: Replace with the specific term supported by EMSA on the Sp-mtmmpb CRM and by ChIP-seq: RNA polymerase II cis-regulatory region sequence-specific DNA binding. Proposed replacements: RNA polymerase II cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:34157281 we show that Alx1 binds directly to several half sites. Moreover, Alx1 forms dimeric complexes at these sites |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Nuclear localization inferred from the homeodomain. Consistent with genome-wide chromatin occupancy of Alx1 detected by ChIP-seq in PMCs; no direct immunolocalization is cached, but the location is not in doubt for a chromatin-bound transcription factor. Reason: Expected and functionally required location of a sequence-specific transcription factor with demonstrated in vivo chromatin binding. Supporting Evidence: PMID:31331943 we used genome-wide ChIP-seq to identify ... Alx1-binding sites and direct gene targets |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Generic InterPro2GO process term. Correct but redundant with the more specific RNA polymerase II term also present, and with the positive-regulation term proposed as NEW. Reason: True by parentage of the demonstrated function; the informative terms are the RNA polymerase II-specific child and the lineage-specification process. |
| GO:0006357 regulation of transcription by RNA polymerase II | IEA GO_REF:0000108 | ACCEPT | Summary: Inter-ontology inference from GO:0000981. Alx1 both activates target genes (its own gene, PMC regulatory and biomineralization genes) and, by perturbation evidence, represses gcm and auto-represses at high levels, so the sign-neutral parent term is an accurate summary of the demonstrated activity. Reason: Supported by direct cis-regulatory and ChIP-seq evidence; a positive-regulation child is proposed as NEW for the well-established activating role. Supporting Evidence: PMID:21723273 we demonstrate that Alx1 protein is both an immediate, direct, auto-activator, and at higher concentrations an auto-repressor PMID:18413610 a role of the micromere lineage regulator alx1 is to repress gcm in that lineage |
| GO:0048513 animal organ development | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA rule generalized from vertebrate Alx-family proteins, which act in limb and craniofacial organ development. The sea urchin Alx1 acts in specification of an embryonic mesenchymal cell lineage and in formation of the larval calcite endoskeleton, which is not an animal organ in the GO sense; the term is not wrong by propagation but is a poor and over-general description of what this protein does. Reason: Vertebrate-derived generalization; the specific processes (mesodermal cell fate specification, regulation of biomineral tissue development) are proposed as NEW and capture the actual biology. Supporting Evidence: PMID:12756175 that Alx1 is essential at an early stage of specification and controls ... downstream genes required for epithelial-mesenchymal transition and |
| GO:1990837 sequence-specific double-stranded DNA binding | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA-derived sequence-specific DNA binding. Directly confirmed by EMSA with purified Alx1 on TAAT half sites and palindromic sites in an S. purpuratus cis-regulatory module, and by genome-wide ChIP-seq. Reason: Experimentally confirmed; the cis-regulatory-region child term is proposed as the more informative replacement for the generic DNA binding row. Supporting Evidence: PMID:34157281 Alx1 and Alx4 contain glutamine-50 ... paired-type homeodomains, which interact preferentially with palindromic binding sites |
| GO:0001228 DNA-binding transcription activator activity, RNA polymerase II-specific | IDA PMID:21723273 Precise cis-regulatory control of spatial and temporal expre... | NEW | Summary: Alx1 is a direct transcriptional activator: cis-regulatory analysis of the alx1 locus shows that Alx1 protein is an immediate, direct auto-activator of its own gene, and ChIP-seq-derived CRMs of effector genes depend on Alx1 binding sites (a conserved palindromic site is essential in a representative CRM; two half sites are essential for the PMC-specific Sp-mtmmpb CRM). Reason: Direct reporter/cis-regulatory evidence for activator function in S. purpuratus; the sign is shown, so the activator child of GO:0000981 is warranted. Supporting Evidence: PMID:21723273 we demonstrate that Alx1 protein is both an immediate, direct, auto-activator, and at higher concentrations an auto-repressor PMID:31331943 Detailed analysis of a representative CRM confirmed that a conserved, ... palindromic Alx1-binding site was essential for expression. PMID:34157281 positively regulates the transcription of most biomineralization genes expressed by |
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IDA PMID:34157281 Analysis of the DNA-binding properties of Alx1, an evolution... | NEW | Summary: EMSA with purified Alx1 shows direct, specific binding to TAAT half sites and to the palindromic site in the Sp-mtmmpb cis-regulatory module; genome-wide ChIP-seq places Alx1 on cis-regulatory modules of PMC regulatory and effector genes. Reason: Direct biochemical and in vivo occupancy evidence for cis-regulatory sequence-specific binding (proposed as the replacement for generic DNA binding). Supporting Evidence: PMID:34157281 we show that Alx1 binds directly to several half sites. Moreover, Alx1 forms dimeric complexes at these sites PMID:31331943 we used genome-wide ChIP-seq to identify ... Alx1-binding sites and direct gene targets |
| GO:0042803 protein homodimerization activity | IDA PMID:34157281 Analysis of the DNA-binding properties of Alx1, an evolution... | NEW | Summary: Alx1 forms dimeric complexes on DNA, both on palindromic sites (the classical paired-class Q50 homeodomain cooperative dimer) and, by a distinct mechanism, on TAAT half sites. Dimerization is also the proposed basis of alx1 auto-repression at high Alx1 concentration. Reason: Direct EMSA evidence of dimeric Alx1-DNA complexes with purified protein; mechanistically informative (dimer-dependent binding and auto-regulation) rather than a bare protein-binding claim. Supporting Evidence: PMID:34157281 We find that Alx1 forms dimeric complexes on TAAT-containing half sites by PMID:21723273 A synthetic experiment indicates that this behavior is probably due to dimerization. |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IDA PMID:21723273 Precise cis-regulatory control of spatial and temporal expre... | NEW | Summary: Alx1 activates transcription of its own gene (direct auto-activation in reporter assays) and of a large set of PMC effector genes, including most biomineralization genes, which carry Alx1-bound CRMs. Reason: Process counterpart of the activator activity; supported by cis-regulatory analysis and ChIP-seq/reporter validation in S. purpuratus. Supporting Evidence: PMID:21723273 we demonstrate that Alx1 protein is both an immediate, direct, auto-activator, and at higher concentrations an auto-repressor PMID:31331943 terminal differentiation genes receive direct transcriptional inputs from Alx1. |
| GO:0007501 mesodermal cell fate specification | IMP PMID:12756175 Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-cl... | NEW | Summary: Alx1 is the key lineage-specific transcription factor that specifies the skeletogenic mesoderm (large micromere / primary mesenchyme cell) fate. Morpholino knockdown blocks PMC specification at an early stage; the micromere domain instead expresses gcm and adopts non-skeletogenic mesoderm/pigment fate; ectopic alx1 (or global derepression through the Pmar1/HesC gate) activates the skeletogenic program in other lineages. The gene product performs the regulatory step itself, as a direct activator of the PMC regulatory state and repressor of the alternative fate. Reason: Participation test passed: Alx1 is the transcription factor that institutes the lineage regulatory state, not merely a required input. GO:0007501 (mesodermal cell fate specification) is used because the PMC lineage is the skeletogenic mesoderm of the sea urchin embryo; no more specific echinoderm skeletogenic-mesenchyme term exists. Supporting Evidence: PMID:12756175 that Alx1 is essential at an early stage of specification and controls PMID:18413610 a role of the micromere lineage regulator alx1 is to repress gcm in that lineage. Thus if alx1 expression is blocked, expression of gcm and pks genes spreads across the vegetal plate, including the micromere domain PMID:29154754 Perturbation of alx1 function using antisense morpholinos (MOs) blocks PMC specification while misexpression of alx1 results in the ectopic activation of the skeletogenic program in non-PMC lineages |
| GO:0010718 positive regulation of epithelial to mesenchymal transition | IMP PMID:12756175 Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-cl... | NEW | Summary: Alx1 controls the downstream genes required for the epithelial-mesenchymal transition (ingression) of the skeletogenic cells; Alx1 morphants fail to ingress, and Alx1 target genes are required for ingression. Reason: Regulatory (not executional) role, hence the regulation term rather than GO:0001837 itself. Supported by morpholino phenotype and by the direct inputs of Alx1 into effector genes identified by ChIP-seq. Supporting Evidence: PMID:12756175 downstream genes required for epithelial-mesenchymal transition and PMID:18413610 both it and alx1 target genes are required for ingression of the skeletogenic cells |
| GO:0070169 positive regulation of biomineral tissue development | IMP PMID:12756175 Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-cl... | NEW | Summary: Alx1 positively regulates the transcription of most biomineralization genes expressed by PMCs, many of which receive direct Alx1 inputs; knockdown produces a skeleton-minus phenotype (no calcite endoskeleton). Reason: Alx1 is a transcriptional regulator of the biomineralization gene battery, not an executor of mineral deposition, so the positive-regulation term is used rather than GO:0031214. Supporting Evidence: PMID:12756175 downstream genes required for epithelial-mesenchymal transition and ... biomineralization. PMID:34157281 positively regulates the transcription of most biomineralization genes expressed by PMID:18413610 interference with their expression produces a ... skeleton-minus phenotype |
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Download this section (compressed HTML)Q: Is Alx1 a direct transcriptional repressor of gcm (and of its own gene at high concentration), which would justify GO:0001227 / GO:0000122 annotations, or is the exclusion of the non-skeletogenic mesoderm fate mediated indirectly through Alx1-activated intermediates?
Q: Does the D2 domain, which is required for skeletogenic function and alters DNA-binding properties, act by changing dimerization/half-site binding or by recruiting a specific coactivator?
Experiment: Mutational dissection of the gcm cis-regulatory module (which already has defined Su(H) sites) for Alx1 binding sites, combined with Alx1 ChIP-qPCR, to test direct repression by Alx1 in the micromere lineage.
Experiment: Co-immunoprecipitation or proximity labeling of tagged Sp-Alx1 in PMCs, with and without the D2 domain, to identify the cofactors that distinguish Alx1 from Alx4 in skeletogenic function.
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