EN2

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0005634 nucleus	IBA GO_REF:0000033	ACCEPT	Summary: EN2 is active in the nucleus, where its homeodomain binds cis-regulatory DNA and regulates transcription. This phylogenetic inference agrees with reviewed UniProt localization and the nuclear-extract/promoter assays in PMID:12642491. Reason: Nuclear activity is integral to EN2's conserved DNA-binding transcription-factor function. The IBA is biologically coherent and independently supported by human localization annotations. Supporting Evidence: PMID:12642491 the promoter domain containing HF1 and HF2 is recognized by cerebellum nuclear extracts containing Engrailed and Foxa2
GO:0030182 neuron differentiation	IBA GO_REF:0000033	KEEP AS NON CORE	Summary: Conserved Engrailed-family genetics supports roles in neuronal differentiation, maturation, and maintenance. Mouse En2 is expressed in multiple developing cerebellar neuronal populations, although expression does not by itself define a direct molecular mechanism. Reason: The phylogenetic annotation is plausible and consistent with vertebrate studies, but neuron differentiation is a developmental outcome of EN2 transcriptional regulation rather than the protein's intrinsic core activity. Supporting Evidence: PMID:21431469 both En1 and En2 are expressed in many neuronal cell types in the cerebellum, and expression persists until at least P21
GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding	IBA GO_REF:0000033	ACCEPT	Summary: EN2 is a homeodomain protein that recognizes sequence-specific regulatory DNA. Engrailed binds conserved homeoprotein sites in the MAP1B promoter, and human EN2 was included in the direct human TF DNA-binding dataset of PMID:28473536. Reason: Sequence-specific binding to Pol II cis-regulatory DNA is a core EN2 molecular function, supported by family conservation, source-protein experiments, and direct human high-throughput DNA-binding evidence. Supporting Evidence: PMID:11331364 one promoter domain that contains all ATTA homeoprotein cognate binding sites common to the rat and human genes is an essential element of this regulation PMID:28473536 By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment)
GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific	IBA GO_REF:0000033	ACCEPT	Summary: EN2 combines sequence-specific homeodomain DNA binding with regulation of target promoters, including the experimentally studied MAP1B promoter. Reason: This is the best high-level GO molecular-function representation of EN2's core activity and is supported by phylogeny, conserved domains, direct DNA binding, and reporter assays. Supporting Evidence: PMID:11331364 These experiments demonstrate that MAP1B promoter is regulated by Engrailed in vivo.
GO:0006357 regulation of transcription by RNA polymerase II	IBA GO_REF:0000033	ACCEPT	Summary: EN2 regulates transcription from cis-regulatory regions, with activation or repression determined by promoter, dose, and partner context. Reason: This process directly corresponds to EN2's conserved DNA-binding transcription-factor activity without imposing an unsupported universal direction of effect. Supporting Evidence: PMID:12642491 Foxa2 antagonizes the Engrailed-driven regulation of the MAP1B promoter, and vice versa.
GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific	IEA GO_REF:0000002	ACCEPT	Summary: InterPro assigns this activity from the conserved homeobox transcription-factor domain architecture. It independently duplicates the accepted IBA activity. Reason: The domain-based inference is correct for EN2 and is supported by experimental promoter binding and regulation. The distinct GOA row and evidence source are retained. Supporting Evidence: PMID:11331364 MAP1B promoter is regulated by Engrailed in vivo
GO:0003677 DNA binding	IEA GO_REF:0000002	MODIFY	Summary: EN2 does bind DNA, but the generic parent term omits its sequence specificity and cis-regulatory transcriptional context. Reason: Replace the uninformative broad term with the experimentally and phylogenetically supported sequence-specific Pol II cis-regulatory DNA-binding activity. Proposed replacements: RNA polymerase II cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:11331364 The promoter region of MAP1B was cut into several subdomains, and each subdomain was tested for its ability to bind Engrailed and EnHD
GO:0005634 nucleus	IEA GO_REF:0000120	ACCEPT	Summary: Combined automated methods place EN2 in the nucleus, consistent with UniProt, HPA nucleoplasm localization, and its DNA-binding transcription-factor role. Reason: The localization is correct. This row is retained independently of the IBA is-active-in annotation because its evidence and qualifier differ.
GO:0006355 regulation of DNA-templated transcription	IEA GO_REF:0000002	MODIFY	Summary: The InterPro mapping captures EN2 transcriptional regulation but is broader than the Pol II-specific process already supported by IBA and promoter experiments. Reason: EN2 is a Pol II-specific DNA-binding transcription factor, so GO:0006357 is the more informative and already supported replacement. Proposed replacements: regulation of transcription by RNA polymerase II Supporting Evidence: PMID:11331364 MAP1B, a cytoskeleton protein involved in neuronal growth and regeneration, is under homeoprotein transcriptional regulation
GO:0043524 negative regulation of neuron apoptotic process	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: This Ensembl Compara transfer derives from mouse En2. Mouse En1/En2 double loss and acute knockdown cause caspase-3-positive death of mesencephalic dopaminergic neurons, establishing an Engrailed-dependent survival role. Reason: The orthology transfer is biologically well supported, but neuronal survival is a context-specific developmental/maintenance outcome rather than EN2's intrinsic molecular activity. The mouse study assesses combined Engrailed function, so the human EN2-specific contribution should not be overstated. Supporting Evidence: PMID:15175251 Gene silencing in the postmitotic neurons by RNA interference activates caspase 3 and induces apoptosis in less than 24 hours.
GO:1990403 embryonic brain development	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Ensembl transferred this process from mouse En2. En2 coding sequence rescues the mouse En1 mid-hindbrain deletion, while En2 mutants have cerebellar abnormalities, supporting a conserved role in embryonic brain development. Reason: The annotation is appropriate as an orthology-supported developmental outcome, but it is broader than the specific cerebellar and midbrain contexts and is not a core molecular activity. Supporting Evidence: PMID:7624797 En-1 coding sequences were replaced with En-2 sequences by gene targeting. This rescued all En-1 mutant defects
GO:0001650 fibrillar center	IDA GO_REF:0000052	KEEP AS NON CORE	Summary: Human Protein Atlas immunofluorescence curation observed EN2 in the fibrillar center, a nucleolar subcompartment. Reason: Retain the directly observed localization, but there is no evidence in the review set that the fibrillar center is where EN2 performs its defining transcriptional regulatory activity.
GO:0005654 nucleoplasm	IDA GO_REF:0000052	ACCEPT	Summary: Human Protein Atlas immunofluorescence places EN2 in the nucleoplasm. Reason: Nucleoplasmic localization is directly observed and is consistent with EN2's sequence-specific DNA-binding transcription-factor activity.
GO:0005730 nucleolus	IDA GO_REF:0000052	KEEP AS NON CORE	Summary: Human Protein Atlas immunofluorescence observed EN2 in the nucleolus. Reason: The experimental localization should be retained, but a nucleolus-specific EN2 function has not been established; it is not used in the core-function synthesis.
GO:0000122 negative regulation of transcription by RNA polymerase II	ISS PMID:12642491 Joint regulation of the MAP1B promoter by HNF3beta/Foxa2 and...	ACCEPT	Summary: The annotation is transferred from chicken EN2. Source experiments show that EN2 can repress MAP1B promoter output in the chick neural tube and can antagonize FOXA2-dependent promoter activation. Reason: The source-protein experiments establish context-dependent repression, and the Engrailed homeodomain/regulatory regions are conserved in human EN2. This is a valid secondary transcriptional mode, not EN2's universal direction of action. Supporting Evidence: PMID:12642491 Foxa2 antagonizes the Engrailed-driven regulation of the MAP1B promoter, and vice versa. PMID:11331364 These experiments demonstrate that MAP1B promoter is regulated by Engrailed in vivo.
GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding	ISS PMID:12642491 Joint regulation of the MAP1B promoter by HNF3beta/Foxa2 and...	ACCEPT	Summary: Recombinant chicken EN2 and Engrailed-containing cerebellar extracts recognize conserved homeoprotein sites in the MAP1B cis-regulatory region. Reason: The source annotation is experimentally grounded and transfers across a highly conserved homeodomain. It is independently supported by the human EN2 DNA-binding dataset in PMID:28473536. Supporting Evidence: PMID:12642491 The MAP1B (Mtap1b) promoter presents two evolutionary conserved overlapping homeoproteins and Hepatocyte nuclear factor 3beta (HNF3beta/Foxa2) cognate binding sites
GO:0001228 DNA-binding transcription activator activity, RNA polymerase II-specific	ISS PMID:12642491 Joint regulation of the MAP1B promoter by HNF3beta/Foxa2 and...	ACCEPT	Summary: The source chicken EN2 protein activates a MAP1B promoter reporter in a dose-dependent manner; FOXA2 modulates that activation. Reason: The source experiment supports an activator mode that is transferable through the conserved Engrailed domains. It is retained as a context-dependent activity, while the polarity-neutral GO:0000981 is used for the core synthesis. Supporting Evidence: PMID:12642491 Transfection experiments further demonstrate that Engrailed and Foxa2 interact physiologically in a dose-dependent manner PMID:11331364 the activity, and regulation by Engrailed, of each subdomain and of the entire promoter were evaluated in vivo by electroporation in the chick embryo neural tube
GO:0003714 transcription corepressor activity	ISS PMID:12642491 Joint regulation of the MAP1B promoter by HNF3beta/Foxa2 and...	ACCEPT	Summary: Chicken EN2 binds FOXA2 directly and, at the MAP1B promoter, each factor can antagonize the other's transcriptional activation depending on dose. Reason: Direct binding to a DNA-binding transcription factor with decreased target-promoter output supports a context-specific corepressor activity. It is a secondary mode and is not substituted for EN2's primary DNA-binding transcription-factor activity. Supporting Evidence: PMID:12642491 Direct interaction was confirmed by pull-down experiments, and the regions participating in this interaction were identified. PMID:12642491 Foxa2 antagonizes the Engrailed-driven regulation of the MAP1B promoter, and vice versa.
GO:0045944 positive regulation of transcription by RNA polymerase II	ISS PMID:12642491 Joint regulation of the MAP1B promoter by HNF3beta/Foxa2 and...	ACCEPT	Summary: Chicken EN2 activates the MAP1B promoter in reporter assays, providing the source evidence for this similarity transfer. Reason: Context-dependent positive transcriptional regulation is experimentally supported for the ortholog and is consistent with the conserved human EN2 protein. It is not treated as EN2's sole or universal transcriptional effect. Supporting Evidence: PMID:12642491 the promoter domain containing HF1 and HF2 is recognized by cerebellum nuclear extracts containing Engrailed and Foxa2 and has regulatory functions
GO:0061629 RNA polymerase II-specific DNA-binding transcription factor binding	ISS PMID:12642491 Joint regulation of the MAP1B promoter by HNF3beta/Foxa2 and...	ACCEPT	Summary: Chicken EN2 binds FOXA2 directly in pull-down experiments. The EN2 homeodomain and a region containing its PBX-binding domain independently contribute to FOXA2 binding. Reason: This is an informative, specific interaction activity rather than generic protein binding. The interacting Engrailed domains are conserved in human EN2. Supporting Evidence: PMID:12642491 In Engrailed, two independent interacting domains exist: the homeodomain and a region that includes the Pbx-binding domain.
GO:1990837 sequence-specific double-stranded DNA binding	IDA PMID:28473536 Impact of cytosine methylation on DNA binding specificities ...	ACCEPT	Summary: Human EN2 was assayed in the study's methylation-sensitive SELEX compendium of human transcription factors, supplying direct sequence-specific double-stranded DNA-binding evidence. Reason: The experimental annotation is consistent with EN2's homeodomain and the more transcription-specific cis-regulatory DNA-binding annotations. The cached narrative describes the 542-human-TF assay but does not reproduce EN2's supplemental motif row, so no motif or methylation preference beyond the curated GO claim is inferred. Supporting Evidence: PMID:28473536 By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment)
GO:0000785 chromatin	ISA GO_REF:0000113	ACCEPT	Summary: TFClass-based inference places EN2 on chromatin, consistent with its sequence-specific cis-regulatory DNA binding and promoter regulation. Reason: Chromatin is a functionally appropriate localization for this homeobox transcription factor. The class inference is corroborated by direct DNA-binding evidence. Supporting Evidence: PMID:28473536 This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity
GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific	ISA GO_REF:0000113	ACCEPT	Summary: TFClass assigns EN2 to the human sequence-specific DNA-binding transcription-factor class. This duplicates the core activity with a distinct classification-based source. Reason: The inference is correct and independently corroborated by IBA, InterPro, promoter, and human DNA-binding evidence. The separate GOA row is preserved. Supporting Evidence: PMID:11331364 MAP1B, a cytoskeleton protein involved in neuronal growth and regeneration, is under homeoprotein transcriptional regulation
GO:0021587 cerebellum morphogenesis	ISS PMID:20081196 The Engrailed homeobox genes determine the different foliati...	NEW	Summary: Proposed new annotation by similarity to mouse En2. Temporal conditional En1/En2 mutants establish a late requirement for correct regional cerebellar foliation, granule-cell-precursor layer growth, and fissure positioning. Reason: Cerebellum morphogenesis captures the experimentally supported regional-foliation outcome used in the top-down cerebellum-development module. This is an orthology-based, context-specific developmental annotation and is deliberately excluded from the EN2 core molecular-function synthesis. Supporting Evidence: PMID:20081196 both En genes are required to ensure that folia exclusive to the vermis or hemispheres form in the appropriate mediolateral position

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Existing Annotations Review

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