ERF

Summary: Human ERF directly represses transcription of ETS-site-containing reporters.

Reason: The original PMID:7588608 Results “Functional analysis of ERF,” Figure 2A–D and Libraries/plasmids, Cell lines/transfection and EMSA Methods were read. Full-length human ERF (548 residues) represses CAT reporters in HeLa cells. Removing an ETS-binding site from the GATA1 promoter reduces ERF repression without eliminating GATA1-driven activation; GAL4 tethering separately establishes an active C-terminal repression region rather than promoter competition alone. Figure 2 pixels confirm the reporter comparisons and GAL4-fusion binding controls. CAT measurements were made in the linear range and normalized to cotransfected beta-galactosidase. ERF itself supplies DNA recognition and repression, so this is direct transcriptional regulation rather than a downstream proliferation phenotype. The assays establish the canonical protein; they do not establish equivalent function of the natural 473-residue product.

Summary: ERF associates with chromatin through its sequence-specific transcriptional role.

Reason: The author-hosted TFClass classification explicitly places ERF/P50548 in Ets-related family 3.5.2; that classification is a source inference, not a localization assay. Independent PMID:23354439 Results/Figure 3h and ChIP-Seq Methods directly recover mouse Erf-associated chromatin from E13.5 fibroblasts, with input controls and serum-dependent binding. The same paper tests human wild-type ERF repression of ETS-containing reporters in HeLa cells (Figure 4b; promoter-assay Methods). These complementary observations support the conserved chromatin-associated transcriptional role without calling the mouse ChIP a human experiment or implying constitutive chromosome residence. These gene-level conclusions do not establish equivalent activity of the 473-residue product, which lacks canonical residues 1–75 and therefore part of the mapped ETS domain.

Summary: The inherited DNA-binding transcription-factor activity is more specifically repressor activity.

Reason: Verified PAINT ancestry places human ERF below the asserted PTN000218930 node, with an exact canonical-sequence alignment and no target-path NOT. The target contributes legitimate experimental support to that ancestral judgment. The family-wide assertion is biologically sound, but human ERF has the more precise activity already represented by the GO:0001227 IDA annotation. PMID:23354439 Figure 4b and promoter-assay Methods show ERF reducing an ETS/RUNX reporter below basal activity in HeLa cells; the human disease-variant reporter assays distinguish loss of repression by ETS-domain mutants. Refine to GO:0001227 without claiming that every ETS-family descendant is a repressor. The independently recovered original PMID:7588608 Results “Functional analysis of ERF,” Figure 2 and Methods additionally test the full-length human ORF in HeLa CAT assays: ETS-site deletion reduces repression and GAL4 tethering demonstrates an active C-terminal repression region, with reporter normalization and binding controls.

Proposed replacements: DNA-binding transcription repressor activity, RNA polymerase II-specific

Summary: ERF is a DNA-binding transcriptional repressor within the TFClass ETS family.

Reason: The author-hosted TFClass draft explicitly links ERF/P50548 and its reported products to Ets-related family 3.5.2. Membership supports the general transcription-factor assertion but does not determine regulatory sign or prove activity for every product. Human ERF repression in PMID:23354439 Figure 4b and promoter-assay Methods supports the existing GO:0001227 repressor term in the GO:0001227 IDA annotation. Refine the broad factor activity to that measured function. The independently recovered original PMID:7588608 Results “Functional analysis of ERF,” Figure 2 and Methods additionally test the full-length human ORF in HeLa CAT assays: ETS-site deletion reduces repression and GAL4 tethering demonstrates an active C-terminal repression region, with reporter normalization and binding controls.

Proposed replacements: DNA-binding transcription repressor activity, RNA polymerase II-specific

Summary: ERF is an ETS-site-dependent transcriptional repressor with an active repression region.

Reason: The original PMID:7588608 Results “Functional analysis of ERF,” Figure 2A–D and Libraries/plasmids, Cell lines/transfection and EMSA Methods were read. Full-length human ERF (548 residues) represses CAT reporters in HeLa cells. Removing an ETS-binding site from the GATA1 promoter reduces ERF repression without eliminating GATA1-driven activation; GAL4 tethering separately establishes an active C-terminal repression region rather than promoter competition alone. Figure 2 pixels confirm the reporter comparisons and GAL4-fusion binding controls. CAT measurements were made in the linear range and normalized to cotransfected beta-galactosidase. The DNA-binding-domain deletion and heterologous GAL4-tethering experiments distinguish sequence recognition from the repression domain. The original maps the active repression region to approximately residues 472–530. Retain the repressor activity; no independent transcriptional activator activity or equivalent shorter-product activity is inferred.

Summary: The ETS-domain/family mapping can be refined to ERF’s measured repressor activity.

Reason: Current InterPro records map IPR000418 to GO:0003700 and match canonical P50548 residues 26–111; IPR046328 matches residues 11–112 and also maps to DNA-binding transcription-factor activity. These are genuine source mappings, not a mistaken family assignment. Human ERF in PMID:23354439 Figure 4b and promoter-assay Methods represses an ETS-containing reporter, supporting the more precise GO:0001227 already present in the GO:0001227 IDA annotation. The independently recovered original PMID:7588608 Results “Functional analysis of ERF,” Figure 2 and Methods additionally test the full-length human ORF in HeLa CAT assays: ETS-site deletion reduces repression and GAL4 tethering demonstrates an active C-terminal repression region, with reporter normalization and binding controls.

Proposed replacements: DNA-binding transcription repressor activity, RNA polymerase II-specific

Summary: ERF binds the TLE5/AES corepressor-class protein Q08117-2 in the HuRI two-hybrid assay.

Reason: The deposited HuRI records identify ERF P50548-1, clone 2592, paired with TLE5/AES Q08117-2, clone 53886, in screen 8. The two-hybrid array, prey-pooling and validated two-hybrid records are method records for this pair, not three independent physiological interactions. Current reviewed UniProt Q08117 classifies TLE5 as a transcriptional corepressor; its alternative-products record identifies the tested partner as AES-2, with an altered N terminus and retention of canonical residues 10–197. Combining that curated protein-class assignment with the direct pair record supports GO:0001222 transcription corepressor binding. This is a bounded classification inference for the reported partner, not a new demonstration that the 264-residue AES-2 product represses transcription in this assay. TLE5 can have context-dependent effects on other repressors, and the interaction does not establish that it mediates ERF repression or is recruited by ERF’s C-terminal region. No target-pair mammalian validation or endogenous-complex mechanism was recovered. The source guide links the exact official product record and assay provenance.

Proposed replacements: transcription corepressor binding

Summary: The reported ERF interaction with Q6FHY5 (MEOX2) is retained as non-core binding.

Reason: HuRI uses human ERF P50548-1, ORFeome clone 2592, in yeast two-hybrid assays. IntAct explicitly records ERF–Q6FHY5 under PMID:32296183 (IM-25472-32649, IM-25472-7688 and IM-25472-118431). The author deposit labels the same MEOX2 clone 824 pair P50222 and reports screen 2. Current Q6FHY5 and P50222 are 303 and 304 residues and differ by one residue in a repetitive region, so the partner accessions are not declared sequence-identical and the original GOA accession is retained. The pair has positive physical-interaction evidence and is retained as non-core under the project’s documented binding policy. The method records do not establish an endogenous ERF complex or show that this partner changes ERF transcriptional activity. Complete supplementary Methods and target-pair mammalian validation were not recovered; the binding result is not discarded on that account.

Summary: The reported ERF interaction with Q96EZ8 (MCRS1) is retained as non-core binding.

Reason: HuRI uses human ERF P50548-1, ORFeome clone 2592, in yeast two-hybrid assays. The author deposit records Q96EZ8-1, ORFeome clone 6901, in screens 9 and 7; this is the product-level counterpart of the gene-level GOA partner. The pair has positive physical-interaction evidence and is retained as non-core under the project’s documented binding policy. The method records do not establish an endogenous ERF complex or show that this partner changes ERF transcriptional activity. Complete supplementary Methods and target-pair mammalian validation were not recovered; the binding result is not discarded on that account.

Summary: The reported ERF interaction with Q9UGI0 (ZRANB1) is retained as non-core binding.

Reason: HuRI uses human ERF P50548-1, ORFeome clone 2592, in yeast two-hybrid assays. The exact Q9UGI0 partner is recorded as ORFeome clone 100016009, found in screens 1, 2 and 3. The pair has positive physical-interaction evidence and is retained as non-core under the project’s documented binding policy. The method records do not establish an endogenous ERF complex or show that this partner changes ERF transcriptional activity. Complete supplementary Methods and target-pair mammalian validation were not recovered; the binding result is not discarded on that account.

Summary: The broad nuclear location is refined to the experimentally supported nucleoplasmic pool.

Reason: The verified PAINT target path supports inherited nuclear localization; the target’s experimental evidence is a legitimate source of the ancestral judgment. Current HPA ICC/IF identifies nucleoplasm with HPA058532, HPA067952 and HPA072144, and Reactome explicitly represents human P50548 residues 1–548 in nucleoplasm (entities 2534321 and 3209166). Human-cDNA GFP-ERF constructs in PMID:14729966 Methods and Figures 1–5 demonstrate nuclear accumulation and regulated export in Ref-1 cells. Refine to the nucleoplasm term already present in the GO:0005654 HPA and Reactome annotations. The separate nucleolar observation remains contextual; the two C-terminal/shared HPA epitopes do not resolve the natural products. This refinement describes the supported nucleoplasmic pool, while the chromatin-associated repression context is represented separately. It does not equate diffuse nuclear staining with demonstration of transcriptional activity.

Proposed replacements: nucleoplasm

Summary: The broad nuclear location is refined to the experimentally supported nucleoplasmic pool.

Reason: SL-0191 is the official nucleus location and its GO:0005634 mapping is correct. Current HPA ICC/IF identifies nucleoplasm with HPA058532, HPA067952 and HPA072144, and Reactome explicitly represents human P50548 residues 1–548 in nucleoplasm (entities 2534321 and 3209166). Human-cDNA GFP-ERF constructs in PMID:14729966 Methods and Figures 1–5 demonstrate nuclear accumulation and regulated export in Ref-1 cells. Refine to the nucleoplasm term already present in the GO:0005654 HPA and Reactome annotations. The separate nucleolar observation remains contextual; the two C-terminal/shared HPA epitopes do not resolve the natural products. This refinement describes the supported nucleoplasmic pool, while the chromatin-associated repression context is represented separately. It does not equate diffuse nuclear staining with demonstration of transcriptional activity.

Proposed replacements: nucleoplasm

Summary: Human ERF has a supported nucleoplasmic pool.

Reason: The current HPA ERF record assigns nucleoplasm as its main location with supported overall reliability. HPA058532 and HPA067952 both show nucleoplasmic staining in MCF-7 cells; HPA072144 also records nucleoplasm in A-549 and U2OS. Antigens map respectively to canonical residues 465–515, 168–232 and 11–36. The first two sequences are shared with the 473-residue product, while the third is absent from it; staining therefore does not establish the localization of both products separately. Independent human-cDNA shuttling experiments in PMID:14729966 and the explicit human Reactome entities support the nuclear pool. Antibody specificity qualifications remain part of the source scope.

Summary: The pathway explicitly places full-length human ERF in nucleoplasm.

Reason: R-HSA-3209159 reaches ERF entity 3209166 through its input. The participant has referenceEntity 54440/P50548, human species, coordinates 1–548 and GO:0005654 nucleoplasm. The compartment is taken from the ERF participant, not inferred merely from the event label. This agrees with HPA target localization and the human ERF-cDNA nuclear shuttling experiments in PMID:14729966 Methods and Figures 1–5. Acceptance concerns the participant location, not independent proof of every reaction mechanism or localization of the shorter product.

Summary: The pathway explicitly places full-length human ERF in nucleoplasm.

Reason: R-HSA-3209177 reaches ERF entity 2534321 through its input. The participant has referenceEntity 54440/P50548, human species, coordinates 1–548 and GO:0005654 nucleoplasm. The compartment is taken from the ERF participant, not inferred merely from the event label. This agrees with HPA target localization and the human ERF-cDNA nuclear shuttling experiments in PMID:14729966 Methods and Figures 1–5. Acceptance concerns the participant location, not independent proof of every reaction mechanism or localization of the shorter product.

Summary: The pathway explicitly places full-length human ERF in nucleoplasm.

Reason: R-NUL-3245934 reaches ERF entity 2534321 through its input. The participant has referenceEntity 54440/P50548, human species, coordinates 1–548 and GO:0005654 nucleoplasm. This mixed-species event uses rat Mapk1 as the kinase and human ERF as its substrate; the R-NUL prefix does not make the ERF participant nonhuman. No kinase activity is assigned to ERF. This agrees with HPA target localization and the human ERF-cDNA nuclear shuttling experiments in PMID:14729966 Methods and Figures 1–5. Acceptance concerns the participant location, not independent proof of every reaction mechanism or localization of the shorter product.

Summary: HPA supports contextual nucleolar staining, without establishing a nucleolar biochemical role.

Reason: HPA072144 records nucleoli in A-549, HBEC3-KT and U2OS, with approved ICC/IF validation; its selected A-549 image was inspected and shows nuclear staining with brighter intranuclear bodies. The antibody antigen maps to canonical residues 11–36 and is absent from the 473-residue product. HPA also records a protein-array low-specificity qualification, so this is antibody-defined localization with a stated limitation, not proof of product-specific nucleolar function or universal residence. Retain the positive HPA observation as non-core; no ribosome-biogenesis or rRNA-processing activity is inferred.

Summary: ERF has a regulated cytosolic pool during nuclear export.

Reason: R-HSA-3209159 has output entity 3209167, human P50548 residues 1–548, explicitly in GO:0005829 cytosol. PMID:14729966 Methods uses human ERF-cDNA GFP constructs, and Figures 1–5 combine fluorescence, phosphorylation mutants, leptomycin B and fractionation to support nuclear/cytoplasmic shuttling in Ref-1 cells. The original shows multisite regulation and a nuanced T526 contribution; T526 phosphorylation alone is not asserted to be a sufficient export switch. HPA independently records cytosol in A-549 or HBEC3-KT depending on antibody. Retain this regulated pool outside the principal chromatin-associated repression context, without assigning an export-catalyst function to the transported ERF.

Summary: ERF’s measured transcriptional effect supports the more specific negative-regulation term.

Reason: IPR000418 maps the canonical ETS domain to GO:0006355. PMID:23354439 Figure 4b and promoter-assay Methods show human ERF suppressing reporter transcription in HeLa cells; the ETS-domain mutant comparisons connect repression to the DNA-binding region. The unsigned parent is correct but less informative than GO:0000122, already imported in the GO:0000122 IDA annotation. Refine to negative regulation of transcription by RNA polymerase II without extending the target-specific regulatory sign to the entire ETS family or claiming that every downstream differentiation phenotype is direct repression. The independently recovered original PMID:7588608 Results “Functional analysis of ERF,” Figure 2 and Methods additionally test the full-length human ORF in HeLa CAT assays: ETS-site deletion reduces repression and GAL4 tethering demonstrates an active C-terminal repression region, with reporter normalization and binding controls.

Proposed replacements: negative regulation of transcription by RNA polymerase II

Summary: ERF’s measured transcriptional effect supports the more specific negative-regulation term.

Reason: Verified PAINT ancestry places human ERF below the asserted PTN000218930 node, with an exact canonical-sequence alignment and no target-path NOT. The target contributes legitimate experimental support to that ancestral judgment. PMID:23354439 Figure 4b and promoter-assay Methods show human ERF suppressing reporter transcription in HeLa cells; the ETS-domain mutant comparisons connect repression to the DNA-binding region. The unsigned parent is correct but less informative than GO:0000122, already imported in the GO:0000122 IDA annotation. Refine to negative regulation of transcription by RNA polymerase II without extending the target-specific regulatory sign to the entire ETS family or claiming that every downstream differentiation phenotype is direct repression. The independently recovered original PMID:7588608 Results “Functional analysis of ERF,” Figure 2 and Methods additionally test the full-length human ORF in HeLa CAT assays: ETS-site deletion reduces repression and GAL4 tethering demonstrates an active C-terminal repression region, with reporter normalization and binding controls.

Proposed replacements: negative regulation of transcription by RNA polymerase II

Summary: ERF’s measured transcriptional effect supports the more specific negative-regulation term.

Reason: IPR046328 maps the canonical ETS-family match to GO:0006357. PMID:23354439 Figure 4b and promoter-assay Methods show human ERF suppressing reporter transcription in HeLa cells; the ETS-domain mutant comparisons connect repression to the DNA-binding region. The unsigned parent is correct but less informative than GO:0000122, already imported in the GO:0000122 IDA annotation. Refine to negative regulation of transcription by RNA polymerase II without extending the target-specific regulatory sign to the entire ETS family or claiming that every downstream differentiation phenotype is direct repression. The independently recovered original PMID:7588608 Results “Functional analysis of ERF,” Figure 2 and Methods additionally test the full-length human ORF in HeLa CAT assays: ETS-site deletion reduces repression and GAL4 tethering demonstrates an active C-terminal repression region, with reporter normalization and binding controls.

Proposed replacements: negative regulation of transcription by RNA polymerase II

Summary: Conserved ERF function supports chorionic trophoblast differentiation, with direct evidence from mouse Erf.

Reason: The verified PAINT tree and alignment place human ERF under the asserted PTN000218930 node; the differentiation IBD includes mouse Erf MGI:109637/P70459. The actual PMID:17502352 targeting, cell-culture and in-situ-hybridization Methods and Results/Figures 5–7 show persistent immature chorion, retained Errβ and absent Gcm1 expression before normal chorioallantoic attachment. Four Erf-null trophoblast stem-cell lines also differentiate with delayed marker changes after growth-factor withdrawal. These are stage-specific differentiation phenotypes, not expression alone. GO:0060718 chorionic trophoblast cell differentiation is a current child of cell differentiation and captures the in-vivo lineage more precisely than the general term or initial trophectoderm specification. ERF’s conserved transcriptional-repressor role supports the ortholog-based contribution, without representing the experiment as a human assay or claiming an identified universal target program. The cultured cells can form giant cells and do not differentiate into syncytiotrophoblasts in these conditions; that culture assay is not used to assert the terminal syncytial subtype. Equivalent function of the shorter human product remains unestablished.

Proposed replacements: chorionic trophoblast cell differentiation

Summary: Sequence-specific DNA binding is a well-supported biochemical property of ERF.

Reason: JASPAR profile MA0760.1 explicitly identifies human ERF/P50548, HT-SELEX and source PMID:23332764, with its deposited position-frequency matrix. This verifies the target-specific assay attribution; the 2013 full Methods, construct boundaries and supplement were not recovered, so no claim is made about its exact tested product. Independent original PMID:7588608 cDNA-isolation Results, functional reporter assays and EMSA Methods establish ETS-site recognition: the human clone was selected with an ETS2 H1 oligonucleotide, site removal reduces reporter repression, and in-vitro-produced ERF was assayed with the ETS2 H28 probe. The initial Southwestern result is reported as data not shown, and the inspected Figure 2D gel is GAL4 binding, not an ERF–H28 gel. Taken together with the curated direct annotation and target-specific deposited motif, these support the binding capacity without assuming the natural shorter product has the same specificity.

Summary: ERF recognizes ETS sites in RNA polymerase II cis-regulatory DNA.

Reason: ARBA00026779 condition set 0 genuinely matches the target’s ETS-domain signatures and Primates lineage, but its generic sequence-specific binding output can be refined using target evidence. Original PMID:7588608 cDNA-isolation Results and Libraries/plasmids Methods select human ERF with the ETS2-promoter H1 element. Functional analysis and Figure 2A–B show promoter-dependent repression and reduced repression after removal of an upstream GATA1 ETS site, while EMSA Methods use the ETS2 H28 probe. Independent PMID:23354439 promoter-assay Methods and Figure 4b test human ERF against ETS-containing cis-regulatory reporters. These observations support GO:0000978, the current more specific child of GO:0000977 for upstream cis-regulatory recognition. The initial Southwestern experiment was reported as data not shown, and Figure 2D is a GAL4-binding control; neither is misrepresented as a displayed ERF–H28 gel. The separate PMID:23332764 HT-SELEX annotation remains a correctly bounded statement of motif recognition on selection DNA, without assigning its unresolved construct or naked-DNA assay a promoter context. This refinement does not establish equivalent DNA recognition by the natural 473-residue product.

Proposed replacements: RNA polymerase II cis-regulatory region sequence-specific DNA binding