ALX4

Summary: ALX4 acts on chromatin as a developmental transcription factor.

Reason: TFClass supplies the classification source. PMID:38262408 directly measures ALX4 occupancy and its redistribution of TWIST1 binding in human neural-crest-derived mesenchymal cells. This is positive chromatin association, not an inference from a nuclear-localization motif alone.

Summary: Sequence-specific regulatory-region DNA binding is supported.

Reason: The PAINT assertion is retained at its stated regulatory-DNA scope. Human ALX4 binds sequence-specific regulatory DNA and modulates RNA-polymerase-II reporter output. PMID:22829454 assays human constructs in human calvarial osteoblasts, and PMID:40410151 separately tests full-length human ALX4 in HEK293T reporter assays. The latter study uses purified mouse DNA-binding regions explicitly identical to the corresponding human regions for its structural/biophysical work; those preparations are not conflated with the human full-length reporter. PMID:38262408 adds endogenous human cranial-neural-crest chromatin occupancy and transcriptional perturbation evidence. The exact ancestral IBD/tree placement was not independently reconstructed. The supplied PAINT node and descendant evidence are preserved; donor count is not a quality criterion, and independent human evidence supports retention without inventing the node history.

Summary: The mouse-derived regulatory-DNA assertion is corroborated in human ALX4.

Reason: O35137 is mouse Alx4 and its supplied Ensembl protein is retained. Human ALX4 binds sequence-specific regulatory DNA and modulates RNA-polymerase-II reporter output. PMID:22829454 assays human constructs in human calvarial osteoblasts, and PMID:40410151 separately tests full-length human ALX4 in HEK293T reporter assays. The latter study uses purified mouse DNA-binding regions explicitly identical to the corresponding human regions for its structural/biophysical work; those preparations are not conflated with the human full-length reporter. PMID:38262408 adds endogenous human cranial-neural-crest chromatin occupancy and transcriptional perturbation evidence. The human target evidence supports the transferred function independently of an assumed pairwise similarity score.

Summary: ALX4 is a DNA-binding RNA-polymerase-II transcription factor.

Reason: The homeobox-domain mapping is supported by target-specific experiments. Human ALX4 binds sequence-specific regulatory DNA and modulates RNA-polymerase-II reporter output. PMID:22829454 assays human constructs in human calvarial osteoblasts, and PMID:40410151 separately tests full-length human ALX4 in HEK293T reporter assays. The latter study uses purified mouse DNA-binding regions explicitly identical to the corresponding human regions for its structural/biophysical work; those preparations are not conflated with the human full-length reporter. PMID:38262408 adds endogenous human cranial-neural-crest chromatin occupancy and transcriptional perturbation evidence. Retain the general TF activity because output also depends on partner, dose and regulatory context; it is not restricted to activation in every setting.

Summary: TFClass correctly captures the experimentally supported transcription-factor role.

Reason: Human ALX4 binds sequence-specific regulatory DNA and modulates RNA-polymerase-II reporter output. PMID:22829454 assays human constructs in human calvarial osteoblasts, and PMID:40410151 separately tests full-length human ALX4 in HEK293T reporter assays. The latter study uses purified mouse DNA-binding regions explicitly identical to the corresponding human regions for its structural/biophysical work; those preparations are not conflated with the human full-length reporter. PMID:38262408 adds endogenous human cranial-neural-crest chromatin occupancy and transcriptional perturbation evidence. The broader TF activity includes the observed context-dependent output; no cofactor-only or DNA-independent mechanism is substituted.

Summary: ALX4 has directly measured transcriptional activator activity.

Reason: The mouse-descendant PAINT node PTN002803099 is retained. Human wild-type and variant reporter experiments in PMID:22829454 and PMID:40410151 demonstrate activation. Context-dependent repression in a LEF1/promoter experiment does not negate this positive capability. The exact ancestral IBD/tree placement was not independently reconstructed. The supplied PAINT node and descendant evidence are preserved; donor count is not a quality criterion, and independent human evidence supports retention without inventing the node history.

Summary: Human target experiments support the mouse-derived activator assertion.

Reason: Human ALX4 binds sequence-specific regulatory DNA and modulates RNA-polymerase-II reporter output. PMID:22829454 assays human constructs in human calvarial osteoblasts, and PMID:40410151 separately tests full-length human ALX4 in HEK293T reporter assays. The latter study uses purified mouse DNA-binding regions explicitly identical to the corresponding human regions for its structural/biophysical work; those preparations are not conflated with the human full-length reporter. PMID:38262408 adds endogenous human cranial-neural-crest chromatin occupancy and transcriptional perturbation evidence. The assertion describes a demonstrated activity, not uniform activation of every ALX4-bound gene or every mutant in all hosts.

Summary: ALX4 participates in skeletal development through developmental gene regulation.

Reason: The original human study identifies pathogenic ALX4 variation in families with skull ossification defects. That clinical evidence is combined with direct human mesenchymal chromatin/transcription experiments and lineage-specific mouse Alx4 studies, supporting the gene product doing regulatory work during skeletal development. The broad skeletal term is appropriate; no particular bone-patterning step is inferred solely from phenotype.

Summary: Human ALX4 dysfunction disrupts follicular differentiation.

Reason: Original PMID:19692347 patient/control skin histology shows that follicle-like structures initiate but differentiate abnormally. This supports development at the term's resolution and does not mean ALX4 initiates every follicle. The independently read mouse conditional study PMID:38481039 corroborates lineage-dependent hair phenotypes. No direct Wnt or cell-adhesion activity is inferred from accompanying beta-catenin changes.

Summary: Refine generic DNA binding to the measured regulatory-region specificity.

Reason: The homeobox-domain inference is biologically correct but unnecessarily generic for the independently demonstrated ALX4 regulatory-DNA activity. Human ALX4 binds sequence-specific regulatory DNA and modulates RNA-polymerase-II reporter output. PMID:22829454 assays human constructs in human calvarial osteoblasts, and PMID:40410151 separately tests full-length human ALX4 in HEK293T reporter assays. The latter study uses purified mouse DNA-binding regions explicitly identical to the corresponding human regions for its structural/biophysical work; those preparations are not conflated with the human full-length reporter. PMID:38262408 adds endogenous human cranial-neural-crest chromatin occupancy and transcriptional perturbation evidence.

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

Summary: Use the established regulatory-region DNA-binding subtype.

Reason: The historical NAS citation identifies human ALX4 as a paired-related homeodomain transcription factor; its original full text was not recovered. This is not a rejection or misattribution claim. Independent human reporter/chromatin evidence now supports the more informative regulatory-region DNA-binding term.

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

Summary: Resolve the broad TF term to RNA-polymerase-II transcription-factor activity.

Reason: Human ALX4 binds sequence-specific regulatory DNA and modulates RNA-polymerase-II reporter output. PMID:22829454 assays human constructs in human calvarial osteoblasts, and PMID:40410151 separately tests full-length human ALX4 in HEK293T reporter assays. The latter study uses purified mouse DNA-binding regions explicitly identical to the corresponding human regions for its structural/biophysical work; those preparations are not conflated with the human full-length reporter. PMID:38262408 adds endogenous human cranial-neural-crest chromatin occupancy and transcriptional perturbation evidence. The refinement concerns the target's measured transcription system, not a claim that the mouse donor or broader source term is false.

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

Summary: The ALX4–IQUB interaction remains unadjudicated at pair-level source resolution.

Reason: The supplied partner Q8NA54 is IQUB. PMID:32296183 main Methods establish the human binary-interaction assay class, and UniProt/IntAct records the pair with NbExp=3. Neither establishes the pair-specific controls or functional interpretation, which remain unexamined because the relevant supplementary evidence was not recovered. Generic protein binding is uninformative, but no evidence-backed specific replacement can yet be selected. Retain UNDECIDED under the inaccessible-evidence rule rather than remove an unadjudicated experimental annotation. Usual subcellular locations alone do not establish that the reported pair is false or cannot coexist.

Summary: The ALX4–PARVG interaction remains unadjudicated at pair-level source resolution.

Reason: The supplied partner Q9HBI0 is PARVG. The HuRI main Methods and UniProt/IntAct NbExp=3 entry establish an interaction record, not an independent review of this pair's controls or functional scope. The relevant supplementary evidence remains unavailable. Retain UNDECIDED under the inaccessible-evidence rule: generic binding is uninformative, but a specific molecular-function replacement cannot be inferred from the database pair record. Typical compartment assignments do not establish that the reported interaction is false or biologically impossible.

Summary: Nuclear residence is supported independently in human ALX4.

Reason: The original full PMID:19692347 Methods/Results show tagged wild-type human ALX4 in COS7 nuclei, with altered localization of R265X; COS7 is a nonhuman host. Separate HEK293T experiments assay protein stability. Failure of the available antibody to detect endogenous protein in either control or patient osteoblasts is not a localization experiment. Independent human chromatin studies and current Human Protein Atlas nucleoplasmic staining corroborate nuclear residence. The target Q9H161 appearing among PAINT descendants is legitimate target experimental grounding, not circularity. The exact ancestral IBD/tree placement was not independently reconstructed. The supplied PAINT node and descendant evidence are preserved; donor count is not a quality criterion, and independent human evidence supports retention without inventing the node history. In PMID:40410151, HA-tagged full-length human wild-type ALX4 is nuclear in HEK293T immunostaining; the V241R comparator retains similar nuclear localization and protein abundance. This is separate from that paper's purified mouse DNA-binding fragments.

Summary: The original tagged-human-protein experiment supports nuclear localization.

Reason: The original full PMID:19692347 Methods/Results show tagged wild-type human ALX4 in COS7 nuclei, with altered localization of R265X; COS7 is a nonhuman host. Separate HEK293T experiments assay protein stability. Failure of the available antibody to detect endogenous protein in either control or patient osteoblasts is not a localization experiment. Independent human chromatin studies and current Human Protein Atlas nucleoplasmic staining corroborate nuclear residence. In PMID:40410151, HA-tagged full-length human wild-type ALX4 is nuclear in HEK293T immunostaining; the V241R comparator retains similar nuclear localization and protein abundance. This is separate from that paper's purified mouse DNA-binding fragments.

Summary: The combined nuclear inference agrees with target-specific localization.

Reason: The original full PMID:19692347 Methods/Results show tagged wild-type human ALX4 in COS7 nuclei, with altered localization of R265X; COS7 is a nonhuman host. Separate HEK293T experiments assay protein stability. Failure of the available antibody to detect endogenous protein in either control or patient osteoblasts is not a localization experiment. Independent human chromatin studies and current Human Protein Atlas nucleoplasmic staining corroborate nuclear residence. ARBA, mouse orthology and the UniProt subcellular source remain distinct supplied inferences, not additional experiments. In PMID:40410151, HA-tagged full-length human wild-type ALX4 is nuclear in HEK293T immunostaining; the V241R comparator retains similar nuclear localization and protein abundance. This is separate from that paper's purified mouse DNA-binding fragments.

Summary: Retain the historical nuclear assertion with independent positive corroboration.

Reason: The cited human homeobox paper is currently abstract-only, so its detailed localization work is not reconstructed. The original full PMID:19692347 Methods/Results show tagged wild-type human ALX4 in COS7 nuclei, with altered localization of R265X; COS7 is a nonhuman host. Separate HEK293T experiments assay protein stability. Failure of the available antibody to detect endogenous protein in either control or patient osteoblasts is not a localization experiment. Independent human chromatin studies and current Human Protein Atlas nucleoplasmic staining corroborate nuclear residence. This supports curator deference without falsely attributing later microscopy to the historical NAS source. In PMID:40410151, HA-tagged full-length human wild-type ALX4 is nuclear in HEK293T immunostaining; the V241R comparator retains similar nuclear localization and protein abundance. This is separate from that paper's purified mouse DNA-binding fragments.

Summary: Human Protein Atlas directly supports a nucleoplasmic pool.

Reason: The current primary HPA subcellular page reports supported nucleoplasm with antibody HPA001903 in HEK293 and SiHa; U2OS is recorded as no staining. This is a cell-line-scoped immunofluorescence result, not evidence for universal expression. The historical v16 nucleus wording is distinguished from the current nucleoplasm label. The machine GO_REF immunofluorescence source is preserved.

Summary: ALX4 participates in a DNA-associated transcription-regulating assembly.

Reason: The mouse ortholog transfer is supported by positive Alx4–Cart1 and Alx4–LEF1 studies, while PMID:38262408 independently resolves a human TWIST1–TCF4–ALX4 complex on DNA. Methods explicitly identify human DNA-binding fragments TWIST1 101–170, TCF4 565–624 and ALX4 210–277 expressed in E. coli. Cooperative occupancy, structure and EMSA support the complex in its DNA/domain context. Lack of a stable interaction in DNA-minimizing solution extracts does not negate a DNA-associated complex, and no fixed soluble full-length stoichiometry is asserted.

Summary: Refine generic transcription regulation to the measured Pol-II process.

Reason: Human ALX4 binds sequence-specific regulatory DNA and modulates RNA-polymerase-II reporter output. PMID:22829454 assays human constructs in human calvarial osteoblasts, and PMID:40410151 separately tests full-length human ALX4 in HEK293T reporter assays. The latter study uses purified mouse DNA-binding regions explicitly identical to the corresponding human regions for its structural/biophysical work; those preparations are not conflated with the human full-length reporter. PMID:38262408 adds endogenous human cranial-neural-crest chromatin occupancy and transcriptional perturbation evidence. The refinement retains both activating and context-dependent repressive outputs rather than treating every regulatory effect as positive.

Proposed replacements: regulation of transcription by RNA polymerase II

Summary: ALX4 directly regulates Pol-II transcription.

Reason: The ancestral node PTN004692309 is retained. Human ALX4 binds sequence-specific regulatory DNA and modulates RNA-polymerase-II reporter output. PMID:22829454 assays human constructs in human calvarial osteoblasts, and PMID:40410151 separately tests full-length human ALX4 in HEK293T reporter assays. The latter study uses purified mouse DNA-binding regions explicitly identical to the corresponding human regions for its structural/biophysical work; those preparations are not conflated with the human full-length reporter. PMID:38262408 adds endogenous human cranial-neural-crest chromatin occupancy and transcriptional perturbation evidence. The exact ancestral IBD/tree placement was not independently reconstructed. The supplied PAINT node and descendant evidence are preserved; donor count is not a quality criterion, and independent human evidence supports retention without inventing the node history.

Summary: Resolve sequence-specific DNA binding to the established regulatory-region activity.

Reason: The mouse-derived molecular assertion is correct in essence. Independent human chromatin occupancy and reporter assays justify the regulatory-region subtype rather than leaving the source at generic sequence-specific DNA binding.

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

Summary: The positive Pol-II process follows a measured activator activity.

Reason: The proximate GO:0001228 source is supported by human reporter assays. Positive output is actually measured, rather than inferred from an ALX4 knockout phenotype; dose, partner and variant context can additionally support repression without negating this positive activity.

Summary: Broad organ-development participation fits the established developmental regulatory role.

Reason: Human skull and skin/follicle evidence, together with human mesenchymal gene-regulatory experiments, supports participation in core developmental programs. GO:0001942 hair follicle development has part_of ancestry to GO:0048513 through skin development. GO:0060348 bone development is_a animal organ development and part_of skeletal system development; this does not make all skeletal-system development an organ-development subtype. Retain this broad existing umbrella as core participation. Lower specificity does not make the underlying developmental role peripheral, and the compact core adds no separate organ-development assertion or claim about every organ.

Summary: The neuron-development ancestral assertion remains unresolved.

Reason: The exact IBD and experimental chain for PTN001216073 were not reconstructed. ALX4's role in cranial neural-crest-derived mesenchyme is not equivalent to progression of a neuron. In PMID:38262408 the emphasized induction of neuronal markers follows TWIST1 loss, while ALX loss alters facial mesenchymal regional programs. The LEF1/N-CAM promoter study likewise does not alone establish neuron development. These distinctions do not demonstrate evolutionary loss or invalidate the PAINT placement; retain source-specific uncertainty rather than infer absence from the literature inspected.

Summary: Specific LEF1 HMG-domain binding supports this partner-recognition activity.

Reason: The mouse ortholog source is retained. Original PMID:11696550 Methods/Results map Alx4 association to the LEF1 HMG domain by co-IP and deletion constructs, and show partner/dose-dependent promoter effects. This is more specific than generic protein binding and mechanistically connected to transcriptional regulation. COS1/C33A hosts and mouse primary stromal experiments remain explicit; the exact referenced Alx4 cDNA species is not silently re-established as human. No neuron-development or universal activation claim follows from N-CAM promoter regulation.

Summary: Retain sequence-specific double-stranded DNA binding at the assayed aspect.

Reason: The curated IDA is from the human methyl-SELEX study. Its canonical extraction contains abstract and conclusion text but not the ALX4-specific clone/table, so no ALX4 methylation preference is invented. Independent ALX4 DNA structures and human reporter/chromatin experiments strongly corroborate the molecular activity. Preserve the experimental source's DNA-binding aspect with curator deference, rather than replacing its assay claim with an unobserved methylation phenotype.

Summary: The electronic double-stranded DNA-binding assertion is independently corroborated.

Reason: The ARBA source is preserved. ALX4 has direct sequence-dependent DNA association supported by human chromatin/reporters and mouse domains identical in the assayed human region. This atomic binding aspect is compatible with the integrated TF activity; it does not assert an ALX4-specific methyl-CpG preference. Unlike the generic sequence-specific DNA-binding parent GO:0043565, this term preserves the double-stranded substrate aspect. GO:0000977 specifies regulatory-site recognition, a complementary refinement used elsewhere; the electronic source is corroborated by direct target evidence, not treated as another independent assay.