NCU03033 encodes the single Neurospora crassa member of the NF-YA/Hap2 family (314 aa), the sequence-recognition subunit of the heterotrimeric CCAAT-binding transcription factor (CBC; Hap2/3/5, HapB/C/E in Aspergillus, NF-Y in mammals). Its conserved essential core comprises an N-terminal subunit-association segment, which docks onto the Hap3/Hap5 histone-fold dimer, and a C-terminal CCAAT-recognition segment; the remainder of the protein is a Q/P-rich and charged disordered region. As part of the trimer it binds CCAAT boxes in nuclear promoters and regulates RNA polymerase II transcription, acting as an activator or repressor depending on promoter context and associated regulators (e.g. Hap4 or HapX). No N. crassa-specific experiment on this protein has been reported; the function is inferred from the PAINT-placed NF-YA/Hap2 clade, the near-identical core shared with yeast Hap2 and php2, and the functional interchangeability of yeast Hap2, Aspergillus HapB and human NF-YA.
Summary: Core molecular function of the NF-YA/Hap2 clade, inherited from PAINT node PTN000290575.
Reason: PAINT placed this function at PTN000290575, an ancestral NF-YA/Hap2 node whose experimentally characterised descendants include S. cerevisiae HAP2 (SGD:S000003206), human NFYA (P23511) and mouse Nfya. NCU03033 is the only PTHR12632/IPR001289 protein in N. crassa and retains the full essential core (76-78% identity to yeast HAP2 and S. pombe php2 over the subunit-association and DNA-recognition segments; file:NEUCR/NCU03033/NCU03033-bioinformatics/RESULTS-rereview.md), so there is no evidence of divergence from the node. Yeast Hap2, A. nidulans HapB and human NF-YA are functionally interchangeable in complementation tests. The activity is exerted as a subunit of the trimer (all three subunits are needed for DNA binding), which GO captures by the companion CCAAT-binding factor complex annotation.
Moreover, S. cerevisiae Hap2p, A. nidulans HapB and human NF-YA are functionally interchangeable, which was demonstrated by the complementation of an S. cerevisiae hap2 and an A. nidulans hapB deletion mutant by a human NF-YA cDNA (19,20).
Summary: True but general; the subunit confers sequence-specific CCAAT recognition at RNA polymerase II promoters.
Reason: DNA binding is correct but uninformative. The NF-YA/Hap2 DNA-recognition segment (UniProt DNA_BIND 225-250, PROSITE PRU00966) provides sequence-specific recognition of CCAAT boxes in promoters, as part of the trimer. The specific descendant RNA polymerase II cis-regulatory region sequence-specific DNA binding (GO:0000978) is supported; it is implied by the DNA-binding transcription factor activity already accepted from the IBA row.
Component of the sequence-specific heterotrimeric transcription factor (NF-Y) which specifically recognizes a 5'-CCAAT-3' box motif found in the promoters of its target genes.
Summary: Correct but less specific than the RNA polymerase II-specific activity inherited by IBA.
Reason: The NF-YA/Hap2 CCAAT-binding factor acts on RNA polymerase II promoters (yeast CYC1 and respiratory genes; Aspergillus amdS, penicillin genes and HapX-CBC iron-regulated genes). The specific descendant GO:0000981, already carried by this gene from the PAINT IBA, is the appropriate term.
Summary: The CCAAT-binding factor acts on nuclear promoters; nuclear location follows from family membership.
Reason: Nucleus is the site of action of the CCAAT-binding factor and is supported by the curated UniRule (RuleBase RU367155) and the PROSITE NFYA/HAP2 rule. No N. crassa localization experiment exists, and the PAINT CC annotation is the complex term, so nucleus is kept as the appropriate location. Note that in A. nidulans an oxidised partner subunit (HapC) is excluded to the cytoplasm, showing that nuclear localization depends on complex assembly; this is not a Hap2-specific property.
Supporting Evidence:
file:NEUCR/NCU03033/NCU03033-uniprot.txt
Component of the sequence-specific heterotrimeric transcription factor (NF-Y) which specifically recognizes a 5'-CCAAT-3' box motif found in the promoters of its target genes.
Summary: Hap2 regulates transcription; it is not part of the RNA-synthesis machinery.
Reason: GO:0006351 describes the synthesis of RNA by the polymerase. NF-YA/Hap2 is a sequence-specific regulator that binds promoter CCAAT boxes; the correct process is regulation of transcription by RNA polymerase II (GO:0006357), which the gene already carries by IBA. This is a lateral correction rather than a refinement.
Hap2/3/5 p form the core CCAAT-binding complex, which is responsible for DNA binding, while Hap4p is involved in transcriptional activation (11).
GO:0006355 regulation of DNA-templated transcription
IEA GO_REF:0000120
MODIFY
Summary: Correct but general; the specific descendant RNA polymerase II term is supported.
Reason: The NF-YA/Hap2 complex targets RNA polymerase II promoters. Replace with the descendant GO:0006357, which the gene already carries from the PAINT IBA.
Hap2/3/5 p form the core CCAAT-binding complex, which is responsible for DNA binding, while Hap4p is involved in transcriptional activation (11).
GO:0006357 regulation of transcription by RNA polymerase II
IBA GO_REF:0000033
ACCEPT
Summary: Core process of the CCAAT-binding factor, inherited from PTN000290575.
Reason: The PAINT node propagates regulation (not positive regulation) of RNA polymerase II transcription, which is the right level: the fungal CBC both activates (yeast respiratory genes with Hap4; A. nidulans amdS, ipnA, aatA) and represses (A. nidulans lysF, hapB, and iron-consuming genes with HapX) depending on partner regulators. Organism-specific regulons (respiration, cellulases, iron homeostasis) are not transferred to N. crassa, where the regulon is unmapped.
On the other side, AnCF-mediated repression of gene expression was also found: the homoaconitase-encoding lysF and the hapB gene were repressed by AnCF (25,26).
Moreover, S. cerevisiae Hap2p, A. nidulans HapB and human NF-YA are functionally interchangeable, which was demonstrated by the complementation of an S. cerevisiae hap2 and an A. nidulans hapB deletion mutant by a human NF-YA cDNA (19,20).
Summary: NF-YA/Hap2 is an obligate subunit of the CCAAT-binding factor trimer.
Reason: PAINT node PTN000290576 places complex membership at the base of the NF-YA clade, with yeast, human, rat, fly and worm donors. NCU03033 retains the subunit-association segment (UniProt MOTIF 194-217) that mediates docking onto the Hap3/Hap5 dimer. In the closest characterised fungus (A. nidulans), the HapB/HapC/HapE trimer is required for DNA binding.
The corresponding Aspergillus nidulans CCAAT-binding factor (AnCF) consists of the subunits HapB, HapC and HapE. All of the three subunits are necessary for DNA binding.
The corresponding Aspergillus nidulans CCAAT-binding factor (AnCF) consists of the subunits HapB, HapC and HapE. All of the three subunits are necessary for DNA binding.
file:NEUCR/NCU03033/NCU03033-uniprot.txt
Belongs to the NFYA/HAP2 subunit family.
Core Functions
Sequence-recognition (NF-YA/Hap2) subunit of the nuclear CCAAT-binding factor; within the Hap2/3/5 trimer it binds CCAAT boxes in RNA polymerase II promoters and regulates transcription of target genes.
Moreover, S. cerevisiae Hap2p, A. nidulans HapB and human NF-YA are functionally interchangeable, which was demonstrated by the complementation of an S. cerevisiae hap2 and an A. nidulans hapB deletion mutant by a human NF-YA cDNA (19,20).
file:NEUCR/NCU03033/NCU03033-uniprot.txt
Component of the sequence-specific heterotrimeric transcription factor (NF-Y) which specifically recognizes a 5'-CCAAT-3' box motif found in the promoters of its target genes.
Q: Which N. crassa promoters does the NCU03033-containing CBC occupy, and does it act with a Hap4-like activator, a HapX-like bZIP, or both in this species?
Suggested Experiments
Experiment: Endogenous epitope tagging of NCU03033 for nuclear localization, co-purification of the Hap3/Hap5 partners, and ChIP-seq with deletion RNA-seq under respiratory, iron-limited and oxidative conditions to define its direct regulon.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
NCU03033 is the single N. crassa NF-YA/Hap2 subunit of the heterotrimeric CCAAT-binding factor, placed by PAINT in the NF-YA/Hap2 clade whose characterised members (yeast Hap2, A. nidulans HapB, human NF-YA) are functionally interchangeable. Both ProtNLM2 GO predictions are correct and already carried by the gene in GOA: the generic DNA-binding transcription factor activity is less precise than the RNA polymerase II-specific activity inherited by IBA (LSP), and nucleus is an existing, accepted location (CNN).
Review rationale: NCU03033 carries the NF-YA/Hap2 signature (InterPro IPR001289) and the curated RuleBase family assignment, and the family's characterised fungal and human members are interchangeable CCAAT-box-binding transcription factor subunits that act on RNA polymerase II promoters. This is a justified family transfer, not an observation on the N. crassa protein itself. GOA already carries the exact predicted term (IEA), which the gene review modifies to the more specific GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific (is_a descendant of GO:0003700), inherited by IBA from PAINT node PTN000290575 and accepted as the core function. The prediction is correct but less precise than that supported annotation.
PMID:19965775: "Moreover, S. cerevisiae Hap2p, A. nidulans HapB and human NF-YA are functionally interchangeable, which was demonstrated by the complementation of an S. cerevisiae hap2 and an A. nidulans hapB deletion mutant by a human NF-YA cDNA (19,20)."
PMID:19965775: "The corresponding Aspergillus nidulans CCAAT-binding factor (AnCF) consists of the subunits HapB, HapC and HapE. All of the three subunits are necessary for DNA binding."
PMID:25589790: "Initial binding of the CBC to CCAAT boxes is mandatory for DNA recognition of HapX."
Review rationale: Nucleus is the site of action of the CCAAT-binding factor, which binds CCAAT boxes in target promoters; this follows from the curated NF-YA/Hap2 family assignment of NCU03033 and its membership in the CCAAT-binding factor complex (IBA, PTN000290576). In N. crassa itself a heteromeric CCAAT-binding activity was purified from nuclear protein, although that study did not identify NCU03033 as one of its subunits. GOA already carries the exact term (IEA), which the gene review accepts, so the prediction is correct but not novel.
PMID:7500955: "Gel mobility shift analysis indicated that a nuclear protein from Neurospora binds in a specific manner to sequences within the 90 bp fragment."
PMID:7500955: "The purified protein appears as a heteromer on denaturing polyacrylamide gel electrophoresis"