NPAC (Cytokine-like nuclear factor N-PAC, also known as GLYR1/NDF/NP60) is a chromatin-associated nuclear protein in Anopheles gambiae that functions as a nucleosome-destabilizing factor facilitating RNA polymerase II transcription elongation through chromatin. The protein contains an N-terminal PWWP domain that reads histone H3K36me3 marks and binds DNA, and a C-terminal dehydrogenase-like Rossmann fold domain that is catalytically inert but mediates oligomerization. In orthologous species (Drosophila, human, mouse), NPAC is recruited to gene bodies of actively transcribed genes, destabilizes nucleosomes in an ATP-independent manner to facilitate Pol II passage, stimulates H3K56 acetylation by p300, and serves as a cofactor of the histone demethylase LSD2/KDM1B promoting H3K4me1/me2 demethylation. In Drosophila, it also interacts with the MSL histone acetyltransferase complex involved in dosage compensation. All functional annotations for the Anopheles protein are inferred by homology; no direct experimental studies exist for this organism.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0003677
DNA binding
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: This IBA annotation is based on phylogenetic inference from the Drosophila ortholog CG4747 (Q8T079) and human GLYR1 (Q49A26). The PWWP domain of NPAC is known to strongly bind DNA in addition to reading histone marks.
Reason: DNA binding via the PWWP domain is well-established for this protein family. UniProt CC states "The PWWP domain is a H3 reader and strongly binds DNA" (by similarity to Q49A26). The annotation is correct but somewhat generic; nucleosome binding (GO:0031491) is a more informative descriptor of the primary chromatin interaction.
Supporting Evidence:
PMID:29759984
NDF has a PWWP motif, interacts with nucleosomes near the dyad
file:ANOGA/NPAC/NPAC-notes.md
The PWWP domain is a H3 reader and strongly binds DNA
|
|
GO:0003682
chromatin binding
|
IEA
GO_REF:0000117 |
ACCEPT |
Summary: This IEA annotation is from ARBA rule ARBA00027831. Chromatin binding is appropriate for this protein which localizes to and interacts with chromatin through the PWWP domain, but is less specific than the nucleosome binding annotation already present.
Reason: Chromatin binding is correct. NPAC/NDF localizes to chromatin in a H3K36me3-dependent manner and directly interacts with nucleosomes. This is a broad but accurate annotation.
Supporting Evidence:
PMID:29759984
NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner
PMID:33676077
Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes
file:ANOGA/NPAC/NPAC-deep-research-bioreason-sft.md
a chromatin-reading module that recognizes nucleosomal features
|
|
GO:0031491
nucleosome binding
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: This IBA annotation is based on phylogenetic inference from Drosophila CG4747 and human GLYR1. Nucleosome binding is a core molecular function of this protein established in multiple studies.
Reason: Nucleosome binding is one of the best-characterized functions of the NP60 subfamily. UniProt CC states "Binds to mononucleosomes" (by similarity to Q8T079). NDF interacts with nucleosomes near the dyad and destabilizes them to facilitate transcription.
Supporting Evidence:
PMID:29759984
NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner
PMID:23260659
NPAC directly interacts with LSD2 and positively regulates its H3K4 demethylation activity both in vitro and in vivo
|
|
GO:0050661
NADP binding
|
IEA
GO_REF:0000002 |
KEEP AS NON CORE |
Summary: This InterPro2GO annotation derives from IPR006115 (6-phosphogluconate dehydrogenase, NADP-binding domain). The Rossmann fold in NPAC does contain conserved NAD(P)H-binding sites structurally, but the protein lacks a conserved active site and the dehydrogenase domain is catalytically inert.
Reason: The structural capacity to bind NADP is plausible given the Rossmann fold architecture, but the dehydrogenase domain is catalytically inert. Fang et al. (2013) reported "attempts to identify the intrinsic enzymatic activity of NPAC as a potential dehydrogenase were unsuccessful." The binding may be structural or regulatory rather than catalytic, and the functional significance of NADP binding is unclear. This is not a core function of the protein.
Supporting Evidence:
PMID:23260659
attempts to identify the intrinsic enzymatic activity of NPAC as a potential dehydrogenase
file:ANOGA/NPAC/NPAC-deep-research-bioreason-sft.md
the active site is not conserved, the dehydrogenase domain seems to serve as a catalytically inert oligomerization module
|
|
GO:0051287
NAD binding
|
IEA
GO_REF:0000002 |
KEEP AS NON CORE |
Summary: This InterPro2GO annotation derives from IPR029154 (3-hydroxyisobutyrate dehydrogenase-like, NAD-binding domain). Same reasoning as for NADP binding -- the Rossmann fold is structurally present but catalytically inert.
Reason: The structural NAD binding capacity is plausible given the conserved Rossmann fold, but the domain serves as "a catalytically inert oligomerization module" rather than performing redox chemistry. NAD binding is not a core function of this protein.
Supporting Evidence:
PMID:23260659
attempts to identify the intrinsic enzymatic activity of NPAC as a potential dehydrogenase
|
|
GO:0140673
transcription elongation-coupled chromatin remodeling
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: This IBA annotation is based on phylogenetic inference from Drosophila CG4747 and human GLYR1. This is one of the most precisely characterized functions of the protein.
Reason: Transcription elongation-coupled chromatin remodeling is the central biological process of NPAC/NDF. The protein destabilizes nucleosomes to facilitate Pol II transcription through chromatin, interacts with p-TEFb and phosphorylated Pol II, and is recruited to gene bodies upon transcriptional induction. This is a core function.
Supporting Evidence:
PMID:29759984
NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes
PMID:33676077
Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA Pol II Ser2P and Ser5P
|
|
GO:0000785
chromatin
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: This IBA annotation places NPAC as active in chromatin, based on the Drosophila and human orthologs which localize to chromatin.
Reason: NPAC localizes to chromatin in a H3K36me3-dependent manner. In Drosophila, it localizes to polytene chromosome interbands (open chromatin). The protein is active at chromatin where it destabilizes nucleosomes and facilitates transcription.
Supporting Evidence:
PMID:33676077
Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes
PMID:29759984
Upon transcriptional induction, NDF is recruited to the transcribed regions of thousands of genes and colocalizes with a subset of H3K36me3-enriched regions
|
|
GO:0005694
chromosome
|
IEA
GO_REF:0000044 |
ACCEPT |
Summary: This IEA annotation is derived from UniProt subcellular location "Chromosome" (by similarity to Q8T079). NPAC localizes to chromosomes.
Reason: Chromosome localization is correct and consistent with the protein's chromatin association. This is a broad but accurate annotation; the more specific "chromatin" (GO:0000785) annotation is also present.
Supporting Evidence:
file:ANOGA/NPAC/NPAC-notes.md
Localization to open chromatin depends on H3K36 trimethylation by Set2
|
|
GO:0140003
histone H3K36me3 reader activity
|
ISS
PMID:29759984 NDF, a nucleosome-destabilizing factor that facilitates tran... |
NEW |
Summary: NPAC reads H3K36me3 marks via its PWWP domain, which is the primary mechanism for its recruitment to actively transcribed gene bodies. This is a more specific and informative annotation than general chromatin binding.
Reason: H3K36me3 reader activity via the PWWP domain is a core molecular function that drives the chromatin localization and biological activity of the protein. This is well-established in both Drosophila and mammalian orthologs and should be annotated for the Anopheles ortholog by sequence similarity.
Supporting Evidence:
PMID:29759984
NDF has a PWWP motif...colocalizes with a subset of H3K36me3-enriched regions
PMID:33676077
Npac, a "reader" of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC
|
Q: Does the Anopheles NPAC ortholog interact with the MSL dosage compensation complex in vivo, as predicted by homology to the Drosophila ortholog?
Q: Does the Anopheles NPAC dehydrogenase domain retain any capacity for NAD(P) binding, and if so, does this have a regulatory role?
Q: Is NPAC involved in any mosquito-specific chromatin regulation processes related to malaria vector biology (e.g., during blood feeding or parasite infection)?
Experiment: ChIP-seq of NPAC in Anopheles gambiae cell lines to determine whether it co-localizes with H3K36me3 marks as in orthologs, confirming the conserved chromatin targeting mechanism.
Hypothesis: NPAC localizes to H3K36me3-enriched gene bodies of actively transcribed genes in Anopheles, consistent with its ortholog function.
Experiment: Co-immunoprecipitation of NPAC with components of the Anopheles MSL complex (MOF) to validate the predicted interaction.
Hypothesis: NPAC interacts with the MSL complex in Anopheles as it does in Drosophila.
The architecture begins with a compact N-terminal PWWP module: IPR000313 (PWWP domain, residues 7–68) is present alongside overlapping, family-specific refinements IPR035501 (Cytokine-like nuclear factor N-PAC, PWWP domain, residues 7–93), IPR000313 (PWWP domain, residues 9–70), and IPR000313 (PWWP domain, residues 10–92). This cluster defines a chromatin-reading module that recognizes nucleosomal features—classically DNA and methylated histone marks—thereby causing nucleosome binding and direct DNA engagement. Downstream, the polypeptide transitions into a large dehydrogenase-like core: IPR051265 (HIBADH-related NP60 subfamily, residues 167–562) encompasses a Rossmann-like redox fold, with IPR036291 (NAD(P)-binding domain superfamily, residues 281–443) and IPR006115 (6-phosphogluconate dehydrogenase, NADP-binding, residues 282–441) specifying a canonical dinucleotide-binding site. The C-terminal region is further defined by IPR008927 (6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily, residues 442–561) and IPR013328 (6-phosphogluconate dehydrogenase, domain 2, residues 444–563), culminating in IPR029154 (3-hydroxyisobutyrate dehydrogenase-like, NAD-binding domain, residues 444–561). This ordered layout—reader domain followed by a redox fold with a Rossmann site—creates a bifunctional protein: a chromatin-tethered scaffold that can bind nucleosomes and DNA, and a metabolite-sensing redox module.
The PWWP cluster confers molecular functions of nucleosome binding (GO:0031491) and DNA binding (GO:0003677). These reader interactions position the protein on chromatin, where the dehydrogenase-like core can act as a regulatory sensor: binding of NAD(H)/NADP(H) within the Rossmann pocket alters the conformation and interaction surface of the C-terminal lobe, thereby modulating recruitment of chromatin-modifying enzymes. Such a mechanism naturally promotes positive regulation of histone acetylation (GO:0035066), because a chromatin-tethered, metabolite-responsive scaffold enhances acetyltransferase access to nucleosomes. The resulting increase in histone acetylation loosens chromatin and facilitates assembly of the transcriptional machinery, driving positive regulation of transcription by RNA polymerase II (GO:0045944).
The presence of a chromatin reader and the absence of transmembrane segments or secretion signals place the protein in the nuclear compartment, consistent with a chromatin-associated nuclear factor and aligning with the cellular component nucleus (GO:0005634). Within the nucleus, the PWWP domain anchors the protein to specific nucleosomal contexts, while the dehydrogenase-like lobe senses cellular redox or metabolite status via NAD(P) binding, translating metabolic cues into chromatin acetylation and transcriptional outputs.
This mechanistic model suggests interaction partners that either bridge to chromatin-modifying enzymes or reflect metabolic coupling. The chromatin-tethered scaffold is poised to engage acetyltransferase complexes and nucleosome components. The observed associations with enzymes of branched-chain and aldehyde metabolism—3-hydroxyisobutyrate dehydrogenase, 3-hydroxyisobutyryl-CoA hydrolase, probable methylmalonate-semialdehyde dehydrogenase (acylating), aldehyde dehydrogenases (NAD+-dependent), and 4-aminobutyrate aminotransferase—can arise from metabolite-channeling or redox-state signaling: the dehydrogenase-like domain may sense NAD(H) availability and transiently bind metabolic enzymes to coordinate acetylation with metabolic flux. Alanine–glyoxylate aminotransferase 2 and aldehyde dehydrogenase family 7 member A1 fit this theme, potentially linking amino acid and aldehyde metabolism to chromatin regulation. The V-type proton ATPase catalytic subunit A and AGAP009944-PA may represent regulatory or scaffolding contacts that couple cellular energy status to chromatin engagement, indirectly influencing nuclear metabolite pools. Overall, the protein likely functions as a nuclear chromatin reader fused to a redox-sensing dehydrogenase-like module that promotes histone acetylation and transcriptional activation in response to metabolic cues.
## Functional Summary
A nuclear chromatin reader that anchors to nucleosomes and DNA through an N-terminal reader module and uses a C-terminal dehydrogenase-like Rossmann fold to sense cellular redox or metabolite levels. By coupling nucleosome binding with dinucleotide-dependent conformational control, it promotes histone acetylation and activates RNA polymerase II–driven transcription, thereby translating metabolic state into chromatin remodeling and gene expression changes within the nucleus.
## UniProt Summary
Nuclear factor that binds DNA and nucleosomes.
## InterPro Domains
- IPR000313: PWWP domain (domain) [7-68]
- IPR035501: Cytokine-like nuclear factor N-PAC, PWWP domain (domain) [7-93]
- IPR000313: PWWP domain (domain) [9-70]
- IPR000313: PWWP domain (domain) [10-92]
- IPR051265: HIBADH-related NP60 subfamily (family) [167-562]
- IPR036291: NAD(P)-binding domain superfamily (homologous_superfamily) [281-443]
- IPR006115: 6-phosphogluconate dehydrogenase, NADP-binding (domain) [282-441]
- IPR008927: 6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily (homologous_superfamily) [442-561]
- IPR013328: 6-phosphogluconate dehydrogenase, domain 2 (homologous_superfamily) [444-563]
- IPR029154: 3-hydroxyisobutyrate dehydrogenase-like, NAD-binding domain (domain) [444-561]
## GO Term Predictions
### Molecular Function
### Biological Process
### Cellular Component
Fei et al. (2018) PMID:29759984 identified the Drosophila ortholog CG4747 and human GLYR1 as NDF, "a nucleosome-destabilizing factor that facilitates transcription through nucleosomes." Key findings:
- NDF interacts with nucleosomes near the dyad
- Destabilizes nucleosomes in an ATP-independent manner
- Facilitates Pol II transcription through nucleosomes
- Stimulates H3K56 acetylation by p300 on nucleosomal substrates
- Recruited to transcribed regions of thousands of genes upon transcriptional induction
- Colocalizes with H3K36me3-enriched regions
- The PWWP domain recognizes H3K36me3, but H3K36me3 is "necessary but not sufficient" for localization
- Essential in human stem cells
- Overexpressed in ~21% of breast cancers
Fang et al. (2013) PMID:23260659 showed NPAC/GLYR1 is a cofactor of LSD2/KDM1B:
- Stimulates H3K4me1 and H3K4me2 demethylation
- The minimal functional segment is a dodecapeptide (residues 214-225)
- Residue F217 stabilizes enzyme-substrate interaction
- Crystal structures of LSD2 alone and LSD2-NPAC complex were determined
Yu et al. (2021) PMID:33676077 showed in mouse ESCs:
- Npac co-localizes with H3K36me3 in gene bodies of actively transcribed genes
- Interacts with p-TEFb (positive transcription elongation factor b)
- Interacts with Ser2-phosphorylated and Ser5-phosphorylated RNA Pol II
- Depletion disrupts transcriptional elongation of pluripotency genes (Nanog, Rif1)
- Required for mESC pluripotency maintenance
UniProt CC for Q7Q161 states (by similarity to Q8T079):
- "Binds to mononucleosomes"
- "Interacts with male-specific lethal (MSL) histone acetyltransferase complex at least composed of mof, msl-1, msl-2 and msl-3"
The MSL complex in Drosophila acetylates H4K16 for dosage compensation on the male X chromosome.
Fang et al. (2013) PMID:23260659: "attempts to identify the intrinsic enzymatic activity of NPAC as a potential dehydrogenase...were unsuccessful." The dehydrogenase domain instead serves as "a catalytically inert oligomerization module" that forms a stable tetramer.
UniProt CC for Q7Q161: "the active site is not conserved, the dehydrogenase domain seems to serve as a catalytically inert oligomerization module"
UniProt CC (by similarity to Drosophila Q8T079): "Localization to open chromatin depends on H3K36 trimethylation by Set2."
| GO ID | Term | Qualifier | Evidence | Reference |
|---|---|---|---|---|
| GO:0003677 | DNA binding | enables | IBA | GO_REF:0000033 |
| GO:0003682 | chromatin binding | enables | IEA | GO_REF:0000117 (ARBA) |
| GO:0031491 | nucleosome binding | enables | IBA | GO_REF:0000033 |
| GO:0050661 | NADP binding | enables | IEA | GO_REF:0000002 (InterPro:IPR006115) |
| GO:0051287 | NAD binding | enables | IEA | GO_REF:0000002 (InterPro:IPR029154) |
| GO:0140673 | transcription elongation-coupled chromatin remodeling | involved_in | IBA | GO_REF:0000033 |
| GO:0000785 | chromatin | is_active_in | IBA | GO_REF:0000033 |
| GO:0005694 | chromosome | located_in | IEA | GO_REF:0000044 |
Two InterPro2GO annotations:
- IPR006115 -> GO:0050661 (NADP binding): Based on the 6-phosphogluconate dehydrogenase NADP-binding domain
- IPR029154 -> GO:0051287 (NAD binding): Based on the 3-hydroxyisobutyrate dehydrogenase-like NAD-binding domain
These are technically correct structurally (the Rossmann fold does bind NAD(P)), but the dehydrogenase domain is catalytically inert. The NAD(P) binding may be structural/regulatory rather than enzymatic.
NPAC/GLYR1/NDF is a multifunctional chromatin-associated protein whose core biology is well-characterized in human and Drosophila but entirely inferred by homology for the Anopheles ortholog. The protein reads H3K36me3 marks via its PWWP domain, destabilizes nucleosomes to facilitate RNA Pol II transcription elongation, cofactors LSD2-mediated H3K4 demethylation, and stimulates H3K56 acetylation by p300. The dehydrogenase-like C-terminal domain is catalytically inert but serves as an oligomerization platform. In Drosophila, it also interacts with the MSL dosage compensation complex.
Source: NPAC-deep-research-bioreason-sft.md
The BioReason SFT functional summary states:
A nuclear chromatin reader that anchors to nucleosomes and DNA through an N-terminal reader module and uses a C-terminal dehydrogenase-like Rossmann fold to sense cellular redox or metabolite levels. By coupling nucleosome binding with dinucleotide-dependent conformational control, it promotes histone acetylation and activates RNA polymerase II-driven transcription, thereby translating metabolic state into chromatin remodeling and gene expression changes within the nucleus.
The identification of NPAC as a chromatin reader with a PWWP domain and a dehydrogenase-like C-terminal domain is correct. The identification of nucleosome binding and DNA binding as molecular functions, and the association with transcription and histone acetylation as biological processes, are broadly supported.
Significant errors:
"Redox/metabolite sensing" is speculative and unsupported: The summary claims the dehydrogenase-like domain "senses cellular redox or metabolite levels" and enables "dinucleotide-dependent conformational control." This is the central interpretive error. Fang et al. (2013, PMID:23260659) explicitly showed that "attempts to identify the intrinsic enzymatic activity of NPAC as a potential dehydrogenase were unsuccessful." The dehydrogenase domain is catalytically inert and functions as an oligomerization module. There is no evidence that it senses redox state or metabolite levels.
"Promotes histone acetylation" is imprecise: The summary claims NPAC "promotes histone acetylation" as a core function. While Fei et al. (2018, PMID:29759984) showed that NDF stimulates H3K56 acetylation by p300 on nucleosomal substrates, this is a consequence of nucleosome destabilization (making the H3K56 site accessible) rather than an independent acetylation-promoting function. The BioReason thinking trace goes further and claims "positive regulation of histone acetylation (GO:0035066)" as a primary GO prediction, which overstates the evidence. The primary function is nucleosome destabilization, not histone acetylation regulation.
Core function as nucleosome-destabilizing factor omitted: The most important finding from Fei et al. (2018, PMID:29759984) is that NPAC/GLYR1 is NDF -- a nucleosome-destabilizing factor that facilitates Pol II transcription through nucleosomes in an ATP-independent manner. The BioReason summary captures "nucleosome binding" but completely misses the destabilization mechanism, instead attributing transcription facilitation to a speculative "metabolite-sensing" mechanism.
LSD2/KDM1B cofactor role omitted: Fang et al. (2013, PMID:23260659) showed NPAC is a specific cofactor of the histone demethylase LSD2/KDM1B, stimulating H3K4me1/me2 demethylation. This is a well-characterized function with crystal structures available, but is not mentioned in the functional summary.
H3K36me3 reader specificity understated: The summary refers to a generic "reader module" but does not specify that the PWWP domain specifically reads H3K36me3 marks, which is the chromatin mark that recruits the protein to actively transcribed gene bodies (PMID:33676077).
MSL complex interaction absent: The Drosophila ortholog interacts with the MSL histone acetyltransferase complex (mof, msl-1, msl-2, msl-3), which is relevant for dosage compensation. This is absent from the summary.
p-TEFb interaction absent: Yu et al. (2021, PMID:33676077) showed NPAC interacts with the positive transcription elongation factor b (p-TEFb) and phosphorylated RNA Pol II to regulate transcription elongation. This mechanistic detail is absent.
What is correct:
- The domain architecture description (PWWP + dehydrogenase-like Rossmann fold) is accurate
- Nucleosome binding and DNA binding as molecular functions
- Nuclear/chromatin localization
- Association with RNA Pol II transcription
Comparison with interpro2go:
The two InterPro2GO annotations (GO_REF:0000002) are:
- GO:0050661 (NADP binding) from IPR006115
- GO:0051287 (NAD binding) from IPR029154
BioReason's summary essentially elaborates on these same InterPro domain hits, constructing a narrative around NAD(P) binding as a "metabolite/redox sensing" function. This is a direct extrapolation from the InterPro2GO signals but takes them in the wrong direction. The interpro2go annotations are structurally defensible (the Rossmann fold does have NAD(P)-binding capacity), but BioReason inflates their functional significance by building a speculative "redox sensor" model. In contrast, the actual biology shows the dehydrogenase domain is catalytically inert and serves for oligomerization.
The IBA annotations (nucleosome binding, DNA binding, chromatin, transcription elongation-coupled chromatin remodeling) provide a much better picture of the protein's actual function than either the InterPro2GO annotations or BioReason's extrapolation from them. BioReason does not add insight beyond what InterPro2GO provides and in fact leads to an incorrect functional narrative.
The thinking trace correctly identifies all InterPro domains and describes the PWWP-dehydrogenase architecture. However, it makes an unsupported inferential leap from "NAD(P)-binding Rossmann fold" to "metabolite-sensing redox module." The trace states the dehydrogenase core "can act as a regulatory sensor: binding of NAD(H)/NADP(H) within the Rossmann pocket alters the conformation and interaction surface of the C-terminal lobe, thereby modulating recruitment of chromatin-modifying enzymes." There is no experimental evidence for this mechanism. The actual literature shows the dehydrogenase domain is catalytically inert and functions as an oligomerization scaffold.
The trace also lists interaction partners including "3-hydroxyisobutyrate dehydrogenase, 3-hydroxyisobutyryl-CoA hydrolase, probable methylmalonate-semialdehyde dehydrogenase" -- these appear to be STRING database neighbors based on domain homology rather than experimentally validated interactions for this protein. The actual known interaction partners are LSD2/KDM1B, the MSL complex, p-TEFb, and RNA Pol II.
The BioReason trace does not cite any PMIDs or literature references, making it impossible to trace the provenance of its claims. All functional inferences appear to be derived from InterPro domain annotations and the UniProt summary, with speculative extrapolation.
id: Q7Q161
gene_symbol: NPAC
product_type: PROTEIN
status: COMPLETE
taxon:
id: NCBITaxon:7165
label: Anopheles gambiae
description: >-
NPAC (Cytokine-like nuclear factor N-PAC, also known as GLYR1/NDF/NP60) is a
chromatin-associated nuclear protein in Anopheles gambiae that functions as a
nucleosome-destabilizing factor facilitating RNA polymerase II transcription
elongation through chromatin. The protein contains an N-terminal PWWP domain that
reads histone H3K36me3 marks and binds DNA, and a C-terminal dehydrogenase-like
Rossmann fold domain that is catalytically inert but mediates oligomerization. In
orthologous species (Drosophila, human, mouse), NPAC is recruited to gene bodies
of actively transcribed genes, destabilizes nucleosomes in an ATP-independent
manner to facilitate Pol II passage, stimulates H3K56 acetylation by p300, and
serves as a cofactor of the histone demethylase LSD2/KDM1B promoting H3K4me1/me2
demethylation. In Drosophila, it also interacts with the MSL histone acetyltransferase
complex involved in dosage compensation. All functional annotations for the Anopheles
protein are inferred by homology; no direct experimental studies exist for this
organism.
existing_annotations:
- term:
id: GO:0003677
label: DNA binding
qualifier: enables
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: >-
This IBA annotation is based on phylogenetic inference from the Drosophila
ortholog CG4747 (Q8T079) and human GLYR1 (Q49A26). The PWWP domain of NPAC
is known to strongly bind DNA in addition to reading histone marks.
action: ACCEPT
reason: >-
DNA binding via the PWWP domain is well-established for this protein family.
UniProt CC states "The PWWP domain is a H3 reader and strongly binds DNA"
(by similarity to Q49A26). The annotation is correct but somewhat generic;
nucleosome binding (GO:0031491) is a more informative descriptor of the
primary chromatin interaction.
supported_by:
- reference_id: PMID:29759984
supporting_text: "NDF has a PWWP motif, interacts with nucleosomes near the dyad"
- reference_id: file:ANOGA/NPAC/NPAC-notes.md
supporting_text: "The PWWP domain is a H3 reader and strongly binds DNA"
- term:
id: GO:0003682
label: chromatin binding
qualifier: enables
evidence_type: IEA
original_reference_id: GO_REF:0000117
review:
summary: >-
This IEA annotation is from ARBA rule ARBA00027831. Chromatin binding is
appropriate for this protein which localizes to and interacts with chromatin
through the PWWP domain, but is less specific than the nucleosome binding
annotation already present.
action: ACCEPT
reason: >-
Chromatin binding is correct. NPAC/NDF localizes to chromatin in a
H3K36me3-dependent manner and directly interacts with nucleosomes.
This is a broad but accurate annotation.
supported_by:
- reference_id: PMID:29759984
supporting_text: "NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner"
- reference_id: PMID:33676077
supporting_text: "Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes"
- reference_id: file:ANOGA/NPAC/NPAC-deep-research-bioreason-sft.md
supporting_text: "a chromatin-reading module that recognizes nucleosomal features"
- term:
id: GO:0031491
label: nucleosome binding
qualifier: enables
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: >-
This IBA annotation is based on phylogenetic inference from Drosophila CG4747
and human GLYR1. Nucleosome binding is a core molecular function of this
protein established in multiple studies.
action: ACCEPT
reason: >-
Nucleosome binding is one of the best-characterized functions of the NP60
subfamily. UniProt CC states "Binds to mononucleosomes" (by similarity to
Q8T079). NDF interacts with nucleosomes near the dyad and destabilizes them
to facilitate transcription.
supported_by:
- reference_id: PMID:29759984
supporting_text: "NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner"
- reference_id: PMID:23260659
supporting_text: "NPAC directly interacts with LSD2 and positively regulates its H3K4 demethylation activity both in vitro and in vivo"
- term:
id: GO:0050661
label: NADP binding
qualifier: enables
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This InterPro2GO annotation derives from IPR006115 (6-phosphogluconate
dehydrogenase, NADP-binding domain). The Rossmann fold in NPAC does contain
conserved NAD(P)H-binding sites structurally, but the protein lacks a conserved
active site and the dehydrogenase domain is catalytically inert.
action: KEEP_AS_NON_CORE
reason: >-
The structural capacity to bind NADP is plausible given the Rossmann fold
architecture, but the dehydrogenase domain is catalytically inert. Fang et al.
(2013) reported "attempts to identify the intrinsic enzymatic activity of NPAC
as a potential dehydrogenase were unsuccessful." The binding may be structural
or regulatory rather than catalytic, and the functional significance of NADP
binding is unclear. This is not a core function of the protein.
supported_by:
- reference_id: PMID:23260659
supporting_text: "attempts to identify the intrinsic enzymatic activity of NPAC as a potential dehydrogenase"
- reference_id: file:ANOGA/NPAC/NPAC-deep-research-bioreason-sft.md
supporting_text: "the active site is not conserved, the dehydrogenase domain seems to serve as a catalytically inert oligomerization module"
- term:
id: GO:0051287
label: NAD binding
qualifier: enables
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This InterPro2GO annotation derives from IPR029154 (3-hydroxyisobutyrate
dehydrogenase-like, NAD-binding domain). Same reasoning as for NADP binding
-- the Rossmann fold is structurally present but catalytically inert.
action: KEEP_AS_NON_CORE
reason: >-
The structural NAD binding capacity is plausible given the conserved Rossmann
fold, but the domain serves as "a catalytically inert oligomerization module"
rather than performing redox chemistry. NAD binding is not a core function
of this protein.
supported_by:
- reference_id: PMID:23260659
supporting_text: "attempts to identify the intrinsic enzymatic activity of NPAC as a potential dehydrogenase"
- term:
id: GO:0140673
label: transcription elongation-coupled chromatin remodeling
qualifier: involved_in
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: >-
This IBA annotation is based on phylogenetic inference from Drosophila CG4747
and human GLYR1. This is one of the most precisely characterized functions
of the protein.
action: ACCEPT
reason: >-
Transcription elongation-coupled chromatin remodeling is the central biological
process of NPAC/NDF. The protein destabilizes nucleosomes to facilitate Pol II
transcription through chromatin, interacts with p-TEFb and phosphorylated Pol II,
and is recruited to gene bodies upon transcriptional induction. This is a core
function.
supported_by:
- reference_id: PMID:29759984
supporting_text: "NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes"
- reference_id: PMID:33676077
supporting_text: "Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA Pol II Ser2P and Ser5P"
- term:
id: GO:0000785
label: chromatin
qualifier: is_active_in
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: >-
This IBA annotation places NPAC as active in chromatin, based on the Drosophila
and human orthologs which localize to chromatin.
action: ACCEPT
reason: >-
NPAC localizes to chromatin in a H3K36me3-dependent manner. In Drosophila,
it localizes to polytene chromosome interbands (open chromatin). The protein
is active at chromatin where it destabilizes nucleosomes and facilitates
transcription.
supported_by:
- reference_id: PMID:33676077
supporting_text: "Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes"
- reference_id: PMID:29759984
supporting_text: "Upon transcriptional induction, NDF is recruited to the transcribed regions of thousands of genes and colocalizes with a subset of H3K36me3-enriched regions"
- term:
id: GO:0005694
label: chromosome
qualifier: located_in
evidence_type: IEA
original_reference_id: GO_REF:0000044
review:
summary: >-
This IEA annotation is derived from UniProt subcellular location "Chromosome"
(by similarity to Q8T079). NPAC localizes to chromosomes.
action: ACCEPT
reason: >-
Chromosome localization is correct and consistent with the protein's chromatin
association. This is a broad but accurate annotation; the more specific
"chromatin" (GO:0000785) annotation is also present.
supported_by:
- reference_id: file:ANOGA/NPAC/NPAC-notes.md
supporting_text: "Localization to open chromatin depends on H3K36 trimethylation by Set2"
# New annotations suggested based on ortholog evidence
- term:
id: GO:0140003
label: histone H3K36me3 reader activity
qualifier: enables
evidence_type: ISS
original_reference_id: PMID:29759984
review:
summary: >-
NPAC reads H3K36me3 marks via its PWWP domain, which is the primary mechanism
for its recruitment to actively transcribed gene bodies. This is a more
specific and informative annotation than general chromatin binding.
action: NEW
reason: >-
H3K36me3 reader activity via the PWWP domain is a core molecular function
that drives the chromatin localization and biological activity of the protein.
This is well-established in both Drosophila and mammalian orthologs and should
be annotated for the Anopheles ortholog by sequence similarity.
supported_by:
- reference_id: PMID:29759984
supporting_text: "NDF has a PWWP motif...colocalizes with a subset of H3K36me3-enriched regions"
- reference_id: PMID:33676077
supporting_text: "Npac, a \"reader\" of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC"
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings: []
- id: GO_REF:0000033
title: Annotation inferred from sequence similarity using PAINT
findings: []
- id: GO_REF:0000044
title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
findings: []
- id: GO_REF:0000117
title: Gene Ontology annotation based on ARBA rules
findings: []
- id: PMID:12364791
title: The genome sequence of the malaria mosquito Anopheles gambiae.
findings:
- statement: Source of the AGAP009949 gene model
supporting_text: "Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs"
- id: PMID:29759984
title: NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes.
findings:
- statement: NDF/GLYR1 destabilizes nucleosomes and facilitates Pol II transcription
supporting_text: "NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner, and facilitates transcription by Pol II through nucleosomes"
- statement: NDF stimulates H3K56 acetylation by p300 on nucleosomal substrates
supporting_text: "purified recombinant dNDF and hNDF can mediate the stimulation of nucleosomal H3K56 acetylation"
- statement: NDF is recruited to gene bodies upon transcriptional induction
supporting_text: "Upon transcriptional induction, NDF is recruited to the transcribed regions of thousands of genes and colocalizes with a subset of H3K36me3-enriched regions"
- id: PMID:23260659
title: LSD2/KDM1B and its cofactor NPAC/GLYR1 endow a structural and molecular model for regulation of H3K4 demethylation.
findings:
- statement: NPAC is a cofactor of LSD2 that stimulates H3K4 demethylation
supporting_text: "NPAC directly interacts with LSD2 and positively regulates its H3K4 demethylation activity both in vitro and in vivo"
- statement: The dehydrogenase domain is catalytically inert
supporting_text: "attempts to identify the intrinsic enzymatic activity of NPAC as a potential dehydrogenase"
- id: PMID:33676077
title: Npac Is A Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse Embryonic Stem Cells.
findings:
- statement: Npac co-localizes with H3K36me3 in actively transcribed gene bodies
supporting_text: "Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes"
- statement: Npac interacts with p-TEFb and phosphorylated Pol II for elongation
supporting_text: "Npac interacts with positive transcription elongation factor b (p-TEFb), Ser2-phosphorylated RNA Pol II"
core_functions:
- molecular_function:
id: GO:0031491
label: nucleosome binding
description: >-
NPAC functions as a nucleosome-destabilizing factor (NDF) that binds
nucleosomes near the dyad via its PWWP domain and destabilizes them in an
ATP-independent manner, facilitating RNA polymerase II passage through
chromatin during transcription elongation. It reads H3K36me3 marks for
recruitment to actively transcribed gene bodies.
directly_involved_in:
- id: GO:0140673
label: transcription elongation-coupled chromatin remodeling
locations:
- id: GO:0000785
label: chromatin
supported_by:
- reference_id: PMID:29759984
supporting_text: "NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner, and facilitates transcription by Pol II through nucleosomes"
- reference_id: PMID:33676077
supporting_text: "Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA Pol II Ser2P and Ser5P"
proposed_new_terms: []
suggested_questions:
- question: >-
Does the Anopheles NPAC ortholog interact with the MSL dosage compensation
complex in vivo, as predicted by homology to the Drosophila ortholog?
- question: >-
Does the Anopheles NPAC dehydrogenase domain retain any capacity for NAD(P)
binding, and if so, does this have a regulatory role?
- question: >-
Is NPAC involved in any mosquito-specific chromatin regulation processes
related to malaria vector biology (e.g., during blood feeding or parasite
infection)?
suggested_experiments:
- description: >-
ChIP-seq of NPAC in Anopheles gambiae cell lines to determine whether it
co-localizes with H3K36me3 marks as in orthologs, confirming the
conserved chromatin targeting mechanism.
hypothesis: >-
NPAC localizes to H3K36me3-enriched gene bodies of actively transcribed
genes in Anopheles, consistent with its ortholog function.
- description: >-
Co-immunoprecipitation of NPAC with components of the Anopheles MSL
complex (MOF) to validate the predicted interaction.
hypothesis: >-
NPAC interacts with the MSL complex in Anopheles as it does in Drosophila.