Hairy2a (Hes4-A) is a basic helix-loop-helix-Orange (bHLH-O) transcriptional repressor of the Hairy/Enhancer of split family, encoded by the S homeolog (hes4.S) of the allotetraploid frog; its L homeolog is hes4-b/Hairy2b. It binds N-box DNA sites through a proline-containing basic region, dimerises with other bHLH proteins (Hey1, Hes6) and recruits Groucho/TLE corepressors through its C-terminal WRPW motif, acting in the nucleus. hes4-a is a Notch target: Delta1/Notch induces it in the dorsal organiser, where it promotes floor plate over notochord fate, and at the neural plate border, where it also responds to BMP, FGF and Wnt. At the border Hairy2 represses Bmp4, upregulates border genes and keeps border progenitors surviving, cycling and undifferentiated (partly through a DNA-binding-independent Stat3-Delta1 route), working with its antagonist Id3 and with Stat3. It therefore acts as a border/progenitor maintenance factor upstream of the neural crest specifiers rather than as a crest fate specifier. It is also required in the prechordal plate and in pre-placodal ectoderm for lens development (by limiting p27Xic1), and is expressed in stripes in presomitic mesoderm. The two homeologs have partly subfunctionalised: hes4-a is maternally supplied with lower zygotic expression, shares floor plate and prechordal plate roles with hes4-b, and has a disputed contribution to the neural crest, where hes4-b is more strongly expressed. Hes4 is absent from rodents.
Definition: The process by which neural plate border and premigratory neural crest progenitor cells are kept in an undifferentiated, proliferative and multipotent state until neural crest specification.
Justification: GO:0014029 neural crest formation, which these factors currently carry, is proposed for obsoletion (projects/NEURAL_CREST_FORMATION_OBSOLETION.md). Competence factors (Myc, Id3, Hairy2, Pou5f3/Oct25) are required for crest to form, but loss causes progenitor arrest or death rather than a change of fate, and sustained activity blocks differentiation, so neither crest cell differentiation nor fate specification describes their role. GO:0019827 stem cell population maintenance is the parent; the new term says which progenitor population is maintained.
Parent term: stem cell population maintenance
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000122 negative regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference (PAINT node PTN004213585) that HES/HEY bHLH-Orange proteins repress RNA polymerase II transcription. Q90Z12 and its homeolog Q90VV1 are among the descendant evidences. Reason: Transcriptional repression is the defining activity of the Hairy/E(spl) family and is supported for Xenopus Hairy2 directly: Hairy2A represses Bmp4 transcription at the neural plate border, and bHLH-Engrailed repressor fusions mimic wild-type Hairy2 activity. The target appearing in its own WITH/FROM reflects its own experimental grounding. Supporting Evidence: PMID:14681193 Hairy2A acts as a repressor of Bmp4 transcription PMID:25997789 hairy genes function as transcriptional repressors file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs |
| GO:0000122 negative regulation of transcription by RNA polymerase II | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning inference of negative regulation of Pol II transcription. Reason: Correct and consistent with the IBA and experimental rows for this repressor. Supporting Evidence: PMID:14681193 Hairy2A acts as a repressor of Bmp4 transcription file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs |
| GO:0000122 negative regulation of transcription by RNA polymerase II | IMP PMID:10976052 Hes6 acts in a positive feedback loop with the neurogenins t... | ACCEPT | Summary: IMP from Koyano-Nakagawa et al. 2000 (Hes6 paper), which UniProt cites for Hairy2 function, DNA binding and Hes6 interaction. Reason: The cached abstract is about Hes6 and only describes the WRPW-bHLH proteins as a class of transcriptional repressors; the full text (not cached) evidently assayed Hairy2 repression, as UniProt cites this paper for Hairy2 FUNCTION and DNA-BINDING. The term is correct for the gene, so the curator is deferred to. Supporting Evidence: PMID:10976052 a class of transcriptional repressors called the WRPW-bHLH proteins file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs |
| GO:0000122 negative regulation of transcription by RNA polymerase II | IMP PMID:14681193 Interplay between Notch signaling and the homeoprotein Xiro1... | ACCEPT | Summary: IMP from Glavic et al. 2004: Hairy2A represses Bmp4 transcription at the neural plate border downstream of Xiro1 and Delta1/Notch. Reason: Direct statement of Hairy2A acting as a transcriptional repressor of Bmp4. Pol II specificity is appropriate for a protein-coding target. Supporting Evidence: PMID:14681193 Hairy2A acts as a repressor of Bmp4 transcription |
| GO:0000122 negative regulation of transcription by RNA polymerase II | IMP PMID:17724611 Two alloalleles of Xenopus laevis hairy2 gene--evolution of ... | ACCEPT | Summary: IMP from Murato et al. 2007 (homeolog-specific knockdowns of Xhairy2a and Xhairy2b). Reason: The abstract does not describe a repression readout, but the full text is not cached and the gene-level claim is well supported (Glavic 2004, Vega-Lopez 2015, family). Accepted, deferring to the curator. Supporting Evidence: PMID:17724611 Xhairy2a seems not to function in the neural crest, although both of them are required in the floor plate and the prechordal plate PMID:25997789 hairy genes function as transcriptional repressors |
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: sequence-specific binding to Pol II cis-regulatory regions (N-box/class C E-box) inherited from the HES/HEY node. Reason: Hairy-family basic regions carry the helix-interrupting proline that confers N-box (CACNAG) binding; Xenopus Hairy2 DNA binding is supported experimentally (IDA, PMID:10976052) and its DNA-binding-dependent functions at the border are lost in a DNA-binding mutant. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs PMID:18710660 a Hairy2 DNA-binding mutant is unable to promote cell survival and to upregulate the expression of early neural border genes |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: DNA-binding transcription factor activity, RNA polymerase II-specific. Reason: Correct, but more general than the evidence supports: Hairy2 acts as a DNA-binding repressor (WRPW/Groucho, EnR fusions phenocopy). The more precise GO:0001227 is added as NEW rather than replacing this sound IBA. Supporting Evidence: PMID:25997789 hairy genes function as transcriptional repressors file:XENLA/hes4-a/hes4-a-uniprot.txt The C-terminal WRPW motif is a transcriptional repression |
| GO:0002088 lens development in camera-type eye | IMP PMID:19253409 Xhairy2 functions in Xenopus lens development by regulating ... | KEEP AS NON CORE | Summary: IMP from Murato and Hashimoto 2009: Xhairy2 knockdown reduces lens marker expression from the lens-field stage; partially rescued by p27xic1 knockdown. Reason: A real requirement in pre-placodal ectoderm, mechanistically parallel to the border role (holding cells in a cycling, competent state by limiting p27xic1). It is a separate deployment from the core repressor/border-maintenance function. The paper uses unsuffixed Xhairy2, so the a/b homeolog contribution is unresolved; GOA carries the same row on hes4-b. Supporting Evidence: PMID:19253409 Xhairy2 knockdown reduced the expression of lens marker genes at every step of lens determination PMID:19253409 loss of lens field caused by Xhairy2 depletion was partially rescued by simultaneous knockdown of the cell cycle inhibitor gene p27(xic1) |
| GO:0003677 DNA binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based DNA binding. Reason: Correct though general; GO:0000978 and GO:0043565 capture the specificity. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: IBA nucleus. Reason: Nuclear DNA-binding repressor; the expected site of action across the family. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping to nucleus. Reason: Consistent with function; no contrary evidence. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs |
| GO:0005634 nucleus | ISS GO_REF:0000024 | ACCEPT | Summary: ISS nucleus from Drosophila hairy (P14003). Reason: Sound transfer within the Hairy family. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based regulation of DNA-templated transcription. Reason: Correct but general; the direction (repression) is captured by GO:0000122. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs |
| GO:0009952 anterior/posterior pattern specification | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA from node PTN000105428 (Hes/Her segmentation-clock genes such as Hes7, her1, her7) for anterior/posterior pattern specification. Reason: Hairy2 is expressed in an anterior stripe of the presomitic mesoderm before each somite forms, consistent with inheritance of a segmentation-related role, and there is no evidence of loss in hes4. But no Xenopus functional data test it, and it is peripheral to the core border/floor-plate functions. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Plays a role in the patterning of tissue boundaries. |
| GO:0014029 neural crest formation | IMP PMID:14681193 Interplay between Notch signaling and the homeoprotein Xiro1... | MODIFY | Summary: IMP from Glavic et al. 2004: Hairy2A downstream of Xiro1 and Delta1/Notch represses Bmp4 so that border BMP levels permit neural crest specification; Hairy2A rescues loss of crest markers when Xiro1 or Notch is blocked. Reason: Hairy2 sets BMP levels for the neural plate border and maintains border progenitors. It is not a crest fate specifier (early overexpression represses crest markers and induces border genes), so GO:0014036 does not apply. This paper names Hairy2A explicitly and the row sits on hes4-a only. GO:0014029 is proposed for obsoletion (projects/NEURAL_CREST_FORMATION_OBSOLETION.md): its definition describes forming the neural plate border region while its placement is under epithelial to mesenchymal transition. This gene's role is keeping crest progenitors undifferentiated and competent, captured by the proposed term 'neural crest progenitor maintenance' (NTR). Proposed replacements: neural crest progenitor maintenance Supporting Evidence: PMID:14681193 ensuring that levels of Bmp4 optimal for the specification of the neural plate border are attained in this region PMID:14681193 Hairy2A acts as a repressor of Bmp4 transcription |
| GO:0014029 neural crest formation | IMP NOT PMID:17724611 Two alloalleles of Xenopus laevis hairy2 gene--evolution of ... | UNDECIDED | Summary: NOT annotation from Murato et al. 2007: homeolog-specific knockdown showed no neural crest phenotype for Xhairy2a (in contrast to Xhairy2b), and lower Xhairy2a expression in the crest. Reason: GOA now asserts both involved_in and NOT involved_in GO:0014029 for Q90Z12. The NOT rests on a negative morpholino result, which shows hes4-a is not required when hes4-b is present, not that it cannot act in crest formation. Vega-Lopez 2015, resolving hairy2a and hairy2b, reports hairy2a border/crest expression and functional equivalence of the hairy genes in crest induction. Both papers are abstract-only in the cache, so the conflict cannot be settled here; curator resolution is needed (suggested_questions). Supporting Evidence: PMID:17724611 Xhairy2a seems not to function in the neural crest, although both of them are required in the floor plate and the prechordal plate PMID:17724611 Xhairy2b expression in the neural crest is much higher than Xhairy2a expression file:XENLA/hes4-a/hes4-a-uniprot.txt that hes4-B/hairy2b but not hes4-A/hairy2a has a role in neural crest PMID:25997789 hairy genes are required for the induction and migration of the neural crest PMID:25997789 hairy genes have a functional equivalence between them |
| GO:0014029 neural crest formation | IMP PMID:18710660 Hairy2 functions through both DNA-binding and non DNA-bindin... | MODIFY | Summary: IMP from Nichane et al. 2008 (Dev Biol 322:368): Hairy2 acts at the neural plate border through DNA-binding (survival, early border genes) and non-DNA-binding Stat3-Delta1 (proliferation, NC expansion) mechanisms. Reason: Supports a border-maintenance role: Hairy2 keeps border cells surviving and in a mitotic, undifferentiated pre-crest state. The paper uses unsuffixed Hairy2, and GOA attaches the same row to hes4-b; reagent homeolog specificity cannot be checked from the abstract. GO:0014029 is proposed for obsoletion (projects/NEURAL_CREST_FORMATION_OBSOLETION.md): its definition describes forming the neural plate border region while its placement is under epithelial to mesenchymal transition. This gene's role is keeping crest progenitors undifferentiated and competent, captured by the proposed term 'neural crest progenitor maintenance' (NTR). Proposed replacements: neural crest progenitor maintenance Supporting Evidence: PMID:18710660 is essential for neural crest progenitor survival and maintains cells in a mitotic undifferentiated pre-neural crest state PMID:18710660 a Hairy2 DNA-binding mutant is unable to promote cell survival and to upregulate the expression of early neural border genes |
| GO:0014029 neural crest formation | IMP PMID:18721802 Hairy2-Id3 interactions play an essential role in Xenopus ne... | MODIFY | Summary: IMP from Nichane et al. 2008 (Dev Biol 322:355): Hairy2 acts downstream of BMP and FGF at the border, upregulates border genes, represses Id3 via BMP attenuation, and with Id3 maintains undifferentiated NC progenitors. Reason: This is the clearest placement evidence: Hairy2 acts on the border region (border genes, BMP level) and holds progenitors undifferentiated, rather than specifying crest fate. This matches the id3-a review. Unsuffixed Hairy2; same row on hes4-b. GO:0014029 is proposed for obsoletion (projects/NEURAL_CREST_FORMATION_OBSOLETION.md): its definition describes forming the neural plate border region while its placement is under epithelial to mesenchymal transition. This gene's role is keeping crest progenitors undifferentiated and competent, captured by the proposed term 'neural crest progenitor maintenance' (NTR). Proposed replacements: neural crest progenitor maintenance Supporting Evidence: PMID:18721802 Hairy2 acts downstream of FGF and BMP signals at the neural border to maintain cells in an undifferentiated state PMID:18721802 Hairy2 overexpression represses neural crest and upregulates neural border genes at early stages while it expands a subset of them in later embryos |
| GO:0021501 prechordal plate formation | IMP PMID:17724611 Two alloalleles of Xenopus laevis hairy2 gene--evolution of ... | ACCEPT | Summary: IMP from Murato et al. 2007: both hairy2a and hairy2b are required in the prechordal plate. Reason: Homeolog-resolved knockdown supports a hes4-a requirement; hairy2a is expressed in the anterior prechordal plate. One of the better-resolved hes4-a-specific functions. Supporting Evidence: PMID:17724611 Xhairy2a seems not to function in the neural crest, although both of them are required in the floor plate and the prechordal plate |
| GO:0030509 BMP signaling pathway | IMP PMID:14681193 Interplay between Notch signaling and the homeoprotein Xiro1... | MODIFY | Summary: IMP from Glavic et al. 2004 annotated to BMP signaling pathway. Reason: Hairy2A does not transduce BMP signals; it represses Bmp4 transcription and so lowers BMP signalling at the border (and attenuates BMP to downregulate Id3). The process it carries out is negative regulation of the pathway. Proposed replacements: negative regulation of BMP signaling pathway Supporting Evidence: PMID:14681193 Hairy2A acts as a repressor of Bmp4 transcription PMID:18721802 Hairy2 downregulates Id3, another essential HLH neural crest regulator, through attenuation of BMP signaling |
| GO:0033504 floor plate development | IMP PMID:15689375 The Notch-target gene hairy2a impedes the involution of noto... | ACCEPT | Summary: IMP from Lopez et al. 2005: Delta1-Notch activation of hairy2a in the organiser promotes floor plate (notoplate) fate at the expense of notochord. Reason: Explicitly hairy2a; a well-supported developmental role of this homeolog. Supporting Evidence: PMID:15689375 Notch-mediated activation of hairy2a in the surrounding cells within the organiser, impeding their involution through the blastopore and promoting their incorporation into the hairy2a+ notoplate precursors (future floor-plate cells) |
| GO:0033504 floor plate development | IMP PMID:17724611 Two alloalleles of Xenopus laevis hairy2 gene--evolution of ... | ACCEPT | Summary: IMP from Murato et al. 2007: both alloalleles required in the floor plate. Reason: Homeolog-resolved knockdown; consistent with Lopez 2005. Supporting Evidence: PMID:17724611 Xhairy2a seems not to function in the neural crest, although both of them are required in the floor plate and the prechordal plate |
| GO:0043066 negative regulation of apoptotic process | IMP PMID:18710660 Hairy2 functions through both DNA-binding and non DNA-bindin... | KEEP AS NON CORE | Summary: IMP from Nichane et al. 2008: Hairy2 promotes survival of border/NC progenitors through a DNA-binding-dependent mechanism. Reason: Survival is one readout of the progenitor-maintenance role rather than a separate function; kept as non-core. Unsuffixed Hairy2. Supporting Evidence: PMID:18710660 a Hairy2 DNA-binding mutant is unable to promote cell survival and to upregulate the expression of early neural border genes |
| GO:0043425 bHLH transcription factor binding | IPI PMID:10976052 Hes6 acts in a positive feedback loop with the neurogenins t... | KEEP AS NON CORE | Summary: IPI from Koyano-Nakagawa et al. 2000 with A1L3K9 (an unnamed Xenopus bHLH protein; UniProt records the interaction as with Hes6). Reason: bHLH-bHLH heterodimerisation is a genuine property of Hairy proteins and a specific MF term, so preferable to protein binding. The Hes6 interaction is described as inhibiting Hairy2 repressor activity, i.e. a regulatory input on Hairy2 rather than its core activity. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Interacts with the bHLH protein hes6; this interaction may inhibit the transcriptional |
| GO:0043565 sequence-specific DNA binding | IDA PMID:10976052 Hes6 acts in a positive feedback loop with the neurogenins t... | ACCEPT | Summary: IDA DNA binding from Koyano-Nakagawa et al. 2000. Reason: UniProt cites this paper for Hairy2 DNA binding; full text not cached. Correct and consistent with the N-box binding bHLH domain. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs |
| GO:0045892 negative regulation of DNA-templated transcription | IMP PMID:10976052 Hes6 acts in a positive feedback loop with the neurogenins t... | MODIFY | Summary: IMP negative regulation of DNA-templated transcription (PMID:10976052). Reason: Correct, but Hairy2 represses Pol II-transcribed genes; narrowed to GO:0000122 for consistency with the other repression rows and Tier 1 reviews. Proposed replacements: negative regulation of transcription by RNA polymerase II Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs PMID:14681193 Hairy2A acts as a repressor of Bmp4 transcription |
| GO:0045892 negative regulation of DNA-templated transcription | IMP PMID:17724611 Two alloalleles of Xenopus laevis hairy2 gene--evolution of ... | MODIFY | Summary: IMP negative regulation of DNA-templated transcription (PMID:17724611). Reason: As for the PMID:10976052 row: narrowed to the Pol II-specific term. Proposed replacements: negative regulation of transcription by RNA polymerase II Supporting Evidence: PMID:14681193 Hairy2A acts as a repressor of Bmp4 transcription |
| GO:0046983 protein dimerization activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based protein dimerization activity (bHLH domain). Reason: bHLH proteins act as dimers; UniProt records heterodimers with Hey1 and Hes6. Supporting Evidence: file:XENLA/hes4-a/hes4-a-uniprot.txt Binds DNA in the form of a heterodimer with the bHLH protein hey1/hrt1 |
| GO:0050767 regulation of neurogenesis | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA regulation of neurogenesis from the Hes/Her node. Reason: Hes repressors widely restrain neurogenesis downstream of Notch and the inheritance is plausible, but Xenopus Hairy2 evidence concerns border maintenance, floor plate and lens; hairy2a alone induced sox2 in animal caps (deep research). Peripheral to the core role. Supporting Evidence: file:XENLA/hes4-a/hes4-a-deep-research-falcon.md hairy2a alone induced *sox2* and only weak *snail1* |
| GO:0001227 DNA-binding transcription repressor activity, RNA polymerase II-specific | IMP PMID:25997789 Functional analysis of Hairy genes in Xenopus neural crest i... | NEW | Summary: Hairy2a is a DNA-binding repressor of Pol II transcription: it binds N-box sites via its proline-containing basic region and recruits Groucho/TLE via WRPW; bHLH-Engrailed repressor fusions mimic wild-type Hairy2 and activator fusions oppose it. Reason: The precise MF for this gene. Comparator check (QuickGO, recorded in notes): human HES1, HEY1 and HEY2 carry GO:0001227, so it is the family convention and its absence on hes4-a is a coverage gap. Hes4-a itself binds DNA (IDA) and represses Bmp4. Supporting Evidence: PMID:25997789 hairy genes function as transcriptional repressors PMID:14681193 Hairy2A acts as a repressor of Bmp4 transcription file:XENLA/hes4-a/hes4-a-uniprot.txt Transcriptional repressor. Binds DNA on N-box motifs file:XENLA/hes4-a/hes4-a-uniprot.txt The C-terminal WRPW motif is a transcriptional repression |
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Download this section (compressed HTML)Q: GOA asserts both involved_in and NOT involved_in GO:0014029 for hes4-a (Q90Z12). The NOT rests on a negative homeolog-specific morpholino result (Murato 2007), while Vega-Lopez 2015 reports hairy2a border/crest expression and functional equivalence with hairy2b, and the Nichane 2008 rows (unsuffixed Hairy2) were attached to both homeologs. Should the NOT be withdrawn, or the positive rows restricted to hes4-b?
Q: GO has no neural plate border formation/maintenance term. Hairy2 acts on the border region (BMP level, border genes, progenitor maintenance) rather than specifying crest fate. If a border term is created, should Hairy2/Hes4 sit in it alongside Pax3, Zic1, Msx1 and Tfap2a, or in a separate border progenitor maintenance term shared with Id3 and Stat3?
Q: Should Hes4 carry GO:0007219 Notch signaling pathway as a direct Notch target effector (as for other Hes genes), or GO:0045596 negative regulation of cell differentiation for its role in keeping border progenitors undifferentiated?
Q: Is hairy/Hes expression at the neural plate border ancestral (amphioxus, lamprey), and did the Notch-Hairy-Bmp4 border circuit predate the crest? Hes4 has been lost in rodents; is a mammalian paralog (Hes1) covering its border role?
Experiment: Homeolog-specific CRISPR F0 mutants of hes4.S and hes4.L, singly and together, scored for border genes (pax3, zic1, msx1), crest markers (snai2, foxd3, sox10), floor plate and prechordal plate markers, and p27xic1, with cross-rescue by each homeolog mRNA.
Hypothesis: hes4.S (hes4-a) and hes4.L (hes4-b) act redundantly in neural crest formation, with hes4-b dominant because of its higher zygotic border expression.
Experiment: CUT&RUN for tagged Hes4 in neural plate border explants, combined with reporter assays of Bmp4 and p27xic1 regulatory regions with N-box mutations.
Hypothesis: Hairy2 represses Bmp4 and other border targets directly via N-box sites.
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