Cloning, Characterization, and the Complete 56.8-Kilobase DNA Sequence of the Human NOTCH4 Gene

doi:10.1006/geno.1998.5330

Genomics

Volume 51, Issue 1, 1 July 1998, Pages 45-58

https://doi.org/10.1006/geno.1998.5330 Get rights and content

Abstract

The first complete mammalian genomic sequence reported thus far in the Notch gene family, including a putative promoter region and 30 exons of the human NOTCH4 gene spanning 56.8 kb of DNA, were sequenced. The NOTCH4 locus contains a TATA-less promoter with two putative transcription initiation sites (Inr), three RBP-Jκ sites, and two GATA recognition sites. Two cDNA isoforms, NOTCH4(L) and NOTCH4(S),were identified. Whereas the NOTCH4(S) isoform contains the entire coding sequence, the NOTCH4(L) isoform has two unspliced intronic sequences between exons 11 and 12 and exons 20 and 21 and a misspliced exon 6. Consistent with these results, two alternatively spliced isoforms of transcripts of approximately 9.3 and 6.7 kb were detected by Northern blot analysis. The predicted amino acid sequence of the NOTCH4 protein based on the NOTCH4(S) cDNA sequence contains 2003 amino acids and includes the predominant motifs of the Notch family: 29 epidermal growth factor (EGF)-like repeats, 3 Notch/lin-12 repeats, a transmembrane region, 6 cdc10/Ankyrin repeats, and a PEST domain.

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Notch4 reveals a novel mechanism regulating Notch signal transduction
2014, Biochimica et Biophysica Acta - Molecular Cell Research
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It is interesting to note that Notch4 expression is under the transcriptional control of canonical Notch signalling. The Notch4 promoter contains CSL binding sites [84] and Notch4 expression is upregulated by transfection with Notch ICD constructs [40,76]. Thus NOTCH4 may serve to attenuate NOTCH1 signalling in a negative feedback loop in a similar manner to the Notch target Nrarp [33,85].
Notch4 is a divergent member of the Notch family of receptors that is primarily expressed in the vasculature. Its expression implies an important role for Notch4 in the vasculature; however, mice homozygous for the Notch4^d1 knockout allele are viable. Since little is known about the role of Notch4 in the vasculature and how it functions, we further investigated Notch4 in mice and in cultured cells. We found that the Notch4^d1 allele is not null as it expresses a truncated transcript encoding most of the NOTCH4 extracellular domain. In cultured cells, NOTCH4 did not signal in response to ligand. Moreover, NOTCH4 inhibited signalling from the NOTCH1 receptor. This is the first report of cis-inhibition of signalling by another Notch receptor. The NOTCH4 extracellular domain also inhibits NOTCH1 signalling when expressed in cis, raising the possibility that reported Notch4 phenotypes may not be due to loss of NOTCH4 function. To better address the role of NOTCH4 in vivo, we generated a Notch4 null mouse in which the entire coding region was deleted. Notch4 null mice exhibited slightly delayed vessel growth in the retina, consistent with our novel finding that NOTCH4 protein is expressed in the newly formed vasculature. These findings indicate a role of NOTCH4 in fine-tuning the forming vascular plexus.
Genetic evidence for the involvement of NOTCH4 in rheumatoid arthritis and alopecia areata
2013, Immunology Letters
Citation Excerpt :
To date, four Notch receptors (Notch 1–4) and five of their ligands (Delta-like 1, 3, 4 and Jagged 1, 2) have been identified, each of which is located on different chromosome in the human genome [1]. The NOTCH4 gene lies at the centromeric end of MHC class III region approximately 335 kb telomeric of the DRB1 locus [2], and contains 30 exons spanning 56.8 kb of DNA [3]. Protein encoded by this gene is cleaved in the trans-Golgi network, and presented on the cell surface as a heterodimer.
To explore the genetic association of single nucleotide polymorphisms (SNPs) in the coding region of the NOTCH4, exon 3 C+1297T and exon 5 A+3063G, in a case–control analysis of 58 rheumatoid arthritis (RA) and 98 alopecia areata (AA) and 100 ethnically matched healthy subjects. NOTCH4 polymorphisms were genotyped by standard PCR followed by restriction digestion. Analysis of C+1297T SNP revealed a significant association of allele C+1297 (p = 0.03, OR = 1.66, 95%CI 1.04–2.64) and genotype CT (p = 0.002, OR = 2.82, 95%CI 1.42–5.59) with susceptibility to RA. Analysis of A+3063G SNP revealed a significant association of allele A+3063 (p = 0.05, OR = 0.59, 95%CI 0.35–1.008) and genotype AA (p = 0.002, OR = 0.39, 95%CI 0.17–0.87) with RA. Over all analysis between alopecia patients and the studied SNPs failed to show any significant association. Classifying the patients by severity of disease, confined the risk role of CT genotype to the severest form of alopecia universalis (p = 0.006, OR = 3.82, 95%CI 1.39–3.82) and AG genotype to semiuniversalis alopecia (p = 0.004, OR = 4.3, 95%CI 1.5–15.3). Present study is the first to report a statistically significant association between RA and NOTCH4 polymorphisms.
Investigation of NOTCH4 coding region polymorphisms in sporadic inclusion body myositis
2012, Journal of Neuroimmunology
Citation Excerpt :
Genes involved in regulating cellular differentiation, such as NOTCH4, are thus candidates for playing a role in this aspect of sIBM pathology. Given the expression of NOTCH4 in skeletal muscle (Li et al., 1998) and its role in regulating cellular differentiation, NOTCH4 variants could play a part in the pathogenesis of sIBM by inhibiting myofibre regeneration, or by generating antigenic peptides that are specific to muscle. Genetic variations within NOTCH4 could also influence susceptibility to sIBM through other mechanisms.
The NOTCH4 gene, located within the MHC region, is involved in cellular differentiation and has varying effects dependent on tissue type. Coding region polymorphisms haplotypic of the sIBM-associated 8.1 ancestral haplotype were identified in NOTCH4 and genotyped in two different Caucasian sIBM cohorts. In both cohorts the frequency of the minor allele of rs422951 and the 12-repeat variation for rs72555375 was increased and was higher than the frequency of the sIBM-associated allele HLA-DRB1*0301. These NOTCH4 polymorphisms can be considered to be markers for sIBM susceptibility, but require further investigation to determine whether they are directly involved in the disease pathogenesis.
Inducible nitric oxide synthase up-regulates Notch-1 in mouse cholangiocytes: Implications for carcinogenesis
2005, Gastroenterology
Background & Aims: Inflammatory mediators and cell fate genes, such as the Notch gene family, both have been implicated in cancer biology. Because cholangiocarcinomas arise in a background of inflammation and express the inflammatory mediator inducible nitric oxide synthase (iNOS), we aimed to determine whether iNOS expression alters Notch expression and signaling. Methods: Notch receptor and ligand expression in human liver was evaluated by immunohistochemistry. The effect of iNOS and NO on Notch-1 expression was examined in cell lines. Results: Notch-1, but not other Notch receptors, were up-regulated by cholangiocytes in primary sclerosing cholangitis and cholangiocarcinoma. The colocalization of Notch-1 and iNOS also was observed in large bile ducts from the hilar region of primary sclerosing cholangitis patients. Notch-1 expression in murine cholangiocytes was iNOS dependent. iNOS expression also facilitated Notch signaling by inducing the nuclear translocation of its intracellular domain and the expression of a transcriptional target, hairy and enhancer of split (Hes)-1. The γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl)-S-phenylglycine]-t-butyl ester, which blocks Notch signaling, enhanced tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cholangiocarcinoma cells. Conclusions These data implicate a direct link between the inflammatory mediator iNOS and Notch signaling, and have implications for the development and progression of cholangiocarcinoma.
Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo
2004, Blood
Citation Excerpt :
The role of Notch4 in the hematopoietic system has not been studied. However, Notch4 has GATA recognition sites in its promoter region, transcription factors that play a role in lineage commitment in hematopoiesis,25,26 suggesting that Notch4 could play a role in hematopoiesis.5 Notch4 expression in hematopoiesis has so far been reported only in maturing macrophages with the use of single-cell polymerase chain reaction (PCR).27
Notch transmembrane receptors are known to play a critical role in cell-fate decisions, with Notch1 shown to enhance self-renewal of hematopoietic stem cells and cause T-cell leukemia. Four Notch receptors exist, and the extent of redundancy and overlap in their function is unknown. Notch4 is structurally distinct from Notch1 through Notch3 and has not been extensively studied in hematopoiesis. By polymerase chain reaction (PCR) we find Notch4 transcript expression in human marrow cells and in both CD34⁺ and CD34^– populations. When constitutively active Notch1 or Notch4 was overexpressed in normal human marrow or cord cells, we found reduced colony-forming and short-term proliferative ability while the primitive progenitor content of myeloid long-term cultures was significantly increased. Notch4–intracellular domain (Notch4-IC)–transduced cord cells transplanted into β₂-microglobulin^–/– nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in significantly higher levels of engraftment of both green fluorescent protein–positive (GFP⁺) and GFP^– populations as compared with controls. GFP⁺ cells in bone marrow and spleen of animals that had received transplants gave rise to an immature CD4⁺CD8⁺ T-cell population, whereas B-cell development was blocked. These results indicate that activation of Notch4 results in enhanced stem cell activity, reduced differentiation, and altered lymphoid development, suggesting it may influence both stem cells and the fate of the common lymphoid progenitor.

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^☆: Sequence data described in this paper have been deposited with the EMBL/GenBank Data Libraries under Accession No. U89335 for the genomic sequence and Accession No. U95299 for the cDNA sequence of human NOTCH4.

^☆☆: M. D. AdamsC. FieldsJ. C. Venter

¹: L. Li and G. M. Huang made equal contributions to this work.

²: Present address: Pangea Systems Inc., 1999 Harrison Street, Suite 1100, Oakland, CA 94612.

³: To whom correspondence should be addressed at Department of Molecular Biotechnology, University of Washington, Box 357730, Seattle, WA 98195. Telephone: (206) 616-5104. Fax: (206) 685-7301.

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Regular ArticleCloning, Characterization, and the Complete 56.8-Kilobase DNA Sequence of the Human NOTCH4 Gene☆,☆☆

Abstract

Trends Genet.

J. Mol. Biol.

Cell

Trends Biochem. Sci.

Cell

Immunol. Today

Cell

Cell

Cell

Cell

Cell

Immunity

Neuron

Blood

Cell

Dev. Biol.

Cell

Cell

Cell

Cell

Genomics

Gene

Curr. Biol.

Cell

Cell

Cell

Notch signaling

Science

Trinucleotide repeat expansions and human genetic disease

BioEssays

Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3′end of a transcript encoding a protein kinase family member

Cell

Notch is required for successive cell decisions in the developingDrosophila

Genes Dev.

Lag-1, a gene required for Lin-12 and Glp-1 signaling in C. elegans, is homologous to human CBF1 and Drosophila Su(H)

Development

Xotch, theXenopusDrosophila

Science

Feed-back mechanism affecting Notch activation at the dorsoventral boundary in the Drosophila wing

Development

An erythrocyte-specific DNA binding factor recognizes a regulatory sequence common to all chicken globin genes

Proc. Natl. Acad. Sci. USA

Complex spatial and temporal regulation of Notch expression during embryonic and imaginal development ofDrosophila:

J. Cell. Biol.

An activated Notch receptor blocks cell-fate commitment in the developingDrosophila

Nature

Expression pattern of Motch, a mouse homolog ofDrosophila

Development

Regular Article
Cloning, Characterization, and the Complete 56.8-Kilobase DNA Sequence of the Human NOTCH4 Gene☆,☆☆