Search results for NOTCH1

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Identifier: R-HSA-1980143
Species: Homo sapiens
Compartment: plasma membrane, cytosol, nucleoplasm
NOTCH1 functions as both a transmembrane receptor presented on the cell surface and as a transcriptional regulator in the nucleus.

NOTCH1 receptor presented on the plasma membrane is activated by a membrane bound ligand expressed in trans on the surface of a neighboring cell. In trans, ligand binding triggers proteolytic cleavage of NOTCH1 and results in release of the NOTCH1 intracellular domain, NICD1, into the cytosol.

NICD1 translocates to the nucleus where it associates with RBPJ (also known as CSL or CBF) and mastermind-like (MAML) proteins (MAML1, MAML2 or MAML3; possibly also MAMLD1) to form NOTCH1 coactivator complex. NOTCH1 coactivator complex activates transcription of genes that possess RBPJ binding sites in their promoters.

Identifier: R-HSA-2644603
Species: Homo sapiens
Human NOTCH1 was cloned as a chromosome 9 gene, translocated to the T-cell beta receptor (TCBR) promoter on chromosome 7 in T-cell acute lymphoblastic leukemia (T-ALL) (Ellisen et al. 1991). This translocation, present in only a small percentage of T-ALL patients, results in the overexpression of a truncated NOTCH1 receptor, which lacks almost the entire extracellular domain, in T lymphocytes. Oncogenic NOTCH1 mutations were subsequently found to be present in >50% of T-ALL patients, with hotspots in the heterodimerization domain (HD domain) and PEST domain of NOTCH1 (Weng et al. 2004).

Normal NOTCH1 becomes activated by binding DLL (DLL1 or DLL4) or JAG (JAG1 or JAG2) ligands expressed on the surface of a neighboring cell, which leads to proteolytic cleavage of NOTCH1 by ADAM10/17 and gamma-secretase, and release of the NOTCH1 intracellular domain (NICD1) which regulates expression of genes that play important roles in the development of T lymphocytes (Washburn et al. 1997. Radtke et al. 1999, Maillard et al. 2004, Sambandam et al. 2005, Tan et al. 2005). Mutations in the HD domain, responsible for association of NOTCH1 extracellular and transmembrane regions after furin-mediated cleavage of NOTCH1 precursor, as well as the truncation of the NOTCH1 extracellular domain by the rare T-ALL translocation, enable constitutive production of NICD1, in the absence of ligand binding (Malecki et al. 2006, Ellisen et al. 1991).

Mutations in the NOTCH1 PEST domain interfere with FBXW7 (FBW7)-mediated ubiquitination and degradation of NICD1, resulting in prolonged half-life and increased transcriptional activity of NICD1, which promotes growth and division of T-lymphocytes (Weng et al. 2004, Thompson et al. 2007, O'Neil et al. 2007).

Mutations in the HD domain and PEST domain of NOTCH1 are frequently found in cis in T-ALL. While HD mutations alone result in up to ~10-fold increase in NOTCH1 transcriptional activity and PEST domain mutations alone result in up to ~2-fold increase in NOTCH1 transcriptional activity, in cis mutations of HD and PEST domains act synergistically, increasing NOTCH1 transcriptional activity up to ~40-fold (Weng et al. 2004).

FBXW7 (FBW7), a component of the SCF (SKP1, CUL1, and F-box protein) ubiquitin ligase complex SCF-FBW7 involved in the degradation of NOTCH1 (Oberg et al. 2001, Wu et al. 2001, Fryer et al. 2004), is subject to loss of function mutations in T-ALL (Akhoondi et al. 2007, Thompson et al. 2007, O'Neil et al. 2007) which are mutually exclusive with NOTCH1 PEST domain mutations (Thompson et al. 2007, O'Neil et al. 2007).

Although gamma-secretase inhibitors (GSIs) are successfully used in vitro to inhibit NOTCH1 signaling in T-ALL cell lines, the gamma-secretase complex has many other substrates besides NOTCH. The specificity of GSIs is therefore limited and, as they are not considered to be particularly promising drugs for the clinical treatment of T-ALL (reviewed by Purow, 2012), they have not been annotated.

For a recent review of NOTCH1 signaling in cancer, please refer to Grabher et al. 2006.
Identifier: R-HSA-2644605
Species: Homo sapiens
FBXW7 (FBW7) is a component of the SCF (SKP1, CUL1, and F-box protein) ubiquitin ligase complex SCF-FBW7 which is involved in the degradation of NOTCH1 (Oberg et al. 2001, Wu et al. 2001, Fryer et al. 2004). Loss of function mutations in FBXW7 are frequently found in T-cell acute lymphoblastic leukemia (Akhoondi et al. 2007, Thompson et al. 2007, O'Neil et al. 2007) and are mutually exclusive with NOTCH1 PEST domain mutations (Thompson et al. 2007, O'Neil et al. 2007).
Identifier: R-HSA-2660825
Species: Homo sapiens
Human NOTCH1 was cloned as a chromosome 9 gene, translocated to the T-cell beta receptor (TCBR) promoter on chromosome 7 in T-cell acute lymphoblastic leukemia (T-ALL) (Ellisen et al. 1991). The translocated gene was found to be homologous to Drosophila Notch, and was initially named TAN-1 (translocation-associated Notch homolog). Although the translocation t(7;9)(q34;q34.3) is present in a small percentage of T-ALL patients, the mutant protein is highly oncogenic and its overexpression causes T-ALL-like illness in mice (Pear et al. 1996).
Identifier: R-HSA-2122948
Species: Homo sapiens
Compartment: plasma membrane, cytosol, nucleoplasm
Mature NOTCH1 heterodimer on the cell surface is activated by one of its ligands: DLL1 (Cordle et al. 2008, Jarriault et al. 1998), DLL4 (Benedito et al. 2009), JAG1 (Li et al. 1998, Benedito et al. 2009) or JAG2 (Luo et al. 1997, Shimizu et al. 2000), expressed in trans on a neighboring cell. Thus, a ligand-expressing cell is a signal-sending cell, while the NOTCH1 expressing cell is a signal-receiving cell. If NOTCH1 has undergone Fringe modification in the Golgi, it is preferentially activated by Delta ligands (Yang et al. 2005), DLL1 and DLL4.


Upon binding to NOTCH1 on a neighboring cell, NOTCH ligands are ubiquitinated by Mindbomb (MIB1 and MIB2) and/or Neuralized (NEURL and NEURL1B) E3 ubiquitin ligases and endocytosed (Koo et al. 2007, Koo et al. 2005, Itoh et al. 2003, Lai et al. 2001, Koutelou et al. 2008, Song et al. 2006). Endocytosis of ubiquitinated ligands is thought to mechanically stretch the bound NOTCH1 receptor, exposing a cleavage site S2 that is recognized by ADAM10 and/or ADAM17 metalloprotease (van Tetering et al. 2009, Brou et al. 2000, Hartmann et al. 2002, Pan et al. 1997). S2 cleavage of NOTCH1 produces the NEXT1 fragment which is further cleaved at an S3 cleavage site by the gamma-secretase complex, resulting in release of the NOTCH1 intracellular domain (NICD1) into the cytosol (de Strooper et al. 1999, Schroeter et al. 1998, Huppert et al. 2000). NICD1 produced by activation of NOTCH1 in response to in trans presented Delta and Jagged ligands (DLL/JAG) traffics to the nucleus where it acts as a transcription regulator.


NOTCH1 signaling can also be activated by ligands other than DLL1, DLL4, JAG1 and JAG2. CNTN1 (Contactin-1), transiently expressed during central and peripheral nervous system development, activates NOTCH1 and NOTCH2 in trans, promoting oligodendrocyte maturation and myelination (Hu et al. 2003). DNER (Delta and Notch-like epidermal growth factor-related receptor) is a transmembrane protein specifically expressed in dendrites and cell bodies of postmitotic neurons. Activation of NOTCH1 by DNER in trans may play an important role in development of the central nervous system by influencing differentiation of astrocytes (Eiraku et al. 2005). Activation of NOTCH1 by both CNTN1 and DNER is Deltex (DTX)-dependent and results in gamma-secretase mediated release of NICD1. Three members of the Deltex protein family: DTX1, DTX2 and DTX4 possess a domain involved in binding cdc10/ankyrin repeats of NOTCH. DTX proteins are considered as positive regulators of NOTCH signaling, although the exact mechanism has not been elucidated (Matsuno et al. 1998, Kishi et al. 2001).In addition, DTX can mediate downregulation of NOTCH signaling by recruiting non-visual beta-arrestins to NOTCH (Mukherjee et al. 2005), thereby trigerring NOTCH ubiquitination. DTX proteins are negatively regulated by ITCH (AIP4) ubiquitin ligase (Chastagner et al. 2006).

NOTCH1 signaling in the signal-receiving cell can be turned off in cis by expression of NOTCH ligands DLL/JAG (Cordle et al. 2008, Sprinzak et al. 2010), as well as DLK1 (Baladron et al. 2005, Bray et al. 2008). Formation of NOTCH1:ligand complexes in cis prevents interaction of NOTCH1 with ligands expressed in trans, resulting in the inhibition of NOTCH signaling. In the signal-sending cell, NOTCH signaling can be negatively regulated by the protein NUMB, which is asymmetrically distributed during cell division (Rhyu et al. 1994). NUMB recruits ITCH ubiquitin ligase to NOTCH1 and promotes sorting of NOTCH1 through late endosomes for degradation (McGill et al. 2009, Chastagner et al. 2008).
Identifier: R-HSA-2122947
Species: Homo sapiens
Compartment: nucleoplasm
NICD1 produced by activation of NOTCH1 in response to Delta and Jagged ligands (DLL/JAG) presented in trans, traffics to the nucleus where it acts as a transcription regulator. In the nucleus, NICD1 displaces the NCOR corepressor complex from RBPJ (CSL). When bound to the co-repressor complex that includes NCOR proteins (NCOR1 and NCOR2) and HDAC histone deacetylases, RBPJ (CSL) represses transcription of NOTCH target genes (Kao et al. 1998, Zhou et al. 2000, Perissi et al. 2004, Perissi et al. 2008). Once the co-repressor complex is displaced, NICD1 recruits MAML (mastermind-like) to RBPJ, while MAML recruits histone acetyltransferases EP300 (p300) and PCAF, resulting in formation of the NOTCH coactivator complex that activates transcription from NOTCH regulatory elements. The minimal functional NOTCH coactivator complex that activates transcription from NOTCH regulatory elements is a heterotrimer composed of NICD, MAML and RBPJ (Fryer et al. 2002, Wallberg et al. 2002, Nam et al. 2006).


NOTCH1 coactivator complex is known to activate transcription of HES1 (Jarriault et al. 1995), HES5 (Arnett et al. 2010), HEY genes (Fischer et al. 2004, Leimeister et al. 2000, Maier et al. 2000, Arnett et al. 2010) and MYC (Palomero et al. 2006) and likely regulates transcription of many other genes (Wang et al. 2011). NOTCH1 coactivator complex on any specific regulatory element may involve additional transcriptional regulatory proteins. HES1 binds TLE proteins, forming an evolutionarily conserved transcriptional corepressor involved in regulation of neurogenesis, segmentation and sex determination (Grbavec et al. 1996, Fisher et al. 1996, Paroush et al. 1994).

After NOTCH1 coactivator complex is assembled on a NOTCH-responsive promoter, MAML (mastermind-like) recruits CDK8 in complex with cyclin C, triggering phosphorylation of conserved serine residues in TAD and PEST domains of NICD1 by CDK8. Phosphorylated NICD1 is recognized by the E3 ubiquitin ligase FBXW7 which ubiquitinates NICD1, leading to degradation of NICD1 and downregulation of NOTCH1 signaling. FBXW7-mediated ubiquitination and degradation of NOTCH1 depend on C-terminally located PEST domain sequences in NOTCH1 (Fryer et al. 2004, Oberg et al. 2001, Wu et al. 2001). The PEST domain of NOTCH1 and the substrate binding WD40 domain of FBXW7 are frequent targets of mutations in T-cell acute lymphoblastic leukemia - T-ALL (Welcker and Clurman 2008).

NICD1, which normally has a short half-life, can be stabilized by binding to the hypoxia-inducable factor 1-alpha (HIF1A) which accumulates in the nucleus when oxygen levels are low. This results in HIF1A-induced inhibition of cellular differentiation that is NOTCH-dependent (Gustafsson et al. 2005).
Identifier: R-HSA-2660826
Species: Homo sapiens
NOTCH1 t(7;9)(NOTCH1:M1580_K2555) mutant is expressed in a small subset of T-cell acute lymphoblastic leukemia (T-ALL) patients. This mutant protein results from a translocation that joins a portion of intron 24 of the NOTCH1 gene to the promoter sequence of T-cell receptor beta (TCRB), leading to overexpression of a truncated NOTCH1 protein in T-cells and their precursors. The truncated NOTCH1 contains amino acids 1580 to 2555 of the wild-type NOTCH1, lacking almost the entire extracellular domain, including EGF and LIN12 repeats (Ellisen et al. 1991). As EGF repeats are needed for NOTCH1 interaction with its ligands (DLL1, DLL4, JAG1, JAG2), the mutant NOTCH1 t(7;9)(NOTCH1:M1580_K2555) does not bind a ligand. The constitutive activity of NOTCH1 t(7;9)(NOTCH1:M1580_K2555) is based on its constitutive proteolytic processing into NOTCH1 intracellular domain (NICD1) by ADAM10/17 protease and gamma-secretase complex, as proteolytic cleavage sites are exposed in the absence of ligand binding in the mutant protein. Constitutively produced NICD1 accumulates in the nucleus, leading to aberrant activation of NOTCH1 target genes which play important roles in the development of T lymphocytes (Washburn et al. 1997. Radtke et al. 1999, Maillard et al. 2004, Sambandam et al. 2005, Tan et al. 2005). Infection of bone marrow cells with recombinant retroviruses that code for truncated NOTCH1 that resembles t(7;9)(NOTCH1:M1580_K2555) resulted in T-ALL-like illness in a portion of mice that received the infected bone-marrow transplant, with all tumors overexpressing truncated forms of NOTCH1 (Pear et al. 1996).
Identifier: R-HSA-2691232
Species: Homo sapiens
The heterodimerization (HD) domain of NOTCH1, responsible for association of NOTCH1 extracellular and transmembrane regions after furin-mediated cleavage of NOTCH1 precursor, is one of the hotspots for gain-of-function NOTCH1 mutations in T-cell acute lymphoblastic leukemia (T-ALL) (Weng et al. 2004). NOTCH1 HD domain mutants are responsive to ligand binding, but the activation (through cleavage of S2 and S3 sites and release of the intracellular domain NICD1) also happens spontaneously, in the absence of DLL and JAG ligands (Malecki et al. 2006). The following NOTCH1 HD domain mutants were directly functionally studied by Malecki et al.: NOTCH1 V1576E, NOTCH1 F1592S, NOTCH1 L1593P, NOTCH1 L1596H, NOTCH1 R1598P, NOTCH1 I1616N, NOTCH1 I1616T, NOTCH1 V1676D, NOTCH1 L1678P, NOTCH1 I1680N, NOTCH1 A1701P and NOTCH1 I1718T; other frequent NOTCH1 HD domain mutants (NOTCH1 L1574P, NOTCH1 L1574Q and NOTCH1 L1600P) are assumed to behave in a similar way.
Identifier: R-HSA-2691230
Species: Homo sapiens
NOTCH1 heterodimerization domain mutations are frequently found in T-cell acute lymphoblastic leukemia (T-ALL) (Weng et al. 2004) and result in constitutive activity of NOTCH1 mutants (Malecki et al. 2006).
Identifier: R-HSA-2644606
Species: Homo sapiens
As NOTCH1 PEST domain is intracellular, NOTCH1 PEST domain mutants are expected to behave as the wild-type NOTCH1 with respect to ligand binding and proteolytic cleavage mediated activation of signaling. However, once the NICD1 fragment of NOTCH1 is released, PEST domain mutations prolong its half-life and transcriptional activity through interference with FBXW7 (FBW7)-mediated ubiquitination and degradation of NICD1 (Weng et al. 2004, Thompson et al. 2007, O'Neil et al. 2007). All NOTCH1 PEST domain mutants annotated here (NOTCH1 Q2395*, NOTCH1 Q2440*, NOTCH1 P2474Afs*4 and NOTCH1 P2514Rfs*4) either have a truncated PEST domain or lack the PEST domain completely.
Identifier: R-HSA-2644602
Species: Homo sapiens
NOTCH1 PEST domain mutations are frequently found in T-cell acute lymphoblastic leukemia (T-ALL). PEST domain mutations interfere with ubiquitination-mediated NOTCH1 downregulation and result in prolonged half-life of the intracellular NOTCH1 fragment, NICD1, and increased NICD1 transcriptional activity (Weng et al. 2004, Thompson et al. 2007, O'Neil et al. 2007).
Identifier: R-HSA-2644607
Species: Homo sapiens
Loss of function mutations found in FBXW7 in T-cell acute lymphoblastic leukemia are predominantly dominant negative missense mutations that target one of the three highly conserved arginine residues in the WD repeats of FBXW7 (Thompson et al. 2007, O'Neil et al. 2007). These three arginine residues are part of the FBXW7 substrate binding pocket and each one of them contacts the phosphorylated threonine residue in the conserved substrate phosphodegron region (Orlicky et al. 2003). Specifically, FBXW7 interacts with the PEST domain of NOTCH1 upon phosphorylation of the PEST domain by CDK8 (Fryer et al. 2004). FBXW7 mutants are therefore unable to bind and promote ubiquitination of the NOTCH1 intracellular domain (NICD1), leading to prolonged NICD1 transcriptional activity (Thompson et al. 2007, O'Neil et al. 2007).
Identifier: R-HSA-2894858
Species: Homo sapiens
Mutations in the heterodimerization domain (HD) and PEST domain of NOTCH1 are frequently found in cis in T-cell acute lymphoblastic leukemia. While HD mutations alone result in up to ~10-fold increase in NOTCH1 transcriptional activity and PEST domain mutations alone result in up to ~2-fold increase in NOTCH1 transcriptional activity, in cis mutations of HD and PEST domains act synergistically, increasing NOTCH1 transcriptional activity up to ~40-fold (Weng et al. 2004).
Identifier: R-HSA-2894862
Species: Homo sapiens
When found in cis, HD and PEST domain mutations act synergistically, increasing NOTCH1 transcriptional activity up to ~40-fold, compared with up to ~10-fold and up to ~2-fold increase with HD mutations alone and PEST domain mutations alone, respectively (Weng et al. 2004). HD domain mutations enable spontaneous, ligand-independent, proteolytic release of the NICD1 fragment, although mutants remain responsive to ligand binding (Malecki et al. 2006), while PEST domain mutations prolong NICD1 half-life and transcriptional activity through interference with FBXW7 (FBW7)-mediated ubiquitination and degradation (Thompson et al. 2007, O'Neil et al. 2007). NOTCH1 HD+PEST domain mutants annotated here are NOTCH1 L1600P;P2514Rfs*4, NOTCH1 L1600P;Q2440*, NOTCH1 L1600P;Q2395* and NOTCH1 L1574P;P2474Afs*4.
Identifier: R-HSA-8941856
Species: Homo sapiens
RUNX3 negatively regulates NOTCH signaling, which contributes to the tumor suppressor role of RUNX3 in hepatocellular carcinoma. RUNX3 binds the promoter of the JAG1 gene, encoding NOTCH ligand JAG1 and inhibits its transcription (Nishina et al. 2011). In addition, RUNX3 also binds to the NOTCH1 coactivator complex at the promoter of HES1, a NOTCH target gene, and inhibits HES1 transcription (Gao et al. 2010).
Identifier: R-HSA-9013700
Species: Homo sapiens
NOTCH4 is co-expressed with DLL4 (Delta-4) and JAG1 (Jagged-1) in the vascular system (Shutter et al. 2000, Uyttendaele et al. 2000). NOTCH4 can be activated by DLL4 and JAG1 when HMVECd cells (human primary endothelial cell line derived from neonatal dermal microvasculature) or HUVEC cells (human umbilical venous endothelial cell line) expressing recombinant mouse Notch4 are co-cultured with HMVECd or HUVEC cells expressing recombinant human or mouse DLL4 (Shawber et al. 2003, Shawber et al. 2007) or mouse Jag1 (Shawber et al. 2007). Activation of NOTCH4 by DLL4 and JAG1 could not be reproduced when the mouse fibroblast cell line NIH 3T3 or human embryonic kidney cell line HEK293 was transduced with Notch4- or either Dll4- or Jag1-expressing vectors and used in co-culture experiments (Aste-Amezaga et al. 2010, James et al. 2014).

Signaling by NOTCH4, similar to other NOTCH family proteins, involves proteolytic cleavage of the membrane-bound NOTCH4 receptor and release of the NOTCH4 intracellular domain fragment (NICD4) into the cytosol (Saxena et al. 2001, Das et al. 2004). NICD4 traffics from the cytosol to the nucleus, where it acts as a transcription factor (Lin et al. 2002).
Identifier: R-HSA-1912422
Species: Homo sapiens
Compartment: nucleoplasm, cytosol, endoplasmic reticulum membrane, endoplasmic reticulum lumen, Golgi membrane, Golgi lumen, plasma membrane
In humans and other mammals the NOTCH gene family has four members, NOTCH1, NOTCH2, NOTCH3 and NOTCH4, encoded on four different chromosomes. Their transcription is developmentally regulated and tissue specific, but very little information exists on molecular mechanisms of transcriptional regulation. Translation of NOTCH mRNAs is negatively regulated by a number of recently discovered microRNAs (Li et al. 2009, Pang et al.2010, Ji et al. 2009, Kong et al. 2010, Marcet et al. 2011, Ghisi et al. 2011, Song et al. 2009, Hashimoto et al. 2010, Costa et al. 2009).

The nascent forms of NOTCH precursors, Pre-NOTCH1, Pre-NOTCH2, Pre-NOTCH3 and Pre-NOTCH4, undergo extensive posttranslational modifications in the endoplasmic reticulum and Golgi apparatus to become functional. In the endoplasmic reticulum, conserved serine and threonine residues in the EGF repeats of NOTCH extracellular domain are fucosylated and glucosylated by POFUT1 and POGLUT1, respectively (Yao et al. 2011, Stahl et al. 2008, Wang et al. 2001, Shao et al. 2003, Acar et al. 2008, Fernandez Valdivia et al. 2011).

In the Golgi apparatus, fucose groups attached to NOTCH EGF repeats can be elongated by additional glycosylation steps initiated by fringe enzymes (Bruckner et al. 2000, Moloney et al. 2000, Cohen et al. 1997, Johnston et al. 1997, Chen et al. 2001). Fringe-mediated modification modulates NOTCH signaling but is not an obligatory step in Pre-NOTCH processing. Typically, processing of Pre-NOTCH in the Golgi involves cleavage by FURIN convertase (Blaumueller et al. 1997, Logeat et al. 1998, Gordon et al. 2009, Rand et al. 2000, Chan et al. 1998). The cleavage of NOTCH results in formation of mature NOTCH heterodimers that consist of NOTCH extracellular domain (NEC i.e. NECD) and NOTCH transmembrane and intracellular domain (NTM i.e. NTMICD). NOTCH heterodimers translocate to the cell surface where they function in cell to cell signaling.
Identifier: R-HSA-9012852
Species: Homo sapiens
Similar to NOTCH1, NOTCH3 is activated by delta-like and jagged ligands (DLL/JAG) expressed in trans on a neighboring cell. The activation triggers cleavage of NOTCH3, first by ADAM10 at the S2 cleavage site, then by gamma-secretase at the S3 cleavage site, resulting in the release of the intracellular domain of NOTCH3, NICD3, into the cytosol. NICD3 subsequently traffics to the nucleus where it acts as a transcriptional regulator. NOTCH3 expression pattern is more restricted than the expression patterns of NOTCH1 and NOTCH2, with predominant expression of NOTCH3 in vascular smooth muscle cells, lymphocytes and the nervous system (reviewed by Bellavia et al. 2008). Based on the study of Notch3 knockout mice, Notch3 is not essential for embryonic development or fertility (Krebs et al. 2003).

Germline gain-of-function NOTCH3 mutations are an underlying cause of the CADASIL syndrome - cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. CADASIL is characterized by degeneration and loss of vascular smooth muscle cells from the arterial wall, predisposing affected individuals to an early onset stroke (Storkebaum et al. 2011). NOTCH3 promotes survival of vascular smooth muscle cells at least in part by induction of CFLAR (c FLIP), an inhibitor of FASLG activated death receptor signaling. The mechanism of NOTCH3 mediated upregulation of CFLAR is unknown; it is independent of the NOTCH3 coactivator complex and involves an unelucidated crosstalk with the RAS/RAF/MAPK pathway (Wang et al. 2002).

In rat brain, NOTCH3 and NOTCH1 are expressed at sites of adult neurogenesis, such as the dentate gyrus (Irvin et al. 2001). NOTCH3, similar to NOTCH1, promotes differentiation of the rat adult hippocampus derived multipotent neuronal progenitors into astroglia (Tanigaki et al. 2001). NOTCH1, NOTCH2, NOTCH3, and their ligand DLL1 are expressed in neuroepithelial precursor cells in the neural tube of mouse embryos. Together, they signal to inhibit neuronal differentiation of neuroepithelial precursors. Expression of NOTCH3 in mouse neuroepithelial precursors is stimulated by growth factors BMP2, FGF2, Xenopus TGF beta5 - homologous to TGFB1, LIF, and NTF3 (Faux et al. 2001).

In mouse telencephalon, NOTCH3, similar to NOTCH1, promotes radial glia and neuronal progenitor phenotype. This can, at least in part be attributed to NOTCH mediated activation of RBPJ-dependent and HES5-dependent transcription (Dang et al. 2006).

In mouse spinal cord, Notch3 is involved in neuronal differentiation and maturation. Notch3 knockout mice have a decreased number of mature inhibitory interneurons in the spinal cord, which may be involved in chronic pain conditions (Rusanescu and Mao 2014).

NOTCH3 amplification was reported in breast cancer, where NOTCH3 promotes proliferation and survival of ERBB2 negative breast cancer cells (Yamaguchi et al. 2008), and it has also been reported in ovarian cancer (Park et al. 2006). NOTCH3 signaling is involved in TGF beta (TGFB1) signaling-induced eptihelial to mesenchimal transition (EMT) (Ohashi et al. 2011, Liu et al. 2014)

NOTCH3 indirectly promotes development of regulatory T cells (Tregs). NOTCH3 signaling activates pre-TCR-dependent and PKC-theta (PRKCQ)-dependent NF-kappaB (NFKB) activation, resulting in induction of FOXP3 expression (Barbarulo et al. 2011). Deregulated NOTCH3 and pre-TCR signaling contributes to development of leukemia and lymphoma (Bellavia et al. 2000, Bellavia et al. 2002).

Identifier: R-HSA-2197563
Species: Homo sapiens
Compartment: nucleoplasm
In the nucleus, NICD2 forms a complex with RBPJ (CBF1, CSL) and MAML (mastermind). NICD2:RBPJ:MAML complex activates transcription from RBPJ-binding promoter elements (RBEs) (Wu et al. 2000). Besides NICD2, RBPJ and MAML, NOTCH2 coactivator complex likely includes other proteins, shown as components of the NOTCH1 coactivator complex.

NOTCH2 coactivator complex directly stimulates transcription of HES1 and HES5 genes (Shimizu et al. 2002), both of which are known NOTCH1 targets.

The promoter of FCER2 (CD23A) contains several RBEs that are occupied by NOTCH2 but not NOTCH1 coactivator complexes, and NOTCH2 activation stimulates FCER2 transcription. Overexpression of FCER2 (CD23A) is a hallmark of B-cell chronic lymphocytic leukemia (B-CLL) and correlates with the malfunction of apoptosis, which is thought be an underlying mechanism of B-CLL development. The Epstein-Barr virus protein EBNA2 can also activate FCER2 transcription through RBEs, possibly by mimicking NOTCH2 signaling (Hubmann et al. 2002).

NOTCH2 coactivator complex occupies the proximal RBE of the GZMB (granzyme B) promoter and at the same time interacts with phosphorylated CREB1, bound to an adjacent CRE site. EP300 transcriptional coactivator is also recruited to this complex through association with CREB1 (Maekawa et al. 2008). NOTCH2 coactivator complex together with CREBP1 and EP300 stimulates transcription of GZMB (granzyme B), which is important for the cytotoxic function of CD8+ T-cells (Maekawa et al. 2008).

There are indications that NOTCH2 genetically interacts with hepatocyte nuclear factor 1-beta (HNF1B) in kidney development (Massa et al. 2013, Heliot et al. 2013) and with hepatocyte nuclear factor 6 (HNF6) in bile duct formation (Vanderpool et al. 2012), but the exact nature of these genetic interactions has not been defined.

Identifier: R-HSA-1912399
Species: Homo sapiens
Compartment: endoplasmic reticulum membrane, endoplasmic reticulum lumen
In the endoplasmic reticulum, glycosyl transferases modify NOTCH precursors by glycosylating conserved serine and threonine residues in EGF repeats of NOTCH.

O-fucosyl transferase POFUT1 fucosylates NOTCH serine and threonine residues that conform to the consensus sequence C2-X(4-5)-S/T-C3, where C2 and C3 are the second and third cysteine residue within the EGF repeat, and X(4-5) is four to five amino acid residues of any type (Yao et al. 2011, Stahl et al. 2008, Wang et al. 2001, Shao et al. 2003).

O-glucosyl transferase POGLUT1, mammalian homolog of the Drosophila enzyme Rumi, adds a glucosyl group to conserved serine residues within the EGF repeats of NOTCH. The consensus sequence for POGLUT1-mediated glucosylation is C1-X-S-X-P-C2, where C1 and C2 are the first and second cysteine residue in the EGF repeat, respectively, while X represents any amino acid (Acar et al. 2008, Fernandez-Valdivia et al. 2011). Both fucosylation and glucosylation of NOTCH receptor precursors are essential for functionality.
Identifier: R-HSA-1980145
Species: Homo sapiens
Compartment: plasma membrane, cytosol, nucleoplasm
NOTCH2 is activated by binding Delta-like and Jagged ligands (DLL/JAG) expressed in trans on neighboring cells (Shimizu et al. 1999, Shimizu et al. 2000, Hicks et al. 2000, Ji et al. 2004). In trans ligand-receptor binding is followed by ADAM10 mediated (Gibb et al. 2010, Shimizu et al. 2000) and gamma secretase complex mediated cleavage of NOTCH2 (Saxena et al. 2001, De Strooper et al. 1999), resulting in the release of the intracellular domain of NOTCH2, NICD2, into the cytosol. NICD2 traffics to the nucleus where it acts as a transcriptional regulator. For a recent review of the cannonical NOTCH signaling, please refer to Kopan and Ilagan 2009, D'Souza et al. 2010, Kovall and Blacklow 2010. CNTN1 (contactin 1), a protein involved in oligodendrocyte maturation (Hu et al. 2003) and MDK (midkine) (Huang et al. 2008, Gungor et al. 2011), which plays an important role in epithelial-to-mesenchymal transition, can also bind NOTCH2 and activate NOTCH2 signaling.

In the nucleus, NICD2 forms a complex with RBPJ (CBF1, CSL) and MAML (mastermind). The NICD2:RBPJ:MAML complex activates transcription from RBPJ binding promoter elements (RBEs) (Wu et al. 2000). NOTCH2 coactivator complexes directly stimulate transcription of HES1 and HES5 genes (Shimizu et al. 2002), both of which are known NOTCH1 targets. NOTCH2 but not NOTCH1 coactivator complexes, stimulate FCER2 transcription. Overexpression of FCER2 (CD23A) is a hallmark of B-cell chronic lymphocytic leukemia (B-CLL) and correlates with the malfunction of apoptosis, which is thought be an underlying mechanism of B-CLL development (Hubmann et al. 2002). NOTCH2 coactivator complexes together with CREBP1 and EP300 stimulate transcription of GZMB (granzyme B), which is important for the cytotoxic function of CD8+ T cells (Maekawa et al. 2008).

NOTCH2 gene expression is differentially regulated during human B-cell development, with NOTCH2 transcripts appearing at late developmental stages (Bertrand et al. 2000).

NOTCH2 mutations are a rare cause of Alagille syndrome (AGS). AGS is a dominant congenital multisystem disorder characterized mainly by hepatic bile duct abnormalities. Craniofacial, heart and kidney abnormalities are also frequently observed in the Alagille spectrum (Alagille et al. 1975). AGS is predominantly caused by mutations in JAG1, a NOTCH2 ligand (Oda et al. 1997, Li et al. 1997), but it can also be caused by mutations in NOTCH2 (McDaniell et al. 2006).


Hajdu-Cheney syndrome, an autosomal dominant disorder characterized by severe and progressive bone loss, is caused by NOTCH2 mutations that result in premature C-terminal NOTCH2 truncation, probably leading to increased NOTCH2 signaling (Simpson et al. 2011, Isidor et al. 2011, Majewski et al. 2011).
Identifier: R-HSA-1912420
Species: Homo sapiens
Compartment: Golgi membrane, endoplasmic reticulum membrane, Golgi lumen, plasma membrane
NOTCH undergoes final posttranslational processing in the Golgi apparatus (Lardelli et al. 1994, Blaumueller et al. 1997, Weinmaster et al. 1991, Weinmaster et al. 1992, Uyttendaele et al. 1996). Movement of NOTCH precursors from the endoplasmic reticulum to Golgi is controlled by SEL1L protein, a homolog of C. elegans sel-1. SEL1L localizes to the endoplasmic reticulum membrane and prevents translocation of misfolded proteins, therefore serving as a quality control check (Li et al. 2010, Sundaram et al. 1993, Francisco et al. 2010). Similarly, C. elegans sel-9 and its mammalian homolog TMED2 are Golgi membrane proteins that participate in quality control of proteins transported from Golgi to the plasma membrane. Translocation of a mutant C. elegans NOTCH homolog lin-12 from the Golgi to the plasma membrane is negatively regulated by sel-9 (Wen et al. 1999). A GTPase RAB6 positively controls NOTCH trafficking through Golgi (Purcell et al. 1999).


Processing of mammalian NOTCH precursors in the Golgi typically involves the cleavage by FURIN convertase. Pre-NOTCH is a ~300 kDa protein, and cleavage by FURIN produces two fragments with approximate sizes of 110 kDa and 180 kDa. The 110 kDa fragment contains the transmembrane and intracellular domains of NOTCH and is known as NTM or NTMICD. The 189 kDa fragment contains NOTCH extracellular sequence and is known as NEC or NECD. The NTM and NEC fragments heterodimerize (Blaumueller et al. 1997, Logeat et al. 1998, Chan et al. 1998) and are held together by disulfide bonds and calcium ions (Rand et al. 2000, Gordon et al. 2009).


An optional step in Pre-NOTCH processing in the Golgi is modification by fringe enzymes. Fringe enzymes are glycosyl transferases that initiate elongation of O-linked fucose on fucosylated peptides by addition of a beta 1,3 N-acetylglucosaminyl group, resulting in formation of disaccharide chains on NOTCH EGF repeats (GlcNAc-bet1,3-fucitol). Three fringe enzymes are known in mammals: LFNG (lunatic fringe), MFNG (manic fringe) and RFNG (radical fringe). LFNG shows the highest catalytic activity in modifying NOTCH (Bruckner et al. 2000, Moloney et al. 2000). Fringe-created disaccharide chains on NOTCH EGF repeats are further extended by B4GALT1 (beta-1,4-galactosyltransferase 1), which adds galactose to the N-acetylglucosaminyl group, resulting in formation of trisaccharide Gal-beta1,4-GlcNAc-beta1,3-fucitol chains (Moloney et al. 2000, Chen et al. 2001). Formation of trisaccharide chains is the minimum requirement for fringe-mediated modulation of NOTCH signaling, although fringe-modified NOTCH expressed on the cell surface predominantly contains tetrasaccharide chains on EGF repeats. The tetrasaccharide chains are formed by sialyltransferase(s) that add sialic acid to galactose, resulting in formation of Sia-alpha2,3-Gal-beta1,4-GlcNAc-beta1,3-fucitol (Moloney et al. 2000). Three known Golgi membrane sialyltransferases could be performing this function: ST3GAL3, ST3GAL4 and ST3GAL6 (Harduin-Lepers et al. 2001). The modification of NOTCH by fringe enzymes modulates NOTCH-signaling by increasing the affinity of NOTCH receptors for delta-like ligands, DLL1 and DLL4, while decreasing affinity for jagged ligands, JAG1 and JAG2.
Identifier: R-HSA-1912408
Species: Homo sapiens
Compartment: nucleoplasm, cytosol, endoplasmic reticulum membrane
In humans, the NOTCH protein family has four members: NOTCH1, NOTCH2, NOTCH3 and NOTCH4. NOTCH1 protein was identified first, as the product of a chromosome 9 gene translocated in T-cell acute lymphoblastic leukemia that was homologous to Drosophila Notch (Ellisen et al. 1991). At the same time, rat Notch1 was cloned (Weinmaster et al. 1991), followed by cloning of mouse Notch1, named Motch (Del Amo et al. 1992). NOTCH2 protein is the product of a gene on chromosome 1 (Larsson et al. 1994). NOTCH2 expression is differentially regulated during B-cell development (Bertrand et al. 2000). NOTCH2 mutations are a rare cause of Alagille syndrome (McDaniell et al. 2006). NOTCH3 is the product of a gene on chromosome 19. NOTCH3 mutations are the underlying cause of CADASIL, cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (Joutel et al. 1996). NOTCH4, the last NOTCH protein discovered, is the product of a gene on chromosome 6 (Li et al. 1998).

MicroRNAs play an important negative role in translation and/or stability of NOTCH mRNAs. MicroRNAs miR-34 (miR-34A, miR-34B and mi-R34C), whose transcription is directly induced by the tumor suppressor protein p53 (Chang et al. 2007, Raver-Shapira et al. 2007, He et al. 2007, Corney et al. 2007) bind and negatively regulate translation of NOTCH1 mRNA (Li et al. 2009, Pang et al. 2010, Ji et al. 2009) and NOTCH2 mRNA (Li et al. 2009). NOTCH1 mRNA translation is also negatively regulated by microRNAs miR-200B and miR-200C (Kong et al. 2010), as well as miR-449A, miR-449B and miR-449C (Marcet et al. 2011). Translation of NOTCH3 mRNA is negatively regulated by microRNAs miR-150 (Ghisi et al. 2011) and miR-206 (Song et al. 2009). Translation of NOTCH4 mRNA is negatively regulated by microRNAs miR-181C (Hashimoto et al. 2010) and miR-302A (Costa et al. 2009).

Nascent NOTCH peptides are co-translationally targeted to the endoplasmic reticulum for further processing, followed by modification in the Golgi apparatus, before trafficking to the plasma membrane. Endoplasmic reticulum calcium ATPases, positively regulate NOTCH trafficking, possibly by contributing to accurate folding of NOTCH precursors (Periz et al. 1999).
Identifier: R-HSA-2979096
Species: Homo sapiens
Similar to NOTCH1, NOTCH2 is activated by Delta-like and Jagged ligands (DLL/JAG) expressed in trans on a neighboring cell (Shimizu et al. 1999, Shimizu et al. 2000, Hicks et al. 2000, Ji et al. 2004). The activation triggers cleavage of NOTCH2, first by ADAM10 at the S2 cleavage site (Gibb et al. 2010, Shimizu et al. 2000), then by gamma-secretase at the S3 cleavage site (Saxena et al. 2001, De Strooper et al. 1999), resulting in the release of the intracellular domain of NOTCH2, NICD2, into the cytosol. NICD2 subsequently traffics to the nucleus where it acts as a transcription regulator.

While DLL and JAG ligands are well established, canonical NOTCH2 ligands, there is limited evidence that NOTCH2, similar to NOTCH1, can be activated by CNTN1 (contactin 1), a protein involved in oligodendrocyte maturation (Hu et al. 2003). MDK (midkine), which plays an important role in epithelial to mesenchymal transition, can also activate NOTCH2 signaling and is able to bind to the extracellular domain of NOTCH2, but the exact mechanism of MDK-induced NOTCH2 activation has not been elucidated (Huang et al. 2008, Gungor et al. 2011).
Identifier: R-HSA-9013694
Species: Homo sapiens
The NOTCH4 gene locus was discovered as a frequent site of insertion for the proviral genome of the mouse mammary tumor virus (MMTV) (Gallahan and Callahan 1987). MMTV-insertion results in aberrant expression of the mouse mammary tumor gene int-3, which was subsequently discovered to represent the intracellular domain of Notch4 (Robbins et al. 1992, Uyttendaele et al. 1996).

NOTCH4 is prevalently expressed in endothelial cells (Uyttendaele et al. 1996). DLL4 and JAG1 act as ligands for NOTCH4 in human endothelial cells (Shawber et al. 2003, Shawber et al. 2007), but DLL4- and JAG1-mediated activation of NOTCH4 have not been confirmed in all cell types tested (Aste-Amezaga et al. 2010, James et al. 2014). The gamma secretase complex cleaves activated NOTCH4 receptor to release the intracellular domain fragment (NICD4) (Saxena et al. 2001, Das et al. 2004). NICD4 traffics to the nucleus where it acts as a transcription factor and stimulates expression of NOTCH target genes HES1, HES5, HEY1 and HEY2, as well as VEGFR3 and ACTA2 (Lin et al. 2002, Raafat et al.2004, Tsunematsu et al. 2004, Shawber et al. 2007, Tang et al. 2008, Bargo et al. 2010). NOTCH4 signaling can be downregulated by AKT1 phosphorylation-induced cytoplasmic retention (Ramakrishnan et al. 2015) as well as proteasome-dependent degradation upon FBXW7-mediated ubiquitination (Wu et al. 2001, Tsunematsu et al. 2004).

NOTCH4 was reported to inhibit NOTCH1 signaling in-cis, by binding to NOTCH1 and interfering with the S1 cleavage of NOTCH1, thus preventing production of functional NOTCH1 heterodimers at the cell surface (James et al. 2014).

NOTCH4 is involved in development of the vascular system. Overexpression of constitutively active Notch4 in mouse embryonic vasculature results in abnormal vessel structure and patterning (Uyttendaele et al. 2001). NOTCH4 may act to inhibit apoptosis of endothelial cells (MacKenzie et al. 2004).

Expression of int-3 interferes with normal mammary gland development in mice and promotes tumorigenesis. The phenotype of mice expressing int-3 in mammary glands is dependent on the presence of Rbpj (Raafat et al. 2009). JAG1 and NOTCH4 are upregulated in human ER+ breast cancers resistant to anti-estrogen therapy, which correlates with elevated expression of NOTCH target genes HES1, HEY1 and HEY2, and is associated with increased population of breast cancer stem cells and distant metastases (Simoes et al. 2015). Development of int-3-induced mammary tumours in mice depends on Kit and Pdgfra signaling (Raafat et al. 2006) and on int-3-induced activaton of NFKB signaling (Raafat et al. 2017). In head and neck squamous cell carcinoma (HNSCC), high NOTCH4 expression correlates with elevated HEY1 levels, increased cell proliferation and survival, epithelial-to-mesenchymal transition (EMT) phenotype and cisplatin resistance (Fukusumi et al. 2018). In melanoma, however, exogenous NOTCH4 expression correlates with mesenchymal-to-epithelial-like transition and reduced invasiveness (Bonyadi Rad et al. 2016). NOTCH4 is frequently overexpressed in gastric cancer. Increased NOTCH4 levels correlate with activation of WNT signaling and gastric cancer progression (Qian et al. 2015).

NOTCH4 is expressed in adipocytes and may promote adipocyte differentiation (Lai et al. 2013).

During Dengue virus infection, DLL1, DLL4, NOTCH4 and HES1 are upregulated in interferon-beta (INFB) dependent manner (Li et al. 2015). NOTCH4 signaling may be affected by Epstein-Barr virus (EBV) infection, as the EBV protein BARF0 binds to NOTCH4 (Kusano and Raab-Traub 2001).
Identifier: R-HSA-9013508
Species: Homo sapiens
In the nucleus, NICD3 forms a complex with RBPJ (CBF1, CSL) and MAML (mastermind) proteins MAML1, MAML2 or MAML3 (possibly also MAMLD1). NICD3:RBPJ:MAML complex, also known as the NOTCH3 coactivator complex, activates transcription from RBPJ-binding promoter elements (Lin et al. 2002). While NOTCH1 prefers paired RBPJ binding sites, NOTCH3 preferentially binds to single RBPJ binding sites (Ong et al. 2006).


NOTCH3 coactivator complex induces transcription of the well established NOTCH target genes HES1 (Lin et al. 2002, Boelens et al. 2014), HEYL (Maier and Gessler 2000, Geimer Le Lay et al. 2014), HES5 (Lin te al. 2002, Shimizu et al. 2002), and HEY2 (Wang et al. 2002).

NOTCH3 positively regulates transcription of the pre-T-cell receptor alpha chain (PTCRA, commonly known as pT-alpha or pre-TCRalpha) (Talora et al. 2003, Bellavia et al. 2007). IK1, splicing isoform of the transcription factor Ikaros (IKZF1), competes with RBPJ for binding to the PTCRA promoter and inhibits PTCRA transcription. NOTCH3, through pre-TCR signaling, stimulates expression of the RNA binding protein HuD, which promotes splicing of IKZF1 into dominant negative isoforms. These dominant negative isoforms of IKZF1 heterodimerize with IK1, preventing its binding to target DNA sequences and thus contributing to sustained transcription of PTCRA (Bellavia et al. 2007, reviewed by Bellavia, Mecarrozzi, Campese, Grazioli, Gulino and Screpanti 2007).

NOTCH3-triggered pre-TCR-signaling downregulates the activity of the transcription factor TCF3 (E2A), through ERK-dependent induction of ID1. Inhibition of TCF3-mediated transcription downstream of NOTCH3 contributes to development of T-cell lymphomas in transgenic mice expressing NICD3 (Talora et al. 2003). Activation of ERKs downstream of NOTCH3-stimulated pre-TCR signaling leads to phosphorylation of the transcription factor TAL1, formation of the TAL1:SP1 complex, and activation of cyclin D1 (CCND1) transcription, which stimulates cell division (Talora et al. 2006).

NOTCH3 signaling can activate NF-kappaB (NFKB)-mediate transcription either indirectly, through activation of pre-TCR signaling, or directly, through association of NOTCH3 with IKKA. NFKB is constitutively active in T lymphoma cells derived from NOTCH3 transgenic mice (Vacca et al. 2006).

Transcription of the PLXND1 gene, encoding the semaphorin receptor Plexin D1, is directly stimulated by NOTCH1 and NOTCH3 coactivator complexes. PLXND1 is involved in neuronal migration and cancer cell invasiveness (Rehman et al. 2016). Expression of FABP7 (BLBP) in radial glia is positively regulated by NOTCH1 and NOTCH3 during neuronal migration (Anthony et al. 2005, Keilani and Sugaya 2008).


NOTCH3 gene is frequently amplified in ovarian cancer (Park et al. 2006). NOTCH3 coactivator complex directly stimulates DLGAP5 transcription. DLGAP5 is involved in G2/M transition and is overexpressed in ovarian cancer cells. (Chen et al. 2012). Another gene overexpressed in ovarian cancer whose transcription is directly stimulated by NOTCH3 is PBX1 (Park et al. 2008). The NOTCH3 coactivator complex directly stimulates WWC1 gene transcription. WWC1 gene encodes protein Kibra, involved in Hippo signaling. NOTCH3-mediated induction of WWC1 positively regulates Hippo signaling and inhibits epithelial-to-mesenchymal transition (EMT) in triple negative breast cancer cells (Zhang et al. 2016).

Identifier: R-HSA-8878159
Species: Homo sapiens
The transcription factor RUNX3 is a RUNX family member. All RUNX family members, RUNX1, RUNX2 and RUNX3, possess a highly conserved Runt domain, involved in DNA binding. For a more detailed description of the structure of RUNX proteins, please refer to the pathway 'Transcriptional regulation by RUNX1'. Similar to RUNX1 and RUNX2, RUNX3 forms a transcriptionally active heterodimer with CBFB (CBF-beta). Studies in mice have shown that RUNX3 plays a role in neurogenesis and development of T lymphocytes. RUNX3 is implicated as a tumor suppressor gene in various human malignancies.
During nervous system formation, the Cbfb:Runx3 complex is involved in development of mouse proprioceptive dorsal root ganglion neurons by regulating expression of Ntrk3 (Neurotrophic tyrosine kinase receptor type 3) and possibly other genes (Inoue et al. 2002, Kramer et al. 2006, Nakamura et al. 2008, Dykes et al. 2011, Ogihara et al. 2016). It is not yet known whether RUNX3 is involved in human neuronal development and neuronal disorders.
RUNX3 plays a major role in immune response. RUNX3 regulates development of T lymphocytes. In mouse hematopoietic stem cells, expression of Runx3 is regulated by the transcription factor TAL1 (Landry et al. 2008). RUNX3 promotes the CD8+ lineage fate in developing thymocytes. In the CD4+ thymocyte lineage in mice, the transcription factor ThPOK induces transcription of SOCS family members, which repress Runx3 expression (Luckey et al. 2014). RUNX3, along with RUNX1 and ETS1, is implicated in regulation of transcription of the CD6 gene, encoding a lymphocyte surface receptor expressed on developing and mature T cells (Arman et al. 2009). RUNX3 and ThPOK regulate intestinal CD4+ T cell immunity in a TGF-beta and retinoic acid-dependent manner, which is important for cellular defense against intestinal pathogens (Reis et al. 2013). Besides T lymphocytes, RUNX3 is a key transcription factor in the commitment of innate lymphoid cells ILC1 and ILC3 (Ebihara et al. 2015). RUNX3 regulates expression of CD11A and CD49D integrin genes, involved in immune and inflammatory responses (Dominguez-Soto et al. 2005). RUNX3 is involved in mouse TGF-beta-mediated dendritic cell function and its deficiency is linked to airway inflammation (Fainaru et al. 2004).
In addition to its developmental role, RUNX3 is implicated as a tumor suppressor. The loss of RUNX3 expression and function was first causally linked to the genesis and progression of human gastric cancer (Li et al. 2002). Expression of RUNX3 increases in human pancreatic islet of Langerhans cells but not in pancreatic adenocarcinoma cells in response to differentiation stimulus (serum withdrawal) (Levkovitz et al. 2010). Hypermethylation of the RUNX3 gene is associated with an increased risk for progression of Barrett's esophagus to esophageal adenocarcinoma (Schulmann et al. 2005). Hypermethylation-mediated silencing of the RUNX3 gene expression is also frequent in granulosa cell tumors (Dhillon et al. 2004) and has also been reported in colon cancer (Weisenberger et al. 2006), breast cancer (Lau et al. 2006, Huang et al. 2012), bladder cancer (Wolff et al. 2008) and gastric cancer (Li et al. 2002). In colorectal cancer, RUNX3 is one of the five markers in a gene panel used to classify CpG island methylator phenotype (CIMP+) (Weisenberger et al. 2006).
RUNX3 and CBFB are frequently downregulated in gastric cancer. RUNX3 cooperates with TGF-beta to maintain homeostasis in the stomach and is involved in TGF-beta-induced cell cycle arrest of stomach epithelial cells. Runx3 knockout mice exhibit decreased sensitivity to TGF-beta and develop gastric epithelial hyperplasia (Li et al. 2002, Chi et al. 2005). RUNX3-mediated inhibition of binding of TEADs:YAP1 complexes to target promoters is also implicated in gastric cancer suppression (Qiao et al. 2016).
RUNX3 is a negative regulator of NOTCH signaling and RUNX3-mediated inhibition of NOTCH activity may play a tumor suppressor role in hepatocellular carcinoma (Gao et al. 2010, Nishina et al. 2011).
In addition to RUNX3 silencing through promoter hypermethylation in breast cancer (Lau et al. 2006), Runx3+/- mice are predisposed to breast cancer development. RUNX3 downregulates estrogen receptor alpha (ESR1) protein levels in a proteasome-dependent manner (Huang et al. 2012).
Besides its tumor suppressor role, mainly manifested through its negative effect on cell proliferation, RUNX3 can promote cancer cell invasion by stimulating expression of genes involved in metastasis, such as osteopontin (SPP1) (Whittle et al. 2015).
Identifier: R-HSA-9793380
Species: Homo sapiens
Skeletal tissues originate from paraxial mesoderm, lateral plate mesoderm, and neural crest. Paraxial mesoderm is produced by invagination of cells through the primitive streak and is the precursor of somites, which are spheres of mesenchyme bounded by epithelium that bud at fixed intervals from the anterior paraxial mesoderm in a process termed somitogenesis (reviewed in Tam and Trainor 1994, Pourquie 2003). Somites give rise to the axial skeleton and skeletal muscles.
Paraxial mesoderm becomes specified at a lower level of BMP signaling (Xi et al. 2017) that results from the interaction of BMP4, produced by the lateral plate mesoderm, with NOGGIN (NOG), a negative regulator of BMP signaling produced by the notochord (reviewed in Tani et al. 2020). WNT signaling by WNT3A that activates beta‑catenin (CTNNB1), FGF signaling that acts though FGFR1, and TBXT activate expression of TBX6 and Mesogenin 1 (MSGN1). MSGN1 binds and activates SNAI1 to promote epithelial-mesenchymal transitions (EMT). TBX6 activates MSGN1, and MSGN1 activates TBX6, to establish a positive feedback loop that ensures commitment to the paraxial mesoderm lineage. TBX6 and MSGN1 act with WNT signaling to activate expression of MSGN1, and the NOTCH ligand Delta‑like 1 (DLL1), which enhances NOTCH signaling. MSGN1 binds and activates expression of DLL1, DLL3, NOTCH1, and NOTCH2, and binds to Clock enhancers that regulate periodic expression of LFNG during somitogenesis in the anterior paraxial mesoderm. The counterbalancing DLL3 protein inhibits NOTCH signaling by binding NOTCH1 in endosomes and targeting NOTCH1 for lysosomal degradation.
TBX6 alone is capable of reprogramming pluripotent stem cells to paraxial mesoderm (Sadahiro et al. 2018) and acts in a regulatory loop with MESP2 to create the boundaries of nascent somites (Oginuma et al. 2011): TBX6 activates expression of MESP2 which then represses TBX6 by targeting TBX6 for degradation, leaving MESP2 alone at the segmental boundary.
Identifier: R-HSA-9022699
Species: Homo sapiens
Receptors directly transcriptionally regulated by MECP2 include glutamate receptor GRIA2 (Qiu et al. 2012), NMDA receptor subunits GRIN2A (Durand et al. 2012) and GRIN2B (Lee et al. 2008), opioid receptors OPRK1 (Chahrour et al. 2008) and OPRM1 (Hwang et al. 2009, Hwang et al. 2010, Samaco et al. 2012), GPRIN1 (Chahrour et al. 2008), MET (Plummer et al. 2013), and NOTCH1 (Li et al. 2014). Channels/transporters regulated by MECP2 include TRPC3 (Li et al. 2012) and SLC2A3 (Chen et al. 2013). MECP2 also regulates transcription of FKBP5, involved in trafficking of glucocorticoid receptors (Nuber et al. 2005, Urdinguio et al. 2008) and is implicated in regulation of expression of SEMA3F (semaphorin 3F) in mouse olfactory neurons (Degano et al. 2009). In zebrafish, Mecp2 is implicated in sensory axon guidance by direct stimulation of transcription of Sema5b and Robo2 (Leong et al. 2015). MECP2 may indirectly regulate signaling by neuronal receptor tyrosine kinases by regulating transcription of protein tyrosine phosphatases, PTPN1 (Krishnan et al. 2015) and PTPN4 (Williamson et al. 2015).
Identifier: R-HSA-8986944
Species: Homo sapiens
MECP2 is an X chromosome gene whose loss-of-function mutations are an underlying cause of the majority of Rett syndrome cases. The MECP2 gene locus consists of four exons. Both exon 1 and exon 2 contain translation start sites. Alternative splicing of the second exon results in expression of two MECP2 transcript isoforms, MECP2_e1 (MECP2B or MECP2alpha) and MECP2_e2 (MECP2A or MECP2beta). The N-terminus of the MECP2_e1 isoform, in which exon 2 is spliced out, is encoded by exon 1. The N-terminus of the MECP2_e2 isoforms, which includes both exon 1 and exon 2, is encoded by exon 2, as the exon 2 translation start site is used. Exons 3 and 4 are present in both isoforms. The MECP2_e2 isoform was cloned first and is therefore more extensively studied. The MECP2_e1 isoform is more abundant in the brain (Mnatzakanian et al. 2004, Kriaucionis and Bird 2004, Kaddoum et al. 2013). Mecp2 isoforms show different expression patterns during mouse brain development and in adult brain regions (Dragich et al. 2007, Olson et al. 2014). While Rett syndrome mutations mainly occur in exons 3 and 4 of MECP2, thereby affecting both MECP2 isoforms (Mnatzakanian et al. 2004), some mutations occur in exon 1, affecting MECP2_e1 only. No mutations have been described in exon 2 (Gianakopoulos et al. 2012). Knockout of Mecp2_e1 isoform in mice, through a naturally occurring Rett syndrome point mutation which affects the first translation codon of MECP2_e1, recapitulates Rett-like phenotype. Knockout of Mecp2_e2 isoform in mice does not result in impairment of neurologic functions (Yasui et al. 2014). In Mecp2 null mice, transgenic expression of either Mecp2_e1 or Mecp2_e2 prevents development of Rett-like phenotype, with Mecp2_e1 rescuing more Rett-like symptoms than Mecp2_e2. This indicates that both splice variants can fulfill basic Mecp2 functions in the mouse brain (Kerr et al. 2012). Changes in gene expression upon over-expression of either MECP2_e1 or MECP2_e2 imply overlapping as well as distinct target genes (Orlic-Milacic et al. 2014).

Methyl-CpG-binding protein 2 encoded by the MECP2 gene binds to methylated CpG sequences in the DNA. The binding is not generic, however, but is affected by the underlying DNA sequence (Yoon et al. 2003). MECP2 binds to DNA containing 5 methylcytosine (5mC DNA), a DNA modification associated with transcriptional repression (Mellen et al. 2012), both in the context of CpG islands and outside of CpG islands (Chen et al. 2015). In addition, MECP2 binds to DNA containing 5 hydroxymethylcytosine (5hmC DNA), a DNA modification associated with transcriptional activation (Mellen et al. 2012). MECP2 binds to DNA as a monomer, occupying about 11 bp of the DNA. Binding of one MECP2 molecule facilitates binding of the second MECP2 molecule, and therefore clustering can occur at target sites. MECP2 binding to chromatin may be facilitated by nucleosome methylation (Ghosh et al. 2010).

MECP2 was initially proposed to act as a generic repressor of gene transcription. However, high throughput studies of MECP2-induced changes in gene expression in mouse hippocampus (Chahrour et al. 2008), and mouse and human cell lines (Orlic-Milacic et al. 2014) indicate that more genes are up-regulated than down-regulated when MECP2 is overexpressed. At least for some genes directly upregulated by MECP2, it was shown that a complex of MECP2 and CREB1 was involved in transcriptional stimulation (Chahrour et al. 2008, Chen et al. 2013).

MECP2 expression is the highest in postmitotic neurons compared to other cell types, with MECP2 being almost as abundant as core histones. Phosphorylation of MECP2 in response to neuronal activity regulates binding of MECP2 to DNA, suggesting that MECP2 may remodel chromatin in a neuronal activity-dependent manner. The resulting changes in gene expression would then modulate synaptic plasticity and behavior (reviewed by Ebert and Greenberg 2013). In human embryonic stem cell derived Rett syndrome neurons, loss of MECP2 is associated with a significant reduction in transcription of neuronally active genes, as well as the reduction in nascent protein synthesis. The reduction in nascent protein synthesis can at least in part be attributed to the decreased activity of the PI3K/AKT/mTOR signaling pathway. Neuronal morphology (reduced soma size) and the level of protein synthesis in Rett neurons can be ameliorated by treating the cells with growth factors which activate the PI3K/AKT/mTOR cascade or by inhibition of PTEN, the negative regulator of AKT activation. Mitochondrial gene expression is also downregulated in Rett neurons, which is associated with a reduced capacity of the mitochondrial electron transport chain (Ricciardi et al. 2011, Li et al. 2013). Treatment of Mecp2 null mice with IGF1 (insulin-like growth factor 1) reverses or ameliorates some Rett-like features such as locomotion, respiratory difficulties and irregular heart rate (Tropea et al. 2009).

MECP2 regulates expression of a number of ligands and receptors involved in neuronal development and function. Ligands regulated by MECP2 include BDNF (reviewed by Li and Pozzo-Miller 2014, and KhorshidAhmad et al. 2016), CRH (McGill et al. 2006, Samaco et al. 2012), SST (Somatostatin) (Chahrour et al. 2008), and DLL1 (Li et al. 2014). MECP2 also regulates transcription of genes involved in the synthesis of the neurotransmitter GABA – GAD1 (Chao et al. 2010) and GAD2 (Chao et al. 2010, He et al. 2014). MECP2 may be involved in direct stimulation of transcription from the GLUD1 gene promoter, encoding mitochondrial glutamate dehydrogenase 1, which may be involved in the turnover of the neurotransmitter glutamate (Livide et al. 2015). Receptors regulated by MECP2 include glutamate receptor GRIA2 (Qiu et al. 2012), NMDA receptor subunits GRIN2A (Durand et al. 2012) and GRIN2B (Lee et al. 2008), opioid receptors OPRK1 (Chahrour et al. 2008) and OPRM1 (Hwang et al. 2009, Hwang et al. 2010, Samaco et al. 2012), GPRIN1 (Chahrour et al. 2008), MET (Plummer et al. 2013), NOTCH1 (Li et al. 2014). Channels/transporters regulated by MECP2 include TRPC3 (Li et al. 2012) and SLC2A3 (Chen et al. 2013). MECP2 regulates transcription of FKBP5, involved in trafficking of glucocorticoid receptors (Nuber et al. 2005, Urdinguio et al. 2008). MECP2 is implicated in regulation of expression of SEMA3F (semaphorin 3F) in mouse olfactory neurons (Degano et al. 2009). In zebrafish, Mecp2 is implicated in sensory axon guidance by direct stimulation of transcription of Sema5b and Robo2 (Leong et al. 2015). MECP2 may indirectly regulate signaling by neuronal receptor tyrosine kinases by regulating transcription of protein tyrosine phosphatases, PTPN1 (Krishnan et al. 2015) and PTPN4 (Williamson et al. 2015).

MECP2 regulates transcription of several transcription factors involved in functioning of the nervous system, such as CREB1, MEF2C, RBFOX1 (Chahrour et al. 2008) and PPARG (Mann et al. 2010, Joss-Moore et al. 2011).

MECP2 associates with transcription and chromatin remodeling factors, such as CREB1 (Chahrour et al. 2008, Chen et al. 2013), the HDAC1/2-containing SIN3A co-repressor complex (Nan et al. 1998), and the NCoR/SMRT complex (Lyst et al. 2013, Ebert et al. 2013). There are contradictory reports on the interaction of MECP2 with the SWI/SNF chromatin-remodeling complex (Harikrishnan et al. 2005, Hu et al. 2006). Interaction of MECP2 with the DNA methyltransferase DNMT1 has been reported, with a concomitant increase in enzymatic activity of DNMT1 (Kimura and Shiota 2003).

In addition to DNA binding-dependent regulation of gene expression by MECP2, MECP2 may influence gene expression by interaction with components of the DROSHA microprocessor complex and the consequent change in the levels of mature microRNAs (Cheng et al. 2014, Tsujimura et al. 2015).

Increased MECP2 promoter methylation is observed in both male and female autism patients (Nagarajan et al. 2008). Regulatory elements that undergo methylation are found in the promoter and the first intron of MECP2 and their methylation was shown to regulate Mecp2 expression in mice (Liyanage et al. 2013). Mouse Mecp2 promoter methylation was shown to be affected by stress (Franklin et al. 2010).

The Rett-like phenotype of Mecp2 null mice is reversible (Guy et al. 2007), but appropriate levels of Mecp2 expression need to be achieved (Alvarez-Saavedra et al. 2007). When Mecp2 expression is restored in astrocytes of Mecp2 null mice, amelioration of Rett symptoms occurs, involving non-cell-autonomous positive effect on mutant neurons and increasing level of the excitatory glutamate transporter VGLUT1 (Lioy et al. 2011). Microglia derived from Mecp2 null mice releases higher than normal levels of glutamate, which has toxic effect on neurons. Increased glutamate secretion may be due to increased levels of glutaminase (Gls), involved in glutamate synthesis, and increased levels of connexin-32 (Gjb1), involved in glutamate release, in Mecp2 null microglia (Maezawa and Jin 2010). Targeted deletion of Mecp2 from Sim1-expressing neurons of the mouse hypothalamus recapitulates some Rett syndrome-like features and highlights the role of Mecp2 in feeding behavior and response to stress (Fyffe et al. 2008).

Mecp2 overexpression, similar to MECP2 duplication syndrome, causes neurologic phenotype similar to Rett (Collins et al. 2004, Luikenhuis et al. 2004, Van Esch et al. 2005, Alvarez-Saavedra 2007, Van Esch et al. 2012). The phenotype of the mouse model of the MECP2 duplication syndrome in adult mice is reversible when Mecp2 expression levels are corrected (Sztainberg et al. 2015).

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