Suppressor of Hairless


RBP-Jkappa interaction with the transcriptional apparatus and other nuclear factors

RBP is a cellular protein that functions as a transcriptional repressor in mammalian cells. RBP has elicited great interest recently because of its established roles in regulating gene expression, in Drosophila and mouse development, and as a component of the Notch signal transduction pathway. This report focuses on the mechanism by which RBP represses transcription and thereby regulates expression of a relatively simple, but natural, promoter. The results show that, irrespective of the close proximity between RBP and other transcription factors bound to the promoter, RBP does not occlude binding by these other transcription factors. Instead, RBP interacts with two transcriptional coactivators to repress transcription: dTAFII110, a subunit of TFIID, and TFIIA. The domain of dTAFII110 targeted by RBP is the same domain that interacts with TFIIA, but is disparate from the domain that interacts with Sp1. Repression can be thwarted when stable transcription preinitiation complexes are formed before RBP addition, suggesting that RBP interaction with TFIIA and TFIID perturbs optimal interactions between these coactivators. Consistent with this, interaction between RBP and TFIIA precludes interaction with dTAFII110. This is the first report of a repressor specifically targeting these two coactivators to subvert activated transcription (Olave, 1998).

The RBP-J/Su(H) DNA-binding protein plays a key role in transcriptional regulation by targeting to specific promoters the Epstein-Barr virus nuclear antigen 2 (EBNA2) and the intracellular portions of Notch receptors. Using the yeast two-hybrid system, a LIM-only protein, KyoT (See Drosophila Muscle LIM protein at 60A), has been isolated that physically interacts with RBP-J. Differential splicing gives rise to two transcripts of the KyoT gene, KyoT1 and KyoT2, that encode proteins with four and two LIM domains, respectively. With differential splicing resulting in deletion of an exon, KyoT2 lacks two LIM domains from the C terminus and has a frameshift in the last exon, creating the RBP-J-binding region in the C terminus. KyoT1 has a negligible level of interaction with RBP-J. Strong expression of KyoT mRNAs is detected in skeletal muscle and lung, with a predominance of KyoT1 mRNA. When expressed in F9 embryonal carcinoma cells, KyoT1 and KyoT2 are localized in the cytoplasm and the nucleus, respectively. The binding site of KyoT2 on RBP-J overlaps those of EBNA2 and Notch1 but is distinct from that of Hairless, the negative regulator of RBP-J-mediated transcription in Drosophila. KyoT2 (but not KyoT1) represses the RBP-J-mediated transcriptional activation by EBNA2 and Notch1 by competing with them for binding to RBP-J and by dislocating RBP-J from DNA. KyoT2 is a novel negative regulatory molecule for RBP-J-mediated transcription in mammalian systems (Taniguchi, 1998).

Within the human ERBB-2 gene promoter, a 100-base pair region 5' to the TATA box enhances basal transcription 200-fold. Two palindromes present within this 100-base pair region are important for transcription. The palindrome binding protein was purified to homogeneity and found to be identical to RBPJkappa, the mammalian homolog of Drosophila Suppressor of Hairless (Su[H]). Recombinant RBPJkappa binds the ERBB-2 promoter with affinity comparable to that seen with well characterized RBPJkappa binding sites. RBPJkappa activates an ERBB-2 palindrome-containing promoter in 293 cells. Because in Drosophila Su(H) acts downstream of NOTCH and because the NOTCH.Su(H)/RBPJkappa protein complex stimulates transcription from target promoters, NOTCH-IC, a constitutively active form of NOTCH, was tested for effects on the ERBB-2 palindrome. NOTCH-IC further increases RBPJkappa-mediated transcription on wild type but not mutant ERBB-2 palindrome. Thus, RBPJkappa can activate ERBB-2 transcription and serve as an anchor to mediate NOTCH function on the ERBB-2 gene (Chen, 1997).

The whole Notch pathway is evolutionarily conserved in vertebrates. Activated forms of mouse Notch (mNotch) associate with the human analogue of Suppressor of Hairless, KBF2/RBP-Jkappa, and act as transcriptional activator of the Hairy enhancer of split locus, acting through the KBF2-binding site (Jarriault, 1996).

Epstein-Barr virus proteins interact with the mammalian homolog of Su(H). The ability of Epstein-Barr virus (EBV) latent infection nuclear protein EBNA3C to activate transcription of two EBNA2-responsive genes and to inhibit EBNA2 activation of transcription in transient-transfection assays appears to be due to its ability to interact with RBPJkappa, a cell protein that links EBNA2 to its response elements. EBNA3A and EBNA3B are similar to EBNA3C in binding to RBPJkappa. EBNA3A and EBNA3B can also inhibit the interaction of RBPJkappa with cognate DNA in vitro. Although EBNA3 open reading frames are each close to 1,000 codons long, EBNA3A amino acids 1 to 138, EBNA3B amino acids 1 to 311, and EBNA3C amino acids 1 to 183 are sufficient for RBPJkappa interaction, while EBNA3B amino acids I to 109 have less or no binding. The RBPJkappa interacting domains overlap with the most highly conserved domain (amino acids 90 to 320) among the EBNA3 proteins. Thus, the EBNA3 gene family appears to have evolved to differentially regulate promoters with RBPJkappa binding sites. EBNA2, EBNA3A, and EBNA3C are important in EBV transformation of primary human B lymphocytes. Their interaction with RBPJkappa links EBV transformation to the notch signaling pathway and the effects of activated Notch in T-cell leukemogenesis (Robertson, 1966).

Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (DeltaE) or consisting only of the intracellular domain (ICN) are found to activate transcription in cultured cells, presumably through RBP-Jkappa response elements within DNA. Both truncated forms also bind to the transcription factor RBP-Jkappa in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation requires the presence of a weak RBP-Jkappa-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jkappa-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jkappa, is not needed for oncogenesis or for transcriptional activation. While ICN appears primarily in the nucleus, DeltaE localizes to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreases nuclear localization and increases transcriptional activation by membrane-bound DeltaE. Transcriptional activation by this mutant form of membrane-bound DeltaE is approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jkappa at promoter sites (Aster, 1997).

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) is a transcriptional activator that is essential for EBV-driven B cell immortalization. EBNA2 is targeted to responsive promoters through interaction with a cellular DNA binding protein, C promoter binding factor 1 (CBF1). A transcriptional repression domain has been identified within CBF1. This domain also interacts with EBNA2, and repression is masked by EBNA2 binding. Thus, EBNA2 acts by countering transcriptional repression. Mutation at amino acid 233 of CBF1 abolishes repression and correlates with a loss-of-function mutation in the Drosophila homolog Su(H) (Hsieh, 1995).

Mammalian Notch intracellular domain (NotchIC) interacts with the transcriptional repressor CBF1, which is the human homolog of Drosophila Suppressor of Hairless. The N-terminal 114-amino-acid region of mouse NotchIC contains the CBF1 interactive domain and the cdc10/ankyrin repeats are not essential for this interaction. This result was confirmed in immunoprecipation assays in which the N-terminal 114-amino-acid segment of NotchIC, but not the ankyrin repeat region, coprecipitated with CBF1. Mouse NotchIC itself is targeted to the transcriptional repression domain (aa179 to 361) of CBF1. NotchIC transactivates gene expression via CBF1 tethering to DNA. Transactivation by NotchIC occurs partially through abolition of CBF1-mediated repession. This same mechanism is used by Epstein-Barr virus EBNA2. Thus, mimicry of Notch signal transduction is involved in Epstein-Barr virus-driven immortalization (Hsieh, 1996).

EBNA2 is essential for immortalization of B cells by Epstein-Barr virus. EBNA2 is tethered to responsive promoters through a cellular factor, CBF1. CBF1 also binds to the activated form of mammalian Notch1, providing a linkage between EBNA2 function and Notch signaling. However, Notch2 is the predominant form expressed in spleen. The degree to which these Notch homologs are functionally convergent is not known. Evidence is presented that Notch2 also signals through CBF1. As is the case for Notch1, Notch2 interacts with the minimal repression domain of CBF1 and is targeted to CBF1 through the intracellular, subtransmembrane domain. Additional characterization suggests that the interaction domain of Notch may be bipartite. The intracellular domain of Notch2 (Notch2IC) locates to the nucleus. This activated form of Notch2 transactivates expression of a target gene containing upstream CBF1 binding sites. The use of CBF1 mutants carrying amino acid substitutions in the transcriptional repression domain reveals that activation of gene expression by Notch2 is also based on masking of CBF1-mediated repression. Targeting of Notch1 and targeting of Notch2 are found to be identical and distinguishable from targeting by EBNA2. Mutation of CBF1 at codons 249 to 251 abolishes interaction with both Notch proteins but not with EBNA2. In a biological examination of Notch2 function in muscle cells, Notch2IC activates endogenous HES-1 gene expression and blocks muscle cell differentiation. Overall, the data imply that at least a subset of the intracellular events following signaling in cells expressing Notch2 are common to those in Notch1-expressing cells. The concept that EBNA2 functions by mimicking Notch signaling is therefore viable whether cells are expressing Notch1 or Notch2 (Hsieh, 1997).

CBF1 is a member of the CSL family of DNA binding factors, which mediate either transcriptional repression or transcriptional activation. CSL proteins play a central role in Notch signaling and in Epstein-Barr virus-induced immortalization. Notch is a transmembrane protein involved in cell-fate decisions, and the cytoplasmic domain of Notch (NotchIC) targets CBF1. The Epstein-Barr virus-immortalizing protein EBNA2 activates both cellular and viral gene expression by targeting CBF1 and mimicking NotchIC. The mechanism of CBF1-mediated repression has been examined and shows that CBF1 binds to a unique corepressor, CBF1 interacting corepressor (CIR: Drosophila homolog CG6843). A CIR homolog is encoded by Caenorhabditis elegans, indicating that CIR is evolutionarily conserved. Two CBF1 mutants that are unable to bind CIR do not function as repressors, suggesting that targeting of CIR to CBF1 is an important component of repression. When expressed as a Gal4 fusion protein, CIR represses reporter gene expression. CIR binds to histone deacetylase and to SAP30 and serves as a linker between CBF1 and the histone deacetylase complex (Hsieh, 1999).

CBF1/RBP-Jkappa, the mammalian homolog of Drosophila Suppressor of Hairless [Su(H)], switches from a transcriptional repressor to an activator upon Notch activation. The mechanism by which Notch regulates this switch is not clear. Prior to induction, CBF1/RBP-Jkappa interacts with a corepressor complex containing SMRT (silencing mediator of retinoid and thyroid hormone receptors) and the histone deacetylase HDAC-1. This complex binds via the CBF1 repression domain, and mutants defective in repression fail to interact with the complex. Activation by Notch disrupts the formation of the repressor complex, thus establishing a molecular basis for the Notch switch. ESR-1, a Xenopus gene activated by Notch and X-Su(H), is induced in animal caps treated with TSA, an inhibitor of HDAC-1. The functional role for the SMRT/HDAC-1 complex in CBF1/RBP-Jkappa regulation reveals a novel genetic switch in which extracellular ligands control the status of critical nuclear cofactor complexes (Kao, 1998).

SMRT has been shown to associate with mSin3A (see Drosophila Sin3A) and the histone deacetylase HDAC1 as part of a large corepressor complex. It was reasoned that CBF1-mediated repression should be compromised in the presence of an inhibitor of histone deacetylase such as trichostatin A (TSA). This was tested in Xenopus animal caps, which are known to respond to the Notch signaling pathway. Specifically, the effects of TSA were assessed on the expression of ESR-1, an E(spl)-related gene from Xenopus. Expression of ESR-1, like similar genes in Drosophila, is induced in neural tissue by activated (cytoplasmic) forms of Xenopus Notch or by the Notch ligand, X-Delta-1. Because induction of ESR-1 expression by Notch appears to be mediated by Xenopus Su(H) [X-Su(H)], it was asked whether induction is enhanced by TSA. Such a result would support the idea that X-Su(H) might inhibit the expression of Notch target genes such as ESR-1 via the SMRT deacetylase complex. Expression of ESR-1 in neuralized animal caps is induced by X-Delta-1 in a dose-dependent manner, with the effects of X-Delta-1 on ESR-1 expression saturating at 1 ng of injected RNA. In the presence of TSA, the induction of ESR-1 transcripts in response to X-Delta-1 is enhanced two- to threefold at each dose. In the presence of TSA, the lowest dose of X-Delta-1 RNA (0.25 ng) induces levels of ESR-1 mRNA comparable to the saturating level induced in the absence of TSA. These results are consistent with the prediction that Notch target genes are derepressed by treatment with TSA, possibly through the inhibition of SMRT-associated histone deacetylases. To assay whether HDAC-1 associates with CBF1 in vivo, coimmunoprecipitation experiments were performed. Cells were transfected with Flag-CBF1 in the presence or absence of HDAC-1 and immunoprecipitated with Flag antibody. The HDAC-1 is coimmunoprecipiated only in the presence of CBF1. An interaction between CBF1 and HDAC-1 was also assessed through GST pull-down assays. HDAC-1 interacts with GST-CBF1. As with SMRT, HDAC-1 does not interact with a repression-defective mutant, CBF1. On the basis of these results, a model is proposed to explain this switch in which CBF1/RBP-Jkappa mediates repression of genes through the recruitment of a corepressor complex containing SMRT and histone deacetylase activity. In the absence of any positive acting factor, that is, TAN-1 (translocation-associated Notch, a truncated form of Notch1 that contains only the cytoplasmic domain), the CBF1/RBP-Jkappa exists as a corepressor complex. In the presence of activated Notch signaling, the intracellular domain of Notch would translocate to the nucleus and displace the corepressor complex. It remains to be tested whether a Notch/CBF1 complex recruits a coactivating complex (Kao, 1998).

EBNA2 is essential for Epstein-Barr virus (EBV) immortalization of B lymphocytes. EBNA2 functions as a transcriptional activator and targets responsive promoters through interaction with the cellular DNA binding protein CBF1. The mechanism whereby EBNA2 overcomes CBF1-mediated transcriptional repression has been examined. A yeast two-hybrid screen performed using CBF1 as the bait identified a protein, SKIP (Ski-interacting protein: Drosophila homolog Bx42), which had not previously been recognized as a CBF1-associated protein. Protein-protein interaction assays demonstrate contacts between SKIP and the SMRT, CIR, Sin3A, and HDAC2 proteins of the CBF1 corepressor complex. Interestingly, EBNA2 also interacts with SKIP in glutathione S-transferase affinity and mammalian two-hybrid assays and colocalizes with SKIP in immunofluorescence assays. Interaction with SKIP is not affected by mutation of EBNA2 conserved region 6, the CBF1 interaction region, but is abolished by mutation of conserved region 5. Mutation of conserved region 5 also severely impairs EBNA2 activation of a reporter containing CBF1 binding sites. Thus, interaction with both CBF1 and SKIP is necessary for efficient promoter activation by EBNA2. A model is presented in which EBNA2 competes with the SMRT-corepressor complex for contacts on SKIP and CBF1 (Zhou, 2000a).

The activated intracellular form of Notch, NotchIC, translocates to the nucleus, where it targets the DNA binding protein CBF1. CBF1 mediates transcriptional repression through the recruitment of an SMRT-histone deacetylase-containing corepressor complex. The mechanism whereby NotchIC overcomes CBF1-mediated transcriptional repression has been examined. SKIP has been identified as a CBF1 binding protein in a yeast two-hybrid screen. Both CBF1 and SKIP are highly conserved evolutionarily, and the SKIP-CBF1 interaction is also conserved in assays using the Caenorhabditis elegans and Drosophila melanogaster SKIP homologs. Protein-protein interaction assays demonstrate interaction between SKIP and the corepressor SMRT. Even more surprising, SKIP also interacts with NotchIC. The SMRT and NotchIC interactions are mutually exclusive. In competition binding experiments SMRT displaces NotchIC from CBF1 and from SKIP. Contact with SKIP is required for biological activity of NotchIC. A mutation in the fourth ankyrin repeat that abolishes Notch signal transduction does not affect interaction with CBF1 but abolishes interaction with SKIP. Further, NotchIC is unable to block muscle cell differentiation in myoblasts expressing antisense SKIP. The results suggest a model in which NotchIC activates responsive promoters by competing with the SMRT-corepressor complex for contacts on both CBF1 and SKIP (Zhou, 2000b).

Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans. In Drosophila, loss-of-function mutations of Notch produce a 'neurogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus, and interacts directly with the DNA-binding protein Suppressor of hairless [Su(H)] in flies, or recombination signal binding protein Jkappa (RBP-Jkappa) in mammals, to activate gene transcription. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila. MAML1, a human homolog of the Drosophila gene Mastermind, has been cloned; it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jkappa, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signaling (Wu, 2000).

Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus, where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-Jk. The mammalian Notch receptors are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. This report explores the molecular basis for this functional difference between Notch 1 and Notch 3 IC. Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-Jk on the HES promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-Jk in vivo, and that the difference in activation capacity instead stems from structural differences in the two ICs when positioned on RBP-Jk. Two distinct regions in the Notch IC are critical for the difference between the Notch 1 and Notch 3 IC; (1) the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators; (2) a novel important region has been identified in the Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region, and is required for Notch 1 IC's ability to activate and for Notch 3 IC's ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors (Beatus, 2001).

The Lin12/Notch receptors regulate cell fate during embryogenesis by activating the expression of downstream target genes. These receptors signal via their intracellular domain (ICD), which is released from the plasma membrane by proteolytic processing and associates in the nucleus with the CSL family of DNA-binding proteins to form a transcriptional activator. How the CSL/ICD complex activates transcription and how this complex is regulated during development remains poorly understood. Nrarp is a new intracellular component of the Notch signaling pathway in Xenopus embryos. Nrarp is a member of the Delta-Notch synexpression group and encodes a small protein containing two ankyrin repeats. Nrarp expression is activated in Xenopus embryos by the CSL-dependent Notch pathway. Conversely, overexpression of Nrarp in embryos blocks Notch signaling and inhibits the activation of Notch target genes by ICD. Nrarp forms a ternary complex with the ICD of XNotch1 and the CSL protein XSu(H) and in embryos Nrarp promotes the loss of ICD. By down-regulating ICD levels, Nrarp could function as a negative feedback regulator of Notch signaling that attenuates ICD-mediated transcription (Lamar, 2001).

Both Nrarp and Mastermind form ternary complexes with the CSL proteins and ICD. It was asked, therefore, whether the binding of Nrarp and Mastermind to Su(H) and ICD is mutually exclusive or whether these proteins can exist in a complex together. Binding of human Mastermind (hMM) to XSu(H) and ICD from XNotch1 was examined first by co-IP analysis in which extracts were prepared from embryos injected with RNA encoding Flag-tagged XSu(H), myc-tagged hMM, and myc-tagged ICDDeltaC. Flag-tagged XSu(H) was recovered from total extracts, and associated proteins were analyzed by Western analysis. The results show that hMM is co-IPed detectably with XSu(H), but only in the presence of ICDDeltaC. Moreover, the amounts of ICDDeltaC associated with XSu(H) in a co-IP complex increase markedly in the presence of hMM. Both of these results are consistent with the idea that hMM binds to XSu(H) and ICD in a ternary complex, as reported by others. Myc-tagged Nrarp is also co-IPed with XSu(H) in the presence of hMM and ICDDeltaC, consistent with the formation of multimeric complexes. However, this finding is inconclusive as to whether these proteins can form a quaternary complex. To address this issue, embryos were injected with RNA encoding Flag-tagged Nrarp along with myc-tagged hMM, XSu(H), and ICD. Flag-tagged Nrarp was recovered from extracts by immunoprecipitation, and associated proteins were analyzed by Western analysis using an alpha-myc antibody. The results show that the immunoprecipitation of Nrarp recovers not only XSu(H) and ICD, but hMM as well, indicating that Nrarp and hMM can bind in tandem to XSu(H)/ICD to form a quaternary complex (Lamar, 2001).

Although Nrarp may contribute to the formation of an active Su(H)/ICD transcriptional complex, it is proposed that the primary role of Narp is to regulate Notch signaling by promoting the degradation of ICD. Several lines of evidence indicate that only very small amounts of ICD need to be released from the membrane to produce maximal stimulation of Notch target genes. For example, ICD is very difficult to detect in the nucleus of cells undergoing Notch signaling even though these levels of ICD are apparently sufficient to produce significant changes in gene expression. One would predict, therefore, that under normal physiological conditions Nrarp is likely to have a dramatic effect on ICD-mediated transcription, in which a low level of ICD is already a rate-limiting factor. Moreover, Nrarp binds to ICD only when it is part of a complex with Su(H), suggesting that only after ICD forms an active transcriptional complex does it become a target of degradation via Nrarp. This mechanism is consistent with recent findings showing that activation of some transcription factors is coupled to their degradation. For example, the melanocyte factor Mi is phosphorylated in response to c-Kit signaling, resulting in both recruitment of coactivator proteins and ubiquitination and subsequent degradation of Mi. Similarly, ligand binding converts the estrogen receptor into a transcriptional activator but also promotes its degradation (Lamar, 2001).

The ability of Nrarp to regulate the levels of ICD may constitute a negative feedback loop that attenuates Notch signaling, assuring that activation of Notch target genes is transient. Notch is used ubiquitously to amplify differences between cells, thus allowing certain patterns of cell fate to be established. As a result, Notch signaling is often used in rapid succession to control multiple cell fate decisions in the same lineage. A mechanism for down-regulating the levels of ICD produced after each decision by Nrarp would allow each decision to remain separate from the next. This rapid resetting of the Notch signaling pathway is particularly evident during segmentation of the paraxial mesoderm in vertebrate embryos, in which the transcription of a number of Notch pathway genes has been shown to oscillate in hourly intervals with a pattern suggestive of a segmental clock. The role of Nrarp in the formation of this periodic pattern of gene expression is of particular interest based on its expression in the presomitic mesoderm and its potential role in down-regulating the levels of ICD required for Notch-mediated transcription (Lamar, 2001).

The RTA protein of the Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is responsible for the switch from latency to lytic replication, a reaction essential for viral spread and KS pathogenesis. RTA is a sequence-specific transcriptional activator, but the diversity of its target sites suggests it may act via interaction with host DNA-binding proteins as well. KSHV RTA interacts with the RBP-Jkappa protein, the primary target of the Notch signaling pathway. This interaction targets RTA to RBP-Jkappa recognition sites on DNA and results in the replacement of RBP-Jkappa's intrinsic repressive action with activation mediated by the C-terminal domain of RTA. Mutation of such sites in target promoters strongly impairs RTA responsiveness. Similarly, such target genes are induced poorly or not at all by RTA in fibroblasts derived from RBP-Jkappa-/- mice, a defect that can be reversed by expression of RBP-Jkappa. In vitro, RTA binds to two adjacent regions of RBP-Jkappa, one of which is identical to the central repression domain that binds the Notch effector fragment. These results indicate that KSHV has evolved a ligand-independent mechanism for constitutive activation of the Notch pathway as a part of its strategy for reactivation from latency (Liang, 2002).

In the absence of Notch signaling, RBP-J/CBF-1 acts as a transcriptional repressor through the recruitment of histone deacetylase (HDAC) corepressor complexes. SHARP, a homolog of Drosophila Split ends, has been identified as an RBP-J/CBF-1-interacting corepressor in a yeast two-hybrid screen. In cotransfection experiments, SHARP-mediated repression is sensitive to the HDAC inhibitor TSA and is facilitated by SKIP, a highly conserved SMRT and RBP-J-interacting protein. SHARP represses Hairy/Enhancer of split (HES)-1 promoter activity, inhibits Notch-1-mediated transactivation and rescues Notch-1-induced inhibition of primary neurogenesis in Xenopus laevis embryos. Based on these data, a model is proposed in which SHARP is a novel component of the HDAC corepressor complex, recruited by RBP-J to repress transcription of target genes in the absence of activated Notch (Oswald, 2002).

Thus SHARP is a novel RBP-Jkappa/CBF-1-interacting protein. Functional analyses revealed that SHARP acts as a corepressor, and sequence analysis revealed the presence of four putative RNA recognition motifs (RRMs) and five putative nuclear localization signals. The interaction domain of SHARP with RBP-Jkappa maps to amino acids 2803–2817. SHARP belongs to the Spen (split ends) family of proteins, which contains RRMs in their N-terminal part and a highly conserved C-terminal domain called the SPOC domain. The SPOC domain has been shown to be essential for Spen function in Drosophila. Spen-like proteins have been identified in Caenorhabditis elegans, Drosophila, mouse and human. Spen proteins are detectable as early as the cellular blastoderm in Drosophila and are ubiquitously nuclear during early development. Expression of SHARP overlaps with expression of Notch-1; however, SHARP is also expressed in other tissues, suggesting that SHARP may have other functions in addition to modulation of Notch signaling during embryogenesis (Oswald, 2002).

SHARP reduces transactivation mediated by RBP–VP16. This reduction is sensitive to TSA, indicating that in vivo SHARP may act as a repressor. This is consistent with the ability of SHARP to interact with HDACs and components of the NuRD complex. SHARP also represses the activity of a luciferase construct derived from the human HES–1 promoter. In this case, repression is dependent on functional RBP-binding sites. Two models of RBP-Jkappa-mediated repression have been postulated. RBP-Jkappa has been shown to interact with the transcriptional coactivators TFIIA and dTAFII110, a subunit of TFIID. The domain of dTAFII110 targeted by RBP-Jkappa is the same domain that interacts with TFIIA, but is different from the domain that interacts with SP1. Repression can be thwarted when stable transcription pre-initiation complexes are formed before RBP-Jkappa addition, suggesting that RBP-Jkappa interaction with TFIIA and TFIID perturbs optimal interactions between these cofactors. In contrast to the specific targeting of the basal transcription machinery by RBP-Jkappa, others have favored the model of transcriptional repression via chromatin remodelling by recruiting HDAC corepressor complexes to RBP-Jkappa. RBP-Jkappa has been shown to interact with a corepressor complex containing SMRT and the histone deacetylase HDAC-1. In addition, CBF1-interacting corepressor (CIR) was isolated in a two-hybrid screen using RBP-Jkappa/CBF-1 as a bait. CIR acted as a repressor in transient transfection assays, and binds to histone deacetylase and SAP30 (Oswald, 2002).

Does SHARP play a role in Notch signaling? An important function of Notch signaling is to limit the number of neurons developing from neural precursor cells via lateral inhibition. Overexpression of a dominant active form of Notch results in a reduction of primary neurons in Xenopus embryogenesis. Conversely, inhibition of Notch signaling results in the production of more primary neurons in the neural plate. Coinjection of SHARP mRNA with Notch-1DeltaE rescued the formation of primary neurons normally blocked by excessive Notch signaling. Additionally, overexpression of SHARP alone induced a neurogenic phenotype. Taken together, the data suggest that SHARP might be a novel molecular component of the switch from repression to transactivation of target genes during Notch signaling (Oswald, 2002).

Protein–protein interaction and DNA-binding studies suggest that RBP interacts either with SHARP or with Notch-IC, supporting the idea that formation of SHARP-containing or Notch-containing RBP complexes could be exclusive. A model is presented in which different DNA-bound RBP complexes mediate transcriptional repression versus transactivation. Gel filtration experiments have identified a 1.5 MDa Notch-IC 'enhancer complex', indicating that the activator (and probably also repressor complex discussed here) contains many as yet not identified component proteins. However, SHARP may act as a molecular switch for the conversion of the activator into the repressor in the absence of activated Notch. In this model, SHARP would function as an adapter to link RBP with the HDAC chromatin remodelling machinery. This is supported by the reported interaction between SHARP and SMRT, the colocalization of SHARP and RBP as well as SHARP and SKIP, and the fact that SHARP function is sensitive to an HDAC inhibitor. SHARP was originally identified as a corepressor involved in steroid receptor signaling. The results reported here support an additional role for SHARP as a corepressor in Notch signaling. In addition, it is suggested that SHARP may play a general role as a molecular switch to create repression complexes in Notch-independent signaling pathways (Oswald, 2002).

Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After ligand binding, proteolytic cleavage steps occur and the intracellular part of Notch translocates to the nucleus, where it targets the DNA-binding protein RBP-Jkappa/CBF1. In the absence of Notch, RBP-Jkappa represses Notch target genes through the recruitment of a corepressor complex. SHARP has been identified as a component of this complex. This study functionally demonstrates that the SHARP repression domain is necessary and sufficient to repress transcription and that the absence of this domain causes a dominant negative Notch-like phenotype. The CtIP and CtBP corepressors were identified as novel components of the human RBP-Jkappa/SHARP-corepressor complex; CtIP binds directly to the SHARP repression domain. Functionally, CtIP and CtBP augment SHARP-mediated repression. Transcriptional repression of the Notch target gene Hey1 is abolished in CtBP-deficient cells or after the functional knockout of CtBP. Furthermore, the endogenous Hey1 promoter is derepressed in CtBP-deficient cells. It is proposed that a corepressor complex containing CtIP/CtBP facilitates RBP-Jkappa/SHARP-mediated repression of Notch target genes (Oswald, 2005).

Assembly of a Notch transcriptional activation complex requires multimerization

Notch transmembrane receptors direct essential cellular processes, such as proliferation and differentiation, through direct cell-to-cell interactions. Inappropriate release of the intracellular domain of Notch (NICD) from the plasma membrane results in the accumulation of deregulated nuclear NICD that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear NICD forms a transcriptional activation complex by interacting with the coactivator protein Mastermind-like 1 and the DNA binding protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) to regulate target gene expression. Although it is well understood that NICD forms a transcriptional activation complex, little is known about how the complex is assembled. This study demonstrates that ICD multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly, it was demonstrated that the assembly is mediated by NICD multimers interacting with Skip (the human homolog of Drosophila Bx42). and Mastermind. These interactions form a preactivation complex that is then resolved by CSL to form the Notch transcriptional activation complex on DNA (Vasquez-Del Carpio, 2011).

Previous studies have demonstrated that NICD forms two distinct protein complexes in cells. One complex is predominately localized in the nucleus and is composed of NICD, Maml1, and CSL. This complex is thought to be the transcriptionally active form of Notch on DNA. In addition, a smaller Notch-containing complex is also detected in cells. This complex is primarily localized in the cytoplasm and does not contain either Maml1 or CSL. The nature and function of this complex has remained unclear. This study demonstrates that prior to forming a transcriptionally active complex, NICD forms multimers, and these multimers serve as precursors to the assembly of Notch activation complexes. Evidence is provided for a stepwise assembly of the Notch activation complex that is mediated by Skip and Maml1. It includes the formation of a preactivation complex composed of Skip, Maml1, and NICD multimers. This intermediate complex is then resolved by interaction with CSL, resulting in the formation of the Notch activation complex consisting of monomeric NICD, Maml1, and CSL (Vasquez-Del Carpio, 2011).

Maml1 is an essential component in Notch signaling, forming a stable complex with NICD and CSL and functioning as a 'coactivator'. A critical role of this protein in the active complex was observed when Maml1 deletion mutants that bind NICD but lack the C-terminal region inhibit Notch transactivation and can act as dominant negatives in Notch signaling. Therefore, it is thought that Maml1 functions to recruit other factors to drive Notch function. Although it is clear from the crystal structure that Maml1 makes formal contacts with Notch and CSL, purified Notch and Maml1 do not interact. In fact, using purified proteins, Maml1 does not interact with either NICD or CSL alone. Maml1 can do so only in the presence of all three proteins. Therefore, a question that remains to be resolved is how Maml1 is incorporated into the Notch activation complex (Vasquez-Del Carpio, 2011).

Skip was initially identified as a cofactor for the Ski oncoprotein. Subsequently, Skip has been reported to act both as a corepressor in association with the CSL corepressors SMRT/NCoR and Sharp and as an enhancer or coactivator of the Notch signaling pathway. The mechanistic details of how Skip works in Notch transcriptional activation are not known. How does Skip potentiate Notch signaling? Based on the current results, it is proposed that the role of Skip in Notch transactivation is to initiate complex assembly by binding to Notch multimers and to recruit Maml1 to form a preactivation complex. It was demonstrated that Skip preferentially binds to NICD multimers, and since NICD monomers and not multimers are in the Notch activation complex, it is suggested that Skip is likely involved in the early events of Notch activation complex assembly. Moreover, it was shown that in the presence of Skip, a protein complex containing NICD multimer, Maml1 and Skip can be detected. Therefore, it appears that the NICD multimer-Skip complex is assembled to provide a docking site for Maml1 to form a preactivation complex. The data indicating that NICD, Maml1, and Skip assemble into a complex prior to interacting with CSL provide a mechanism for previous observations showing that both Maml1 and Skip are found at the HES-1 promoter only when NICD is present. Based on these data, it is predicted that by preventing the NICD multimer-Skip interaction, Maml1 would not be efficiently recruited to the activation complex and thus the intensity of Notch signaling would be decreased (Vasquez-Del Carpio, 2011).

CSL appears to be the mediator involved in the conversion of a preactivation complex to the Notch transcriptional activation complex. The interaction between the preactivation complex and CSL essentially loads NICD and Maml1 onto CSL bound to DNA and initiates transcriptional activation. How does CSL perform this conversion? It appears that CSL is involved in destabilizing the interaction between the NICD molecules. Previous studies demonstrated that CSL interacts with a 4-amino-acid motif (PhiWPhiP) found within the RAM domain of NICD. This study shows that the RAM domain also interacts with a region between amino acids 2203 and 2216 of NICD, which is here defined as the C-terminal multimerization region (CTM). C-terminal deletion mutants of Notch that terminate at amino acid 2240 still form multimers, but deletion mutants that terminate at amino acid 2202 are monomeric. Furthermore, a deletion mutant that is monomeric is severely compromised for transcriptional activation. This is not simply due to the loss of a transactivation domain, since activity can be restored by rescue with a multimerization-competent, transactivation-deficient mutant of Notch. The interaction between the RAM domain and CTM does not require a functional PhiWPhiP motif. Therefore, it is possible to physically separate the RAM domain of NICD into an N-terminal multimerization (NTM), amino acids 1820 to 1847, and a CSL binding region (PhiWPhiP motif). Since the RAM domain has two distinct components, it is proposed that the RAM domain functions as a switch between the preactivation complex and the Notch activation complex. In this model, a region of the RAM domain (NTM) C-terminal to the PhiWPhiP motif interacts with the CTM. Thus, the NTM and CTM are the main sites of interaction for multimer formation, although other low-affinity sites might be present and contribute to the overall stability of the multimer, like the ankyrin repeats. Later, when CSL interacts with the PhiWPhiP motif, a conformational change likely occurs in the RAM domain. This results in the RAM domain no longer interacting with the CTM, which drives the conversion from the preactivation complex to the Notch activation complex. Moreover, during the transition from preactivation complex to activation complex, it is not difficult to envision how CSL can displace Skip by steric hindrance from the complex, since both interact with the ankyrin repeat of Notch. Depending on Notch presence or absence, it has been reported that Skip can be found forming part of a CSL-repressor complex or a transcriptional activation complex. The data that support the model in which Skip is associated with the Notch activation complex come from chromatin immunoprecipitation (ChIP) analysis, in which Skip and other proteins can be detected sitting on the chromatin when Notch is present. In this model, Skip will be displaced from the repressor complex and be recruited again once the Notch activation complex is formed. In the current model, Skip is displaced during the transition from the preactivation complex to the transcriptional activation complex by CSL. Considering the results provided, the possibility cannot be excluded that Skip may form part of the final Notch activation complex on DNA, either by staying in the complex upon CSL interaction or by rerecruitment after the transcriptional activation complex is formed. This issue cannot be resolved by ChIP assay, since the technique provides a snapshot in a certain time frame of proteins interacting and not the dynamics of complex formation or transcription (Vasquez-Del Carpio, 2011).

Biochemical and biophysical studies have been mostly focused on the ankyrin repeat domain of NICD. However, these studies did not detect multimeric forms of NICD. Why were Notch multimers not detected? This study has demonstrated that the C-terminal region of NICD is required for multimer formation. Deletion of the C-terminal region of NICD impairs the formation of NICD multimers. Moreover experiments using only the ankyrin repeats showed that this domain is not sufficient for multimerization, although its contribution to the stability of the multimer may be important. Furthermore, the truncated forms of NICD utilized in these biophysical studies did not contain the RAM domain or the C-terminal region of NICD, which this study has demonstrated to be necessary for the formation of NICD multimers (Vasquez-Del Carpio, 2011).

NICD has been shown to form dimeric activation complexes with Mastermind and CSL on DNA that contained two CSL binding sites positioned in a head-to-head arrangement. In this crystal structure, the dimeric complexes are stabilized by the interaction of the ankyrin domain from one NICD with the ankyrin domain of another NICD. The mutation of a residue within the ankyrin domain involved in the dimer formation into alanine (R1985A) prevented the formation of this dimeric structure on DNA. Is this dimerization on DNA similar to the multimerization that was observe? To determine if arginine at position 1985 of NICD plays a role in multimerization, the residue was mutated, and it was determined if NICD still formed multimers. The NICDR1985A mutant still retains reporter activity on a promoter that contains a tandem array of CSL binding sites, 8x CSL promoter, but not on a promoter that contains two CSL binding sites positioned head to head, Hes-1 promoter. These data indicate that the mutant is functioning similarly to the published mutant. To determine if NICDR1985A forms multimers, 293T cells were cotransfected with NICDR1985A carrying Flag (NICDR1985AF) and Myc (NICDR1985AM) epitope tags. When NICDR1985AF was immunoprecipitated, NICDR1985AM coimmunoprecipitated. These data indicated that the multimerization observed does not involve the R1985 residue found within the ankyrin domain of Notch. Furthermore the observed dimeric complexes in the crystal structure result from cooperative binding of transcriptional complexes on DNA and not from an intrinsic multimerization property of Notch that was described for complex assembly (Vasquez-Del Carpio, 2011).

Why do Notch proteins multimerize with each other? It is proposed that multimerization has evolved to regulate Notch function by controlling the timing/duration of Notch signaling (Vasquez-Del Carpio, 2011).

How does multimerization regulate the timing/duration of Notch signaling? Once released from the plasma membrane, it is proposed that NICD forms multimers. This establishes the initial step in regulation, because multimerization is a function of free (monomeric) NICD concentration. NICD multimer formation is necessary to form a complex with Skip. This provides a second step of regulation in Notch activation, because the interaction between NICD multimers and Skip is required to recruit Maml1 to form the preactivation complex. Therefore, Skip availability is predicted to be a limiting factor in Notch signaling. Once the preactivation complex is assembled, the formation of an activation complex with DNA-bound CSL is thought to be rapid. Interaction of the preactivation complex with CSL triggers the loading of NICD and Maml1 to form the activation complex with CSL and concomitantly the release of NICD and Skip. In this step, the NICD multimer is disassociated by CSL, resulting in the retention of only one NICD molecule in the activation complex. The released NICD monomer is then free to multimerize and initiate another round of activation complex assembly. Once in the activation complex, NICD is rapidly degraded following the initiation of transcription. Therefore, it is proposed that the duration of Notch signaling is a function of the rate of assembly and subsequent destruction of the Notch activation complex and that the cycling of NICD monomers and multimers may provide a mechanism for the oscillation of Notch transcriptional activity (Vasquez-Del Carpio, 2011).

Rbm15 modulates Notch-induced transcriptional activation and affects myeloid differentiation

RBM15 is the fusion partner with MKL in the t(1;22) translocation of acute megakaryoblastic leukemia. To understand the role of the RBM15-MKL1 fusion protein in leukemia, the normal functions of RBM15 and MKL must be understood. A role for Rbm15 in myelopoiesis is demonstrated in this study. Rbm15 is expressed at highest levels in hematopoietic stem cells and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Decreasing Rbm15 levels with RNA interference enhances differentiation of the 32DWT18 myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits 32DWT18 differentiation. Rbm15 alters Notch-induced HES1 promoter activity in a cell type-specific manner. Rbm15 inhibits Notch-induced HES1 transcription in nonhematopoietic cells but stimulates this activity in hematopoietic cell lines, including 32DWT18 and human erythroleukemia cells. Moreover, the N terminus of Rbm15 coimmunoprecipitates with RBPJkappa, a critical factor in Notch signaling, and the Rbm15 N terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full-length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJkappa (Ma, 2007).

After ligand binding, Notch receptors are cleaved to release their intracellular domains. The intracellular domains, the activated form of Notch receptors, are then translocated into the nucleus where they interact with other transcriptional machinery to regulate the expression of cellular genes. To dissect the molecular mechanisms of Notch signaling, the cellular targets that interact with Notch1 receptor intracellular domain (N1IC) were screened. Endogenous transcription factor Ying Yang 1 (YY1) is associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin (ANK) domain of N1IC and zinc finger domains of YY1 are essential for the association of N1IC and YY1 according to the pull-down assay of glutathione S-transferase fusion proteins. Furthermore, both YY1 and N1IC are present in a large complex of the nucleus to suppress the luciferase reporter activity transactivated by Notch signaling. The transcription factor YY1 indirectly regulates the transcriptional activity of the wild-type CBF1-response elements via the direct interaction of N1IC and CBF1. The association between endogenous N1IC and intrinsic YY1 has also been demonstrated in human acute T-cell lymphoblastic leukemia cell lines. Taken together, these results indicate that transcription factor YY1 may modulate Notch signaling via association with the high molecular weight Notch complex (Yeh, 2003).

The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Evidence is provided that the polycomb group protein Sex combs extra/RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, it was also shown that, though RING1 and RBP-J do not associate directly, the two molecules can be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, overexpression of RING1 together with KyoT2 in cells inhibits transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 is abrogated by overexpression of KBP1, a molecule that competes with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 is dependent on its associating with KyoT2. Taken together, these data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J: competition for binding sites with transactivators, and recruitment of suppressors such as RING1 (Qin, 2004).

Notch signaling mediates communication between cells and is essential for proper embryonic patterning and development. CSL is a DNA binding transcription factor that regulates transcription of Notch target genes by interacting with coregulators. Transcriptional activation requires the displacement of corepressors from CSL by the intracellular portion of the receptor Notch (NotchIC) and the recruitment of the coactivator protein Mastermind to the complex. This study reports the 3.1 Å structure of the ternary complex formed by CSL, NotchIC, and Mastermind bound to DNA. As expected, the RAM domain of Notch interacts with the beta trefoil domain of CSL; however, the C-terminal domain of CSL has an unanticipated central role in the interface formed with the Notch ankyrin repeats and Mastermind. Ternary complex formation induces a substantial conformational change within CSL, suggesting a molecular mechanism for the conversion of CSL from a repressor to an activator (Wilson, 2006)

Neural networks are balanced by inhibitory and excitatory neuronal activity. The formation of these networks is initially generated through neuronal subtype specification controlled by transcription factors. The basic helix-loop-helix (bHLH) transcription factor Ptf1a is essential for the generation of GABAergic inhibitory neurons in the dorsal spinal cord, cerebellum, and retina. The transcription factor Rbpj is a transducer of the Notch signaling pathway that functions to maintain neural progenitor cells. Ptf1a and Rbpj interact in a complex that is required in vivo for specification of the GABAergic neurons, a function that cannot be substituted by the classical form of the bHLH heterodimer with E-protein or Notch signaling through Rbpj. A mutant form of Ptf1a without the ability to bind Rbpj, while retaining its ability to interact with E-protein, is incapable of inducing GABAergic (Pax2)- and suppressing glutamatergic (Tlx3)-expressing cells in the chick and mouse neural tube. Moreover, an Rbpj conditional mutation was used to demonstrate that Rbpj function is essential for GABAergic specification, and that this function is independent of the Notch signaling pathway. Together, these findings demonstrate the requirement for a Ptf1a-Rbpj complex in controlling the balanced formation of inhibitory and excitatory neurons in the developing spinal cord, and point to a novel Notch-independent function for Rbpj in nervous system development (Hori, 2008).

Stem cells are influenced by their surrounding microenvironment, or niche. In the testis, Sertoli cells are the key niche cells directing the population size and differentiation fate of spermatogonial stem cells (SSCs). Failure to properly regulate SSCs leads to infertility or germ cell hyperplasia. Several Sertoli cell-expressed genes, such as Gdnf and Cyp26b1, have been identified as being indispensable for the proper maintenance of SSCs in their niche, but the pathways that modulate their expression have not been identified. Although it has been recently found that constitutively activating NOTCH signaling in Sertoli cells leads to premature differentiation of all prospermatogonia and sterility, suggesting that there is a crucial role for this pathway in the testis stem cell niche, a true physiological function of NOTCH signaling in Sertoli cells has not been demonstrated. To this end, recombination signal binding protein for immunoglobulin kappa J region (Rbpj), a crucial mediator of NOTCH signaling, was conditionally ablated in Sertoli cells using Amh-cre. Rbpj knockout mice had: significantly increased testis sizes; increased expression of niche factors, such as Gdnf and Cyp26b1; significant increases in the number of pre- and post-meiotic germ cells, including SSCs; and, in a significant proportion of mice, testicular failure and atrophy with tubule lithiasis, possibly due to these unsustainable increases in the number of germ cells. Germ cells were identified as the NOTCH ligand-expressing cells. It is concluded that NOTCH signaling in Sertoli cells is required for proper regulation of the testis stem cell niche and is a potential feedback mechanism, based on germ cell input, that governs the expression of factors that control SSC proliferation and differentiation (Garcia, 2014).

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