Suppressor of Hairless


Invertebrate Suppressor of Hairless homologs

The homologous receptors LIN-12 and GLP-1 mediate diverse cell-signaling events during development of the nematode Caenorhabditis elegans. These two receptors appear to be functionally interchangeable and show sequence similarity to Drosophila Notch. The lag-1 gene (lin-12 -and glp-1) plays a central role in LIN-12 and GLP-1-mediated signal transduction. The predicted LAG-1 protein is homologous to two DNA-binding proteins: human C Promoter Binding Factor (CBF1) and Drosophila Suppressor of Hairless (Su[H]). Furthermore, LAG-1 binds specifically to the DNA sequence RTGGGAA, previously identified as a CBF-1/Su(H)-binding site. The 5' flanking regions and first introns of the lin-12, glp-1 and lag-1 genes are enriched for potential LAG-1-binding sites.It is proposed that LAG-1 is a transcriptional regulator that serves as a primary link between the LIN-12 and GLP-1 receptors and downstream target genes in C. elegans. In addition, LAG-1 may be a key component of a positive feedback loop that amplifies activity of the LIN-12/GLP-1 pathway (Christensen, 1996).

In C. elegans, the GLP-1 receptor acts with a downstream transcriptional regulator, LAG-1, to mediate intercellular signaling. GLP-1 and LAG-1 are homologs of Drosophila Notch and Su(H), respectively. The functions of two regions of the GLP-1 intracellular domain were investigated: the ANK repeat domain, which includes six cdc10/ankyrin repeats plus flanking amino acids, and the RAM domain, which spans approximately 60 amino acids just inside the transmembrane domain. Both ANK and RAM domains interact with the LAG-1 transcription factor. The interaction between the ANK domain and LAG-1 is only observed in nematodes by a co-localization assay and, therefore, may be either direct or indirect. By contrast, the interaction between the RAM domain and LAG-1 is likely to be direct, since it is observed by co-precipitation of the proteins in vitro as well as by yeast two-hybrid experiments. The RAM domain, when expressed in nematodes without a functional ANK repeat domain, does not mimic the unregulated receptor in directing cell fates nor does it interfere with signaling by endogenous components. In yeast the ANK repeats are strong transcriptional activators. Furthermore, missense mutations that eliminate receptor activity also abolish transcriptional activation by the GLP-1 ANK repeats in yeast. One possible function for the ANK repeat domain is to act as a transcriptional co-activator with LAG-1 (Roehl, 1996).

The egg-laying system of Caenorhabditis elegans hermaphrodites requires development of the vulva and its precise connection with the uterus. This process is regulated by LET-23-mediated epidermal growth factor signaling and LIN-12-mediated lateral signaling pathways. Among the nuclear factors that act downstream of these pathways, the LIM homeobox gene lin-11 plays a major role. lin-11 mutant animals are egg-laying defective because of the abnormalities in vulval lineage and uterine seam-cell formation. However, the mechanisms providing specificity to lin-11 function are not understood. The regulation of lin-11 during development of the egg-laying system was examined. The tissue-specific expression of lin-11 is controlled by two distinct regulatory elements that function as independent modules and together specify a wild-type egg-laying system. A uterine pi lineage module depends on the LIN-12/Notch signaling, while a vulval module depends on the LIN-17-mediated Wnt signaling. These results provide a unique example of the tissue-specific regulation of a LIM homeobox gene by two evolutionarily conserved signaling pathways. Finally, evidence is provided that the regulation of lin-11 by LIN-12/Notch signaling is directly mediated by the Su(H)/CBF1 family member LAG-1 (Gupta, 2002).

The Caenorhabditis elegans gene hlh-6 is expressed specifically in pharyngeal glands, one of five distinct pharyngeal cell types. Expression of hlh-6 is controlled by a discrete set of cis-regulatory elements, including a negative element called HRL1. This study demonstrates that HRL1 is a functional binding site for LAG-1, the CSL transcriptional effector of Notch in C. elegans, and that regulation of hlh-6 by LAG-1 is direct. Regulation of hlh-6 by LAG-1 is strictly negative: removal of HRL1 or LAG-1 regulation results in ectopic expression of hlh-6, but does not affect expression in pharyngeal glands. Furthermore, direct regulation of hlh-6 expression does not appear to involve Notch signaling, contrary to the canonical mechanism by which CSL factors regulate target genes. An additional cis-regulatory element was identified in the hlh-6 promoter that, together with previously identified elements, is sufficient to overcome repression by LAG-1 and activate hlh-6 expression in pharyngeal glands (Ghai, 2008).

A minimal 434 bp enhancer from the promoter region of the Ciona Brachyury gene (Ci-Bra, Drosophila homolog: brachyenteron) is sufficient to direct a notochord-specific pattern of gene expression. Evidence is presented that a Ciona homolog of snail (Ci-sna) encodes a repressor of the Ci-Bra enhancer in the tail muscles. DNA-binding assays have identified four Ci-Sna-binding sites in the Ci-Bra enhancer, and mutations in these sites cause otherwise normal Ci-Bra/lacZ transgenes to be misexpressed in ectopic tissues, particularly the tail muscles. Selective misexpression of Ci-sna using a heterologous promoter results in the repression of Ci-Bra/lacZ transgenes in the notochord. Moreover, the conversion of the Ci-Sna repressor into an activator results in the ectopic induction of Ci-Bra/lacZ transgenes in the muscles, and also causes an intermixing of notochord and muscle cells during tail morphogenesis. These results suggest that Ci-Sna functions as a boundary repressor, which subdivides the mesoderm into separate notochord and tail muscle lineages. Repression appears to depend on tight linkage between sna1 and sna2 sites and Su(H) activator sites, located on the minimal enhancer. The insertion of spacer sequences between sna1 and Su(H)1 or between Su(H)2 and sna2 results in a severe derepression of Ci-Bra/lacZ transgenes in the tail muscles. Intact sna1 and sna2 sites appear to be required for the repression of Ci-Bra in the tail muscles. The function of Ci-Snail in creating a boundary between notochord and muscle is likened to the function of Snail in Drosophila in creating a boundary between neuroectoderm and mesoderm (Fujiwara, 1998).

Ancestral and conserved cis-regulatory architectures in developmental control genes

Among developmental control genes, transcription factor-target gene 'linkages'-- the direct connections between target genes and the factors that control their patterns of expression--can show remarkable evolutionary stability. However, the specific binding sites that mediate and define these regulatory connections are themselves often subject to rapid turnover. This paper describes several instances in which particular transcription factor binding motif combinations have evidently been conserved upstream of orthologous target genes for extraordinarily long evolutionary periods. This occurs against a backdrop in which other binding sites for the same factors are coming and going rapidly. These examples include a particular Dpp Silencer Element upstream of insect brinker genes, in combination with a novel motif referred to as the Downstream Element; combinations of a Suppressor of Hairless Paired Site (SPS) and a specific proneural protein binding site associated with arthropod Notch pathway target genes; and a three-motif combination, also including an SPS, upstream of deuterostome Hes repressor genes, which are also Notch targets. It is proposed that these stable motif architectures have been conserved intact from a deep ancestor, in part because they mediate a special mode of regulation that cannot be supplied by the other, unstable motif instances (Rebeiz, 2012).

Previous studies described the phylogenetically widespread occurrence of single, high-affinity bHLH repressor (R) binding sites (a consensus GGCACGCGCC, with variants in the last two bases) upstream of bilaterian proneural genes (Rebeiz, 2005). The possibility could not be ruled out that only the 'linkage' (direct transcription factor-target gene relationship) has been maintained, and that the binding site itself has been replaced repeatedly in the course of animal evolution. However, several lines of evidence suggest that these R sites have been conserved from a deep common ancestor. These included the stability of the precise 10-bp sequence of the site over very long intervals, and the strong conservation of both the motif and flanking sequences in some instances, clearly suggesting that the sites are indeed orthologous (Rebeiz, 2012).

The present report substantially expands the inventory of such apparently ancient and conserved cis-regulatory motifs in developmental control genes. This study describes five additional cases in which specific motif combinations have evidently been retained over hundreds of millions of years of evolution. With the exception of two novel elements [the insect brk Downstream Element (DE) and the deuterostome Hes XE], these motifs represent high-affinity binding sites for known transcription factors. The retention of these specific motif instances is especially striking when considered against the background of rapid appearance and disappearance of other binding sites for the same factors (Rebeiz, 2012).

The conservation of the distinctive SE + DE motif (SE: GRCGNCN5GTCTG) combination upstream of insect brk genes extends over perhaps 270-300 My, reflecting the fact that the brk gene itself is found only in insects. A similar (minimum) age can be assigned to the P + SPS architecture found upstream of insect bHLH repressor genes, while the E + SPS + P combination associated with arthropod BFM genes is even older, in excess of 400 My, in view of its occurrence in the crustacean D. pulex. Finally, it is likely that the X + R + SPS ensemble upstream of deuterostome Hes1 genes was present in the common ancestor, over 500 My ago. It is also possible that an SPS element was associated with an ancestral bilaterian Hes repressor gene, which would make this feature close to 600 My old (Rebeiz, 2012).

This analyses do not permit the discerning of the population genetic/microevolutionary processes by which the distinctive cis-regulatory architectures first arose and became fixed in an ancestral population. However, it is believed that some useful insights can be offered into why these architectures have endured over such lengthy timescales (Rebeiz, 2012).

What characteristics of ancient and conserved motifs drive their long-term preservation by selection, even as other binding sites for the same factors come and go rapidly in evolution? An earlier proposal is first reiterated that such deeply conserved motifs mediate abstract or generic regulatory functions of fundamental utility to all or most members of an ancient clade (Rebeiz, 2005). It is certainly plausible that, once established, the capacity to repress brk transcription in response to a Dpp signal remained of great utility to all the descendants of the common insect ancestor, as diverse as they became. Similarly, the abstract ability to activate a Hes repressor gene via Notch signaling would remain of exceptional utility to descendants of a bilaterian (or earlier) ancestor that had evolved it. Finally, a generic capability for autorepression of a Hes bHLH repressor gene might very well be retained by descendants of a deuterostome ancestor (Rebeiz, 2012).

But it is certainly sensible to argue that, to retain such abstract and valuable regulatory capabilities, it would suffice to preserve only the linkage between the appropriate transcription factors and their targets. In this view, individual factor-binding motifs need not be retained; they would be free to turn over during evolution. However, the examples described in this study suggest a second important reason for the long-term evolutionary retention of particular motifs or motif combinations. It is proposed that these conserved sequence elements mediate a distinctive regulatory capability not conferred by other instances of the same motif or motifs. In the case of the SPS element, considerable confident can be had that this perspective is correct. The SPS has been shown to mediate cooperative binding of two Su(H)/Mam/NICD trimers, thus conferring on the associated target gene unusually high sensitivity to Notch signaling. While two 'lone' Su(H) sites are indeed able to contribute to a target gene's response to activated Notch, they would not do so in a cooperative manner. In a similar vein, it seems plausible to suggest that while all SE motifs may be able to participate in signal-dependent repression of brk, the SE + DE combination offers a unique and valuable version of this capability (e.g., greater signal sensitivity), possibly conferring a fitness advantage. It is hypothesized that in both cases, once the specialized motif architecture (SPS or SE + DE) had evolved to confer a distinctive capacity, it would be selectively retained in evolution. As is known, other instances of the SE or Su(H) binding motifs do arise and become fixed in individual clades, but these would not be expected to exhibit the same durability, since (according to the hypothesis) they confer no unique capability. The foregoing interpretation is particularly supported by the frequent observation that if only one element mediating a particular response [either SE or Su(H) site] is present upstream of an orthologous gene in a given species, it is of the 'special' type (SE + DE or SPS). Examples include the SE + DE combination in T. castaneum brk and the SPS motifs in the A. gambiae bHLHR1 gene, the A. mellifera BFM gene, and H. sapiens HES1 (Rebeiz, 2012).

Another factor that may contribute to the long-term evolutionary conservation of the specialized motif architectures this study has considered is their very complexity. Both the SE + DE unit and the SPS represent unusually extended and constrained motif combinations. While in principle this does not prevent them from turning over by duplication/degeneration, they are unlikely to evolve de novo (Rebeiz, 2012).

Finally, an intriguing feature is noted of the conserved motif architectures described in this study that involve the SPS: the apparently conserved order and even orientation of the individual sequence elements. The arthropod BFM genes are associated with a 'lower-strand' E motif followed by an SPS followed by a 'lower-strand' P site; insect Hes repressor genes bear an 'upper-strand' P site followed by an SPS; and deuterostome Hes1 genes have an 'upper-strand' X site followed by an 'upper-strand' R site followed by an SPS, which also has fixed orientation. Inter-site distances are often not conserved; consider the varying separation of the SPS and the P site in the BFM genes, or the different distances between the X + R combination and the SPS in the deuterostome Hes1 genes. Evidently, the motif order and orientation of these architectures have functional significance, consistent with an 'enhanceosome' model for the structure of these regions. Alternatively, these features may suggest the existence of a 'scanning' mechanism for optimal enhancer-promoter interaction. Such a property might be a particular characteristic of promoter-proximal cis-regulatory modules such as these, as contrasted with more distal enhancers. In the latter case, interaction with the promoter by 'looping' may impose fewer architectural constraints (Rebeiz, 2012).

This study has proposed that the distinctive cis-regulatory architectures ancient ones that have been conserved from a deep ancestor. However, it also seems likely that, because of their very complexity, they may not represent the 'original' version of their respective regulatory linkages. These two realizations can be reconciled via the following general evolutionary scenario (Rebeiz, 2012).

The direct linkage of an ancestral Hes gene to Su(H) and the Notch pathway evidently originated in a deep metazoan ancestor, and was very likely mediated by a lone Su(H) binding site or sites. The genome of the demosponge A. queenslandica includes one member of the closely related Hey repressor family, but no Hes genes; this Amphimedon Hey gene has one high-affinity Su(H) site 600 bp upstream of the transcription start site. The placozoan T. adhaerens has one Hey ortholog, one Hey-related gene, and one Hes gene. The Hey ortholog has three high-affinity Su(H) sites in the first 800 bp upstream of the ATG start codon, while the Hes gene includes a single such site within 500 bp of its ATG. The genome of the cnidarian N. vectensis (sea anemone) is endowed with a large paralogous family of 11 Hes genes, many of them with multiple lone Su(H) sites immediately upstream. Likewise, the Nematostella Hey ortholog has two upstream Su(H) sites. The SPS evidently did not appear upstream of a Hey/Hes gene until after the cnidarian-bilaterian divergence, but this association is now widespread among both protostomes and deuterostomes (Rebeiz, 2012).

It is suggested, then, that what appeared first was the simple capacity to regulate a Hey/Hes gene directly by Su(H) (presumably linked to the Notch pathway), via one or more lone Su(H) binding sites. Then, in a bilaterian ancestor, an SPS came into being upstream of an individual Hes gene, making possible a cooperative and thus highly sensitive response to Notch-activated Su(H). Once this novel regulatory capacity was established, it bestowed a sufficient selective advantage to ensure its subsequent retention in a wide variety of bilaterian taxa. Such a scenario can account for the phylogenetic distribution of the SPS-containing cis-regulatory architectures described. More complex histories cannot be ruled out, including the possibility that the SPS arose independently more than once in association with Hes genes (Rebeiz, 2012).

It is important to note the finding that, in the case of target genes that are part of paralogous families (Hes repressor and BFMs), only one particular paralog in a given species is typically associated with the conserved motif architectures described. This is true even if other paralogs make use of the same overall cis-regulatory 'code' (combination of transcription factor binding sites) to direct a similar expression specificity. For example, of the seven unambiguous Hes repressor paralogs in H. sapiens, only HES1 bears the X + R + SPS motif combination, though four others have upstream S sites and two of these also have upstream R sites. Likewise, the D. melanogaster genome includes nine BFM genes, most of which employ the S + P code, but only one, E(spl)m4, is associated with an SPS + P combination. It seems likely that, while the distinctive regulatory capability conferred by an ancient and conserved motif combination is of long-term selective value, it suffices for a single paralog in the genome to retain it (Rebeiz, 2012).

This observation is consistent with a duplication-divergence model for the evolution of Hes and BFM paralogs. The special cis-regulatory architectures this study has described, along with the associated protein coding sequences, comprise functional units that have been conserved from deep common ancestors because of the unique regulatory capabilities they confer. Paralogous genes that arise by duplication within various taxa (this is a widespread phenomenon in the case of Hes genes) would not be subject to the same stringent constraints on their cis-regulatory architecture, since the ancestral gene would be present to provide the distinctive capabilities. The paralogs would thus be free to evolve their cis-regulatory motifs according to other selective pressures or genetic drift, yielding the many variations on a basic theme (e.g., S + P) that is observed within a single species today (Rebeiz, 2012).

Suppressor of Hairless vertebrate homolog: RBP-Jkappa

Su(H) is 82% identical to the mouse protein (Furukawa, 1992 and Schwesguth, 1995). Su(H) is also a homolog of the mammalian C-promoter-binding factor1 (CBF1) gene (Fortini, 1994).

A truncated, active form of Notch1 binds CBF1, also known as JkappaRBP, the mammalian homolog of Suppressor of Hairless and activates transcription through a CBF1 response element containing promoter. One model for the function of mammalian Notch assumes that Notch is cleaved by a membrane protease, and the released membrane domain is translocated to the nucleus. This model is supported by the observation that the untethered intracellular domain of Notch is as activate as truncated Notch and is located predominantlyh in the nucleus, and by the observation that small amounts of CBF1 are associated with Notch1 in the nucleus (Lu, 1996 and references).

The X-Notch-1 receptor, and its putative ligand, X-Delta-1, are thought to mediate an inhibitory cell-cell interaction, called lateral inhibition, that limits the number of primary neurons that form in Xenopus embryos. The expression of Xenopus ESR-1, a gene related to Drosophila Enhancer of split, appears to be induced by Notch signaling during this process. To determine how the activation of X-Notch-1 induces ESR-1 expression and regulates primary neurogenesis, the Xenopus homolog of Drosophila Suppressor of Hairless ) was isolated. X-Su(H) binds to the RAM21 region of X-Notch-1, just N-terminal of a ankyrin repeat domain and homologous to a similar site in Drosophila Notch. X-Su(H), when modified by the C-terminal covalent addition of ankyrin repeats, induces ESR-1 expression, perhaps directly. Using a DNA binding mutant of X-Su(H), it is shown that X-Su(H) activity is required for induction of ESR-1. Expression of the DNA binding mutant in embryos leads to a neurogenic phenotype (a higher density of primary neurons) as well as increased expression of both X-Delta-1 and XNGNR1, a proneural gene expressed during primary neurogenesis. These results suggest that activation of X-Su(H) is a key step in the Notch signaling pathway during primary neurogenesis in Xenopus embryos (Wettstein, 1997).

Notch is involved in the cell fate determination of many cell lineages. The intracellular region (RAMIC) of Notch1 transactivates genes by interaction with a DNA binding protein RBP-J. The activities of mouse RAMIC and its derivatives were compared in transactivation and differentiation suppression of myogenic precursor cells. RAMIC comprises two separate domains: IC for transactivation (the IC domain includes the whole intracellular domain exclusive of the RAM domain) and RAM (immediately C-terminal to the transmembrane region) for RBP-J binding. Although the physical interaction of ankyrin repeats within IC with RBP-J is much weaker than is RAM interaction with RBP-J, transactivation activity of IC is shown to involve RBP-J by using an RBP-J null mutant cell line. IC shows differentiation suppression activity that is generally comparable to its transactivation activity. The RBP-J-VP16 fusion protein, which has strong transactivation activity, also suppresses myogenesis of C2C12 myogenic precursor cells. The RAM domain, which has no other activity than binding to RBP-J, synergistically stimulates transactivation activity of IC to the level of RAMIC. The RAM domain is proposed to compete with a putative co-repressor for binding to RBP-J because the RAM domain can also stimulate the activity of RBP-J-VP16. Taken together, these results indicate that differentiation suppression of myogenic precursor cells by Notch signalling is due to the transactivation of genes carrying RBP-J binding motifs (Kato, 1997).

Activation of Notch by its ligand Serrate apportions myogenic and non-myogenic cell fates within the early Xenopus heart field. The crescent-shaped field of heart mesoderm is specified initially as cardiomyogenic. While the ventral region of the field forms the myocardial tube, the dorsolateral portions lose myogenic potency and form the dorsal mesocardium and pericardial roof. The local interactions that establish or maintain the distinct myocardial and non-myocardial domains have never been described. Xenopus Notch1 (Xotch) and Serrate1 are expressed in overlapping patterns in the early heart field. Conditional activation or inhibition of the Notch pathway with inducible dominant negative or active forms of the RBP-J/Suppressor of Hairless [Su(H)] transcription factor indicates that activation of Notch feeds back on Serrate1 gene expression to localize transcripts more dorsolaterally than those of Notch1, with overlap in the region of the developing mesocardium. Moreover, Notch pathway activation decreases myocardial gene expression and increases expression of a marker of the mesocardium and pericardial roof, whereas inhibition of Notch signaling has the opposite effect. Activation or inhibition of Notch also regulates contribution of individual cells to the myocardium. Importantly, expression of Nkx2.5 and Gata4 remains largely unaffected, indicating that Notch signaling functions downstream of heart field specification. It is concluded that Notch signaling through Su(H) suppresses cardiomyogenesis and that this activity is essential for the correct specification of myocardial and non-myocardial cell fates (Rones, 2000).

Thermodynamic and structural insights into CSL-DNA complexes

The Notch pathway is an intercellular signaling mechanism that plays important roles in cell fates decisions throughout the developing and adult organism. Extracellular complexation of Notch receptors with ligands ultimately results in changes in gene expression, which is regulated by the nuclear effector of the pathway, CSL [C-promoter binding factor 1 (CBF-1), suppressor of hairless (Su(H)), lin-12 and glp-1 (Lag-1)]. CSL is a DNA binding protein that is involved in both repression and activation of transcription from genes that are responsive to Notch signaling. One well-characterized Notch target gene is hairy and enhancer of split-1 (HES-1), which is regulated by a promoter element consisting of two CSL binding sites oriented in a head-to-head arrangement. Although previous studies have identified in vivo and consensus binding sites for CSL, and crystal structures of these complexes have been determined, to date, a quantitative description of the energetics that underlie CSL-DNA binding is unknown. This study provides a thermodynamic and structural analysis of the interaction between CSL and the two individual sites that comprise the HES-1 promoter element. Comprehensive studies that analyze binding as a function of temperature, salt, and pH reveal moderate, but distinct, differences in the affinities of CSL for the two HES-1 binding sites. Similarly, structural results indicate that overall CSL binds both DNA sites in a similar manner; however, minor changes are observed in both the conformation of CSL and DNA. Taken together, these results provide a quantitative and biophysical basis for understanding how CSL interacts with DNA sites in vivo (Friedmann, 2010).

Given the small differences in affinity that were observed for CSL binding the 5'consensus and 3' nonconsensus sites of the HES1 SPS, it was interested to know why previous studies that identified the consensus binding site for CSL revealed a strong preference for a G/C base step at this position (-C/tGTGGGAA-), as opposed to A/T, C/G, and T/A base steps. Previous structures have shown that in some, but not all CSL-DNA complexes, the side chain of an absolutely conserved glutamine residue makes a water-mediated contact with the guanine base in the major groove, providing some explanation for the specificity and tolerance for purine bases at this position. Despite these structural results, there is relatively strong conservation for a T/A base step, that is, pyrimidine base, at this position in the HES-1 SPS found in mammals, Xenopus, and Zebrafish. Although the T→A mutation of the nonconsensus site (-CGTGTGAA-) actually enhanced binding similar to the consensus site, strikingly, the T→C mutation had a profound reduction in binding. Taken together, these results suggest that the identity of this base step is important for the affinity and specificity of CSL binding; however, a satisfactory molecular explanation for the observed differences in binding are still lacking (Friedmann, 2010).

RBP-Jkappa mutation

Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency causes hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region (Yamamoto, 2003).

The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. Vascular defects are described in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling (Krebs, 2004).

Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between the endocardium and myocardium. Ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1, and BMP10 expression and signaling, and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression, and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro-cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. It is suggested that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease (Grego-Bessa, 2007).

Constitutive activation of the Notch pathway can promote gliogenesis by peripheral (PNS) and central (CNS) nervous system progenitors. This raises the question of whether physiological Notch signaling regulates gliogenesis in vivo. To test this, Rbpsuh (Rbpj) was conditionally deleted from mouse PNS or CNS progenitors using Wnt1-Cre or Nestin-Cre. Rbpsuh encodes a DNA-binding protein (RBP/J) that is required for canonical signaling by all Notch receptors. In most regions of the developing PNS and spinal cord, Rbpsuh deletion causes only mild defects in neurogenesis, but severe defects in gliogenesis. These resulted from defects in glial specification or differentiation, not premature depletion of neural progenitors, because undifferentiated progenitors from the PNS and spinal cord could be cultured despite their failure to form glia in vivo. In spinal cord progenitors, Rbpsuh was required to maintain Sox9 expression during gliogenesis, demonstrating that Notch signaling promotes the expression of a glial-specification gene. These results demonstrate that physiological Notch signaling is required for gliogenesis in vivo, independent of the role of Notch in the maintenance of undifferentiated neural progenitors (Taylor, 2007).

The Notch pathway has been implicated in mesenchymal progenitor cell (MPC) differentiation from bone marrow-derived progenitors. However, whether Notch regulates MPC differentiation in an RBPjkappa-dependent manner, specifies a particular MPC cell fate, regulates MPC proliferation and differentiation during early skeletal development or controls specific Notch target genes to regulate these processes remains unclear. To determine the exact role and mode of action for the Notch pathway in MPCs during skeletal development, tissue-specific loss-of-function [Prx1Cre; Rbpjk(f/f)], gain-of-function [Prx1Cre; Rosa-NICD(f/+)] and RBPjkappa-independent Notch gain-of-function [Prx1Cre; Rosa-NICD(f/+); Rbpjk(f/f)] mice were analyzed for defects in MPC proliferation and differentiation. These data demonstrate for the first time that the RBPjkappa-dependent Notch signaling pathway is a crucial regulator of MPC proliferation and differentiation during skeletal development. This study also implicates the Notch pathway as a general suppressor of MPC differentiation that does not bias lineage allocation. Finally, Hes1 was identified as an RBPjkappa-dependent Notch target gene important for MPC maintenance and the suppression of in vitro chondrogenesis (Dong, 2010).

RBP-Jkappa and stem cell renewal

Neural stem cells, which exhibit self-renewal and multipotentiality, are generated in early embryonic brains and maintained throughout the lifespan. The mechanisms of their generation and maintenance are largely unknown. This study shows, by using RBP-Jkappa-/- embryonic stem cells in an embryonic stem cell-derived neurosphere assay, that neural stem cells are generated independent of RBP-Jkappa, a key molecule in Notch signaling. However, Notch pathway molecules are essential for the maintenance of neural stem cells; stem cells are depleted in the early embryonic brains of RBP-Jkappa-/- or Notch1-/- mice. Neural stem cells also are depleted in embryonic brains deficient for the presenilin1 (PS1) gene, a key regulator in Notch signaling, and are reduced in PS1+/- adult brains. Both neuronal and glial differentiation in vitro are enhanced by attenuation of Notch signaling and suppressed by expressing an active form of Notch1. These data are consistent with a role for Notch signaling in the maintenance of the neural stem cell, and inconsistent with a role in a neuronal/glial fate switch (Hitoshi, 2002).

Historically, Notch signaling in Drosophila was thought to maintain cells in an undifferentiated state. More recently, gain-of-function evidence in mammals has suggested that Notch signaling directly and instructively induces glial differentiation. Some Notch-signaling loss-of-function studies in mammals seem consistent with this neuronal/glial fate switch idea, in that there is a premature appearance and increased number of postmitotic neurons expressing MAP2 or ßIII tubulin between E10.5 and E13.5 in the PS1-/- brain. Similarly, mice with mutations in other Notch-signaling molecules such as Notch1, RBP-Jkappa, or Hes1/5 have revealed premature neuronal differentiation. However, it is worth noting that such mice with null mutations in Notch-signaling genes die in mid-to-late embryogenesis, when neurogenesis predominates over gliogenesis in vivo. A clonal analysis of E10 cortical cells in vitro shows that neuronal differentiation from single neural stem cells precedes gliogenesis in clonal cell colonies. Thus, the in vivo analyses of Notch mutants may not allow sufficient time to assess whether gliogenesis is increased or decreased (Hitoshi, 2002).

During brain development, neurons and glia are generated from a germinal zone containing both neural stem cells (NSCs) and more limited intermediate neural progenitors (INPs). The signalling events that distinguish between these two proliferative neural cell types remain poorly understood. The Notch signalling pathway is known to maintain NSC character and to inhibit neurogenesis, although little is known about the role of Notch signalling in INPs. This study shows that both NSCs and INPs respond to Notch receptor activation, but that NSCs signal through the canonical Notch effector C-promoter binding factor 1 (CBF1), whereas INPs have attenuated CBF1 signalling. Furthermore, whereas knockdown of CBF1 promotes the conversion of NSCs to INPs, activation of CBF1 is insufficient to convert INPs back to NSCs. Using both transgenic and transient in vivo reporter assays this study shows that NSCs and INPs coexist in the telencephalic ventricular zone and that they can be prospectively separated on the basis of CBF1 activity. Furthermore, using in vivo transplantation it was shown that whereas NSCs generate neurons, astrocytes and oligodendrocytes at similar frequencies, INPs are predominantly neurogenic. Together with previous work on haematopoietic stem cells, this study suggests that the use or blockade of the CBF1 cascade downstream of Notch is a general feature distinguishing stem cells from more limited progenitors in a variety of tissues (Mizutani, 2007).

Cbfa1/RBP-Jkappa transcriptional regulation

During endochondral bone development, both the chondrogenic differentiation of mesenchyme and the hypertrophic differentiation of chondrocytes coincide with the proliferative arrest of the differentiating cells. However, the mechanisms by which differentiation is coordinated with cell cycle withdrawal, and the importance of this coordination for skeletal development, have not been defined. Through analysis of mice lacking the pRB-related p107 and p130 proteins, it was found that p107 is required in prechondrogenic condensations for cell cycle withdrawal and for quantitatively normal alpha1(II) collagen expression. Remarkably, the p107-dependent proliferative arrest of mesenchymal cells is not needed for qualitative changes that are associated with chondrogenic differentiation, including production of Alcian blue-staining matrix and expression of the collagen IIB isoform. In chondrocytes, both p107 and p130 contribute to cell cycle exit, and p107 and p130 loss is accompanied by deregulated proliferation, reduced expression of Cbfa1, and reduced expression of Cbfa1-dependent genes that are associated with hypertrophic differentiation. Moreover, Cbfa1 is detected, and hypertrophic differentiation occurs, only in chondrocytes that have undergone or are undergoing a proliferative arrest. The results suggest that Cbfa1 links a p107- and p130-mediated cell cycle arrest to chondrocyte terminal differentiation (Rossi, 2002).

RBP-Jkappa transcriptional targets

In the mouse, targeted mutation of the Notch pathway genes Notch1 and RBP-Jk (Drosophila homolog Suppressor of hairless) has demonstrated a role for these genes in somite segmentation. These mutations lead to altered expression of the Notch signalling pathway homologs Hes-5 (Drosophila homologs Hairy and Enhancer of split), Mash-1 (Drosophila homologs: Achaete and Atonal) and Dll1 (Drosophila homolog: Delta), resulting in enhanced neurogenesis. Precocious neuronal differentiation is indicated by the expanded expression domains of Math4A, neuroD and NSCL-1 (a bHLH transcription factor expressed in the nervous system). The RBP-Jk mutation has stronger effects on expression of these genes than does the Notch1 mutation, consistent with functional redundancy of Notch genes in neurogenesis. In the neural tube, Dll1 is expressed in individual, isolated cells in a basal position in the neural epithelium, in cells that are thought to be committed neuronal precursors. Dll1 expression in the neural tube is increased in both RBP-Jk and Notch1 mutants. In Drosophila, a connection between the up-regulation of E(spl) and the downregulation of Delta, after activation of Notch signaling, may be provided by genes of the achaete-scute complex. Mash-1 is upregulated by Notch1 and RBP-Jk mutation in midbrain/hindbrain region and in the anterior neural tube region. Math-4A is upregulated in the midbrain and spinal cord. Neuro-D is upregulated in the midbrain and spinal cord and in the trigeminal and geniculate ganglia. Hes-5 is downregulated by Notch1 and RBP-Jk in the midbrain, hindbrain, spinal cord and presomitic mesoderm. Thus it appears that the murine Notch signaling pathway is involved in the regulaion of neural stem cell differentiation (de la Pompa, 1997).

Analysis by electrophoretic mobility shift assays (EMSA) of the different proteins associated with the kappaB sequence of the interleukin-6 (IL-6) promoter (IL6-kappaB) detected a specific complex formed with the recombination signal sequence binding protein Jkappa (RBP-Jkappa). Single-base exchanges within the oligonucleotide sequence defines the critical base pairs involved in the interaction between RBP-Jkappa and the IL6-kappaB motif. Binding analysis suggests that the amount of RBP-Jkappa protein present in the nucleus is severalfold higher than the total amount of inducible NF-kappaB complexes but that the latter bind DNA with a 10-fold-higher affinity. A reporter gene study was performed to determine the functional implication of this binding. It was found that the constitutive occupancy of the IL6-kappaB site by the RBP-Jkappa protein is responsible for the low basal levels of IL-6 promoter activity in L929sA fibrosarcoma cells and that RBP-Jkappa partially blocks access of NF-kappaB complexes to the IL-6 promoter. It is proposed that such a mechanism could be involved in the constitutive repression of the IL-6 gene under normal physiological conditions (Plaisance, 1997).

The cellular interleukin-6 (IL-6) gene contains a target site for the mammalian transcriptional repressor RBP. The target site is contained within the interleukin response element (ILRE), which mediates IL-6 activation by NF-kappa B. RBP represses activated transcription from the IL-6 gene. The presence and position of the RBP target site are both crucial in mediating repression by RBP. While RBP binds within the ILRE, it does not target NF-kappa B alone; nonetheless, NF-kappa B binding to the ILRE is required for repression. These results indicate that RBP represses coactivation by NF-kappa B and another cellular transcription factor, C/EBP-beta (Kannabiran, 1998).

NF-kappaB2 (p100/p52), a member of the NF-kappaB/Rel family of transcription factors (see Drosophila Dorsal), is involved in the regulation of a variety of genes important for immune function. The NF-kappaB2 gene is regulated both postively and negatively. Two kappaB elements within the NF-kappaB2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, there exists a transcriptional repression in the absence of NF-kappaB. To identify a DNA binding activity responsible for this transcriptional repression, a nuclear complex, named Rep-kappaB has been partially purified. Detailed examination of Rep-kappaB-DNA interaction reveals the sequence requirements for binding are almost identical to those of recombination signal binding protein Jkappa (RBP-Jkappa), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su(H)]. In electromobility shift assays, Rep-kappaB binding activity is recognized by an antibody directed against RBP-Jkappa. Human RBP-Jkappa represses basal as well as RelA (p65)-stimulated NF-kappaB2 promoter activity. Studies in Drosophila melanogaster have shown that Su(H) is implicated in the Notch signaling pathway, which regulate cell fate decisions. In transient-transfection assays, it has been shown that truncated Notch-1 strongly induces NF-kappaB2 promoter activity. In summary, these data clearly demonstrate that Rep-kappaB is closely related or identical to RBP-Jkappa. RBP-Jkappa is a strong transcriptional repressor of NF-kappaB2. This repression can be overcome by activated Notch-1, suggesting that NF-kappaB2 is a novel putative Notch target gene (Oswald, 1998).

A DNA-binding protein, HS2NF5, that binds tightly to a conserved region within hypersensitive site 2 (HS2) of the human beta-globin locus control region (LCR) has been characterized. The beta-globin LCR controls the chromatin structure, transcription, and replication of the beta-globin genes. HS2NF5 has been purified to near-homogeneity from fetal bovine thymus. Two polypeptides of 56 and 61 kDa have been copurified with the DNA binding activity. The two proteins bind to the LCR recognition site with an affinity (3.1 nM) and specificity similar to mouse erythroleukemia cell HS2NF5. The amino acid sequences of tryptic peptides of purified HS2NF5 reveal it to be identical to the murine homolog of the Suppressor of hairless transcription factor, also known as recombination signal binding protein Jkappa or C promoter binding factor 1 (CBF1). The CBF1 site within HS2 resides near sites for hematopoietic regulators such as GATA-1, NF-E2, and TAL1. An additional conserved, high affinity CBF1 site is localized within HS4 of the LCR. Since CBF1 is a downstream target of the Notch signaling pathway, it is proposed that Notch may modulate LCR activity during hematopoiesis (Lam, 1998).

The Notch signaling pathway is important for cellular differentiation. The current view is that the Notch receptor is cleaved intracellularly upon ligand activation. The intracellular Notch domain then translocates to the nucleus, binds to Suppressor of Hairless (RBP-Jk in mammals), and acts as a transactivator of Enhancer of Split (HES in mammals) gene expression. The Notch 3 intracellular domain (IC), in contrast to all other analyzed Notch ICs, is a poor activator, and in fact acts as a repressor by blocking the ability of the Notch 1 IC to activate expression through the HES-1 and HES-5 promoters. A model is presented in which Notch 3 IC interferes with Notch 1 IC-mediated activation at two levels. (1) Notch 3 IC competes with Notch 1 IC for access to RBP-Jk and does not activate transcription when positioned close to a promoter. (2) Notch 3 IC appears to compete with Notch 1 IC for a common coactivator present in limiting amounts. Further support for the existence of a coactivator comes from the finding that the Notch 3 ankyrin repeat construct, which lacks the strong RBP-Jk-binding RAM23 domain, is still able to repress Notch 1 IC-mediated activation. The common coactivator is most likely not required for all transcriptional complexes, since activation via the GAL4/VP16 fusion protein is not inhibited by Notch 3 IC. In keeping with this, cotransfection of the general coactivators TIF2, SRC1 and p300, does not neutralize Notch 3 IC’s repressor activity. In conclusion, this is the first example of a Notch IC that functions as a repressor in Enhancer of Split/HES upregulation, and shows that mammalian Notch receptors have acquired distinct functions during evolution (Beatus, 1999).

What is the structural basis for the difference in transactivating capacity between Notch 1 IC and Notch 3 IC? All Notch receptors, including Notch 3, are highly structurally related in the intracellular domains, in particular in the ankyrin repeat region. The ankyrin repeat region is important for the transactivating activity in Drosophila Notch, LIN-12 and Notch1. The high degree of conservation between Notch 1 IC and Notch 3 IC in this domain may at first seem paradoxical. It should however be noted that relatively subtle mutations in the ankyrin repeat region can dramatically alter its transactivation competence. The RAM23 region is conserved to a somewhat lesser extent, but apparently the conservation is sufficient for both Notch 1 IC and Notch 3 IC to bind to RBP-Jk. The most obvious differences between Notch 1 IC and Notch 3 IC are found at the C-terminal end, where Notch 3 IC is shorter and lacks the OPA repeats found in other Notch homologs. It remains to be tested, however, whether this region plays a role in transactivation (Beatus, 1999).

What is the role of Notch3 IIC in vivo? A partial reduction of HES-5 expression in the rhombomere region was observed in a nestinp/Notch 3 IC transgenic mouse embryo with a distinct CNS phenotype. This suggests that Notch 3 IC also acts as a repressor of HES expression in vivo. Downregulation of HES-5 is evident around the rhombic lip and in the myelencephalic region, but not in more anterior and posterior CNS regions. Interestingly, this is reminiscent of the situation in RBP-Jk and Notch 1 -/- mice, in which HES-5 expression is also reduced in this region. This further supports a role of Notch 3 IC as a repressor of Notch 1-signaling, but also suggests that HES-5 expression is, at least in part, regulated by other factors in other regions of the CNS. A role for Notch 3 as a repressor of HES expression in vivo receives further support from comparisons of the phenotypes resulting from targeting of HES-1 and overexpression of Notch 3 IC in transgenic mice. Expression of Notch 3 IC in the developing CNS of transgenic mouse embryos produces an embryonically lethal phenotype. The transgenic embryos have an undulating spinal cord, fail to close the anterior neural pore and exhibit protrusions of neural tissue from the anterior neural pore region. Although initially interpreted differently, the latter phenotype may be a consequence of the open neural pore, in particular considering that the transgenic embryos show a relatively modest increase in proliferative rate in the CNS. Embryos lacking the HES-1 gene die just after birth, and show a kinked neural tube, open anterior neural pore and an everted neuroepithelium. Thus, the HES-1 -/- phenotype shows clear similarities to those observed in embryos overexpressing Notch 3 IC in the early CNS (Beatus, 1999 and references).

The finding that Notch 3 IC acts as a negative modulator of HES expression will be important for understanding of the CADASIL (Cerebral Autosomal Dominant with Arteriopathy and Subcortical Infarcts with Leukoencephalopathy). CADASIL is a familial disease which leads to migraine, subcortical brain infarcts and dementia and is caused by missense mutations in the EGF-repeat region of the human Notch 3 gene. CADASIL is a dominant disease, but it is not yet known whether the mutations in Notch 3 lead to haploinsufficiency (a condition whereby normal function is impaired by loss of one functional allele), or if the CADASIL mutations result in gain-of-function receptors. Since Notch 1, 2 and 3, and HES genes are expressed in the adult brain, it is conceivable that the function of Notch 3 as a negative modulator of HES expression may be affected in the disease. Thus, if CADASIL mutations produce gain-of-function Notch 3 receptors, this would result in decreased HES expression. Conversely, in the haploinsufficiency scenario, repression of HES expression would be reduced (Beatus, 1999 and references).

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. Keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation (Rangarajan, 2001).

The Notch signaling pathway regulates the commitment and early development of T lymphocytes. Notch-mediated induction of the pre-T cell receptor alpha (pTa) gene, a T-cell-specific transcriptional target of Notch, was studied. pTa encodes a transmembrane protein that pairs with the newly rearranged TCRß chain to form the essential pre-TCR signaling complex in the developing T cells. The pTa gene is expressed in immature T cells, and its up-regulation coincides with irreversible T cell lineage commitment in both murine and human thymic precursors. Moreover, pTa is required for alphaß but not gammadelta T cell development, and may facilitate the alphaß lineage commitment by providing an instructive signal from the pre-TCR. The pTa enhancer is activated by Notch signaling and contains binding sites for its nuclear effector, CSL. Mutation of the CSL-binding sites abolishes enhancer induction by Notch and delays the up-regulation of pTa transgene expression during T cell lineage commitment. These results show a direct mechanism of stage- and tissue-specific gene induction by the mammalian Notch/CSL signaling pathway (Reizis, 2002).

Generation of left-right asymmetry is an integral part of the establishment of the vertebrate body plan. The Notch signaling pathway plays a primary role in the establishment of left-right asymmetry in mice by directly regulating expression of the Nodal gene. Embryos mutant for the Notch ligand Dll1 or doubly mutant for the Notch1 and Notch2 receptors exhibit multiple defects in left-right asymmetry. Analysis of the enhancer regulating node-specific Nodal expression reveals the presence of two consensus binding sites (T/CGTGGGAA) for the RBP-J protein. Electrophoretic mobility shift assays confirms that these consensus RBP-Ji sites bind recombinant RBP-J protein. Analysis of RBP-J RNA expression during mouse embryogenesis reveals that RBP-J is expressed ubiquitously, including in node cells that express the Nodal gene. Mutation of the RBP-J-binding sites destroys the ability of this enhancer to direct node-specific gene expression in transgenic mice. These results demonstrate that Dll1-mediated Notch signaling is essential for generation of left-right asymmetry, and that the Notch pathway acts upstream of Nodal expression during left-right asymmetry determination in mice (Luke, 2003).

Definitive hematopoiesis in the mouse embryo originates from the aortic floor in the P-Sp/AGM region in close association with endothelial cells. An important role for Notch1 in the control of hematopoietic ontogeny has been established, although its mechanism of action is poorly understood. Detailed analysis was performed of Notch family gene expression in the aorta endothelium between embryonic day (E) 9.5 and E10.5. Since Notch requires binding to RBPjkappa transcription factor to activate transcription, the aorta of the para-aortic splanchnopleura/AGM in RBPjkappa mutant embryos was examined. Specific patterns of expression of Notch receptors, ligands and Hes genes were found that were lost in RBPjkappa mutants. Analysis of these mutants revealed the absence of hematopoietic progenitors, accompanied by the lack of expression of the hematopoietic transcription factors Aml1/Runx1, Gata2 and Scl/Tal1. In wild-type embryos, a few cells lining the aorta endothelium at E9.5 simultaneously expressed Notch1 and Gata2, and it was demonstrate by chromatin immunoprecipitation that Notch1 specifically associates with the Gata2 promoter in E9.5 wild-type embryos and 32D myeloid cells, an interaction lost in RBPjkappa mutants. Consistent with a role for Notch1 in regulating Gata2, increased expression of this gene was observed in 32D cells expressing activated Notch1. Taken together, these data strongly suggest that activation of Gata2 expression by Notch1/RBPjkappa is a crucial event for the onset of definitive hematopoiesis in the embryo (Robert-Moreno, 2005).

Radial glia function during CNS development both as neural progenitors and as a scaffolding supporting neuronal migration. To elucidate pathways involved in these functions, the promoter for Blbp, a radial glial gene, was mapped in vivo. A binding site for the Notch effector CBF1 is essential for all Blbp transcription in radial glia, and BLBP expression is significantly reduced in the forebrains of mice lacking the Notch1 and Notch3 receptors. These results identify Blbp as the first predominantly CNS-specific Notch target gene and suggest that it mediates some aspects of Notch signaling in radial glia (Anthony, 2004).

The identification of Blbp as a Notch target indicates that the role of Notch signaling in neural progenitors varies as development proceeds, and that the spectrum of downstream target genes change. Blbp transcription in the neocortex does not begin until the onset of neurogenesis at E10.5, and significant levels of BLBP protein are not detectable in this region until E12.5. In contrast, neocortical expression of Hes5 is detectable as early as E9.5, and high levels of both Notch1 and Hes5 are present by E10.5. These data demonstrate that distinct developmental stages are accompanied by distinct patterns of Notch target gene expression. These shifts in Notch target gene expression appear to be mediated by additional regulatory factors that interact with the CBF1 coactivator complex; the existence of these additional factors is evident from findings that the CBF1-binding site within the Blbp promoter is necessary but not sufficient for transcription, and that multiple promoter elements mediate transcription of Blbp at different times and places. POU domain transcription factors have been implicated in regulating Blbp transcription in the embryonic forebrain, and thus represent one possible class of CBF1-interacting proteins that function in radial glia. Interestingly, Notch and the POU domain protein Nubbin positively interact to promote gliogenesis in certain Drosophila cell lineages. The fact that constitutively active Notch could promote glial fate in many but not all murine radial glia may reflect a dependence of Notch signaling on other factors such as POU domain proteins. Additional candidates include factors downstream of Neuregulin and Reelin; these signaling molecules have been shown to induce radial glial expression of BLBP (Anthony, 2004).

Finally, it is noted that whereas radial glia serve as neuronal progenitors, Bergmann glia do not. This suggests that in addition to its well-documented role as a cell fate regulator, Notch signaling may also function to support neuronal migration. Previous studies have demonstrated that Notch signaling promotes a radial glial phenotype in the forebrain, radializes cerebellar astrocytes, and induces expression of ErbB2, a receptor implicated in radial glial differentiation. Since antibody blocking experiments have implicated BLBP in regulating glial morphology, the available data suggest that Notch signaling may induce and/or maintain the radial glial scaffold, and that it does so in part through its induction of BLBP. Further insight into the mechanisms regulating radial glial function will likely be gained by the elucidation of BLBP function as well as the identification of additional radial glial Notch target genes (Anthony, 2004).

The HES family of bHLH repressors plays a key role in regulating the differentiation of neural precursors in the vertebrate embryo. Members of the HES gene family are expressed in neural precursors as targets of the Notch signaling pathway, but how this occurs in the context of neurogenesis is not known. This issue is addressed by identifying enhancers driving Notch-dependent gene expression of two Hes5-like genes expressed in Xenopus called Esr1 and Esr10. Using frog transgenesis, enhancer elements were identified driving expression of Esr1 and Esr10 in neural precursors or in response to ectopic expression of the proneural protein, Xngnr1. Using deletion and mutation analysis, motifs required for enhancer activity of both genes were defined, namely Notch-responsive elements and, in the case of Esr10, E-box motifs. Esr1 and Esr10 are differentially regulated both in terms of Notch input and its interaction with heterologous factors. These studies reveal inputs required for proneural expression of genes encoding bHLH repressors in the developing vertebrate nervous system (Lamar, 2005).

The data indicates that proneural bHLH input to the Esr10 enhancer is both indirect (through Notch) and direct. The Notch intracellular domain (ICD) and Xngnr1 synergistically upregulate transcription in transfection assays, Xngnr1 binds to the Esr10 downstream E-box in vitro, and the Esr10 proneural enhancer with mutant E-boxes shows marked loss of activity in vivo, which cannot be rescued by exogenous Xngnr1. These findings extend observations in Drosophila that proneural proteins synergize with Notch in activating E(spl) genes in larval discs. The data also support analysis of the Drosophila E(spl) gene m8. In that case, E boxes and Su(H) sites only in the configuration of a classical SPS enabled synergy between ICD and bHLH proteins, and enhancer activity was lost when one Su(H) site was mutant or oriented incorrectly. The SPS motif is a bipartite binding site for the Suppressor of hairless protein. The binding sites are separated by 30 or 29 nucleotides in the promoters of E(spl) genes of Drosophila melanogaster and higher vertebrates, respectively. One of the binding sites occurs in a reverse orientation to the other. Furthermore, a hexamer motif, which lies between or within the motifs, has a functional aspect. The Esr10 proneural enhancer behaves similarly in transgenics and provides the first example of such a required architecture among vertebrate Notch targets (Lamar, 2005).

By contrast, Esr1 is not directly regulated by proneural proteins. Although Esr1/RV has three E-boxes, E3 is not conserved in X. tropicalis, E1 is not conserved in the proneural enhancer of the closely related Esr7 gene, and neither E1 nor E3 fits the RCAGSTG consensus required for high-affinity binding of Drosophila proneural proteins to E-boxes. However, the CACCTG motif seen in E2 is targeted by Drosophila proneural proteins, a CACCTG E-box is required for retinal expression of Xenopus Ath5, and CACCTG binds MyoD in vitro and in vivo. Furthermore, E2 is embedded in a 13-base homology extending beyond the E-box in numerous Hes5 orthologs, although it is not seen in the Esr10 promoter. E2 was mutated using two strategies and no effect was seen on transgene expression in vivo. Further mutation may be required to evaluate the contribution of this motif to Esr1 expression. Nonetheless that E2 is contained within the Esr1 enhancer rules out the possibility that any factor binding to E2 is sufficient (with Notch acting through S1) to activate robust enhancer activity (Lamar, 2005).

Sites required for proneural Esr1 expression other than Su(H) sites have not been identified. Su(H) sites could be sufficient to activate Esr1, and tissue-specific responses to Notch might be due either to tissue-specific repressors or to the spacing of Su(H) sites providing a distinct platform for co-activators. Alternatively, Su(H) sites in the Esr1 enhancer could synergize with heterologous (non-bHLH) factors induced by Xngnr1, which, unlike direct bHLH input to either Esr10 or m8, interact with Notch through an S1-S4 configuration of Su(H) sites. Finally, enhancer activity could require input from both Notch (dependent on Xngnr1) and neural factors not dependent on Xngnr1. Although all three scenarios are possible, observation of attenuated but spatially appropriate GFP expression driven by the Esr1 enhancer argues against Su(H) site spacing as the sole determinant of specificity and suggests rather that tissue specific input to Esr1 requires sequences downstream of Hin3 (Lamar, 2005).

Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken stomach development

During development of the chicken proventriculus (glandular stomach), gut endoderm differentiates into glandular and luminal epithelium. Delta1-expressing cells, undifferentiated cells and Notch-activated cells colocalize within the endodermal epithelium during early gland formation. Inhibition of Notch signaling using Numb or dominant-negative form of Su(H) results in a luminal differentiation, while forced activation of Notch signaling promotes the specification of immature glandular cells, but prevents the subsequent differentiation and the invagination of the glands. These results suggest that Delta1-mediated Notch signaling among endodermal cells functions as a binary switch for determination of glandular and luminal fates, and regulates patterned differentiation of glands in the chicken proventriculus (Matsuda, 2005).

Cooperative assembly of higher-order Notch complexes functions as a switch to induce transcription

Notch receptors control differentiation and contribute to pathologic states such as cancer by interacting directly with a transcription factor called CSL (for CBF-1/Suppressor of Hairless/Lag-1) to induce expression of target genes. A number of Notch-regulated targets, including genes of the hairy/enhancer-of-split family in organisms ranging from Drosophila to humans, are characterized by paired CSL-binding sites in a characteristic head-to-head arrangement. Using a combination of structural and molecular approaches, it has been establish that cooperative formation of dimeric Notch transcription complexes on promoters with paired sites is required to activate transcription. These findings identify a mechanistic step that can account for the exquisite sensitivity of Notch target genes to variation in signal strength and developmental context, enable new strategies for sensitive and reliable identification of Notch target genes, and lay the groundwork for the development of Notch pathway inhibitors that are active on target genes containing paired sites (Nam, 2007).

Cocrystals of a human Notch transcriptional activation complex (NTC) core, which consists of an N-terminal MAML-1 peptide, the ANK domain of human Notch1, and CSL on a DNA duplex derived from the HES-1 promoter, contain contacts between the convex surfaces of ANK domains from adjacent unit cells that also are seen in crystals of the ANK domain solved in isolation in several different crystallization conditions. These contacts lie near a twofold symmetry axis in the crystals, such that the interacting complexes are positioned head-to-head at a distance roughly equal to that needed to occupy both recognition elements of an SPS. Primary sequence alignment of Notch ANK domains from different homologs shows that the key contacts are evolutionarily conserved. These conserved residues are not engaged in contacts within an individual MAML1/ANK/CSL/DNA complex, suggesting that the observed conservation reflects functional importance in mediating dimerization at SPS sites. The conservation among the four mammalian Notch receptors also predicts that each receptor should be capable of making interactions like those between the adjacent Notch1 complexes (Nam, 2007).

The ANK-ANK contacts primarily are electrostatic and lie in the second and third ankyrin repeats. Key interactions consist of contacts between the guanidino group of Arg-1985 and at least three backbone carbonyl oxygen atoms, as well as interactions between Glu-1950 and Lys-1946. Arg-1983 also forms hydrogen bonds with Ser-1952 and a backbone carbonyl. In addition to homotypic interactions between the ANK domains, unmodeled electron density in the MAML-1/ANK/CSL/DNA complex also suggests the existence of interactions between the ANK domain of one complex and the N-terminal end of MAML-1 in the second complex. Based on the architecture of the complex, and the evolutionary conservation of SPSs and the crystal contact residues, it is postulated that the ANK domains of Notch receptors mediate dimerization of ternary complexes on SPSs found in Notch target gene promoters (Nam, 2007).

To test whether residues engaged in ANK-ANK contacts in the crystal contribute to transcriptional activation of SPS-bearing promoters, the ability of different forms of ICN to induce transcription of a luciferase gene under control of the HES-1 promoter, which has a functionally important SPS element, was tested. In contrast to normal ICN1, mutations that disrupt the predicted dimerization interface either abrogate (R1985A) or diminish (K1946E and E1950K) the ability of ICN1 to induce expression of the HES-1 reporter gene. Combining the K1946E and E1950K mutations in cis, however, rescues the defect in transcriptional activation, indicating that the putative dimerization interface is functionally important in regulating transcriptional activity at a promoter that contains an SPS. In addition, when coexpressed with ICN1, the R1985A mutation dominantly interferes with activation of the HES-1 promoter element by normal ICN1. Importantly, when these mutants are scored on an artificial reporter that contains four CSL-binding sites oriented in the same direction and in tandem, there is no change in the ability of the mutants to activate transcription. Moreover, in cotransfected cells, all ICN1 polypeptides with mutations that disrupt the predicted dimerization interface are expressed at similar levels to normal ICN1, and they coimmunoprecipitate in similar amounts with CSL and MAML-1. Together, these findings indicate that the ability to form monomeric ternary complexes with MAML-1 and CSL is not affected by these mutations (Nam, 2007).

To establish directly whether NTCs (consisting of one molecule each of MAML-1, ICN, and CSL) can cooperatively dimerize on DNA, electrophoretic mobility shift assays (EMSAs) were carried on an oligonucleotide probe containing the HES-1 promoter SPS. Without Notch or MAML-1, CSL binds to each of the two sites independently. When present in excess, most probes bind a single CSL molecule, a finding consistent with previous studies showing that CSL binds its recognition element as a monomer without detectable cooperativity at paired sites. Adding RAMANK from Notch1 does not change the stoichiometric distribution of complexes bound per probe molecule. However, when MAML-1 is added, the stoichiometric distribution of the complexes changes dramatically: all of the probe is either free or bound by NTC dimers, indicating that NTC loading at one site leads to cooperative loading of the second site. As predicted, cooperative loading is abrogated by the R1985A mutation, which instead produces a smear corresponding to an ensemble of species that likely results from a weak residual tendency to self-associate. In contrast, the R1985A mutation does not detectably affect ternary complex formation on a probe containing only a single CSL-binding site, indicating that the R1985A mutation is a cooperativity mutant that specifically interferes with dimerization. The partial loss of activity of the K1946E and E1950K mutants in the HES-1 reporter assays is echoed in EMSA titrations, where the proteins undergo a cooperative transition to form dimers at a concentration ~4-fold greater than normal ICN1 or the K1946E/E1950K double mutant (Nam, 2007).

To test whether higher-order complexes exhibit specificity for the SPS architecture, additional EMSA assays were carried out on variant DNA sequences that eliminate the integrity of one of the SPS sites, flip the site orientation, or alter the spacing between the sites by a half-turn of helix. When either site A or site B is mutated so that it no longer corresponds to a CSL consensus site (YGTGDGAA), cooperative assembly of the dimer is no longer observed. Moreover, cooperative dimerization is no longer detected when the second site is inverted, and it is dramatically diminished when the second site is moved by a 5-base insertion. Because the intrinsic affinity of a single ternary complex for DNA is not altered under the conditions of inversion or insertion, these studies show that the proper spatial arrangement of the two individual binding sites is needed for cooperative dimerization to occur (Nam, 2007).

It was next asked what range of spacer lengths between sites is compatible with cooperative loading of dimeric complexes. The optimal spacing between consensus sites for cooperative dimerization is 16 bp, but cooperative dimerization still can occur on templates with spacers varying from 15 to 19 bp, implying that two NTCs can adjust their positions relative to each other to accommodate a modest range of spacer lengths between sites. This inferred flexibility is consistent with the different conformations of CSL seen in the crystal structures of the Notch ternary complexes formed with the human and worm proteins and with the enrichment of adenosine and thymidine in the spacer between the paired sites (Nam, 2007).

To determine whether the assembly of NTCs and their cooperative dimers is general among the human Notch homologues, the ability of the RAMANK domains of Notch1-4 to form complexes on single and sequence-paired sites was tested. Despite qualitative differences in mobility on the EMSA, all four purified RAMANK polypeptides bind to CSL independent of MAML-1 and then recruit MAML-1 to ternary complexes on a single site probe. When the longer, paired site probe is provided, all RAMANK polypeptides mediate cooperative dimerization, as predicted from the conservation in primary sequence at the dimerization interface. Thus, a similar series of events takes place to assemble single and dimeric NTCs in all four mammalian Notch homologues (Nam, 2007).

Genome-wide analysis reveals conserved and divergent features of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells

Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is oncogenic in murine and human T-cell progenitors. Using ChIP-Seq, this study found that in human and murine T-lymphoblastic leukemia (TLL) genomes Notch1 binds preferentially to promoters, to RBPJ binding sites, and near imputed ZNF143, ETS, and RUNX sites. ChIP-Seq confirmed that ZNF143 binds to ∼40% of Notch1 sites. Notch1/ZNF143 sites are characterized by high Notch1 and ZNF143 signals, frequent cobinding of RBPJ (generally through sites embedded within ZNF143 motifs), strong promoter bias, and relatively low mean levels of activating chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in cells. K-means clustering of Notch1 binding sites and associated motifs identified conserved Notch1-RUNX, Notch1-ETS, Notch1-RBPJ, Notch1-ZNF143, and Notch1-ZNF143-ETS clusters with different genomic distributions and levels of chromatin marks. Although Notch1 binds mainly to gene promoters, ∼75% of direct target genes lack promoter binding and are presumably regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R, and the GIMAP cluster. Human and murine TLL genomes also have many sites that bind only RBPJ. Murine RBPJ-only sites are highly enriched for imputed REST (a DNA-binding transcriptional repressor) sites, whereas human RPBJ-only sites lack REST motifs and are more highly enriched for imputed CREB sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in TLL cells, as well as unique classes of divergent RBPJ-only sites that also likely regulate transcription (Wang, 2011).

Su(H) and somitogenesis

Vertebrate hairy genes are expressed in patterns thought to be readouts of a 'segmentation clock' in the presomitic mesoderm (PSM). Transgenic Xenopus embryos were used to show that two types of regulatory elements are required to reconstitute the segmental pattern of Xenopus hairy2. The first is a promoter element containing two binding sites for Xenopus Su(H), a transcriptional activator of Notch target genes. The second is a short sequence in the hairy2 3' untranslated region (UTR), which most likely functions posttranscriptionally to modulate hairy2 RNA levels. 3' UTRs of other hairy-related, segmentally expressed genes can substitute for that of hairy2. These results demonstrate a novel mechanism regulating the segmental patterns of Notch target genes and suggest that vertebrate segmentation requires the intersection of two regulatory pathways (Davis, 2001).

A schematic is presented of the molecular targets that control the hairy2a pattern, and, by implication, the targets of the segmentation clock. There are at least three types of cis regulatory inputs. The first one is represented by the two Su(H) binding sites, which are presumably bound by a Su(H) containing complex that represses the hairy2a promoter, until Notch signaling switches Su(H) to an activator. The second one is represented by the conserved hexamer between the Su(H) binding sites. Although mutation of the hexamer lowers expression levels in tissues other than the PSM, the possibility that the hexamer has a specific function in modulating the paired Su(H) motif function in the PSM cannot be ruled out. The central role of the paired Su(H) motif for transcriptional activation of hairy2a suggests that Notch signaling must be a fundamental component of the segmentation clock. The third input occurs through the 3' UTR, in particular through the 25 bp motif. The 3' UTR confers global instability on the hairy2a RNA, but apparently local stability in the anterior PSM. How inputs through these three types of small sequences are integrated to control the dynamic hairy2a PSM pattern is a major question for understanding the molecular mechanisms of segmentation (Davis, 2001).

A molecular oscillator regulates the pace of vertebrate segmentation. The oscillator (clock) controls cyclic initiation of transcription in the unsegmented presomitic mesoderm (PSM). An evolutionarily conserved 2.3 kb region has been identified in the murine Lunatic fringe (Lfng) promoter that drives periodic expression in the PSM. This region includes conserved blocks required for enhancing and repressing cyclic Lfng transcription, and to prevent continued expression in formed somites. Dynamic expression in the cycling PSM is lost in the total absence of Notch signaling, and Notch signaling acts directly via CBF1/RBP-Jkappa binding sites to regulate Lfng. These results are consistent with a model in which oscillatory Notch signaling underlies the segmentation clock and directly activates and indirectly represses Lfng expression (Morales, 2002).

Suppressor of Hairless [Su(H)] codes for a protein that interacts with the intracellular domain of Notch to activate the target genes of the Delta-Notch signalling pathway. The zebrafish homolog of Su(H) has been cloned and characterized and its function has been analyzed by morpholino mediated knockdown. While there are at least four notch and four delta homologs in zebrafish, there appears to be only one complete Su(H) homolog. The function of Su(H) in the somitogenesis process was analyzed and its influence on the expression of notch pathway genes was examined, in particular her1, her7, deltaC and deltaD. The cyclic expression of her1, her7 and deltaC in the presomitic mesoderm is disrupted by the Su(H) knockdown mimicking the expression of these genes in the notch1a mutant deadly seven. deltaD expression is similarly affected by Su(H) knockdown like deltaC but shows in addition an ectopic expression in the developing neural tube. The inactivation of Su(H) in a fss/tbx24 mutant background leads furthermore to a clear breakdown of cyclic her1 and her7 expression, indicating that the Delta-Notch pathway is required for the creation of oscillation and not only for the synchronization between neighboring cells. The strongest phenotypes in the Su(H) knockdown embryos show a loss of all somites posterior to the first five to seven. This phenotype is stronger than the known amorphic phenotypes for notch1 (des) or deltaD (aei) in zebrafish, but mimicks the knockout phenotype of RBP-Jkappa gene in the mouse that is the homolog of Su(H). This suggests that there is some functional redundancy among the Notch and Delta genes. This fact that the first five to seven somites are only weakly affected by Su(H) knockdown indicates that additional genetic pathways may be active in the specification of the most anterior somites (Sieger, 2003).

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

Continued: Suppressor of Hairless Evolutionary homologs part 2/2

Suppressor of Hairless: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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