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Abstract

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

Notch signaling dictates cell fate and critically influences cell proliferation, differentiation, and apoptosis in metazoans. Multiple factors at each step—ligands, receptors, signal transducers and effectors—play critical roles in executing the pleiotropic effects of Notch signaling. Ligand-binding results in proteolytic cleavage of Notch receptors to release the signal-transducing Notch intracellular domain (NICD). NICD migrates into the nucleus and associates with the nuclear proteins of the RBP-Jκ family (also known as CSL or CBF1/Su(H)/Lag-1). RBP-Jκ, when complexed with NICD, acts as a transcriptional activator, and the RBP-Jκ-NICD complex activates expression of primary target genes of Notch signaling such as the HES and enhancer of split [E(spl)] families. HES/E(spl) is a basic helix-loop-helix (bHLH) type of transcriptional repressor, and suppresses expression of downstream target genes such as tissue-specific transcriptional activators. Thus, HES/E(spl) directly affects cell fate decisions as a primary Notch effector. HES/E(spl) had been the only known effector of Notch signaling until a recent discovery of a related but distinct bHLH protein family, termed HERP (HES-related repressor protein, also called Hey/Hesr/HRT/CHF/gridlock). In this review, we summarize the recent data supporting the idea of HERP being a new Notch effector, and provide an overview of the similarities and differences between HES and HERP in their biochemical properties as well as their tissue distribution. One key observation derived from identification of HERP is that HES and HERP form a heterodimer and cooperate for transcriptional repression. The identification of the HERP family as a Notch effector that cooperates with HES/E(spl) family has opened a new avenue to our understanding of the Notch signaling pathway. © 2003 Wiley-Liss, Inc.

The evolutionarily conserved Notch signaling pathway controls cell fate in metazoans through local cell–cell interactions (Egan et al., 1998; Greenwald, 1998; Artavanis-Tsakonas et al., 1999). Notch signaling dictates cell fate and critically influences cell proliferation, differentiation, and apoptosis (Miele and Osborne, 1999). Components in the Notch pathway, such as Notch, bigbrain, Delta, mastermind, neuralized and enhancer of split complex, were isolated originally as neurogenic genes in Drosophila, since embryos lacking the function of these genes showed an increased number of neuroblasts at the expense of epidermal precursors (Egan et al., 1998; Greenwald, 1998; Artavanis-Tsakonas et al., 1999). However, it has subsequently been demonstrated that the Notch pathway is involved not only in neurogenesis but also in the development of many other organs derived from all three germ lines (Hartenstein et al., 1992). In vertebrates also, Notch receptors, ligands and other components are expressed in various organs from all three germ lines. Mutations for Notch receptors and ligands lead to abnormalities in many tissues, including vessels, thymus, craniofacial region, limb, rib, somite, central nervous system, heart, kidney as well as hematopoietic cells (Swiatek et al., 1994; Conlon et al., 1995; de la Pompa et al., 1997; Hrabe de Angelis et al., 1997; Sidow et al., 1997; Jiang et al., 1998; Kusumi et al., 1998; Hamada et al., 1999; Xue et al., 1999; Krebs et al., 2000; McCright et al., 2001; Dunwoodie et al., 2002). Thus, the Notch pathway plays crucial roles in the development of most organs.

Interaction of Notch receptors with their ligands such as the Delta and Jagged families leads to cleavage of the transmembrane Notch receptor, giving rise to the Notch intracellular domain (NICD) that migrates into the nucleus (Fig. 1) (Weinmaster, 1998; Mumm and Kopan, 2000). NICD has a transcriptional activation domain, but no DNA binding domain of its own. In the nucleus, NICD associates with a transcriptional factor, RBP-Jκ (also known as CSL for CBF1/Su(H)/Lag-1) (Egan et al., 1998; Greenwald, 1998; Weinmaster, 1998; Artavanis-Tsakonas et al., 1999; Mumm and Kopan, 2000), and activates transcription from the RBP-Jκ binding DNA site. In the absence of NICD, RBP-Jκ associates with a corepressor complex and acts as a transcriptional repressor from its DNA binding site (GTGGGAA) (Ling et al., 1994; Kao et al., 1998). The NICD-RBP-Jκ complex up-regulates expression of primary target genes of Notch signaling such as HES in mammals, and E(spl) (for Enhancer of Split) in Drosophila [hereafter HES/E(spl)] (Egan et al., 1998; Greenwald, 1998; Artavanis-Tsakonas et al., 1999). The HES/E(spl) family is a basic helix-loop-helix (bHLH) type trancriptional repressor and acts as Notch effectors by negatively regulating expression of downstream target genes such as tissue-specific transcription factors (Ohsako et al., 1994; Van Doren et al., 1994; Ishibashi et al., 1995; Chen et al., 1997a). Consistent with this view, HES1 and HES5, for instance, were shown to be up-regulated by NICD and necessary to prevent neuronal differentiation of neural precursor cells from mouse embryos (Ohtsuka et al., 1999).

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Figure 1. Model of the Notch signaling pathway in neurogenesis in mammals. See the text for detail. NICD, notch intracellular domain, HES: hairy and E(spl), TLE, transducin-like enhancer of split, Mash1: mammalian achaete-scute homolog 1.

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Although the HES family had been the only known effector of Notch in mammals, tissue distribution of Notch ligands and receptors does not always overlap with that of HES, suggesting the existence of yet undetected effectors of Notch signaling. Recently, a new bHLH family has been isolated and named as Hey/Hesr/HRT/CHF/gridlock/HERP (hereafter HERP) (Table 1). The amino acid sequence of HERP as well as its characteristic domains indicate that HERP is most closely related to the HES family among the reported bHLH proteins (Fig. 2). This finding immediately led to the speculation that HERP might be a novel Notch effector. Interestingly, HERP expression is detected in both HES-expressing and non-HES-expressing tissues. Remarkably, HES and HERP may function not only as homodimers but also as HES-HERP heterodimers in those cells co-expressing HES and HERP (Iso et al., 2001b). Although both HES and HERP act as transcriptional repressors, HERP employs different repression mechanisms than does HES (Iso et al., 2001b). HERP could thus play a critical role in mediating Notch effects in both HES-expressing and non-HES-expressing tissues either as a hetero- or homo-dimer.

Table 1. HERP family nomenclature
AbbreviationsaFull nameSpeciesReferences
  • a

    These proteins aligned vertically are identical or homologue of other species.

HERP1HERP2HERP3HES-related repressor proteinMouse, rat, humanIso et al. (2001a, 2002)
Hesr2Hesr1Hesr3Hairy/E(spl)-relatedDrosophila, mouse, humanKokubo et al. (1999), Satow et al. (2001)
Hey2Hey1HeyLHairy/E(spl)-related with YRPWDrosophila, chicken, mouse, humanLeimeister et al. (1999, 2000a), Steidl et al. (2000)
HRT2HRT1HRT3Hairy-related transcription factorMouse, humanNakagawa et al. (1999)
CHF1CHF2 Cardiovascular helix-loop-helix factorMouse, humanChin et al. (2000)
Gridlock   Zebrafish, humanZhong et al. (2000)
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Figure 2. Alignment of HES, HERP, and DEC amino acid sequences. A: Schematic diagram of HES, HERP, and DEC. Conserved domains are marked by distinct colors: Blue for the basic domain, green for the helix-loop-helix domain, orange for the Orange domain, and pink for the tetrapeptide motif. Potential target genes of Notch are listed on the right. DEC is shown because of its similarity to HES and HERP, but there is no data supporting DEC as a Notch target. See text for detail. B and C: Amino acid sequences of the basic helix-loop-helix domain (B) and the carboxyl termini including the tetrapeptide motif (C) were aligned by using ClustalW, and presented by BoxShade. Identical amino acids are in black and conserved residues are in gray. An arrowhead indicates the invariant amino acid residues in the basic domain of HES (proline) and HERP (glycine). Asterisks indicate the tetrapeptide motifs. D: Phylogenetic tree showing the relationship of representative members of bHLH transcription factor families. Shown is a dendrogram created by aligning the sequences of the bHLH domains of the indicated proteins by the ClustalW algorithm.

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CLASSIFICATION OF bHLH PROTEINS

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

The bHLH family of transcriptional regulators plays crucial roles in the development of various organs and cell types including the nervous system, the heart, skeletal muscles, the pancreas, endodermal endocrine organs, and hematocytes (Murre et al., 1994; Massari and Murre, 2000). Over 240 HLH proteins have been identified to date in organisms ranging from yeast to human (Massari and Murre, 2000). The bHLH proteins bind specific DNA sequences as a dimer. The basic and HLH domains have distinct functions. The basic domain is a major determinant of DNA binding specificities (Murre et al., 1994). DNA binding is mediated by a contact between each basic domain of a dimer and a specific half-site of consensus DNA sequences. The HLH domains are characterized by hydrophobic residues that allow them to form a homo- or hetero-dimer (Murre et al., 1994).

bHLH proteins can be classified into several groups according to their structural features and biochemical characteristics (Table 2A) (Murre et al., 1994; Atchley and Fitch, 1997; Fisher and Caudy, 1998; Massari and Murre, 2000). Class A proteins are transcriptional activators such as MyoD and Mash1, and bind class A sites (CANCTG) (Table 2B). Class B proteins are bHLH-lucine zipper type proteins such as Myc and Max. Both class A and B sites (CANGTG) are subtypes of the E box (CANNTG). Class C proteins are transcriptional repressors such as HES in mammals, and hairy and E(spl) in Drosophila, and are characterized by an invariant proline residue at a specific site of the basic domain. Class C proteins bind class C sites (CACGNG) as well as N-box sequences (CACNAG). Class C proteins, HES and hairy, are also known to bind class B sites to some degree but not class A sites (Ohsako et al., 1994; Van Doren et al., 1994; Fisher and Caudy, 1998; Jennings et al., 1999).

Table 2A. Classification of basic helix-loop-helix proteins
Fisher and Caudy (1998)Massari and Murre (2000) 
Class AI, IITranscriptional activators
Class BIII, IVbHLH-lucine zipper proteins
Class CVITranscriptional repressors
 VHLH proteins lacking basic region
 VIIbHLH-PAS proteins
Table 2B. Consensus binding site
ClassificationConsensusExamples
  1. Class A and B are subtypes of E box. Class C and N box are mutually overlapping.

Class ACANCTGCACCTG, CAGCTG
Class BCANGTGCACGTG, CATGTG
Class CCACGNGCACGCG, CACGAG
E boxCANNTGCACCTG, CAGCTG, CACGTG, CATGTG
N boxCACNAGCACGAG, CACAAG

STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

To date, seven HES members (Akazawa et al., 1992; Sasai et al., 1992; Ishibashi et al., 1993; Bae et al., 2000; Hirata et al., 2000; Koyano-Nakagawa et al., 2000; Pissarra et al., 2000; Bessho et al., 2001a), and three HERP members (Kokubo et al., 1999; Leimeister et al., 1999; Nakagawa et al., 1999; Chin et al., 2000; Zhong et al., 2000; Iso et al., 2001a) have been isolated in mammals (Fig. 2). The two families share several common features (Fig. 2). They contain bHLH domain, and another domain, termed the Orange (or helix3-helix4) in the corresponding regions carboxy-terminus to bHLH region. The amino acid sequences of these domains are highly conserved within the respective family, but less so among the two different families. The most remarkable difference that distinguishes HES from HERP is a proline residue in the basic region (Fig. 2B, indicated by an arrowhead). This proline residue is invariant among HES/E(spl) family members across species from Drosophila to human. Because of this proline, HES members are also called proline bHLH proteins. The HERP family has a glycine at the corresponding position and this glycine also is strictly conserved from Drosophila to human HERPs. Thus, these prolines and glycines are hallmarks for the HES and HERP families, respectively. All HES members share the C-terminal tetrapeptide WRPW motif, whereas the HERP family has YRPW or its variants. In addition to the tetrapeptide motif, the HERP family has an additional conserved region carboxyl-terminal to the tetrapeptide motif, TE(V/I)GAF, which is absent in HES (Fig. 2C).

Both HES and HERP function as transcriptional repressors. A phylogenetic tree shows that they form a distinct subgroup in a large bHLH protein family (Fig. 2D) (Vasiliauskas and Stern, 2000; Bessho et al., 2001a; Davis and Turner, 2001; Teramoto et al., 2001). These findings indicate that HERPs are closely related to the HES family belonging to class C protein, but it forms a distinct subgroup (Fig. 2D).

REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

Both the HES and HERP families have been reported to act as transcriptional repressors except HES6, which antagonizes the function of HES1, resulting in derepression (Bae et al., 2000; Koyano-Nakagawa et al., 2000). Although these two families have similar domains, they appear to use different repression mechanisms.

The HES/E(spl) family

Mechanisms for transcriptional repression by HES/E(spl) have been extensively studied genetically and biochemically. Three mechanisms have been proposed. The first mechanism is DNA-binding-dependent transcriptional repression, also known as active repression (Kageyama and Nakanishi, 1997; Kageyama et al., 2000). HES/E(spl) proteins form a homodimer and bind class C or N box consensus DNA sites (Sasai et al., 1992; Tietze et al., 1992; Oellers et al., 1994; Ohsako et al., 1994; Van Doren et al., 1994). They recruit the corepressor Groucho or its mammalian homologue TLE via the C-terminal WRPW motif (Paroush et al., 1994; Fisher et al., 1996; Grbavec and Stifani, 1996). The WRPW motif of HES/E(spl) is both necessary and sufficient to confer repression when expressed as a fusion protein with a heterologous DNA binding domain of Gal4. Chen et al. (1999) have shown in Drosophila that Groucho can recruit the histone deacetylase Rpd3, an orthologue of mammalian HDAC through poorly conseverd glycine/proline-rich domain in the central variable region of Groucho. The histone deacetylase then may repress transcription by altering local chromatin structure. Whether mammalian TLE employs the same mechanism remains to be determined.

The second mechanism is passive repression (Sasai et al., 1992; Hirata et al., 2000) involving protein sequestration. HES1, for instance, can form a non-functional heterodimer with other bHLH factors such as E47, a common heterodimer partner of tissue-specific bHLH factors such as MyoD and Mash1, thereby disrupting the formation of functional heterodimers such as MyoD-E47 and Mash1-E47.

The third mechanism is mediated by the Orange domain/helix3–helix4 (Castella et al., 2000). The Orange domain is essential to repress transcription of its own (HES1) promoter as well as the p21WAF promoter (Castella et al., 2000). This ability of the Orange domain is dependent on the presence of a DNA-binding bHLH domain. An important role of the Orange domain has been demonstrated in a sex determination assay in Drosophila (Dawson et al., 1995). Interestingly, Castella and colleagues show that the Orange domain hardly represses transcription of Gal4 dependent reporter gene constructs when fused to a Gal4 DNA binding domain (Castella et al., 2000). They proposed that the Orange domain, a putative protein interaction motif, is necessary for either the direct recruitment of an unknown corepressor and/or the stabilization or regulation of the WRPW-mediated repression function through intra- or intermolecular interaction (Castella et al., 2000).

Functional dissection of the E(spl) protein has revealed an additional important region between the Orange domain (helix3–helix4) and the WRPW motif for correct bristle development in Drosophila, although molecular mechanisms of how this region functions remain to be clarified (Giebel and Campos-Ortega, 1997).

The HERP family

A study using HERP1 deletion mutants fused with the Gal4-DNA binding domain unexpectedly revealed that the repression activity of HERP resides primarily in the bHLH domain rather than the C-terminal tetrapeptide (YQPW) motif (Iso et al., 2001b). This is in sharp contrast to the well established critical roles of the WRPW motif in the HES/E(spl) family (Wainwright and Ish-Horowicz, 1992; Paroush et al., 1994; Fisher et al., 1996; Giebel and Campos-Ortega, 1997). This indicates that engagement of TLE/Groucho is unlikely a critical component of repression by HERP. Indeed, the bHLH domain of HERP1 is both necessary and sufficient for recruitment of a corepressor complex including N-CoR, mSin3A, and HDAC1 (Iso et al., 2001b). Consistently, Nakagawa et al. (2000) showed that HRT2 (HERP1) represses its own gene expression and this repression required the basic domain, but not the carboxyl-terminal region containing the tetrapeptide motif. They further showed that the transcriptional repression by HRT2 (HERP1) was not affected by the addition of the histone deacetylase inhibitor TSA. The ineffectiveness of TSA is a paradox in view of the recruitment of HDAC to the complex by HERP1 (Nakagawa et al., 2000; Iso et al., 2001b). The reason for the apparent discrepancy is unknown. Perhaps HDAC is involved only in specific contexts (i.e., promoters and cell types), or HDAC functions, other than its inhibition of deacetylation, might be involved in the repression by HRT2.

Our studies show that HERP associates with N-CoR in addition to the Sin3/HDAC1 complex. However, recent data show that the purified mammalian Sin3 complex does not contain N-CoR (Zhang et al., 1997), nor is Sin3 present in the N-CoR-related SMRT complex (Guenther et al., 2000; Li et al., 2000). This suggests that the association of the two co-repressor complexes may not constitutively occur in cells. Whether HERP associates alternately with one co-repressor complex or the other, or HERP has an ability to simultaneously recruit the two co-repressor complexes remains to be clarified. Thus, despite similarities of their domains, HES and HERP appear to employ different repression mechanisms involving heterologous sets of corepressor proteins-Groucho/TLE for HES and N-CoR/mSin3A/HDAC for HERP.

It should be noted that, despite its well established repression role, the WRPW motif of the HES/E(spl) family is not always required. For instance, the WRPW domain of the HES/E(spl) family is dispensible for suppression of neurogenesis in zebrafish, or for suppression of Scute activity in the sex determination pathway in Drosophila (Dawson et al., 1995; Takke et al., 1999). The Orange domain of HES1, but not WRPW, is essential to repress transcription of its own gene and p21WAF promoters (Castella et al., 2000). Thus, the requirement for the WRPW of HES/E(spl) is not absolute, and repression functions of specific domains may be context-dependent. It is conceivable that HERP also might employ different domains for repression in a context-dependent manner, although all the available data thus far indicate that the tetrapeptide motif of HERP is not essential.

Passive repression mechanisms have also been proposed for the HERP family. CHF1 (HERP1) binds the aryl hydrocarbon receptor nuclear translocator (ARNT) and inhibits ARNT-dependent transcription of the VEGF promoter by dissociating the ARNT complex from DNA (Chin et al., 2000). In addition, Sun et al. (2001b) showed that CHF2 (HERP2) inhibits MyoD-dependent transcription of the myogenin promoter as well as muscle conversion, likely by disturbing the binding of MyoD-E47 heterodimers to E-box sites. They further demonstrated that this transcriptional repression by CHF2 requires a hydrophobic carboxyl-terminal region of CHF2 (HERP2) containing the Orange domain but neither the bHLH domain nor the YRPW tetrapeptide motif. Whether this region is involved in the inhibition of DNA binding by MyoD-E47 heterodimers is unknown.

Altogether, these findings illustrate that the HES and HERP families use distinct repression mechanisms. Although they have closely-related domains and motifs, they appear to use distinct domains for transcriptional repression.

DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

HES/E(spl)

It was initially reported that Drosophila E(spl) proteins bind the N box (CACNAG) (Tietze et al., 1992; Oellers et al., 1994). However, the optimal site for the E(spl) proteins determined by in vitro random oligonucleotide binding site selection is a palindromic 12-bp sequence, TGGCACGTG(C/T)(C/T)A, which contains a class B core (CACGTG) and has been termed an ESE box (for E(spl) E box) (Jennings et al., 1999). Although this approach also picked up a few percent of N box (CACGAG) and class C site (CACGCG), the binding activity of E(spl) proteins for these sites are much weaker than that for class B site (Jennings et al., 1999). The in vivo significance of class B site binding by E(spl) is confirmed by the observation that even subtle sequence changes within this class B core or flanking bases have dramatic consequences for lacZ reporter gene expression in transgenic flies (Jennings et al., 1999). Thus, the optimal sites for E(spl) proteins are likely class B sites rather than N box or class C sites in Drosophila.

Another HES/E(spl) family member, Drosophilahairy protein binds both class B and C sites in vitro (Ohsako et al., 1994; Van Doren et al., 1994). The hairy-binding class C site (CACGCG) was found in the promoter region of the achaete gene (Table 3), and the mutation of this promoter site created ectopic sensory hair organs in the fly, as in hairy mutants, strongly suggesting that hairy functions via this class C site in vivo.

Table 3. DNA binding activity for E(spl), hairy, HES, and HERP
 Class AClass BClass CE boxN boxTarget geneReferences
  1. See text and references for details. The data are based on gel mobility shift assay. ++, strong binding; +, weak binding; −, no binding. #1, proteins strongly bound E(spl) E box, or ESE box, but not classical E box. #2, the sequence of class C in hASH gene is a variant, CACGCA.

E(spl)m8    ++E(spl)m8Tietze et al. (1992), Oellers et al. (1994)
E(spl) ++#1+ + Jennings et al. (1999)
Hairy++++  AchaeteOhsako et al. (1994), Van Doren et al. (1994)
HES1+  ++HES1Takebayashi et al. (1994)
   +#2  hASHChen et al. (1997a)
   +  CD4Kim and Siu (1998)
     +Acid α-glucosidaseYan et al. (2001)
HES2   +++ Ishibashi et al. (1993)
HES3   ++ Hirata et al. (2000)
HES5   ++ Akazawa et al. (1992)
HES6    Gao et al. (2001)
  ++#1   Cossins et al. (2002)
HERP1+++++ + Iso et al. (2001b)
 ++  HERP1/HRT2Nakagawa et al. (2000)
HERP2+++++ + Iso et al. (2001b)
HERP3     Nakagawa et al. (2000)

Mammalian homologue HES proteins, HES1, -2, -3, and -5, have also been shown to bind an N box, but in vivo target genes have not been established except for HES1 (Table 3) (Akazawa et al., 1992; Sasai et al., 1992; Ishibashi et al., 1993; Hirata et al., 2000). Several target genes have been proposed for HES1. One such candidate is the HES1 gene itself. In reporter gene assays in cultured mammalian cells, HES1 negatively regulates its own promoter activity. When the N-box sequences in the HES1 promoter were mutated, this negative auto-regulation was diminished, suggesting that HES1 regulates its own gene expression through the N box in a negative feedback loop (Takebayashi et al., 1994). Mash1 is another potential target gene of HES1. Overexpressed HES1 can repress Mash1 transcription by directly binding to the promoter region of the Mash1 via a variant class C site (CACGCA) in cultured cells (Chen et al., 1997a). Consistently, targeted disruption of the HES1 gene in mice upregulates Mash1 mRNA levels (Ishibashi et al., 1995). These data suggest that HES1 functions as a negative regulator of neurogensis by directly repressing a proneural gene, Mash1. CD4 is another candidate target gene for HES1 (Kim and Siu, 1998). HES1 binds an N-box sequence in the CD4 gene promoter in vitro. Overexpression of HES1 leads to N-box-dependent repression of the CD4 promoter as well as downregulation of endogenous CD4 expression in CD4+ CD8 TH cells (Kim and Siu, 1998). The acid α-glucosidase promoter is also repressed by HES1 in a class C site (CACGCG)-dependent manner in hepatoma-derived Hep G2 cells (Yan et al., 2001). Although a cyclin-dependent kinase inhibitor, p21WAF, has also been proposed as a candidate target gene for HES1, the presence of a HES1 binding site in the p21WAF gene promoter has not been confirmed (Castella et al., 2000).

Collectively, these data indicate that class C sites and N boxes are likely critical in vivo binding sites for HES1 in mammals. Whether class B sites are in vivo targets of HES, as they are for E(spl) in Drosophila remains to be determined. Given the high amino acid similarity between Drosophila E(spl) and mammalian HES within the basic domains, however, class B sites seem to be good candidates in mammals as well.

HERP

The DNA binding site for HERP1 has also been determined by electrophoretic mobility shift assays (Iso et al., 2001b). The amino acid sequence in the basic domain of HERP1 is most similar to that of HES1 among reported bHLH proteins (Fig. 2B). Indeed, HERP1, like HES1, can bind both class B and C sites, albeit with different preferences than HES1 (Table 3). Surprisingly, HERP1 also binds class A sites (CAGGTG), raising the possibility that HERP1 may directly compete with tissue-specific bHLH activators for the E box. What are the potential target genes of HERP? It has been reported that HRT2 (HERP1) negatively regulates its own gene expression (Nakagawa et al., 2000). However, no class A, B, or C binding sites were found in the shortest reporter gene construct of the HRT2 promoter, and therefore, the apparent negative feedback regulation by HRT2 toward its own promoter may be mediated either by yet undetermined HRT2 binding sites or by indirect mechanisms.

DNA binding sequences for HERP2 seem essentially identical with those of HERP1, as expected from the very similar amino acid sequences of their DNA binding basic domains (Iso et al., 2001b). If HERP1 and HERP2 bind the same sequences, however, why are two HERPs required? One simple explanation is that HERP1 and HERP2 may be expressed in different cell types. This appears to be the case at least in several tissues and organs. For instance, HERP1 and HERP2 show a mutually complementary expression pattern in subdomains of heart and brain during embryogenesis (Table 4) (Leimeister et al., 1999; Nakagawa et al., 1999). An intriguing possibility derived from this striking mutual exclusivity is that HERP1 and HERP2 may regulate different sets of target genes and thereby contribute to establishment of distinct subdomains (i.e., cardiac atrium vs. ventricle) within a single organ. In addition, given that HERPs are transcriptional repressors, HERP members might repress each others gene expression to create a mutually exclusive expression pattern as we speculate in Figure 3. These possibilities need to be addressed in the future.

Table 4. HERP1 and HERP2 are expressed in distinct domains within individual tissues
 HERP1HERP2References
HeartVentricleAtriumThis paper, Leimeister et al. (1999), Nakagawa et al. (1999)
Craniofacial regionTissue surrounding whisker folliclesWhisker folliclesLeimeister et al. (1999)
RetinaThe outer and inner regions of the inner nuclear layerThe middle region of the inner nuclear layerSatow et al. (2001)
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Figure 3. Model for cardiac chamber formation by HERP1 and HERP2. Cardiac-specific transcriptional activators such as Nkx2.5, MEF2C, GATA4 are expressed in both ventricles and atria, and upregulate expression of cardiac-specific genes (i.e., genes for contractile proteins, genes A to G) in both chambers. Expression of the transcriptional repressors, HERP1 and HERP2, however, are confined to ventricles and atria, respectively. HERP1 expression in the ventricular precursor cells may inhibit expression of atrial-specific genes (B, E, and HERP2). In contrast, HERP2 expression in atrial precursor cells may block expression of ventricle-specific genes expression (C, F, and HERP1). In this manner, HERP1 and HERP2, in combination with the activators, may contribute to defining the atrial and ventricular chambers. HERP1 and HERP2 might mutually repress each others expression.

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The strict conservation of proline and glycine in the basic domains of all the HES and HERP family members, respectively, suggests a potentially important role of these residues (arrowhead in Fig. 2B). However, a role for these residues in the DNA binding of HES/HERP has not been rigorously determined. In one study, a proline to asparagine mutation in a E(spl) protein largely diminishes its DNA binding activity, whereas a mutation of the same proline to threonine appears to have little effect (Tietze et al., 1992; Oellers et al., 1994). In addition, a glycine to proline mutation in CHF2 (HERP2) did not significantly affect repression of the myogenin promoter activity in cultured cells or of myogenic conversion of 10T1/2 cells by MyoD (Sun et al., 2001b). A potential role of the proline/glycine in defining the DNA binding specificity as well as other functions of HES and HERP families remains to be clarified. Hitherto, in vivo target genes for HERP have not been determined. In addition to the above mentioned auto- and cross-regulation between different HERP members, the previously established HES1 target such as Mash1 seems to be a target candidate for the HES1-HERP heterodimer (see below), if both are co-expressed in the same cells.

HERP: A NEW HETERODIMER PARTNER FOR HES

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

HES and HERP are co-expressed in certain cells (summarized in Table 5), and they both can bind the same DNA sequences in vitro. Why then do cells need the two effectors for Notch signaling if they both target the same genes? One possibility is that such occupancy may depend simply on the relative abundance of HES and HERP proteins in a given cell. Alternatively, DNA binding affinities of HES and HERP homodimers may be modulated in vivo, for instance, by post-translational modification and protein–protein interaction. Phosphorylation of HES1 at specific residues in the basic domain results in the loss of its DNA-binding activity (Strom et al., 1997). Since these residues are not present in HERP, such a phosphorylation event in HES1 would selectively lower its DNA binding affinity, and might allow HERP to occupy the promoter.

Table 5. Co-expression of HES and HERP
 HESHERPReferences
  • a

    Although those genes listed in the table seem to be expressed within the same organ, it is not certain whether or not they are expressed within the same cells. Based on results of many different reports, the data are combined. All data are derived from in situ hybridization except HES1 expression in an embryonic heart, which is detected only by RNA protection assay.

Embryonic heartHES1a (whole heart)HERP1 (ventricles) HERP2 (atria)This paper, Sasai et al. (1992), Leimeister et al. (1999), Nakagawa et al. (1999)
Presomitic mesodermHES1, -5, and -7HERP1, -2, and -3Kokubo et al. (1999), Nakagawa et al. (1999), Jouve et al. (2000), Leimeister et al. (2000a), Bessho et al. (2001a), Dunwoodie et al. (2002)
Olfactory epitheliumHES1, -3, -5, and -6HERP1 and -2Lobe (1997), Kokubo et al. (1999), Nakagawa et al. (1999), Leimeister et al. (1999), Bae et al. (2000), Cau et al. (2000), Vasiliauskas and Stern (2000), Pissarra et al. (2000)
Wisker follicleHES3HERP2Lobe (1997), Leimeister et al. (1999)
Dorsal root gangliaHES3 and -6HERP1 and -3Lobe (1997), Nakagawa et al. (1999), Bae et al. (2000), Leimeister et al. (2000a), Pissarra et al. (2000), Vasiliauskas and Stern (2000)
ThymusHES1 and -6HERP3Sasai et al. (1992), Nakagawa et al. (1999), Leimeister et al. (2000a), Pissarra et al. (2000)

A more intriguing possibility is that HES and HERP positively interact with each other to enhance DNA binding. Indeed, HES and HERP associate with each other as a hetero-oligomer both in vitro and in intact cells in the absence of DNA (Leimeister et al., 2000a; Iso et al., 2001b). Gel-shift assays have demonstrated that a heterodimer of HES-HERP binds far more efficiently than the respective homodimers to class C DNA sequences (Fig. 4) (Iso et al., 2001b). Consistently, reporter gene assays using multimerized class C sites showed synergystic repression by HES-HERP heterodimers (Iso et al., 2001b). The marked increase in DNA binding activity shown by HES-HERP heterodimers, accompanied by their functional synergy, strongly suggests that HES-HERP heterodimers are likely to form and act in cells co-expressing them. Heterodimerization of the two Notch effectors provides an efficient means for signal amplification.

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Figure 4. HES1 and HERP1 form a heterodimer. A representative gel shift assay showing HES1-HERP1 heterodimer formation. A gel shift assay was performed using nuclear extracts from the cells transfected with the expression vectors for HERP1 and HES1. These proteins were incubated with a class C DNA probe. Note that nuclear extracts from the cells expressing both HES1 and HERP1 showed a dramatic increase of DNA binding activity (compare lane 5 with lane 1 or 3). This intense band (lane 5) contains a HES1-HERP1 heterodimer since addition of antibodies against either HES1 or HERP1 shifted this band (lanes 6 and 7). The simultaneous addition of the two antibodies generates a further shifted band (not shown). Homodimers of HES1 (lane 1) or HERP1 (lane 3) are not detectable with this short exposure time, although addition of the antibodies do show up marginally visible supershifted bands (lanes 2 and 4).

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MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

Notch signaling is best known for lateral inhibition (Egan et al., 1998; Greenwald, 1998; Artavanis-Tsakonas et al., 1999). Lateral interaction occurs within a population of equivalent cells and results in the generation of different cell types. Once a small difference is created between equivalent cells, a cell that has obtained a specific fate prevents surrounding cells from assuming the same cell fate through cell–cell interaction. This process, termed lateral inhibition or lateral specification, is mediated by Notch signaling and exemplified by C. elegans gonadogenesis and Drosophila sensory organ development (Egan et al., 1998; Greenwald, 1998; Artavanis-Tsakonas et al., 1999).

In mammals, the Notch pathway dictates cell fate decisions of bipotential precursor cells to generate distinct subpopulations, a phenomenon reminiscent of lateral specification. Examples of such mammalian cell fate decision by Notch include T lymphoid versus B lymphoid cells, pancreatic exocrine versus endocrine cells, and arteries versus veins (Apelqvist et al., 1999; Pui et al., 1999; Lawson et al., 2001).

It is tempting to speculate that HERP1 and HERP2, as direct targets of Notch, might also contribute to dictating cell fate dicisions. As discussed in the earlier section, tissue distribution of HERP1 and HERP2 are often observed in a strikingly complementary fashion within single organs (Table 4) (Leimeister et al., 1999; Nakagawa et al., 1999). For instance, expression of HERP1 mRNA in the heart is limited to ventricles whereas that of HERP2 is confined to atria in mouse embryos (Fig. 5) (Leimeister et al., 1999; Nakagawa et al., 1999). In the craniofacial region, HERP2 is observed in the whisker follicles while HERP1 is expressed in the surrounding tissue (Leimeister et al., 1999). Given that HERPs are transcriptional repressors, HERPs might play a direct role in establishing distinct cell fates from equi-potential primordial cells by repressing cell-type specific genes. For instance, HERP1 expressed in heart ventricle might eliminate atria-specific gene expression in cells of a future ventricle-forming region to establish ventricular identity during embryonic heart development (Fig. 3). Unlike the HERP family, the HES family does not show such expression patterns, and hence HERP might play a more critical role than HES in establishing subdomains of an organ.

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Figure 5. In situ analysis of expression of HERP1 and HERP2 mRNA transcripts. Serial horizontal sections of mouse embryos (E12.0) at heart level were prepared. Hematoxylin eosin (HE) staining (left). Dark field micrographs for mRNA expresion of HERP1 (center) and HERP2 (right). Note that HERP1 mRNA is limited to ventricles while HERP2 mRNA is predominantly expressed in the atria.

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What are the upstream signals that restrict expression of one HERP member or another in cells of a specific subdomain of an organ? Given the multiple Notch ligands and receptors, specific ligands and receptors might be responsible for distinct HERP member expression. In this regard, HES1 expression is induced by co-culture of C2C12 myoblast cells with Dll1 (Delta like 1)—but not with Jagged1-expressing cells (Shawber et al., 1996; Jarriault et al., 1998; Kuroda et al., 1999), suggesting ligand-specific regulation for HES1. Are HERP family members also regulated in a ligand-specific manner? In contrast to HES1, both ligands, Dll1 and Jagged1, equally induce HERP2 mRNA expression in C2C12 myoblast (Iso et al., 2001a). However, ligand- and receptor-specific regulation for HERP members have not been extensively addressed and remains a good possibility.

ROLES OF HERP IN DEVELOPMENT

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

HERP in angiogenesis

Evidence is mounting that Notch signaling is involved in the development of the vascular system (Ruchoux et al., 1995; Joutel et al., 1996, 2000; Uyttendaele et al., 1996, 2000, Uyttendaele et al., 2001; Zimrin et al., 1996; Hrabe de Angelis et al., 1997; Wong et al., 1997; Shen et al., 1997a; Xue et al., 1999; Chin et al., 2000; Krebs et al., 2000; Shutter et al., 2000; Zhong et al., 2000, 2001; Henderson et al., 2001; Lawson et al., 2001; Lindner et al., 2001; Villa et al., 2001). Both HERP1 and HERP2 mRNA are highly expressed in the aorta (Chin et al., 2000; our unpublished observation), and another HERP member HeyL (HERP3) mRNA is also detected in aortic smooth muscle layer of the mouse embryo (Leimeister et al., 2000b). Expression of mouse CHF1 (HERP1) is induced during differentiation of neural crest-derived Monc1 cells into vascular smooth muscle cells (Chin et al., 2000). Expression of mRNAs for both HERP1 and HERP2 is induced in cultured smooth muscle cells by stimulation of Notch ligands such as Dll1 and Jagged1 (Iso et al., 2002). In endothelial cells also, Hesr1 (HERP2) mRNA expression is induced during endothelial cell tube formation, a well-characterized in vitro angiogenic process, and gain- and loss-of-function studies show that Hesr1 (HERP2) is involved in proliferation, migration, and network formation of endothelial cells (Henderson et al., 2001).

In zebrafish, the gridlock mutation (grl m145), originally isolated in a large-scale chemical mutagenesis screen for developmental mutations of the zebrafish, shows an abnormal assembly of the aorta (Zhong et al., 2000). The grl gene (HERP1 homologue) is strongly expressed in the dorsal aorta, but not in the axial vein. The grl m145 mutation changes the stop codon to Gly and extends the protein by 44 amino acids at the carboxyl-terminus, resulting in a phenotype showing selective disturbance of assembly of the aorta. Injection of wild type grl RNA in the mutant restores a normal phenotype. In addition, gridlock is required for arterial-venous differentiation during embryonic vascular development (Zhong et al., 2001). Loss of grl function ablates regions of the artery, and expands contiguous regions of the vein.

Collectively, these findings indicate that HERPs are involved in multiple aspects of vascular development including smooth muscle differentiation, angiogenic processes, arterial-venous cell fate determination, and vascular morphogenesis. Given that HERP1 and HERP2 are immediate targets of Notch and that expression of HES genes has not been observed in the vascular system, HERP family members may be the effectors of the Notch pathway in vascular development.

HES and HERP in somitogenesis

Somites appear as epithelial spheres in a head-to-tail order in an oscillating fashion from a mesenchymal unsegmented tissue called the presomitic mesoderm (PSM) (Hartenstein et al., 1992; Maroto and Pourquie, 2001). Somites generate the skeletal muscles of the body as well as the axial skeleton and the dermis of the back.

Mutations for Notch1, Dll1, Dll3 and other components of Notch signaling such as presenilin, RBP-Jκ, and lunatic fringe (Lfng) exhibit defects of somite segmentation (Swiatek et al., 1994; Conlon et al., 1995; Oka et al., 1995; Hrabe de Angelis et al., 1997; Wong et al., 1997; Evrard et al., 1998; Kusumi et al., 1998; Zhang and Gridley, 1998; Donoviel et al., 1999; Huppert et al., 2000; Koizumi et al., 2001; Dunwoodie et al., 2002). Recent studies also demonstrate that Notch signaling regulates the synchronously oscillating gene expression in the PSM. Expression of mRNA for c-hairy1, c-hairy2, HES1, HES5 as well as Lfng oscillates in the PSM (Palmeirim et al., 1997; Forsberg et al., 1998; McGrew et al., 1998; Aulehla and Johnson, 1999; Jouve et al., 2000; Dunwoodie et al., 2002). Oscillations of these genes are likely to be involved in a clock linked to somite segmentation. The dynamic expression of HES1 mRNA as well as compartmentalization of somites was disrupted in the Dll1 null mutant (Hrabe de Angelis et al., 1997; Jouve et al., 2000). Mouse Dll3 null mutants also show delayed and irregular somite formation and the disrupted expression of HES1 and HES5 in PSM (Dunwoodie et al., 2002). These findings suggest that Notch signaling regulates the cyclic expression of HES1 and HES5 mRNA and somite formation. However, mutation of the HES1 gene does not recapitulate the Dll1 mutant phenotype (Ishibashi et al., 1995), suggesting that HES1 may not be required for somitogenesis or the HES1 defect can be compensated by other factors.

It has recently been reported that the expression of the newly isolated HES7 gene oscillates in the PSM and that targeted disruption of the HES7 gene in mice resulted in disorganized segmentation of somites and loss of cyclic expression of Lfng in the PSM (Bessho et al., 2001b), indicating essential functions of HES7 in somitogenesis.

All three HERP members also are expressed in the PSM. HRT1 (HERP2) and HRT3 (HERP3) also are expressed in a dynamic pattern in the PSM and somites, but expression of HRT2 (HERP1) is much weaker than HERP2 and HERP3 (Nakagawa et al., 1999). Leimeister et al. (2000a) also showed that expression of mHey2 (HERP1) in the PSM was severely affected in Dll1 and Notch1 knockout mice. They also found that expression of cHey2, a chicken homologue of HERP1 oscillates in the chicken PSM and overlaps precisely with that of c-hairy1 throughout the PSM (Leimeister et al., 2000a). In addition, Dunwoodie et al. (2002) found that normal expression of mHey1 (HERP2) in PSM is disrupted in the Dll3 null mutant.

These findings suggest that all three HERP family members as well as HES1, HES5, and HES7 are likely to play crucial roles in somitogenesis. Analysis of somite formation in HERP null mutant mice is needed to clarify the roles of HERPs in somitogenesis.

HERP in myogenesis

Given the critical roles of Notch signaling in developing somites (see the preceding section HES and HERP in Somitogenesis), one wonders whether Notch signaling might also be involved in muscle differentiation in the myotomal compartment of the somite. Consistent with this idea, mouse HRT1 (HERP2) mRNA is predominantly detected in the dermomyotome within the somite (Nakagawa et al., 1999).

Coculture of C2C12 myoblast cells with Notch ligand-expressing cells blocks muscle differentiation by inhibiting expression of muscle differentiation markers such as myogenin, myosin light chain 1, -2 and -3, α-myoglobin, and troponin T (Lindsell et al., 1995; Luo et al., 1997; Jarriault et al., 1998; Kuroda et al., 1999). Whereas Notch-ligand stimulation induces strong and continuous HERP2 mRNA expression in C2C12 cells (Iso et al., 2001a), HES1 mRNA expression was only transiently and very weakly induced in these muscle cells (Shawber et al., 1996; Jarriault et al., 1998; Kuroda et al., 1999; Iso et al., 2001a). Furthermore, forced expression of HES1 did not inhibit muscle differentiation of C2C12 cells (Nofziger et al., 1999). These findings suggest that HERP2 may play a more important role than HES1 in inhibiting muscle differentiation. To further support this idea, Sun et al. (2001b) showed that CHF2 (HERP2) mRNA is expressed at a high level in undifferentiated C2C12 myoblast cells, but it declines as muscle differentiation proceeds. They further show that exogenously overexpressed CHF2 may inhibit MyoD-induced myogenic conversion of 10T1/2 cells by associating with MyoD and inhibiting DNA binding of MyoD-E47 heterodimers (Sun et al., 2001b). These data strongly suggest a potential role of HERP2 as a negative regulator of muscle differentiation, although further loss-of-function studies for HERP2 are necessary to demonstrate that HERP2 is a true effector of the Notch pathway in inhibiting muscle differentiation.

HES and HERP in gliogenesis

Notch signaling is generally thought to inhibit the differentiation of neural precursor cells and keep them in an undifferentiated state. However, this notion has recently been challenged (Wang and Barres, 2000; Frisen and Lendahl, 2001). Several groups have reported that Notch1 and its effectors, HES1 and HES5, may play an instructive role in actively promoting gliogenesis rather than simply inhibiting neuronal differentiation (Furukawa et al., 2000; Gaiano et al., 2000; Hojo et al., 2000; Morrison et al., 2000). For example, cultured neural crest stem cells (NCSCs) generate Schwann cells with a high frequency when Notch is activated, either by an active form of Notch or by soluble ligands (Morrison et al., 2000). An instructive role of Notch in gliogenesis has also been reported during retinogenesis. Furukawa et al. (2000) reported that retinal progenitor cells overexpressing either the NICD or HES1 by retroviral infection differentiated into Muller glia cells (MGCs), while forced expression of a dominant-negative HES1 gene reduced the number of glia. In another report, HES5-deficient retina showed a 30–40% decrease in MGC numbers without affecting cell survival, whereas forced expression of HES5 by retrovirus significantly increased the population of glial cells at the expense of neurons (Hojo et al., 2000). These findings suggest an active role of Notch in gliogenesis, which may be mediated by HES family members.

Hesr2 (HERP1) may also mediate Notch signaling in retinal gliogensis (Satow et al., 2001). Hesr2 (HERP1) is predominantly expressed in the middle region of the inner nuclear layer of the retina, which mainly contains MGCs and other cell types. In retinal explant studies, retroviral expression of exogenous Hesr2 (HERP1), but not Hesr1 (HERP2) or Hesr3 (HERP3), promoted gliogenesis while inhibiting neurogenesis (Satow et al., 2001), indicating a specific role of HERP1 in promoting gliogenesis. Consistent with an active role of HERP1 in retinal gliogenesis, a double mutation of neuronal determination genes, Mash1 and Math3, led to an increase in the number of MGCs and upregulation of Hesr2 (HERP1) expression (Satow et al., 2001). These results suggest that Hesr2 (HERP1) may be a Notch effector in promoting retinal gliogenesis.

It should be noted, however, that whether the gliogenesis by Notch is a purely instructive or default mechanism (due to inhibition of neurogenesis), is not completely established. If it turns out to be an instructive effect of Notch, a central issue in the future is to determine the molecular targets and specific effectors of Notch signaling that result in gliogenesis.

REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

HES and HERP as a primary target of notch signaling

Several lines of evidence have suggested that HES-1, -5, -7, and HERP-1, -2, -3 are potential target genes of Notch (Figs. 2A and 6). The promoters of HES1, -5, and -7 as well as those of HERP1, -2, and -3 are activated by the constitutively active NICD in transiently transfected reporter gene assays (Jarriault et al., 1995; Nishimura et al., 1998; Maier and Gessler, 2000; Nakagawa et al., 2000; Bessho et al., 2001a; Iso et al., 2002). In addition, endogenous HERP-1 and -2 mRNA expression (but not HES1) were strongly upregulated after the NICD expression in several different cell types including C2C12, 10T1/2, 293T, and U2OS cells (Iso et al., 2001a). Although these results suggest that these members of the HES and HERP families may be potential targets of Notch, the question of whether they are the direct and physiological targets of the NICD-RBP-Jκ complex remains unsolved by these studies using overexpressed NICD. It is formally possible that HES and HERP expression may be upregulated secondary to expression of other genes after NICD overexpression. Further, overexpressed NICD may cause physiologically irrelevant cellular reactions. This latter concern is substantiated by the observation that endogenous NICD is hardly detected by immunostaining, whereas transfected NICD is expressed at a readily detected level (Lieber et al., 1993; Kopan et al., 1994; Nye et al., 1994; Ahmad et al., 1995; Jarriault et al., 1995; Zagouras et al., 1995; Capobianco et al., 1997).

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Figure 6. A: Constitutively active Notch fails to induce HERP and HES mRNA expression in RBP-Jκ-deficient OT11 cells. RBP-Jκ-deficient OT11 (derived from mice with targeted RBP-Jκ gene disruption) or control OT13 (RBP-Jκ positive) cells were infected with recombinant adenovirus expressing NICD (Adeno-NICD) or control virus (Adeno-empty). Northern blot analysis was performed with RNA from duplicate infections. Note the robust induction of HERP1 and HERP2 mRNA, weak induction of HERP3, and marginal induction of HES1 by Adeno-NICD only in OT13 cells, but not in OT11 cells. B: Restoration of HERP and HES mRNA induction by exogenous RBP-Jκ expression in RBP-Jκ-deficient OT11 cells. OT11 and OT13 cells were infected with adenovirus expression vectors at the indicated multiplicity of infection (MOIs). Note that HERP1 and HERP2 mRNA induction is restored in OT11 cells only when Adeno-RBP-Jκ is co-infected with Adeno-NICD. Expression of HERP3 and HES1 mRNA are less conspicuous in these cells. Thus the expression of RBP-Jκ protein was sufficient to rescue HERP and HES mRNA induction by Notch in RBP-Jκ-deficent OT11 cells.

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To circumvent these potential concerns associated with overexpression studies, several groups used a co-culture approach with Notch ligand-expressing cells, which is considered to generate a more physiological level of Notch signaling (Shawber et al., 1996; Jarriault et al., 1998; Kuroda et al., 1999; Iso et al., 2001a). Use of cyclohexamide, an inhibitor of de novo protein synthesis in combination with the co-culture system eliminates secondary effects by expression of other proteins, and allows one to assess direct effect of Notch signaling on upregulation of HES and HERP mRNA (Kuroda et al., 1999; Iso et al., 2001a, 2002). These studies have now provided strong evidence that HES1, HERP1, and HERP2 are primary targets of Notch in tissue culture.

Tissue-specific expression of HERP1

Expression of HERP1 was not induced by ligand stimulation in any of the several cell types initially tested including C2C12 muscle cells, 10T1/2 fibroblasts, 293T, and P19 teratocarcinoma cells ((Iso et al., 2001a) and our unpublished results). Because of this observation, we initially thought that HERP1 may not be an immediate target of Notch or that the HERP1 gene might be under cell type-specific regulation. The observation that zebrafish gridlock (HERP1) plays a central role in the development of the aorta (Zhong et al., 2000) suggested a cell type specific role for HERP1 in vascular tissue. Consistent with this, when an aortic smooth muscle cell line, A10, was used in coculture studies, expression of endogenous HERP1 (as well as HERP2) mRNA is induced by Notch in the absence of de novo protein synthesis (Iso et al., 2002). Since HERP1 mRNA expression is detected in multiple tissues in mice embryos, its role may not be limited to vascular cells. However, the absence of HERP1 mRNA induction in several different kinds of cells described above suggests that HERP1 might have a more cell-type-restricted role (i.e., vascular tissue) than does HERP2.

Mutual repression between members of the HERP family

Notch stimulation engendered by coculturing cells with Notch ligand-expressing cells can induce expression of both HERP1 and HERP2 mRNA in A10 smooth muscle cells (Iso et al., 2002). Interestingly, the time courses of these induced expression patterns are different between the two. Upon co-culture, both HERP1 and HERP2 mRNAs simultaneously start to accumulate, and yet they accumulate in a very different manner. HERP2 mRNA rapidly accumulates, reaches a plateau at 8 h that is sustained for at least 24 h. In contrast, HERP1 mRNA expression is transient. The HERP1 mRNA level rapidly decreases after reaching a peak at 6 h, and returns to basal levels by 8 h, as if HERP2 suppresses HERP1 mRNA accumulation. The decrease of HERP1 mRNA expression in the face of increasing levels of HERP2 mRNA is somewhat reminiscent of the mutually exclusive expression of HERP1 and HERP2 reported in several subdomains of different organs observed in animals (Leimeister et al., 1999; Nakagawa et al., 1999). These results raise the possibility that HERP1 and HERP2 might mutually suppress each others expression. Consistent with this idea is the finding that overexpressed HRT1 (HERP2) inhibits HRT2 (HERP1) promoter activity in reporter gene assays in transient transfection studies (Nakagawa et al., 2000) (our unpublished data). Further studies are needed to examine whether this attractive model (see also Fig. 3) is valid in vivo.

Linkage among specific Notch ligands, receptors, and effectors

The finding that HERP can be induced in a cell-type specific manner raises a question of how different members of HES and HERP are differentially regulated by Notch. There are six Notch ligands (Dll1–4, Jagged1, and Jagged2), six receptors (Notch 1–6), and six known target genes (HES-1, -5, -7, and HERP 1–3). The existence of such multiple components at each step of Notch signaling leads one to speculate that different ligands, for instance, might be linked to distinct receptors and effectors. However, tissue distribution of these components does not immediately support their specific relationships (Lindsell et al., 1996; de la Pompa et al., 1997; Leimeister et al., 1999; Nakagawa et al., 1999).

As discussed earlier, HERP1 mRNA was induced by Notch ligand stimulation only in A10 aortic smooth muscle cells, but not in several other cells tested, whereas HERP2 mRNA was induced in all these cell lines (Iso et al., 2001a, 2002). All these cells express at least Notch-1, -2, and -3 receptors (Iso et al., 2001a, Iso et al., 2002), and yet only A10 smooth muscle cells express HERP1 mRNA, indicating that the selective induction of HERP1 in A10 cells is not due to selective expression of the three Notch receptors. Other cellular components of Notch signaling necessary to activate HERP1 may be present only in A10 cells but not in the other cells. Recently, additional receptors, Notch-5 and -6 have been isolated in zebrafish (GeneBank accession number; Y10353 for Notch5 and Y10354 for Notch6) (Sawada et al., 2000). It remains to be studied whether mammalian homologues of these new Notch receptors as well as Notch4 might participate in the cell-type specific expression of HERP1.

Analyses of mice deficient for a component of the Notch pathway have also provided insight regarding the relationship between specific Notch ligands, receptors, and effectors. Gene disruption of Notch ligand Dll1 causes a decrease in expression of HERP1, -2, and -3 in mice (Leimeister et al., 2000a,b). In Notch1 deficient mice, expression of HES5, HERP1, -2, and -3 were greatly diminished in various tissues while HES1 expression was not affected in any tissue (de la Pompa et al., 1997; Leimeister et al., 2000a,b). In Notch2 null mutant mice, neither HES1 nor HES5 expression was altered (Hamada et al., 1999). In mice deficient for Dll3, normal expression of HES1, HES5, and Hey1 (HERP2) was disrupted in PSM (Dunwoodie et al., 2002). Mice homozygous for RBP-Jκ gene disruption exhibits reduced expression of HES5, but not HES1 (de la Pompa et al., 1997). These findings indicate that a single Notch ligand or receptor participates in the upregulation of multiple members of the HES and HERP families. It should be kept in mind, however, that expression of HES and HERP genes was not completely abolished in these mutants, suggesting that there are alternative pathway(s) to maintain their expression in animals. The unaltered HES1 expression in most of these mutants mice, despite the well-documented function of HES1 as a Notch effector, suggests that the defects of these genes may be compensated by other receptors, ligands, or an unidentified RBP-Jκ-independent pathway for HES1 expression.

Clarifying these relationships between ligands, receptors, and target genes is one of key issues to understand tissue-specificity in Notch signaling, and continued efforts toward discovery of new Notch components and their characterization are necessary toward this end.

ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

It has recently been shown that RBP-Jκ is a protein component of a large native NICD complex in the nucleus (Jeffries et al., 2002). The activated Notch receptor, NICD has a domain termed RBP-Jκ associated module (RAM), through which NICD associates with RBP-Jκ (Tamura et al., 1995). The RAM domain of NICD is required for activation of target gene promoters in reporter gene assays (Nofziger et al., 1999; Iso et al., 2002). Mutations of RBP-Jκ binding sites in the promoters of HERP1, HERP2, and HES1 genes abolish activation of their promoters by Notch (Jarriault et al., 1995; Nishimura et al., 1998; Maier and Gessler, 2000; Nakagawa et al., 2000; Iso et al., 2002). These findings suggest a critical role of RBP-Jκ in the target genes regulation by Notch.

To further address the role of RBP-Jκ protein in the, expression of endogenous HERP and HES mRNAs, several groups employed a mutant RBP-Jκ (RBP-Jκ R218H) that does not bind DNA and has been considered as a dominant negative mutant (Kato et al., 1997; Nofziger et al., 1999; Nakagawa et al., 2000). In Xenopus oocytes, expression of the dominant negative forms of X-Su(H)1, a Xenopus homologue of RBP-Jκ, leads to a neurogenic phenotype with an increased number of primary neurons, whereas overexpression of wild type X-Su(H)1 did not significantly alter neuronal phenotype in vivo (Wettstein et al., 1997). In cultured mammalian cells also, transactivation of HERP1 and HES1 promoters by NICD was reduced by overexpressing the RBP-Jκ mutants, R218H and RY227GS, which lack DNA binding activity (Iso et al., 2001a). In addition, Xenopus ESR1 (a Notch target closely related to HES5) mRNA induction by Notch was reduced by the dominant negative form of X-Su(H) in oocytes (Wettstein et al., 1997). In these two studies, however, even wild type X-Su(H) or RBP-Jκ/CSL has a similar degree of inhibitory effects. This may be due to uncontrolled expression of RBP-Jκ/X-Su(H) that squelches the signaling molecules and disrupts their stoichiometry, and raises a concern regarding the specificity of the putative dominant negative RBP-Jκ and the role of wild-type RBP-Jκ in expression of HES or HERP.

To consolidate the role of RBP-Jκ protein in HERP and HES mRNA expression, RBP-Jκ deficient cells (OT11) derived from homozygous RBP-Jκ null mice were employed (Iso et al., 2001a). As shown in Figure 6A, constitutively active NICD expressed by recombinant adenovirus vector failed to induce HERPs and HES1 mRNA expression in the RBP-Jκ-deficient OT11 cells. Induction of their expression is restored by simply providing exogenous RBP-Jκ protein in these cells (Fig. 6B). Thus, the absence of HERP and HES mRNA induction in OT11 cells is not due to inadvertent phenotypic changes in these cells; rather it is a direct consequence of the lack of RBP-Jκ. These findings established that RBP-Jκ is essential for HES and HERP expression in response to Notch stimulation.

ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

A group of recently isolated genes, Stra13/SHARP/DEC genes (hereafter DEC family) have amino acid sequences most similar to those of HES among the known bHLH proteins (Fig. 2A) (Boudjelal et al., 1997; Rossner et al., 1997; Shen et al., 1997b; Fujimoto et al., 2001). DEC has a proline residue in the basic domain (not in the same position as HES) and the Orange domain. Davis and Turner named HES, HERP, and DEC families as bHLH-O proteins, as they represent a unique family of bHLH proteins bearing the Orange domain (Davis and Turner, 2001) (Fig. 2D). DEC functions as a transcriptional repressor through class B DNA sequences (Zawel et al., 2002) and is involved in the control of proliferation and/or differentiation of chondrocytes and neurons as well as hematopoiesis (Boudjelal et al., 1997; Rossner et al., 1997; Shen et al., 1997b; Sun et al., 2001a; Seimiya et al., 2002). These features of DEC make one wonder whether DEC might be a primary target of Notch. DEC expression is induced by several stimuli including hypoxia, cAMP, and TGF-β (Shen et al., 1997b, 2001; Ivanova et al., 2001; Yoon et al., 2001; Yun et al., 2002; Zawel et al., 2002). However, there is no evidence that DEC is upregulated by Notch. Nevertheless, given the similarities between DEC and HES, the possibility remains that DEC might be a Notch target in other contexts than have been tested.

Recently, it has been reported that promoter regions of a number of genes other than HES and HERP families contain RBP-Jκ binding sites, including MHC class I, CD23, interleukin6, β-globin, erbB-2, NF-kB2, and cyclin D1 genes (Israel et al., 1989; Ling et al., 1994; Shirakata et al., 1996; Chen et al., 1997b; Kannabiran et al., 1997; Plaisance et al., 1997; Lam and Bresnick, 1998; Oswald et al., 1998; Ronchini and Capobianco, 2001). The cyclin D1 promoter which has a poorly conserved consensus sequence, GCTGAGAT, is bound by RBP-Jκ in electrophoretic mobility shift assay, and cyclin D1 mRNA expression was upregulated by overexpressed NICD (Ronchini and Capobianco, 2001). Thus, the cyclin D1 gene is likely an immediate target of the NICD-RBP-Jκ complex. In the other genes mentioned above, however, the implication of RBP-Jκ in their transcription has been suggested only by electrophoretic mobility shift assays and reporter gene assays, without demonstrating induction of their mRNA expression by Notch. Conversely, induction of endogenous mRNA following overexpression of NICD has been shown in some genes, such as CD21 and NRARP (Notch related ankyrin repeat protein), although promoter analyses of these genes have not yet been reported (Strobl et al., 2000; Krebs et al., 2001; Lamar et al., 2001). Therefore, further studies, including those with ligand stimulation in addition to NICD overexpression, are awaited to determine whether these genes are directly regulated by the NICD-RBP-Jκ complex in vivo, and whether protein products of some of these putative Notch target genes might act as an effector for Notch signaling. Nevertheless, these findings are concordant with the idea that HES and HERP may not be the only primary target or effector for the Notch signaling.

ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

As we have seen, HES1, -5, -7 and HERP1, -2, -3 are likely target genes of Notch. Among these HES and HERP members, HES1 and HES5 are true effectors of Notch in animals. Kageyama's group has shown that inhibitory effect of Notch signaling on neuronal differentiation was abolished in neural precursor cells prepared from HES1–HES5 double deficient mice (HES1−/−/HES5−/−), but not in cells from either HES1 (HES1−/−) or HES5 (HES5−/−) single mutants, indicating that HES1 and HES5 have redundant functions in mediating the Notch effects (Ohtsuka et al., 1999). These findings, together with induction of HES1 and HES5 mRNA by Notch, provide strong evidence that HES1 and HES5 are effectors of Notch.

That HERP may be a physiological Notch effector is supported by the following observations: 1) HERP mRNA expression is directly up-regulated by Notch ligand stimulation in the absence of de novo protein synthesis in cultured cells, 2) HERP has an intrinsic transcriptional repressor activity and forms a heterodimer with HES1, the established Notch effector, and 3) the HES-HERP heterodimer binds the same group of target DNA sequences as HES homodimer but with much higher efficiency. However, physiological relevance of these observations needs to be confirmed in target gene promoters.

CONCLUSIONS AND FUTURE DIRECTIONS

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES

In this review, we summarized similarities and differences between HES and HERP in molecular structure, repression mechanisms and tissue distribution. Several members of both families are primary target genes of Notch (Figs. 2, 6, and 7). They are directly induced by Notch ligand stimulation in the absence of de novo protein synthesis, and some of them in a cell type-specific manner. Although both HES and HERP families possess very similar domains, belong to class C bHLH protein families and function as transcriptional repressors, they utilize distinct domains and corepressors to repress transcription (Figs. 2 and 7).

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Figure 7. Model for HES and HERP cooperation in Notch signaling. Upon notch stimulation, HES and HERP expression may both be induced. In tissues where only HES or HERP is expressed, the respective homodimer binds promoters of target genes. The HES homodimers recruit TLE via their C-terminal WRPW motif, whereas the HERP homodimers recruit an N-CoR/mSin3A/HDAC complex via their bHLH domain. In tissues where both HES and HERP are coexpressed, the HES-HERP heterodimers become the predominant complex that avidly binds a specific DNA site, which may be newly defined by the two heterologous basic domains of HES and HERP. Because of the higher DNA binding activity of the heterodimers, a lower concentration of HES and HERP may be sufficient to achieve repression. Repression by HES-HERP heterodimers may be reinforced by their ability to recruit a more diverse set of corepressors including both TLE and N-CoR/mSin3A/HDAC. The model is based on experimental data derived largely from HES1 and HERP1. Because of the significant similarity among members of each family, however, such a model may be apt for other members including those in human, mouse, and Drosophila. The association of HERP with N-CoR is based on co-transfection and GST-pull down studies (Iso et al., 2001b). N-CoR has been recently shown to associate with HDAC3 (Wen et al., 2000; Guenther et al., 2001), but not with the Sin3/HDAC1 complex (Guenther et al., 2000; Li et al., 2000). Further studies are under way to clarify the significance of the N-CoR interaction with HERP. See text for further discussion.

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The differential expression of HES and HERP in different tissues suggests that HES and HERP may work separately as the respective homodimers. In other tissues where both of them are co-expressed, Notch signaling may rely on heterodimer formation between HES and HERP with distinctive repression mechanisms (Fig. 7). Because both HES and HERP are conserved from Drosophila to human, the heterodimerization of HES-HERP is likely to be conserved throughout evolution. In vivo target genes for HES and HERP have not been firmly established except for a few cases (Ishibashi et al., 1995; Chen et al., 1997a), and identification of target genes for a HES-HERP heterodimer as well as HERP homodimers is among the most important issues to be addressed in the future. We have discussed the possibility that the two HERP isoforms, HERP1 and HERP2, might regulate subdomain-specific gene expression within a single organ, such as cardiac ventricle versus atrium (Fig. 3). Identification of such HERP isoform-specific target genes may greatly facilitate our understanding how distinct subdomains of a single tissue or organ (i.e., cardiac chamber formation) is established.

Ever increasing numbers of isoforms of Notch components—ligands, receptors, and effectors—are certainly adding complexities to Notch signaling. Each ligand isoform, for instance, may be linked to only specific isoforms of receptors, which in turn may be linked to particular effector isoforms. Such a link among specific isoforms of ligand, receptor, and effector might create cell-type specific sub-pathway of Notch signaling, and contribute to generation of distinct cell fates, provided that different effector isoforms regulate distinct sets of target genes.

This hypothesis is supported partly by the tissue-specific distribution of different isoforms of Notch components. For instance, HERP1 appears to be important particularly in the development of vascular tissue, and HERP1 might be regulated by vascular-specific isoforms of ligands and receptors such as Dll4 and Notch4/int-3 that are predominantly expressed in vascular endothelial cells (Uyttendaele et al., 1996; Shirayoshi et al., 1997; Shutter et al., 2000). Distinct functions of each isoform in animals are clearly demonstrated at least for Notch receptors and ligands, by the gene disruption studies for three receptors (Notch1, Notch2, and Notch4) and four ligands (Dll1, Dll3, Jagged1 and Jagged2). Mice with a mutation of one of these genes show different phenotypic changes, indicating distinct roles of the isoforms (Swiatek et al., 1994; Conlon et al., 1995; Hrabe de Angelis et al., 1997; Sidow et al., 1997; de la Pompa et al., 1997; Jiang et al., 1998; Kusumi et al., 1998; Hamada et al., 1999; Xue et al., 1999; Krebs et al., 2000; McCright et al., 2001; Dunwoodie et al., 2002).

The formation of HES-HERP heterodimers might also allow cells to regulate different target genes than the respective homodimers, by redirecting target DNA binding specificity, although such a change was not obvious in gel-shift assays (Iso et al., 2001b). Alternatively, quantitative differences in degrees of affinity of target genes by homodimers versus heterodimers might also lead to different phenotypes.

HES and HERP have been viewed as primary targets/effectors of Notch. However, we have detected a low level of HES1 and HERPs mRNAs even in the absence of RBP-Jκ in cultured cells. Consistently, RBP-Jκ null mice show no change in spatial distribution of HES1 transcripts (de la Pompa et al., 1997). These results make one wonder whether Notch signaling can express these effectors by means other than RBP-Jκ, or whether there are Notch-independent mechanisms for HES and HERP expression. In this regard, there are several examples of cross-talk between Notch and other signaling pathways (Axelrod et al., 1996; Guan et al., 1996; Price et al., 1997; Ordentlich et al., 1998; Oswald et al., 1998; Bash et al., 1999; Carmena et al., 2002; Chu et al., 2002), and such signals other than Notch might be involved in the regulation of HES1 and HERPs expression.

Finally, mutations of Notch components are implicated in human diseases such as acute T cell lymphoblastic leukemia (T-ALL) (Ellisen et al., 1991), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (Joutel et al., 1996), and Alagille syndrome (arteriohepatic dysplasia manifest by a paucity of biliary ducts in the liver and a variety of cardiovascular abnormalities including the great vessels) (Oda et al., 1997). These diseases are due to mutations of Notch ligands and receptors. Unregulated expression of components of Notch is also related to many malignant tumors (Zagouras et al., 1995; Daniel et al., 1997; Chen et al., 1997a; Gray et al., 1999; Jang et al., 2000; Ito et al., 2001; Shou et al., 2001). Given that certain HES and HERP members are Notch target/effectors, one would wonder whether misexpression of HES and HERP genes might also be involved in the etiology of human diseases. HERP genes have been mapped by fluorescence in situ hybridization both in mouse and human, and HERP mutations have already been screened in several candidate diseases whose genetic loci map in the vicinity of the chromosomal location of HERP genes. However, initial screening did not reveal any diagnostic alterations in the coding region (Steidl et al., 2000).

Continued efforts to dissect the regulatory mechanisms of HES and HERP as well as discovery of new factors in the Notch pathway should lead us to a deeper understanding of Notch signaling. Such information would hopefully lead, eventually, to the development of diagnostic and therapeutic modalities for Notch-related diseases. This quest will be greatly facilitated as the Human Genome Project approaches completion over the next several years.

REFERENCES

  1. Top of page
  2. Abstract
  3. CLASSIFICATION OF bHLH PROTEINS
  4. STRUCTURAL SIMILARITIES AND DIFFERENCES AMONG HES AND HERP FAMILIES
  5. REPRESSION MECHANISMS OF THE HES AND HERP FAMILIES: REPRESSION DOMAINS AND COFACTORS
  6. DNA BINDING SITE SPECIFICITY AND TARGET GENES FOR HES AND HERP
  7. HERP: A NEW HETERODIMER PARTNER FOR HES
  8. MUTUALLY EXCLUSIVE EXPRESSION OF HERP1 AND HERP2
  9. ROLES OF HERP IN DEVELOPMENT
  10. REGULATION OF HES AND HERP GENE EXPRESSION BY NOTCH SIGNALING
  11. ROLE OF RBP-Jκ IN HES AND HERP GENE EXPRESSION
  12. ARE HES AND HERP THE ONLY PRIMARY TARGETS OF NOTCH?
  13. ARE HERPs PHYSIOLOGICAL EFFECTORS OF NOTCH?
  14. CONCLUSIONS AND FUTURE DIRECTIONS
  15. Acknowledgements
  16. REFERENCES
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