HES-1 repression of differentiation and proliferation in PC12 cells: role for the helix 3-helix 4 domain in transcription repression.

HES-1 is a Hairy-related basic helix-loop-helix protein with three evolutionarily conserved regions known to define its function as a transcription repressor. The basic region, helix-loop-helix domain, and WRPW motif have been characterized for their molecular function in DNA binding, dimer formation, and corepressor recruitment, respectively. In contrast, the function conferred by a fourth conserved region, the helix 3-helix 4 (H-3/4) domain, is not known. To better understand H-3/4 domain function, we expressed HES-1 variants under tetracycline-inducible control in PC12 cells. As expected, the induced expression of moderate levels of wild-type HES-1 in PC12 cells strongly inhibited nerve growth factor-induced differentiation. This repression was dependent on the H-3/4 domain. Unexpectedly, expression of HES-1 also arrested cell growth, an effect that could be reversed upon down regulation of HES-1. Concomitant with growth arrest, there was a strong reduction in bromodeoxyuridine incorporation and PCNA protein levels, although not in cyclin D1 expression. Expression of a HES-1 protein carrying the H-3/4 domain, but not the WRPW domain, still partially inhibited both proliferation and differentiation. Transcription assays in PC12 cells directly demonstrated that the H-3/4 domain can mediate DNA-binding-dependent transcription repression, even in the absence of corepressor recruitment by the WRPW motif. HES-1 expression strongly repressed transcription of the p21(cip1) promoter, a cyclin-cyclin-dependent kinase inhibitor up regulated during NGF-induced differentiation, and the H-3/4 domain is necessary for this repression. Thus, the H-3/4 domain of HES-1 contributes to transcription repression independently of WRPW function, inhibits neurite formation, and facilitates two distinct and previously uncharacterized roles for HES-1: the inhibition of cell proliferation and the direct transcriptional repression of the NGF-induced gene, p21.

Regulation of hippocampal neuronal differentiation by the basic helix-loop-helix transcription factors HES-1 and MASH-1.

HES-1 is a vertebrate homologue of the Drosophila basic helix-loop-helix (bHLH) protein Hairy, a transcriptional repressor that negatively regulates neuronal differentiation. HES-1 expression in neuronal precursors precedes and represses the expression of the neuronal commitment gene MASH-1, a bHLH activator homologous to the proneural Achaete-Scute genes in Drosophila. Down-regulation of HES-1 expression in developing neuroblasts may be necessary for the induction of a regulatory cascade of bHLH activator proteins that controls the commitment and progression of neuronal differentiation. Here we show that the differentiation of embryonic day-17 rat hippocampal neurons in culture was coincident with a decline in HES-1 expression and DNA binding. Therefore, we examined the effect of forced expression of HES-1 and MASH-1 upon nerve growth factor (NGF) -induced differentiation in TrkA transfected hippocampal neurons. Expression of HES-1 inhibited both the intrinsic and NGF-induced neurite outgrowth, whereas MASH-1 expression increased neurite outgrowth. Strikingly, the increased hippocampal differentiation observed with MASH-1 expression is completely blocked by coexpression of HES-1. Furthermore, both wild-type HES-1 and a non-DNA binding mutant of HES-1 repressed MASH-1-dependent transcription activation. These results suggest that down-regulation of HES-1 is necessary for autonomous, growth factor-induced and MASH-1-activated hippocampal differentiation.

The function of hairy-related bHLH repressor proteins in cell fate decisions.

Hairy-related proteins are a distinct subfamily of basic helix-loop-helix (bHLH) proteins that generally function as DNA-binding transcriptional repressors. These proteins act in opposition to bHLH transcriptional activator proteins such as the proneural and myogenic proteins; together, the activator and repressor genes that encode these proteins have co-evolved as a regulatory gene "cassette" or "module" for controlling cell fate decisions. In the development of the Drosophila peripheral nervous system, Hairy-related genes function at multiple steps during neurogenesis, for example, as positional information genes that establish the "prepattern" that controls where "proneural cluster" equivalence groups will form, and later as nuclear effectors of the Notch signaling pathway to "single out" individual precursor cells within the equivalence group. Hairy-related genes also function in the establishment and restriction of other types of equivalence groups, such as those for muscle and Malphigian tubule precursors. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis.

Groucho-dependent and -independent repression activities of Runt domain proteins.

Runt domain proteins are transcriptional regulators that specify cell fates for processes extending from pattern formation in insects to leukemogenesis in humans. Runt domain family members are defined based on the presence of the 128-amino-acid Runt domain, which is necessary and sufficient for sequence-specific DNA binding. We demonstrate an evolutionarily conserved protein-protein interaction between Runt domain proteins and the corepressor Groucho. The interaction, however, is independent of the Runt domain and can be mapped to a 5-amino-acid sequence, VWRPY, present at the C terminus of all Runt domain proteins. Drosophila melanogaster Runt and Groucho interact genetically; the in vivo repression of a subset of Runt-regulated genes is dependent on the interaction with Groucho and is sensitive to Groucho dosage. Runt's repression of one gene, engrailed, is independent of VWRPY and Groucho, thus demonstrating alternative mechanisms for repression by Runt domain proteins. Unlike other transcriptional regulatory proteins that interact with Groucho, Runt domain proteins are known to activate transcription. This suggests that the Runt domain protein-Groucho interaction may be regulated.

Mediation of NGF signaling by post-translational inhibition of HES-1, a basic helix-loop-helix repressor of neuronal differentiation.

The induction of neurite outgrowth by NGF is a transcription-dependent process in PC12 cells, but the transcription factors that mediate this process are not known. Here we show that the bHLH transcriptional repressor HES-1 is a mediator of this process. Inactivation of endogenous HES-1 by forced expression of a dominant-negative protein induces neurite outgrowth in the absence of NGF and increases response to NGF. In contrast, expression of additional wild-type HES-1 protein represses and delays response to NGF. Endogenous HES-1 DNA-binding activity is post-translationally inhibited during NGF signaling in vivo, and phosphorylation of PKC consensus sites in the HES-1 DNA-binding domain inhibits DNA binding by purified HES-1 in vitro. Mutation of these sites generates a constitutively active protein that strongly and persistently blocks response to NGF. These results suggest that post-translational inhibition of HES-1 is both essential for and partially mediates the induction of neurite outgrowth by NGF signaling.

The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain.

Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.

Hairy function as a DNA-binding helix-loop-helix repressor of Drosophila sensory organ formation.

Sensory organ formation in Drosophila is activated by proneural genes that encode basic-helix-loop-helix (bHLH) transcription factors. These genes are antagonized by hairy and other proline-bHLH proteins. hairy has not been shown to bind to DNA and has been proposed to form inactive heterodimers with proneural activator proteins. Here, we show that hairy does bind to DNA and has novel DNA-binding activity: hairy prefers a noncanonical site, CACGCG, although it also binds to related sites. Mutation of a single CACGCG site in the achaete (ac) proneural gene blocks hairy-mediated repression of ac transcription in cultured Drosophila cells. Moreover, the same CACGCG mutation in an ac minigene transformed into Drosophila creates ectopic sensory hair organs like those seen in hairy mutants. Together these results indicate that hairy represses sensory organ formation by directly repressing transcription of the ac proneural gene.

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence.

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

daughterless, a Drosophila gene essential for both neurogenesis and sex determination, has sequence similarities to myc and the achaete-scute complex.

daughterless (da) has multiple functions in Drosophila embryonic development: maternal da activity is necessary for proper sex determination, and zygotic da activity is necessary for formation of the peripheral nervous system. We have cloned the region containing da and have found that five recessive lethal da mutations map to a single transcription unit. A predicted protein product of this transcription unit has sequence similarities with the oncogene myc, with the gene MyoD1, which is involved in myoblast determination, and with the Drosophila achaete-scute complex, which is involved in neuronal precursor determination. The role of da as a gene controlling cell determination in multiple developmental pathways is discussed.

The maternal sex determination gene daughterless has zygotic activity necessary for the formation of peripheral neurons in Drosophila.

The daughterless (da) gene is known to have separate maternal and zygotic functions: Maternally supplied daughterless activity is required for proper sex determination and dosage compensation in female embryos, whereas loss of zygotically supplied da+ activity causes embryonic lethality in both male and female embryos. We have found that the zygotic da+ activity is necessary for neural development: The use of neuron-specific antibodies and beta-galactosidase-marked X chromosomes has revealed that in both male and female embryos deletions or strong mutations of the da gene remove all peripheral neurons and associated sensory structures without disrupting the epithelium from which they derive. Partial da+ function causes partial removal of peripheral neurons. Our results indicate that da+ is required for the formation of peripheral neurons and their associated sensory structures.

Pioneer growth cone behavior at a differentiating limb segment boundary in the grasshopper embryo.

The first neurons to extend axons through embryonic grasshopper limbs are a pair of sibling pioneer neurons. After migrating proximally along the limb axis, the pioneer growth cones normally make an abrupt ventral turn. In some cases (less than 20%) this turn is directly toward the proximo-ventrally located Cx1 guidepost neurons. However, in the majority of cases (greater than 80%) the pioneer growth cones make a more acute ventral turn along a single circumferential line which lies distal to the Cx1 neurons. Growth cones from other afferent neurons orient along the same line. Growth cones can extend along this line around more than half of the circumference of the limb and can grow in either direction along it. The circumferential line appears to be the prospective trochanter-coxa segment boundary. Afferent axons on the segment boundary leave it and contact the proximo-ventrally located Cx1 neurons. The site at which pioneer growth cones leave the boundary is variable and appears to be the point from which filopodial contact with Cx1 cells is first established. In addition to the trochanter-coxa segment boundary, the pioneer growth cones and axons also respond to the tibia-femur and femur-trochanter segment boundaries. The role of segment boundaries as barriers to growth cone movement and the effect of such barriers on the timing and placement of differentiation of pioneer neurons are discussed.

Epithelial cell specialization at a limb segment boundary in the grasshopper embryo.

Epithelial cells at the developing femur-trochanter limb segment boundary in the grasshopper embryo are specialized with respect to nonboundary cells. They are elongated, with the long axis oriented along the limb circumference. Some cells along the boundary preferentially bind an antibody (anti-HRP), and so are molecularly specialized as well. The specialized cells are the most proximal cells of the more distal segment.

Pioneer growth cone steering along a series of neuronal and non-neuronal cues of different affinities.

We have analyzed the morphology of over 5000 Ti1 pioneer growth cones labeled with anti-HRP, which reveals the disposition of axons, growth cone branches, and filopodia. Ti1 axon pathways typically consist of a sequence of 7 characteristically oriented segments, with a single, distinct reorientation point between each segment. Growth cones exhibit the same orientations and reorientations in a given region as do axon segments at later stages. The single, distinct reorientations suggest that growth cones make discrete switches between guidance cues as they grow. Ti1 growth cones are guided by various types of cues. A set of 3 immature identified neurons serves as nonadjacent guidepost cells and lies at the proximal end of 3 of the axon segments. To form another segment, growth cones reorient along a limb segment boundary within the epithelium. Growth cones also respond consistently to, and orient toward, a specific mesodermal cell, which may be a muscle pioneer. Thus, growth cones respond to at least 3 different types of cells in the leg. Ti1 growth cones exhibit a hierarchy of affinity for these cues. Guidepost neurons are the dominant cues in that contact with them reorients growth cones from guidance by the other types of cues. Growth cone branches are exclusively oriented to specific cues. Growth cones reorient by extending a branch directly to the cue of highest affinity and by withdrawing any branches that are extended to a cue of lesser affinity. A single filopodium in direct contact with a guidepost neuron can reorient a growth cone that still has multiple filopodia or even prominent branches specifically oriented to a previous cue of lesser affinity. These observations suggest that growth cone steering may not result simply from passive adhesion and filopodial traction, but may involve more active processes.

Pioneer growth cone morphologies reveal proximal increases in substrate affinity within leg segments of grasshopper embryos.

We have compared the morphologies of approximately 5000 antibody-labeled afferent pioneer growth cones fixed at various stages of growth along their characteristic path over the epithelium in the legs of grasshopper embryos, and have used growth cone morphology as an indicator of differences in the affinity of the epithelial substrate for pioneer growth cones in vivo. Growth cone morphologies differ markedly between different locations in limb buds, and also in the same location in limbs at different stages of differentiation. Growth cones characteristically extend branches and lamellae circumferentially along segment boundaries, and filopodia and lamellae are retained (or extended) longer there. Where they contact a relatively well-differentiated segment boundary, the growth cones also abruptly reorient circumferentially. In the proximal regions of limb segments, growth cones consistently have a high degree of branching and lamellae; previously formed axons also extend secondary branches and spread there as development progresses. Low incidence of these morphologies is observed at all stages in the distal regions of limb segments. Thus, neuronal morphologies correlate both spatially and temporally with the differentiation of limb segmentation. These results suggest the following: Detailed growth cone morphology is a reliable indicator of differences in extrinsic guidance cues. The affinity of the epithelial substrate for afferent pioneer growth cones increases proximally within segments, with a peak at the segment boundary. (This affinity could be based on surface density of adhesion molecules or on nonadhesive molecules that actively regulate growth cone extension.) Increasing epithelial affinity within segments appears to act as a proximal guidance cue for afferent pioneer growth cones. Pioneer growth cones are observed to navigate proximally in circumstances where proximally located guidepost cells differentiate too late to guide them.

Pioneer axons lose directed growth after selective killing of guidepost cells.

The first nerve cells to appear in the limb buds of embryonic grasshoppers are a pair which lie at the distal tip and project axons along the length of the limb to the central nervous system (CNS). The stereotyped route navigated by these 'pioneer' axons is followed by other neurones and eventually becomes that of a major adult nerve trunk. The guidance cues which delineate this route are unknown, but it has been suggested that guidance is provided by a set of nonadjacent 'guidepost' cells along which the pioneers grow (Fig. 1). We have now tested this suggestion by selectively destroying identified guidepost cells and observing pioneer axon trajectories in their absence. Our results support the guidepost cell hypothesis.