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. HES-1, the Hairy and Enhancer of split homologue 1 (19,52), is a vertebrate member of a highly conserved family of Hairy-related basic helix-loop-helix (bHLH) proteins. Originally described in Drosophila melanogaster, Hairy-related proteins include Hairy (51), Deadpan (3), and the seven bHLH members of the Enhancer of split [E(Spl)] complex (14, 37). Members of this family are DNA-binding transcription repressors that antagonize the function of bHLH activators and repress neuronal development (reviewed in references 6, 21, 35, and 36). The Hairy-related proteins bind to specific DNA sites (class C sites or N-boxes) in target gene promoters by means of the conserved basic region (43,44,52,56,57,59). The DNAbinding function of Hairy has been shown to be essential for the transcriptional repression of its downstream target, achaete, a proneural bHLH activator gene (44, 59). Transcriptional repression of target promoters is thought to occur at least partly by recruitment of a corepressor protein, Groucho, via the WRPW tetrapeptide motif conserved in the C terminus of all family members (24,46,61). Indeed, a fusion of the WRPW motif to the Gal-4 heterologous DNA-binding protein is sufficient by itself to repress transcription (22,25). However, Hairy also binds to another corepressor, dCtBP (48, 65), suggesting that Hairy may have alternative repression functions in addition to the conserved Groucho recruitment mechanism.Additionally, some bHLH repressors do not share the requirement for intrinsic DNA-binding capability to repress neuronal development. A bHLH-deleted version of E(Spl) (m8) has been shown to repress neuronal development despite lacking intrinsic DNA-binding capability (24,41,43). Functional dissection of the E(Spl) protein in Drosophila highlighted the importance of the helix 3-helix 4 (H-3/4) domain (37) and the WRPW motif, as well as the intervening C-terminal region, for correct bristle development (24). The mechanism of repression did not appear ...
N-myc expression in the mouse embryo was examined in its organogenesis period. Northern blot analysis of total RNA of embryos from 9.5 days to 17.5 days of gestation indicated that N-myc mRNA level was the highest at 9.5 days and decreased as development proceeded. Tissue distribution of N-myc expression in 9.5 day embryos was histologically analyzed by in situ hybridization of the transcripts and immunofluorescent staining of N-myc protein. In addition to the central nervous system indicated in previous studies on embryos of different stages, we found N-myc expression in various developing tissues. Neural crest-derived tiss,ues generally expressed N-myc transcripts and proteins to significant levels, e.g. facial primordia, visceral arches and dorsal root ganglia. Among mesodermal tissues, N-myc expression was especially high in the migrating sclerotomes derived from caudal halves of the somites, primitive nephric tubules, and mesenchymes condensed around the digestive tract and in the limb buds. Expression in endodermal tissues, however, was very low. in situ hybridization and immunohistology gave consistent results, confirming the authenticity of the detection of N-myc expression.
We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.There are a number of reported cellular oncogenes whose expression is developmentally regulated (1). This suggests that these genes may be directly or indirectly involved in the developmental process. Those encoding nuclear proteins are of particular interest, since they may participate in gene regulation in a rather straightforward way. The N-myc gene we describe in this report belongs to this category.The N-myc gene was first discovered on the basis of its amplification in some human neuroblastomas (24, 43), but later discovery of its high expression in early mammalian embryos (12,19,57) suggested its role in embryogenesis.As a member of the myc gene family, mammalian N-myc genes share general features with other members: they are composed of three exons, and coding sequences are located in the second and third exons (11,12,18,22,23,25,48,52). In spite of considerable similarities of the gene and protein structure, expression patterns of myc family genes are quite different with respect to the spatiotemporal order (11-13, 18, 19, 23, 33, 42).To elucidate the role of the N-myc gene in the developmental process, we undertook the study of chicken N-myc gene, choosing chicken as the source of the gene for the following reasons. As a classical experimental material, chicken embryo development has been thoroughly described. Since avian embryos are very accessible for experimental manipulations, our knowledge of N-myc expression can be immediately used in embryological experiments. In addition to these aspects, comparison between avian and mammalian genes can reveal regional conservations in the nucleotide and amino acid sequences more clearly than comparison between those of mammals. This will provide a rigid framework for the analysis of N-myc proteins from the aspect of functional domains. Thus, we describe here the molecular cloning, structural analysis, and embryonic expression...
The highest expression of the N-myc gene occurs during embryonic organogenesis in the mouse ontogeny, with the peak of expression around embryonic day 9.5. Homozygous N-myc-deficient mice, produced by germline transmission of a disrupted allele in ES cells, developed normally to day 10.5, indicating dispensability of N-myc expression in the earlier period, but later accumulated organogenic abnormalities and died around day 11.5. The most notable abnormalities were found in the limb bud, visceral organs (lung, stomach, liver and heart) and the central/peripheral nervous systems, and were highly correlated with the site of N-myc expression. The limb buds and the lungs excised from N-myc-deficient mutant embryos were placed in culture to allow their development to stages beyond the point of death of the embryos. Analyses indicated that the mutant limbs failed to develop distal structures and the development of bronchi from the trachea was defective in the lungs. The latter defect was largely corrected by addition of fetal calf serum to the culture medium, suggesting that an activity missing in the mutant lung was replenished by a component of the serum. The phenotype of N-myc-deficient mutant embryos indicated requirement of the N-myc function in many instances of tissue interactions in organogenesis and also in cell-autonomous regulation of tissue maturation.
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