INTRODUCTION

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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 DNA-binding 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 to require the conserved basic and helix-loop-helix (HLH) regions. Moreover, while WRPW and H-3/4 deletions were generally neutral, a bHLH construct retaining just the H-3/4 region was dominant negative for bristle formation, suggesting a functional role for the H-3/4 domain.

The H-3/4 domain of Hairy (37), called the orange domain by Dawson and colleagues (13), was shown to be necessary for Hairy function in a sex determination assay in Drosophila. In the experiments of Dawson et al., the activity of the H-3/4 domain was dependent upon the presence of a DNA-binding bHLH region, but not the WRPW motif. Interestingly, a Hairy protein chimera with the H-3/4 domain replaced with the corresponding region from HES-1 retained function, whereas the corresponding E(Spl) m8 substitution was inactive. Thus, the H-3/4 domain appears to be important for function in both Hairy and E(Spl) proteins, though neither the underlying mechanism nor the basis for the apparent specificity of this region have been established.

The regulation of neuronal differentiation by HES-1 is analogous to the function of Hairy in several key respects. Like Hairy, HES-1 has been shown to be a DNA-binding transcription repressor (52, 55, 56) which recruits Groucho-related TLE family corepressors to DNA at specific sites (22, 54). Also, MASH-1, an achaete-scute homologue necessary for nervous system development (27), is transcriptionally regulated by HES-1 through a specific site (class C site) in the MASH-1 promoter (8), comparable to the transcriptional repression of achaete by Hairy (44, 59). Unlike Drosophila, where the loss of the bHLH repressor leads to additional neuronal cells, deletion of the HES-1 gene in mice results in a marked loss of neurons, apparently due to the premature differentiation of neuronal precursors (33). HES-1 has also been shown to block the transcription-activation and myogenic differentiation properties of the bHLH activator, MyoD (52). In vitro studies suggest that HES-1 interacts with the ubiquitous E2A proteins (E-proteins) E12 and E47, thereby disrupting the formation of MyoD-E-protein heterodimers. A similar inhibition of MASH-1 activity was also reported (52). The functional requirement for the H-3/4 domain in either DNA-binding-dependent or -independent repression by HES-1 has not been determined.

The linkage of bHLH repressors to cell fate specification and proliferation has been most clearly documented in the development of neuronal progenitor cells in the Drosophila peripheral nervous system (reviewed in reference 5). The differentiation of the sensory organ is promoted by bHLH activators and inhibited in the surrounding epithelial cells by the E(Spl) complex bHLH repressors, and it is dependent upon two rounds of additional cell division. The linkage between differentiation and cell cycle control is better established for the bHLH activators (10, 12, 40, 47, 66). Transcription factors such as myogenin (62) and NeuroD (38, 42) are known to coordinate the up regulation of differentiation-specific genes with exit from cell cycle. This is thought to result at least partially from the up regulation of the cyclin-cyclin-dependent kinase (CDK) inhibitor, p21^cip1/WAF-1 (16, 30, 31, 40, 45, 63, 66). While it is not known if HES-1 coordinates any aspect of cell cycle control with the inhibition of differentiation, p21 could be a target for HES-1. In vivo, p21 is expressed predominantly in terminally differentiated neuronal cells, while HES-1 is expressed earlier in the neuronal precursors of the mitotically active ventricular zone (52). The p21 promoter contains multiple bHLH activator-binding sites (E-boxes) which have been shown to be functional in the up regulation of p21 (49). The Id HLH repressor protein (2), which lacks a basic region and forms non-DNA-binding heterodimers with bHLH activators (E-proteins), has also been shown to repress p21 expression (49). Similarly, HES-1 might also repress p21 transcription, either through E-protein interaction or by binding to DNA directly at specific sites.

For the analysis of H-3/4 domain function, we expressed wild-type HES-1 (WT HES-1) and several mutant forms of HES-1 in PC12 cells. PC12 cells are a rat pheochromocytoma cell line (26) that has been extensively studied in the analysis of HES-1 and neuronal differentiation (19, 55) as well as in the regulation of cell cycle by p21 (17, 50, 60, 64). We generated tetracycline-inducible stable cell lines and found that overexpression of WT HES-1-repressed nerve growth factor (NGF)-induced differentiation, as expected from previous studies (55), and that this repression was dependent upon the H-3/4 domain. Unexpectedly, we also found that overexpression of WT HES-1 also repressed proliferation. Repression of proliferation by WT HES-1 was also observed in transiently transfected neuroblastoma cells, and in colony-forming efficiency (CFE) assays in PC12 cells. Furthermore, we identified the promoter of the cyclin-CDK inhibitor, p21, as a direct target for HES-1-mediated transcriptional repression in the inducible PC12 cells, and this repression was also H-3/4 dependent. Transcription assays using the auto-regulated HES-1 promoter (56) and the p21 promoter showed that the H-3/4 domain conferred DNA-binding-dependent transcription repression function to HES-1 independently of the WRPW motif. Thus, the H-3/4 domain of HES-1 is an important component of HES-1-mediated transcription repression and the inhibition of differentiation and growth arrest.

	MATERIALS AND METHODS

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Expression vector construction. The HES-1 expression plasmids used in the transient reporter assays and CFE assays were based on pCDNA3 (Invitrogen, Carlsbad, Calif.). We have previously described Flag epitope-tagged WT and basic region mutant HES-1 constructs (7). R and S HES-1 are deletions made by cutting the full-length construct at the most 5' of the internal RsaI and SmaI sites, respectively and utilizing a stop codon present after the EcoRV 3' cloning site of the pCDNA3 vector. The basic region mutation in a DNA-binding-defective mutant (B* HES-1) is from an equivalent construct previously named DN HES-1 (55), and B*S HES-1 incorporates the same mutation into S HES-1. The 3/4 HES-1 construct was made by partial RsaI and SmaI digestion to remove the H-3/4 coding region. The Gal-4 fusion proteins were generated from the full-length HES-1 pCDNA constructs. An RsaI/EarI-digested fragment was blunt-end cloned into the HindIII-digested pM vector, which contains the Gal-4 DNA-binding domain, to generate pM H3/4/C. An RsaI/SmaI-digested fragment was blunt-end cloned into the HindIII-cut pM vector to generate pM H3/4. To enable blunt-end cloning, the overhangs of the HindIII-digested pM vector were filled in with the Klenow fragment of DNA polymerase I prior to use. The construct pM C was made by first cloning the SmaI/EarI-blunted fragment of HES-1 into the EcoRV site of pCDNA3. The fragment was cut out with an EcoRI/NotI digest (sites in vector) and then blunt-end cloned into the BamHI site in pM, using Klenow to fill in the overhangs prior to ligation. The bHLH expression constructs were derived from the Gal-4 fusion constructs. pM H3/4 was digested with SmaI/XbaI to release the fragment for H-3/4. A partial SmaI digest of XbaI-cut pM C was used to generate the SmaI/XbaI fragment for cloning HES C. HES 3/4/C was generated from the BamHI/XbaI fragment of pM H3/4/C. These fragments were cloned into the NotI (Klenow-filled)/XbaI sites of a pCDNA3 vector that contained the Flag epitope, followed by the nuclear localization signal derived from pVP16.

Cell maintenance. PC12 and tetracycline-inducible PC12 cells were maintained in Dulbecco minimum essential medium (Mediatech, Herndon, Va.) with 10% horse serum antibiotic (Gemini Bio-products, Calabasas, Calif.) and 5% fetal calf serum antibiotic (Gemini Bio-products), P-Gent antibiotic (Gemini Bio-products), and Glutamax (Life Technologies, Gaithersburg, Md.) in 10% CO₂ in a humidified atmosphere at 37°C. Fungizone (Life Technologies) was added to the media during selection of the stable cell lines. PC12 tetracycline-inducible cells were maintained in the presence of 2 mg of tetracycline per ml and 100 µg of G418 per ml (Gemini Bio-products). Hygromycin B (100 µg/ml) (Life Technologies) was used to maintain selection of the cells following stable transfection with the pBI expression vector. Unless otherwise indicated, 2.5S murine NGF (Promega) was added at 100 ng/ml, human recombinant bFGF (Promega) was added at 10 ng/ml, and retinoic acid (Sigma, St. Louis, Mo.) was added to a 1 µM concentration.

Stable cell line generation. The PC12 cells used to produce the stable cell lines were purchased from Clontech and are stably transfected with the tetracycline-sensitive activator protein under neomycin resistance. The PC12 cells were plated in 60-mm-diameter dishes overnight and were then transfected with 1 µg of the pBI vector (empty vector and WT, B*, and S and R HES-1 versions) and 0.1 µg of a Hygromycin B resistance vector using Lipofectamine Plus reagent (Life Technologies) according to the manufacturer's instructions. The cells were allowed to grow for 48 h and were then passaged to 150-mm-diameter plates in the presence of 200 µg of Hygromycin B (Life Technologies) for selection. Twenty-four colonies from each transfection were picked and plated in duplicate, either with or without 2 µg of tetracycline in the media. Clones that in the absence of tetracycline were observed to have green fluorescence under UV light (e.g., expressed enhanced green fluorescent protein [EGFP]) were identified. The corresponding uninduced clone was further analyzed for expression of the Flag epitope-tagged protein by Western analysis. Approximately 33% of the colonies selected had detectable EGFP after 48 h of induction, and all of those clones tested had detectable HES-1 or HES-1 mutant expression using anti-Flag Western analysis. Additional WT HES-1 and control cell lines were generated from a second transfection, and the cells generated were not distinguishable from the first transfection.

Transient transfections. Transient transfections for the hHES p21 and Gal-4-upstream activation sequence (UAS) reporter assays were carried out in six-well tissue culture dishes in duplicate by using the Lipofectamine Plus reagent (Life Technologies) according to the manufacturer's instructions. The hHES reporter was made from the ~1.6-kb BamHI fragment of the genomic DNA, a gift from John Feder (Mercator Genetics, Menlo Park, Calif.), fused to the luciferase gene in PGL2 basic (Promega). The PC12 cells were plated to a density of approximately 50% 24 h prior to transfection. For each transfection, 0.5 µg of the hHES reporter was transfected with 1 µg of pCDNA3 -galactosidase (-Gal) (internal transfection efficiency control) and 1 µg of either pCDNA3 or a pCDNA3 HES-1 construct. The p21-luciferase (p21-luc) construct was obtained from X. H. Sun (New York University, New York) and has been previously described (49). For studies of the p21 promoter, 0.25 µg of p21-luc was used with 1 µg of pCDNA3 -Gal and either 1 µg of pCDNA3 or 0.5 mg of both pCDNA3 MASH-1 (7) and pCDNA3 E47 (a gift from Robert Benezra, Sloan-Kettering Institute, New York, N.Y.). Additionally, either 1 µg of pCDNA3 or a pCDNA3 HES-1 construct was included. For the Gal-4 assay, 1 µg each of a pM vector and the luciferase reporter with five UAS (22) were used per transfection. Transfections were carried out for 5 h in the presence of the lipofectamine (without serum) after which the media was replaced with fresh media (with serum), and the cells were incubated for a total of 72 h prior to assaying. The luciferase assay procedure was carried out as previously detailed (44). The data for the p21-luc and hHES-luc transfections is the average of three to five independent experiments carried out in duplicate, the data for the Gal-4-UAS experiment is for two independent experiments in duplicate. The luciferase data is corrected for -Gal activity. The errors are the standard deviations of the means.

CFE assays. PC12 and neuroblastoma cells plated at approximately 50% density in six-well dishes were transfected with 1 µg of either pCDNA3 or a pCDNA3 HES-1 construct. After 48 h, the cells were passaged to 100-mm-diameter dishes and selected with 400 µg of G418 per ml. Care was taken to ensure that colonies were not rinsed off or disrupted during media changes. After approximately 30 days, the plates were rinsed in phosphate-buffered saline (PBS), and the colonies were fixed and stained with 50% methanol, 10% acetic acid, and 0.05% Coomassie brilliant blue (Sigma). The colonies were counted, and the CFE was determined by dividing the number of colonies on each plate by the number obtained with the control transfection (pCDNA3 vector). The PC12 data represents the average CFE from seven (control, WT, and R and S HES-1), four (B* HES-1), or two (3/4 HES-1) independent transfections; the error is the standard deviation of the mean. The neuroblastoma cell data is from a single experiment, representative of at least three independent transfections of the cell lines.

Western analysis. Western analysis was performed as previously described (7). Anti-Flag monoclonal M5 antibody (Kodak, Rochester, N.Y.) was used at a 1:1,000 dilution. Anti-HES-1 N-terminal polyclonal antibody, a gift from John Feder (Mercator Genetics), has been previously described (55) and was used at a 1:1,000 dilution. Anti-p21 and anti-cyclin D1 monoclonal antibodies are from Santa Cruz (Santa Cruz, Calif.) and were used at a 1:200 dilution. Anti-PCNA was obtained from Novocastra Laboratories (Newcastle upon Tyne, United Kingdom) and was used at a 1:250 dilution. Secondary goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated immunoglobulin G was purchased from Jackson Immunoresearch (West Grove, Pa.) and used at a 1:20,000 dilution. Antibodies were detected by using Pierce (Rockford, Ill.) supersignal chemiluminescent reagent on Kodak Biomax MR film. The data shown is from exposures in the linear range of detection.

Immunocytochemistry. Anti-Flag immunocytochemistry was performed as previously described (7). The anti-Flag antibody was used at a 1:2,500 dilution, the biotin-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch) was diluted to 1:300, and the CY3-conjugated streptavidin (Jackson Immunoresearch) was diluted to 1:1,000.

	RESULTS

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Inducible expression of WT HES-1 and HES-1 deletion proteins in PC12 cells. To investigate the function of the HES-1 H-3/4 domain, we generated stable PC12 cell lines that expressed Flag epitope-tagged WT HES-1 or HES-1 deletion mutants under tetracycline-inducible control (tet-off system; Clontech). We used a bidirectional expression vector (pBI-EGFP; Clontech) that coexpressed EGFP (Clontech) from the same promoter that drove expression of HES-1 (Fig. 1A). Upon withdrawal of tetracycline, the coinduction of HES-1 proteins could be monitored in the living cells by means of green fluorescence under UV light. This enabled the efficient screening of the stable clones we isolated, as only the clones that flouresced upon induction were further analyzed for exogenous HES-1 protein expression. Induction of nuclear-expressed HES-1 protein was verified by anti-Flag immunocytochemistry of the WT HES-1-inducible PC12 cells (Fig. 1F versus D). From Western blot analysis, by using anti-Flag and anti-HES-1 antibodies, we estimate that WT HES-1 levels are increased three- to fivefold over endogenous levels following 3 days of induction (see Fig. 4F and 7 for representative clones). The induction of HES-1 had a noticeable effect upon cell morphology in the absence of NGF, resulting in a noticeable flattening of the cells and an increased adhesion to the tissue culture dish (compare Fig. 1C to 1E and 2F). However, the induction of HES-1 had no apparent effect on cell viability, and the flattening was reversed upon readdition of tetracycline (data not shown). Therefore, the tetracycline-inducible PC12 cells are a useful system for examining the effects of induced expression of exogenous HES-1 protein.

View larger version (67K): [in this window] [in a new window]   FIG. 1.   HES-1-inducible expression in PC12 cells. (A) Schematic of the tetracycline-inducible bidirectional promoter used to coexpress EGFP and HES-1 variants. (B) Schematic of the WT, S, and R HES-1 constructs inducibly expressed in the stable PC12 cell lines. (C to F) Uninduced WT HES-1 cells, maintained in the presence of 2 µg of tetracycline per ml exhibited a normal morphology (C), and little or no Flag epitope was detected by anti-Flag immunocytochemistry (D). Withdrawal of tetracycline-induced Flag-HES-1 expression resulted in a flattened morphology in all eight cell lines examined (as shown for a representative clone in panel E) and localization of the exogenous HES-1 in the nuclei of the cells (F). A gel retardation assay (G) of WT HES-1-inducible PC12 cell nuclear extracts showed an increase in HES-1-specific DNA binding upon induction (compare lanes 2 and 1). The presence of exogenous HES-1 in the retarded complex was confirmed by the addition of anti-Flag antibodies to the binding reaction, which disrupted the binding of Flag-tagged protein (panel G, lane 4). Nuclear-localized expression of the S HES-1 (H and I) and R HES-1 (J and K) proteins was also determined by anti-Flag immunocytochemistry of the induced cells (I and K).

The H-3/4 domain contributes to HES-1-mediated inhibition of NGF response in PC12 cells. We have previously shown that transient and low-level constitutive expression of WT HES-1 inhibits the NGF-induced differentiation of PC12 cells (55). Accordingly, WT HES-1 expression in the inducible cell lines also inhibited the NGF-dependent neurite response. Uninduced WT HES-1 cells respond to NGF by extending neurites (Fig. 2E) indistinguishable from those in control cells (Fig. 2A, bars 1 and 2, and B). Upon the withdrawal of tetracycline and induction of WT HES-1 (as indicated by EGFP expression) (Fig. 2G), the cells no longer become neuritic in the presence of NGF (Fig. 2A, lanes 3 and 4, and 2F and G). In contrast, tetracycline induction of EGFP in control cells had no effect on NGF response (Fig. 2A, lanes 1 and 2, B, and C).

View larger version (102K): [in this window] [in a new window]   FIG. 2.   The H-3/4 domain of HES-1 partially mediates the inhibition of NGF response. The NGF response of tetracycline-inducible PC12 cells is graphed as the percentage of induced (without tetracycline) response divided by the percentage of uninduced (with tetracycline) response following 6 days of treatment with NGF (A). The NGF response for two control cell lines is unaffected by EGFP induction (columns 1 and 2). In contrast, induction of WT HES-1 greatly inhibits NGF response (columns 3 and 4). Expression of S HES-1, which contains the H-3/4 domain but lacks the WRPW motif, significantly inhibits NGF response (columns 5 and 6). Expression of R HES-1, which lacks both the H-3/4 and WRPW regions, does not inhibit NGF response (columns 7 and 8). Control PC12 cells, which expressed only EGFP upon induction, differentiated into a neuronal phenotype following 7 days of treatment with NGF (B to D). Induction of EGFP, seen by fluorescence microscopy in panel D, did not affect neurite outgrowth response to NGF (C). Uninduced WT HES-1 cells (E) had a NGF response similar to that of control cells (B). Upon induction of HES-1 by the removal of tetracycline, the cells no longer responded even after 7 days of NGF treatment (F and G) and have the flattened morphology of the induced cells seen in Fig. 1E. If the induced WT HES-1 cells were subsequently uninduced and then reexposed to NGF, they regained NGF-induced neurite response (data not shown).

HES-1 induction inhibits proliferation in PC12 cells. We unexpectedly found that the induction of moderate levels of WT HES-1 in the inducible cells strongly inhibited proliferation. We quantified this in a proliferation assay: Fig. 3A shows the rate of proliferation for two WT HES-1 clones and two control clones (empty vector, EGFP only), with and without induction. The uninduced WT HES-1 cells had a rate of proliferation similar to uninduced control cells. Induction of EGFP in control cells had no significant effect upon proliferation. In marked contrast, the induction of HES-1 greatly lowered the rate of proliferation of the cells.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Induction of WT HES-1 inhibits PC12 cell proliferation. (A) Two lines of control and two lines of WT HES-1 PC12 cells were maintained either with or without tetracycline for 3 days, then equal numbers of cells were passaged in triplicate to measure proliferation. The cells were maintained up to 9 additional days (with or without tetracycline), and a set of plates for each cell type was counted at the 3-, 5-, 7-, and 9-day time points. The experiment was repeated, and the data for both experiments is shown in the graph as the average fold-increase in cell number with the error as the standard deviation (A). The growth of uninduced WT HES-1 cells was slightly lower than control lines, perhaps due to leakage of the exogenous HES-1 (A, left panel). In contrast, the growth of the WT HES-1 cells was markedly lower upon induction of HES-1 (A, right panel). Induction of EGFP did not greatly affect the growth of control cells. The rate of proliferation of the WT HES-1 cells may be overestimated in the induced panel, since prolonged induction could select for the lower-inducing, faster-growing cells. (B) Western analysis of endogenous HES-1 in parental PC12 cells showed that HES-1 was induced by serum in the media (B, compare the panels of low-serum-level-maintained cells to the panels of normal (high)-serum-level-maintained cells). Addition of the differentiation agents NGF or basic FGF did not appear to affect HES-1 levels in either serum condition. In contrast, retinoic acid (R.A.), which halts PC12 cell proliferation but does not differentiate the cells, did raise HES-1 protein levels slightly.

View larger version (40K): [in this window] [in a new window]   FIG. 4.   The inhibition of proliferation in HES-1-overexpressing cells was confirmed by BrdU incorporation. After 20 h of exposure to BrdU, about 40% of control cell lines had incorporated the BrdU nucleoside analogue into their DNA, as detected by anti-BrdU immunocytochemistry. This fraction of incorporation is consistent with the doubling time for this cell type of about 48 h. The level of incorporation for the three control lines was similar with or without induction of EGFP (A). The incorporation of BrdU into uninduced WT HES-1 PC12 cells was similar to control values. Upon induction of HES-1 (3 days prior to BrdU treatment), the level of BrdU incorporation dropped dramatically to about 5%, consistent with either a lower growth rate or a lower fraction of cells in S phase, and a lack of DNA synthesis (implied by the loss of PCNA). In contrast, induction of R HES-1, a deletion mutant that lacks transcription repression activity (Fig. 7; discussed below) did not lower BrdU incorporation. Examples of the BrdU staining is shown for the WT HES-1 cells, both uninduced (B) and induced (D). Anti-BrdU staining is clearly seen as dark nuclei, which are mostly absent in the induced cells. The WT HES-1-induced cells that incorporated BrdU tended to have a rounded morphology (panel D, arrow), indicating either that they were in S phase or that they may have been low, or nonexpressing, subpopulations. The positions of the nuclei are shown by propidium iodide staining, which was partially obscured in the cells with high levels of BrdU incorporation due to the opacity of the DAB precipitate (C and E). (F) The induction of WT (F, upper panels) and R HES-1 protein (F, lower panels) is shown by the anti-HES-1 and anti-Flag Western blots for three independent cell lines.

The H-3/4 domain mediates HES-1-induced growth inhibition. In contrast to WT HES-1, PC12 cells that expressed the deletion-mutant R HES-1 were not inhibited in proliferation. R HES-1 is deleted C-terminal to the HLH region, and the removal of the WRPW motif in R HES-1 was expected to abolish transcription repression function, as well as any H-3/4 domain function. Indeed, analysis of three independent lines of tetracycline-inducible R HES-1 expressing cells (Fig. 4F) showed a modest increase in BrdU uptake upon induction (Fig. 4A). In addition, induction of R HES-1 resulted in a more rounded cell morphology (data not shown) and a decreased adhesion to the tissue culture plastic surface. This is a phenotype apparently opposite to that resulting from WT HES-1 induction. Analysis of growth rates was complicated by the tendency of the R HES-1-induced cells to detach from the dish and grow as clumps (data not shown). The BrdU incorporation data, however, clearly indicated that the inhibition of proliferation was dependent upon the region of HES-1 C terminal to the HLH domain, which contains the H-3/4 domain and also the WRPW motif.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   The H-3/4 domain mediates the inhibition of proliferation by HES-1. To determine if the growth inhibition phenotype resulting from WT HES-1 overexpression was a function of the H-3/4 domain, we performed CFE assays in PC12 cells using expression vectors encoding WT, R, S, 3/4, and B* HES-1 proteins. The empty expression vector (pCDNA3) was used as the control. The cells were grown in the presence of 200 µg of G418 per ml to select cells containing the expression construct, which has a neomycin resistance gene. The number of colonies present after 1 month of growth was determined and normalized to the control value (1.0). Six separate transfections with the same amount of vector were performed for all except 3/4 HES-1, which was transfected twice.

HES-1 inhibits proliferation in neuroblastoma. To determine if HES-1 also inhibits the growth of other neuronal cell types, we performed CFE assays on three neuroblastoma cell lines, SHEP1, SHSY5Y, and SHIN. Neuroblastoma are neuronal tumor cell lines and, at least for these three types, do not express detectable levels of HES-1 protein (shown for SHEP and SHSY5Y in Fig. 6A). Accordingly, they express mRNA of the HES-1-regulated gene, MASH-1 (Fig. 6B). The expression of exogenous WT HES-1 in these cell lines results in the formation of essentially no stable clones (Fig. 6C and D). Furthermore, we were unable to generate NIH 3T3 cells constitutively expressing HES-1 (data not shown), suggesting that growth inhibition is a general property of HES-1 overexpression.

View larger version (30K): [in this window] [in a new window]   FIG. 6.   WT HES-1 inhibits proliferation in neuroblastoma cell lines. Three neuroblastoma cell lines (SHSY5Y, SHEP1, and SHIN) were tested for CFE following transfection with WT HES-1. These cell lines do not have significant expression of HES-1 protein, as shown by Western analysis of two of the lines in panel A. Transiently expressed HES-1 (lane 1) and endogenous PC12 cell (lane 6) lysates were run as positive controls for the detection of HES-1 protein. Lanes 3 and 5 are from lysates of NGF-treated cells. As might be anticipated by the lack of HES-1, expression of the MASH-1 gene can be detected in these cells, shown by reverse transcription-PCR of the SHSY5Y line in panel B. The (+) lane (lane 1) is a cDNA-positive control, the () lane (lane 2) is a reverse-transcriptase-negative control, and lanes 3 through 8 are serial dilutions of the input reverse transcription reaction for PCR amplification. MASH-1 mRNA has also been detected by reverse transcription-PCR in the SHEP and SHIN cell lines, as well as in LAI 5S, LAI 55N, and BEI YC neuroblastoma cell lines (data not shown). The greatly reduced colony formation of the HES-1-transfected cell lines is shown in panel C, and representative plates with Coomassie blue-stained cell colonies are shown in panel D. The near absence of colonies expressing WT HES shows that the inhibition of cell proliferation is a general property of WT HES-1 overexpression.

The H-3/4 domain mediates HES-1 repression of p21 protein expression in PC12 cells. To determine whether HES-1 represses the expression of cell cycle proteins and, if so, whether the WRPW motif and/or the H-3/4 domain mediate this repression, we compared the effect of expression of WT HES-1, S HES-1, and R HES-1 on the expression of three cell cycle proteins: p21^CIP1, PCNA, and cyclin D1. Expression of the WT HES-1, S HES-1, and R HES-1 proteins in induced cells was verified by anti-HES-1 and anti-Flag Western analysis of cell lysates (Fig. 7, top two panels). The induction of WT HES-1 resulted in a significant loss of the S-phase marker PCNA (Fig. 7, second panel from bottom), consistent with the lack of BrdU incorporation and growth arrest in G₁. The level of the cyclin-CDK inhibitor, p21, was also considerably reduced by HES-1 induction (Fig. 7, bottom panel). In contrast, the level of the G₁ progression cyclin, D1, remained constant (Fig. 7, middle panel).

View larger version (64K): [in this window] [in a new window]   FIG. 7.   The H-3/4 domain of HES-1 mediates the repression of the cell cycle proteins PCNA and p21. Expression of induced, Flag-tagged proteins in the WT, S, and R HES-1 PC12 cell lines was verified by anti-HES-1 and anti-Flag Western analysis (upper two panels). Induction of both WT and S HES-1 reduced the level of PCNA and p21 proteins, whereas induction of the R HES-1 protein had no significant effect on either PCNA or p21 (lower 2 panels). Neither expression of WT HES-1 nor the S and R HES-1 mutant proteins affected the level of cyclin D1 (middle panel).

The H-3/4 domain of HES-1 contributes to HES-1 function in transcription repression. HES-1 has been previously shown to be autoregulatory (56), and, therefore, the HES-1 promoter represents a good model for assaying the transcription repression function of HES-1 proteins. The activity of various HES-1 constructs was assessed by using a HES-1 responsive reporter, incorporating 1.6-kb of human HES-1 genomic DNA (20) upstream of the start codon, fused to a luciferase reporter gene (hHES-luc). In addition to the WT HES-1, S HES-1, R HES-1, 3/4 HES-1, and B* HES-1 constructs described above, we also generated a DNA-binding-defective version of S HES-1 (B*S HES-1) (Fig. 8A). The HES-1 constructs each had a Flag epitope fused at the N terminus, and expression of the constructs was confirmed by Western blots of parallel transfections (data not shown). Equivalent amounts of the pCDNA3 expression vectors were transfected into PC12 cells, and the activity of the hHES-luc promoter was determined. Wild-type HES-1 strongly repressed the hHES promoter (Fig. 8B). The DNA-binding-defective (B*) form of HES-1 only weakly repressed the promoter, confirming the importance of the HES-1 DNA binding for repression of this promoter (Fig. 8B). DNA binding is thought to be necessary to recruit the TLE-related family of corepressors to the promoter, and this is facilitated by the C-terminal WRPW motif of HES-1. Expression of R HES-1, which lacks the WRPW motif and the H-3/4 domain, resulted in mild activation of the promoter. This may result from a competitive inhibition for the DNA-binding sites by R HES-1 protein, which would prevent endogenous HES-1 from occupying the DNA and repressing transcription. In contrast, transfection of S HES-1 resulted in repression of the hHES promoter, albeit less effectively than the wild type (~3-fold compared to ~14-fold repression). The S HES-1 data demonstrates that the H-3/4 domain functions in transcription repression, because S HES-1 differs from R HES-1 only by inclusion of this region. Comparison of the activity between these two mutants showed an approximate sixfold difference in activity due to the H-3/4 domain. The requirement for the H-3/4 domain was supported by the use of an internal-deletion HES-1 mutant (3/4 HES-1). Despite the presence of a WRPW motif (and despite expression at levels comparable to WT HES-1 [data not shown]), the absence of the H-3/4 domain renders this protein inactive as a repressor. Like the R construct, the 3/4 HES-1 mutant is a weak functional activator (or derepressor) of the hHES-1 promoter, although the mechanisms may not be the same in each case.

View larger version (49K): [in this window] [in a new window]   FIG. 8.   Transcriptional analysis of HES-1. The hHES-1 promoter was incorporated upstream of a luciferase reporter gene (hHES-luc) in order to analyze the regulation of the hHES-1 gene by HES-1 in PC12 cells. The luciferase activity of the reporter was corrected for the level of -Gal activity from a cotransfected internal control plasmid and expressed as fold activity, with the control value normalized as 1.0. (A and B) The function of the H-3/4 domain in HES-1-mediated repression. WT HES-1 strongly repressed transcription from the hHES-1 promoter compared to the control transfected with empty expression vector. In comparison, loss of DNA binding due to mutation of the basic region (B* HES-1) resulted in essentially no transcription repression. Furthermore, removal of the H-3/4 domain in  3/4 HES-1 also resulted in a loss of repression activity of the protein, despite the presence of the WRPW motif in the DNA-binding-competent protein. Indeed, HES-1 with the H-3/4 domain (S HES-1), but not the rest of the C terminus (including WRPW), was functional as a modest repressor. The repression activity of this protein was also impaired by the inability to bind to DNA (B*S HES-1), and instead functioned as a weak activator, or derepressor, of the promoter. Similarly, R HES-1, which lacks both the H-3/4 and the WRPW structures, was also a modest functional activator. (C and D) The role of DNA binding in HES-1-mediated repression. Comparison of the transcription activity of bHLH HES-1 constructs to WT HES-1. Expression of the H-3/4 domain alone (HES 3/4), as well as the WRPW-containing constructs (HES 3/4/C and HES C) with a nuclear-localization signal increased the activity of the promoter. (E and F) Repression function of H-3/4 and WRPW heterologous fusion proteins. The bHLH HES-1 constructs were fused to a heterologous Gal-4 DNA-binding protein (E) and tested for their ability to repress a Gal-4-binding-site-containing reporter construct (with five UAS and a simian virus 40 minimal promoter) (F). The WRPW-containing domains of HES-1 (pM-h3/4/C and pM-C) acted as transcription repressors when fused to Gal-4, consistent with the recruitment to DNA of the corepressor, TLE.

HES-1 repression of transcription of the p21 promoter in PC12 cells. The reduced expression of p21 in the stable cell lines following induction of wild-type and S HES-1, but not R HES-1 (Fig. 7), suggests that p21 may be a direct target for transcriptional repression by HES-1. Indeed, the promoter of p21 contains a consensus class C site, as well as several nonconsensus sites to which HES-1 can bind specifically (data not shown). In addition, HES-1 is known to inhibit the bHLH activator from binding to E-boxes, enhancer elements that are also necessary for up regulation of p21 transcription (49). Thus, p21 is a plausible target for HES-1-mediated transcriptional regulation, whether by class C site or E-box-directed mechanisms. To examine the mechanism of p21 regulation by HES-1, we used a p21 promoter construct with 2.4 kb of DNA upstream of the start site, fused to a luciferase reporter gene (49). The constitutive expression of p21 was increased by NGF treatment, and in both instances the transcription activity was strongly repressed by HES-1 (Fig. 9A). The addition of MASH-1 and E47 bHLH activators increased both the basal and the NGF-induced activity several-fold, presumably by binding to the E-box enhancers present in the p21 promoter. Again, in both instances HES-1 expression inhibited the activation to below basal levels.

View larger version (31K): [in this window] [in a new window]   FIG. 9.   HES-1 represses p21 transcription. (A) The endogenous activity of the p21 promoter in PC12 cells (upper half of panel A) was modestly increased by NGF treatment (dark shading), consistent with the induction of p21 by NGF in PC12 cells. Both the endogenous and NGF-induced promoter activities were strongly repressed by HES-1 to below basal levels. Activation of the E-box-enhanced p21 promoter by coexpression of MASH-1 and E47 (lower half of panel A, bracketed) was also further increased by NGF treatment (dark shading). Again, both the MASH-1-E47 and the MASH-1-E47-NGF-activated promoters were strongly repressed to below basal levels by the expression of HES-1. (B) MASH-1-E47-enhanced activation of the p21 promoter (lower section of panel B, bracketed) was repressed by HES-1 in an H-3/4 domain-dependent manner. WT HES-1 strongly, and S HES-1 modestly, repressed the activated p21 promoter, whereas  3/4 HES-1 modestly activated the promoter. R HES-1 conferred no repression activity, and B*S HES-1 was a modest activator.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have demonstrated a functional role for the H-3/4 domain in the class C site (N-box)-dependent transcription repression by HES-1. In addition, we have shown that HES-1 expression inhibits PC12 cell proliferation as well as differentiation and that the H-3/4 domain is important for both of these inhibitory activities. The repression of p21 transcription is unlikely to mediate the inhibition of proliferation by HES-1, since p21 is a cyclin-CDK inhibitor that negatively regulates proliferation. Instead, p21 repression by HES-1 may contribute to the repression of differentiation by HES-1, as discussed below.

In Fig. 10, we present a model to illustrate the potential mechanisms by which the H-3/4 domain contributes to the DNA-binding-dependent regulation of transcription by HES-1. This model contains elements that serve to explain the observed ability of HES-1 or HES-1 mutants to repress, or in some cases to derepress, transcription. We propose that the H-3/4 domain, a putative protein interaction motif (37), is necessary for either (Fig. 10, i) the direct recruitment of an unknown corepressor and/or (Fig. 10, ii) the stabilization or regulation of WRPW-mediated repression function through intra- or intermolecular interaction. While we have suggested that the H-3/4 domain interacts with an unknown corepressor, repression could also result from the interaction of the H-3/4 domain with bHLH activators bound to adjacent sites. This is analogous to a model proposed by Dawson and colleagues (13) in which the corepressor would be the bHLH activator protein, Scute, bound to an E-box. The ability of some HES-1 mutant proteins to derepress the hHES-1 promoter could result from the occupancy of DNA sites (Fig. 10, iii) by proteins deficient in transcription repression activity (i.e., R HES-1 and 3/4 HES-1). Derepression also resulted from the expression of HES-1 mutants that lack an intrinsic DNA-binding function, either due to deletion of the bHLH region (all  bHLH proteins) or due to a basic region mutation (B* and B*S HES-1) that disrupts DNA binding. This derepression is likely to result from the titration of corepressors such as X (or TLE) away from DNA (Fig. 10, iv).

View larger version (27K): [in this window] [in a new window]   FIG. 10.   Model of DNA binding, H-3/4-dependent transcription repression by HES-1. The H-3/4 domain may be required to recruit unknown corepressors (X) to DNA (i) to interact with other promoter-bound proteins to either stabilize repression (ii) or inhibit activators (not shown), or to serve as a heterodimer with endogenous HES-1, providing an active conformation for TLE function (ii). Presumably,  3/4 HES-1, lacking the H-3/4 domain, acts instead to inhibit these functions (data not shown). R HES-1 as a homodimer, or as an inactive heterodimer with endogenous HES-1, could derepress the promoter by binding to DNA without forming repressor complexes (iii). This would also result in the competitive inhibition of any endogenous HES-1 homodimers for the DNA sites. As a bHLH protein, the H-3/4 domain could rerepress the promoter by titrating unknown corepressors (shown as X) away from DNA or by forming DNA-binding-deficient heterodimers with endogenous HES-1 to the same effect (iv).

The C-EBP transcription factor has previously been shown to regulate p21 at the translational rather than transcriptional level (58). While it is possible that HES-1 may also repress p21 at the translational level, the direct transcriptional repression of the p21 promoter by HES-1 (Fig. 9) indicates that this previously characterized transcriptional repressor protein does repress p21 at the transcriptional level.

DNA-binding-dependent, H-3/4-domain-mediated mechanisms of transcription repression. Our results for the DNA-binding and non-DNA-binding HES-1 variants complement each other and together indicate that HES-1-mediated transcription repression is primarily dependent upon binding to cognate class C DNA sites. First, the loss of DNA-binding ability in B* HES-1 and B*S HES-1 results in a substantially impaired ability to repress transcription in comparison to the corresponding DNA-binding-competent protein (WT and S HES-1, respectively) (Fig. 8B). Second, the R and  3/4 HES-1 proteins have the ability to bind to DNA, but since they lack the H-3/4 and/or WRPW regions, they do not facilitate repression (Fig. 8B). The transcription derepression resulting from R HES-1 expression (Fig. 8B) is consistent with the hypothesis that the R HES-1 protein is a competitive inhibitor of DNA binding by endogenous HES-1 (Fig. 10, iii). The R HES-1 protein is essentially the same as a bHLH-only form of HES-1 that we have previously shown binds to HES-1-specific DNA sequences in vitro (55) as well as in nuclear extracts from mammalian cells (data not shown). PC12 cells contain endogenous HES-1 (19, 55), which can inhibit the hHES-1 reporter and lower its basal activity. Exogenous R HES-1 could therefore compete for the same class C sites as the endogenous HES-1 protein. Since R HES-1 lacks the ability to recruit corepressors, occupancy of the class C sites would effectively derepress the system. Furthermore, the lack of repression by 3/4 HES-1 (Fig. 8B), which has a functional WRPW motif and is expressed equivalently to the WT and B* HES-1 proteins in transient transfections (data not shown), suggests that the recruitment of factors to DNA via the H-3/4 domain may be critical to WRPW-mediated repression in this particular assay. A similar loss of function was reported for  H-3/4 deletions in both E(Spl) (24) and in Hairy (13), as determined by genetic assays in Drosophila. Together, the data from the DNA-binding and nonbinding HES-1 variants indicate that direct binding of HES-1 to class C sites is necessary for transcription repression activity.

The repression of a p21-dependent cell cycle exit by HES-1 may inhibit differentiation. The ability of HES-1 to repress NGF-induced neurite outgrowth in PC12 cells is consistent with the role of HES-1 as a repressor of neuronal differentiation. This repression is at least partly dependent upon the H-3/4 domain, suggesting that transcriptional repression of differentiation-specific genes is mediated in part through this domain. In principle, the ability of HES-1 to block proliferation through repression of as-yet-undefined target genes may, in itself, be sufficient to inhibit differentiation by precluding a cell cycle exit program essential for differentiation. In particular, previous studies have suggested that such a differentiation-specific cell cycle arrest program may be mediated by p21: several studies have indicated that the differentiation of certain cell types may require the up regulation of p21 expression to induce exit from cell cycle (28, 39, 45). NGF signaling in PC12 cells induces p21 expression as part of a peripheral-neuron-like differentiation process (64), and overexpression of p21 alone is sufficient to arrest cell cycle in PC12 cells (18). In addition to cell cycle exit, p21 overexpression results in differentiation-specific cell cycle changes and potentiates differentiation in response to growth factor signaling (17). Furthermore, the growth arrest mediated by p21 is a necessary precondition for the differentiation of NGF-treated PC12 cells grown in serum (50, 60). Therefore, expression of p21 may be critical for establishing specific cell cycle exit conditions that are necessary for differentiation. Thus, the prevention of a p21-dependent exit from cell cycle, and any differentiation-specific cell cycle changes associated with p21, may be part of the pathway by which HES-1 inhibits differentiation.

Induction of growth arrest by HES-1. We have found that a moderate overexpression of WT HES-1 strongly inhibits proliferation in PC12 cells. We previously reported our inability to obtain stable HES-1-expressing PC12 cell lines when using an expression vector with a strong promoter (cytomegalovirus) to constitutively express HES-1 (55). HES-1-expressing cell lines were obtained only by transfection with a vector containing a low-activity (uninduced) mouse mammary tumor virus promoter to drive expression, and even these lines were slow growing and difficult to maintain. Subsequently, Issack and Ziff (34) noted an inability to maintain cells transfected with a HES-1 expression vector in culture. By generating tetracycline-inducible stable cell lines, we have now shown that the expression of moderate levels of exogenous HES-1 results in a marked inhibition of proliferation (Fig. 3B and 4A). The induced cells do not undergo increased cell death, and proliferation is restored if the induction is halted. Thus, the effect of HES-1 on proliferation is likely to result from the regulation of cell cycle effector genes and not from either nonspecific, toxic effects or from the induction of apoptosis.