Hes1 directly controls cell proliferation through the transcriptional repression of p27Kip1 - PubMed (original) (raw)

Hes1 directly controls cell proliferation through the transcriptional repression of p27Kip1

Kaoru Murata et al. Mol Cell Biol. 2005 May.

Abstract

A transcriptional regulator, Hes1, plays crucial roles in the control of differentiation and proliferation of neuronal, endocrine, and T-lymphocyte progenitors during development. Mechanisms for the regulation of cell proliferation by Hes1, however, remain to be verified. In embryonic carcinoma cells, endogenous Hes1 expression was repressed by retinoic acid in concord with enhanced p27(Kip1) expression and cell cycle arrest. Conversely, conditional expression of a moderate but not maximal level of Hes1 in HeLa cells by a tetracycline-inducible system resulted in reduced p27(Kip1) expression, which was attributed to decreased basal transcript rather than enhanced proteasomal degradation, with concomitant increases in the growth rate and saturation density. Hes1 induction repressed the promoter activity of a 5' flanking basal enhancer region of p27(Kip1) gene in a manner dependent on Hes1 expression levels, and this was mediated by its binding to class C sites in the promoter region. Finally, hypoplastic fetal thymi, as well as livers and brains of Hes1-deficient mice, showed significantly increased p27(Kip1) transcripts compared with those of control littermates. These results have suggested that Hes1 directly contributes to the promotion of progenitor cell proliferation through transcriptional repression of a cyclin-dependent kinase inhibitor, p27(Kip1).

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Figures

FIG. 1.

FIG. 1.

Down-regulation of the endogenous Hes1 in concord with the increased p27Kip1 expression and arrest of cell cycle progression by retinoic acid treatment in F9 cells. (A) F9 EC cells were cultured in the absence or presence of 1 μM RA for 4 days, and DNA contents were analyzed by flow cytometry. (B) Aliquots of the cells described above were lysed, and either total cell or nuclear extracts were immunoblotted with the indicated antibodies. (C) RNAs were extracted from the aliquots described above, cDNAs were synthesized, and RT-PCR was performed for the indicated genes. Amplified products were Southern blotted with corresponding 32P-labeled cDNA probes. Relative densitometric intensities of RA-treated samples compared to the control samples are indicated.

FIG. 2.

FIG. 2.

Immunostaining analysis for the expression of Hes1 and p27Kip1 in the F9 cells treated with RA. F9 cells were cultured in the absence (above) or presence (below) of 1 μM RA for 4 days, fixed, permeabilized, blocked, and doubly stained with anti-Hes1 (green) and anti-p27Kip1 (red) antibodies, followed by DAPI staining. Aliquots of the cultured cells were also stained with anti-claudin-4 antibody.

FIG. 3.

FIG. 3.

Effects of the conditional expression of Hes1 in HeLa cells on cell proliferation and CDK inhibitors. (A) HeLa/Tet-Hes1 cells maintained in the medium containing Dox (10 ng/ml) were shifted to fresh medium without Dox, and Hes1 expression was examined after indicated periods by immunoblotting (upper panel). HeLa/Tet-Hes1 cells were shifted to fresh media containing indicated doses of Dox, and 2 days later, Hes1 expression was examined by immunoblotting (lower panel). (B) HeLa/Tet-Hes1 cells were cultured at 1 × 105 cells/well of six-well plates, in medium containing 10 ng/ml (○), 0.1 ng/ml (□), or 0 ng/ml (▪) Dox, and the viable cells were counted at the indicated days. The culture medium was replaced with the fresh media containing the corresponding doses of Dox every other day. As a control, parental HeLa cells were cultured similarly in Dox-free medium (•). The means and standard errors (SE) of triplicate cultures are indicated. Asterisks indicate that the majority of cells were dying and detached from the dish. (C) Aliquots of HeLa/Tet-Hes1 cells in the cultures described above were lysed at the indicated days and immunoblotted with the indicated antibodies. The arrowhead indicates the Hes1 band.

FIG. 4.

FIG. 4.

Reduced p27Kip1 protein by Hes1 expression is attributed to the repressed basal transcript rather than the enhanced proteasomal degradation. (A) HeLa/Tet-Hes1 cells were cultured in the presence of 0.1 ng/ml or 10 ng/ml Dox for 4 days, and a proteasome inhibitor MG132 (10 μM), a calpain inhibitor MG′ (10 μM), or a vehicle (dimethyl sulfoxide, 10 μl) was added for 2 h before the harvest. Cells were lysed and immunoblotted with the indicated antibodies. (B) (Left panel) HeLa/Tet-Hes1 cells were cultured in the presence of 0.1 ng/ml or 10 ng/ml Dox for 4 days, RNAs were extracted, and RT-PCR was performed for p27Kip1 and GAPDH. Amplified products were Southern blotted using corresponding 32P-labeled cDNA probes followed by autoradiography. (Right panel) Aliquots of the RNAs described above were amplified in real-time PCR using specific primer sets for p27Kip1 (solid bars) and 36B4 rRNA as an endogenous control (open bars). The mean copy numbers and SE are indicated.

FIG. 5.

FIG. 5.

Hes1 expression represses the promoter activity of the 5′ flanking regulatory region of p27Kip1 gene in a dose-dependent manner. (A) A schematic representation of a 3.5-kb 5′ flanking DNA upstream of the translation start site of the p27Kip1 gene (p27PF). There are multiple potential Hes1 binding sites, three sites compatible with N box and four sites compatible with class C site. Two positive elements, Sp1 and CTF sites, to which transcription factors Sp1 and NF-Y have been shown to bind and enhance the promoter activity, respectively, are also indicated. An ApaI fragment of p27PF (p27PF/ApaI) contains a single class C site (CACGCG), which has been mutated to GTCGAC by site-directed mutagenesis (p27PF/ApaI-Mu). (B) HeLa/Tet-Hes1 cells were transfected with p27PF (solid columns) or p27PF/ApaI (grey columns) plasmid and cultured in the absence or presence of various concentrations of Dox for 2 days, and the luciferase activity was assayed and corrected for the transfection efficiency. The activities were normalized to that of the cells at 10 ng/ml Dox, and the means and SE of three independent experiments are indicated. The mean relative light units (RLUs) of p27PF and p27PF/ApaI at 10 ng/ml Dox were 5.7 × 105 and 45.2 × 105, respectively. Asterisks indicate statistically significant suppression (P < 0.01). Aliquots of the cells were lysed and immunoblotted with anti-Hes1 and anti-p27Kip1 antibodies to ensure their expression profiles in the particular experiments. (C) HeLa/Tet-Hes1 cells were transfected with p27PF/ApaI (open columns) or p27PF/ApaI-Mu (filled columns) plasmid, cultured in the absence of Dox and in the presence of various concentrations of Dox for 2 days, and the luciferase activity was assayed as described above. The activities were normalized to that of the cells at 10 ng/ml Dox, and the means and SE of three independent experiments are indicated. The mean RLUs of p27PF/ApaI and p27PF/ApaI-Mu at 10 ng/ml Dox were 3.6 × 105 and 1.7 × 105, respectively. Asterisks indicate statistically significant suppression (P < 0.05). (D) C3H10T1/2 cells were transfected with a plasmid containing neo, Hes1, or _Hes1_ΔWRPW cDNA, along with p27PF and pRL-TK plasmid, and 2 days later, the luciferase activity was determined. Means of RLUs in triplicate culture corrected for the transfection efficiency are indicated.

FIG. 6.

FIG. 6.

Hes1 binds to the 5′ flanking regulatory region of p27Kip1 gene via class C sites. (A) HeLa/Tet-Hes1 cells were cultured in the absence or presence of Dox (10 ng/ml) for 2 days, and the nuclear extracts were prepared. Although not shown, it was confirmed that the extract in the absence of Dox contained Hes1, and the extract in the presence of Dox barely did. Each extract was incubated with a 32P-labeled class C site-containing DNA oligomer of the p27PF/ApaI or the oligomer with mutation within the C site, and EMSA was performed. As competitors, excess doses (100- and 200-fold) of cold ApaI/C or ApaI/C-Mu oligomer were included. The solid arrowhead indicates the mobility-shifted oligomer band. (B) 293T cells were transfected with C-terminally FLAG-tagged Hes1 cDNA, and the nuclear extracts were incubated with labeled ApaI/C or ApaI/C-Mu oligomer, followed by anti-FLAG antibody in various salt concentrations. The open arrowhead indicates the supershifted band; the solid arrowhead indicates the original mobility-shifted oligomer band. (C) HeLa cells were transfected with Hes1 cDNA or vector alone, cross-linked with formaldehyde, and immunoprecipitated with anti-Hes1 or control rabbit immunoglobulin G (Cont. IgG). DNAs were extracted from the immunocomplexes and PCR amplified by the primer set of p27Kip1 promoter region or a p27Kip1 gene intron. DNA without ChIP served as a control (the first lane).

FIG. 7.

FIG. 7.

Increased p27Kip1 transcript in the thymocytes of _Hes1_−/− embryos compared with Hes1+/− littermates. (A) Hes1+/− mice were mated, and embryos at E14.5 were genotyped. Thymic lobes of the _Hes1_−/− embryo, which lacked eyes (arrows), were severely hypoplastic compared with those of Hes1+/− littermates (inserts). The thymocyte numbers in the former were less than 10% of the latter. (B) (Left panel) RNAs were extracted from the thymocytes of Hes1+/− and _Hes1_−/− embryos, and RT-PCR was performed for Hes1, p27Kip1, and GAPDH, followed by Southern blotting for p27Kip1 and GAPDH, using corresponding 32P-labeled cDNA probes. (Right panel) Means and SE of the radiointensities of p27Kip1 RT-PCR products following Southern blotting normalized by those of GAPDH in the thymocytes of four Hes1+/− and four _Hes1_−/− embryos are indicated. (C) RNAs were extracted from the liver cells and brain tissues of Hes1+/− and _Hes1_−/− embryos, and quantitative real-time RT-PCR was performed using a p27Kip1 primer set. The mean copy numbers and SE of p27Kip1 transcript normalized to those of control cyclophilin transcripts are indicated.

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