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CCAAT/enhancer binding protein alpha uses distinct domains to prolong pituitary cells in the growth 1 and DNA synthesis phases of the cell cycle.

Liu W, Enwright JF, Hyun W, Day RN, Schaufele F - BMC Cell Biol. (2002)

Bottom Line: C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells.Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Metabolic Research Unit, Diabetes Research Center and Department of Medicine, University of California, San Francisco, CA 94143-0540, USA. liu_weiqun@hotmail.com

ABSTRACT

Background: A number of transcription factors coordinate differentiation by simultaneously regulating gene expression and cell proliferation. CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic/leucine zipper transcription factor that integrates transcription with proliferation to regulate the differentiation of tissues involved in energy balance. In the pituitary, C/EBPalpha regulates the transcription of a key metabolic regulator, growth hormone.

Results: We examined the consequences of C/EBPalpha expression on proliferation of the transformed, mouse GHFT1-5 pituitary progenitor cell line. In contrast to mature pituitary cells, GHFT1-5 cells do not contain C/EBPalpha. Ectopic expression of C/EBPalpha in the progenitor cells resulted in prolongation of both growth 1 (G1) and the DNA synthesis (S) phases of the cell cycle. Transcription activation domain 1 and 2 of C/EBPalpha were required for prolongation of G1, but not of S. Some transcriptionally inactive derivatives of C/EBPalpha remained competent for G1 and S phase prolongation. C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.

Conclusion: We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells. G1 and S phase prolongation did not require that C/EBPalpha remained transcriptionally active or retained the ability to dimerize via the leucine zipper. G1, but not S, arrest required a domain overlapping with C/EBPalpha transcription activation functions 1 and 2. Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

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The indicated amino acids of C/EBPα were appended to DBD-GFP (see Fig. 1B) and expressed in GHFT1-5 cells. The proportions of green fluorescent cells in G1, S and M phase in nocodazole-treated cells were determined and plotted as the mean +/- standard deviation from three independent experiments. The DBD + 1–154 mutant was repeated a total six times (not shown), for which the statistical significance remained p < 0.01. Statistically significant increases in the proportion of G1 cells, compared to Sham cells, are indicated (One-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).
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Figure 7: The indicated amino acids of C/EBPα were appended to DBD-GFP (see Fig. 1B) and expressed in GHFT1-5 cells. The proportions of green fluorescent cells in G1, S and M phase in nocodazole-treated cells were determined and plotted as the mean +/- standard deviation from three independent experiments. The DBD + 1–154 mutant was repeated a total six times (not shown), for which the statistical significance remained p < 0.01. Statistically significant increases in the proportion of G1 cells, compared to Sham cells, are indicated (One-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).

Mentions: The C/EBPα DBD therefore was insufficient for prolongation of G1 (Fig. 6B). We next determined which additional domains of C/EBPα were required for G1 prolongation. C/EBPα amino acids 1–154 or 154–257 were appended to the DBD (see Fig. 1B). The addition of amino acids 1–154 (Fig. 7, DBD + 1–154) caused a statistically significant increase, relative to sham-transfected cells, in the proportion of cells in G1 (p << 0.01, n = 6). The proportion of cells in G1 was not statistically different in cells expressing DBD + 1–154 from that in cells expressing full-length C/EBPα-GFP. Adding amino acids 1–125, instead of amino acids 1–154, caused a similar prolongation in G1 (Fig. 7, DBD + 1–125). Thus, an element in the amino terminal domain of C/EBPα was required for G1 prolongation.


CCAAT/enhancer binding protein alpha uses distinct domains to prolong pituitary cells in the growth 1 and DNA synthesis phases of the cell cycle.

Liu W, Enwright JF, Hyun W, Day RN, Schaufele F - BMC Cell Biol. (2002)

The indicated amino acids of C/EBPα were appended to DBD-GFP (see Fig. 1B) and expressed in GHFT1-5 cells. The proportions of green fluorescent cells in G1, S and M phase in nocodazole-treated cells were determined and plotted as the mean +/- standard deviation from three independent experiments. The DBD + 1–154 mutant was repeated a total six times (not shown), for which the statistical significance remained p < 0.01. Statistically significant increases in the proportion of G1 cells, compared to Sham cells, are indicated (One-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC101385&req=5

Figure 7: The indicated amino acids of C/EBPα were appended to DBD-GFP (see Fig. 1B) and expressed in GHFT1-5 cells. The proportions of green fluorescent cells in G1, S and M phase in nocodazole-treated cells were determined and plotted as the mean +/- standard deviation from three independent experiments. The DBD + 1–154 mutant was repeated a total six times (not shown), for which the statistical significance remained p < 0.01. Statistically significant increases in the proportion of G1 cells, compared to Sham cells, are indicated (One-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).
Mentions: The C/EBPα DBD therefore was insufficient for prolongation of G1 (Fig. 6B). We next determined which additional domains of C/EBPα were required for G1 prolongation. C/EBPα amino acids 1–154 or 154–257 were appended to the DBD (see Fig. 1B). The addition of amino acids 1–154 (Fig. 7, DBD + 1–154) caused a statistically significant increase, relative to sham-transfected cells, in the proportion of cells in G1 (p << 0.01, n = 6). The proportion of cells in G1 was not statistically different in cells expressing DBD + 1–154 from that in cells expressing full-length C/EBPα-GFP. Adding amino acids 1–125, instead of amino acids 1–154, caused a similar prolongation in G1 (Fig. 7, DBD + 1–125). Thus, an element in the amino terminal domain of C/EBPα was required for G1 prolongation.

Bottom Line: C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells.Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Metabolic Research Unit, Diabetes Research Center and Department of Medicine, University of California, San Francisco, CA 94143-0540, USA. liu_weiqun@hotmail.com

ABSTRACT

Background: A number of transcription factors coordinate differentiation by simultaneously regulating gene expression and cell proliferation. CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic/leucine zipper transcription factor that integrates transcription with proliferation to regulate the differentiation of tissues involved in energy balance. In the pituitary, C/EBPalpha regulates the transcription of a key metabolic regulator, growth hormone.

Results: We examined the consequences of C/EBPalpha expression on proliferation of the transformed, mouse GHFT1-5 pituitary progenitor cell line. In contrast to mature pituitary cells, GHFT1-5 cells do not contain C/EBPalpha. Ectopic expression of C/EBPalpha in the progenitor cells resulted in prolongation of both growth 1 (G1) and the DNA synthesis (S) phases of the cell cycle. Transcription activation domain 1 and 2 of C/EBPalpha were required for prolongation of G1, but not of S. Some transcriptionally inactive derivatives of C/EBPalpha remained competent for G1 and S phase prolongation. C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.

Conclusion: We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells. G1 and S phase prolongation did not require that C/EBPalpha remained transcriptionally active or retained the ability to dimerize via the leucine zipper. G1, but not S, arrest required a domain overlapping with C/EBPalpha transcription activation functions 1 and 2. Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

Show MeSH
Related in: MedlinePlus