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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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GHFT1-5 cells were transfected with the A, Sham, GFP-C/EBP and ΔLZ expression vectors or B, Sham, C/EBP-GFP and DBD expression vectors and treated with nocodazole. The proportions of cells in G1, S and M phase were determined and plotted as the mean +/- standard deviation from six independent experiments for both A and B. No C/EBP, the subpopulation of cells with background levels of green fluorescence (i.e. did not express GFP-linked C/EBPα). Data from No C/EBP cells are shown adjacent to the green fluorescent cells for each expression construct. Statistically significant differences in the proportion of green fluorescent cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (One-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).
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Figure 6: GHFT1-5 cells were transfected with the A, Sham, GFP-C/EBP and ΔLZ expression vectors or B, Sham, C/EBP-GFP and DBD expression vectors and treated with nocodazole. The proportions of cells in G1, S and M phase were determined and plotted as the mean +/- standard deviation from six independent experiments for both A and B. No C/EBP, the subpopulation of cells with background levels of green fluorescence (i.e. did not express GFP-linked C/EBPα). Data from No C/EBP cells are shown adjacent to the green fluorescent cells for each expression construct. Statistically significant differences in the proportion of green fluorescent cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (One-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).

Mentions: When expressed in GHFT1-5 cells, ΔLZ was as effective as full-length GFP-C/EBPα in prolonging both G1 and S (Fig. 6A). Thus, leucine zipper dimerization, site-specific DNA binding and targeting of C/EBPα to peri-centromeric were not required for C/EBPα prolongation in G1 and S. In contrast, the isolated C/EBPα bZIP DNA binding domain (Fig. 1B, DBD), which still targeted to peri-centromeric chromatin, did not prolong G1 (Fig. 6B). S phase remained prolonged upon DBD expression. The different effects of the isolated DBD on G1 and S blockage indicated that C/EBPα regulation of G1 and S phase arrest was mechanistically distinct. Thus, C/EBPα regulates proliferation by two distinct pathways. Both pathways do not depend upon site-specific DNA binding, which is commonly considered a prerequisite for gene-specific transcription.


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)

GHFT1-5 cells were transfected with the A, Sham, GFP-C/EBP and ΔLZ expression vectors or B, Sham, C/EBP-GFP and DBD expression vectors and treated with nocodazole. The proportions of cells in G1, S and M phase were determined and plotted as the mean +/- standard deviation from six independent experiments for both A and B. No C/EBP, the subpopulation of cells with background levels of green fluorescence (i.e. did not express GFP-linked C/EBPα). Data from No C/EBP cells are shown adjacent to the green fluorescent cells for each expression construct. Statistically significant differences in the proportion of green fluorescent cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (One-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).
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Figure 6: GHFT1-5 cells were transfected with the A, Sham, GFP-C/EBP and ΔLZ expression vectors or B, Sham, C/EBP-GFP and DBD expression vectors and treated with nocodazole. The proportions of cells in G1, S and M phase were determined and plotted as the mean +/- standard deviation from six independent experiments for both A and B. No C/EBP, the subpopulation of cells with background levels of green fluorescence (i.e. did not express GFP-linked C/EBPα). Data from No C/EBP cells are shown adjacent to the green fluorescent cells for each expression construct. Statistically significant differences in the proportion of green fluorescent cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (One-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).
Mentions: When expressed in GHFT1-5 cells, ΔLZ was as effective as full-length GFP-C/EBPα in prolonging both G1 and S (Fig. 6A). Thus, leucine zipper dimerization, site-specific DNA binding and targeting of C/EBPα to peri-centromeric were not required for C/EBPα prolongation in G1 and S. In contrast, the isolated C/EBPα bZIP DNA binding domain (Fig. 1B, DBD), which still targeted to peri-centromeric chromatin, did not prolong G1 (Fig. 6B). S phase remained prolonged upon DBD expression. The different effects of the isolated DBD on G1 and S blockage indicated that C/EBPα regulation of G1 and S phase arrest was mechanistically distinct. Thus, C/EBPα regulates proliferation by two distinct pathways. Both pathways do not depend upon site-specific DNA binding, which is commonly considered a prerequisite for gene-specific transcription.

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