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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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A, Chloramphenicol acetyltransferase activity expressed from a promoter transiently transfected into GHFT1-5 cells together with vectors expressing C/EBPα, fused or not, with GFP. The promoter contained a single C/EBPα binding site upstream of a TATA box [45]. CAT activities were normalized to the activity present in cells transfected with the expression vector not containing the C/EBPα cDNA ("Sham") and plotted as the mean +/- standard deviation from five independent experiments. B, Nuclear extracts from sham-transfected cells and cells expressing C/EBPα, C/EBPα-GFP and GFP-C/EBPα were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a membrane and stained with an antibody directed against the FLAG epitope, which was appended to the amino terminus of C/EBPα in all the constructs. Arrow, expressed C/EBPα-GFP and GFP-C/EBPα. C, Whole cell extracts, from cells transfected with C/EBPα-GFP and GFP-C/EBPα or GFP-C/EBPαΔLZ expression vectors, were incubated with a radiolabeled oligonucleotide containing a high affinity consensus C/EBPα binding site. The observed complexes were competed with a 1, 10 and 100 fold molar excess of unlabeled oligonucleotide or were supershifted with an antibody directed against C/EBPα.
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Figure 2: A, Chloramphenicol acetyltransferase activity expressed from a promoter transiently transfected into GHFT1-5 cells together with vectors expressing C/EBPα, fused or not, with GFP. The promoter contained a single C/EBPα binding site upstream of a TATA box [45]. CAT activities were normalized to the activity present in cells transfected with the expression vector not containing the C/EBPα cDNA ("Sham") and plotted as the mean +/- standard deviation from five independent experiments. B, Nuclear extracts from sham-transfected cells and cells expressing C/EBPα, C/EBPα-GFP and GFP-C/EBPα were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a membrane and stained with an antibody directed against the FLAG epitope, which was appended to the amino terminus of C/EBPα in all the constructs. Arrow, expressed C/EBPα-GFP and GFP-C/EBPα. C, Whole cell extracts, from cells transfected with C/EBPα-GFP and GFP-C/EBPα or GFP-C/EBPαΔLZ expression vectors, were incubated with a radiolabeled oligonucleotide containing a high affinity consensus C/EBPα binding site. The observed complexes were competed with a 1, 10 and 100 fold molar excess of unlabeled oligonucleotide or were supershifted with an antibody directed against C/EBPα.

Mentions: We initially characterized the abilities of the C/EBPα-GFP and GFP-C/EBPα fusion proteins to bind DNA and activate transcription in GHFT1-5 cells (Fig. 2). The C/EBPα-GFP or GFP-C/EBPα expression vectors were transfected into GHFT1-5 cells with a promoter consisting of a single C/EBPα binding site upstream of the growth hormone TATA box. This minimal promoter was specifically responsive to C/EBPα expression in GHFT1-5 cells [45,46]. Cells transfected with the C/EBPα-GFP expression vector showed a statistically significant (p<0.05, n = 5) 9.66 +/- 6.08-fold higher promoter activity than did cells sham-transfected with the same expression vector deleted of the C/EBPα cDNA (Fig. 2A). In contrast, activation by GFP-C/EBPα was a statistically insignificant 1.88 +/- 1.48-fold (Fig. 2A). Promoter activation by C/EBPα-GFP was reproducibly less than promoter activation by unfused C/EBPα. Western blots of nuclear extracts from the transfected cells showed that unfused C/EBPα was expressed at marginally higher levels (Fig. 2B).


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)

A, Chloramphenicol acetyltransferase activity expressed from a promoter transiently transfected into GHFT1-5 cells together with vectors expressing C/EBPα, fused or not, with GFP. The promoter contained a single C/EBPα binding site upstream of a TATA box [45]. CAT activities were normalized to the activity present in cells transfected with the expression vector not containing the C/EBPα cDNA ("Sham") and plotted as the mean +/- standard deviation from five independent experiments. B, Nuclear extracts from sham-transfected cells and cells expressing C/EBPα, C/EBPα-GFP and GFP-C/EBPα were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a membrane and stained with an antibody directed against the FLAG epitope, which was appended to the amino terminus of C/EBPα in all the constructs. Arrow, expressed C/EBPα-GFP and GFP-C/EBPα. C, Whole cell extracts, from cells transfected with C/EBPα-GFP and GFP-C/EBPα or GFP-C/EBPαΔLZ expression vectors, were incubated with a radiolabeled oligonucleotide containing a high affinity consensus C/EBPα binding site. The observed complexes were competed with a 1, 10 and 100 fold molar excess of unlabeled oligonucleotide or were supershifted with an antibody directed against C/EBPα.
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Related In: Results  -  Collection

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Figure 2: A, Chloramphenicol acetyltransferase activity expressed from a promoter transiently transfected into GHFT1-5 cells together with vectors expressing C/EBPα, fused or not, with GFP. The promoter contained a single C/EBPα binding site upstream of a TATA box [45]. CAT activities were normalized to the activity present in cells transfected with the expression vector not containing the C/EBPα cDNA ("Sham") and plotted as the mean +/- standard deviation from five independent experiments. B, Nuclear extracts from sham-transfected cells and cells expressing C/EBPα, C/EBPα-GFP and GFP-C/EBPα were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a membrane and stained with an antibody directed against the FLAG epitope, which was appended to the amino terminus of C/EBPα in all the constructs. Arrow, expressed C/EBPα-GFP and GFP-C/EBPα. C, Whole cell extracts, from cells transfected with C/EBPα-GFP and GFP-C/EBPα or GFP-C/EBPαΔLZ expression vectors, were incubated with a radiolabeled oligonucleotide containing a high affinity consensus C/EBPα binding site. The observed complexes were competed with a 1, 10 and 100 fold molar excess of unlabeled oligonucleotide or were supershifted with an antibody directed against C/EBPα.
Mentions: We initially characterized the abilities of the C/EBPα-GFP and GFP-C/EBPα fusion proteins to bind DNA and activate transcription in GHFT1-5 cells (Fig. 2). The C/EBPα-GFP or GFP-C/EBPα expression vectors were transfected into GHFT1-5 cells with a promoter consisting of a single C/EBPα binding site upstream of the growth hormone TATA box. This minimal promoter was specifically responsive to C/EBPα expression in GHFT1-5 cells [45,46]. Cells transfected with the C/EBPα-GFP expression vector showed a statistically significant (p<0.05, n = 5) 9.66 +/- 6.08-fold higher promoter activity than did cells sham-transfected with the same expression vector deleted of the C/EBPα cDNA (Fig. 2A). In contrast, activation by GFP-C/EBPα was a statistically insignificant 1.88 +/- 1.48-fold (Fig. 2A). Promoter activation by C/EBPα-GFP was reproducibly less than promoter activation by unfused C/EBPα. Western blots of nuclear extracts from the transfected cells showed that unfused C/EBPα was expressed at marginally higher levels (Fig. 2B).

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