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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, GFP-C/EBP or, B, GFP-C/EBP deleted of amino acids 310 to 358 (ΔLZ) were expressed in GHFT1-5 cells. The ΔLZ construct is depicted in figure 1B. The transfected cells were counter-stained with the blue fluorescent, DNA-binding dye Hoechst 33342 immediately before analysis by fluorescence microscopy. Green fluorescent (left panels) and blue fluorescent (right panels) images were collected from a single cell using filter sets that selectively discriminate GFP and Hoechst 33342 fluorescence (see Materials and Methods). The subnuclear position of GFP-C/EBPα and ΔLZ were compared to the subnuclear position of the peri-centromeric chromatin stained by Hoechst 33342. Representative images are shown.
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Figure 5: A, GFP-C/EBP or, B, GFP-C/EBP deleted of amino acids 310 to 358 (ΔLZ) were expressed in GHFT1-5 cells. The ΔLZ construct is depicted in figure 1B. The transfected cells were counter-stained with the blue fluorescent, DNA-binding dye Hoechst 33342 immediately before analysis by fluorescence microscopy. Green fluorescent (left panels) and blue fluorescent (right panels) images were collected from a single cell using filter sets that selectively discriminate GFP and Hoechst 33342 fluorescence (see Materials and Methods). The subnuclear position of GFP-C/EBPα and ΔLZ were compared to the subnuclear position of the peri-centromeric chromatin stained by Hoechst 33342. Representative images are shown.

Mentions: Our prior fluorescence microscopy studies showed that C/EBPα-GFP, GFP-C/EBPα, and antibody-stained C/EBPα [45,46], concentrated at specific intranuclear domains in GHFT1-5 cells (see Fig. 5A, GFP-C/EBPα). These domains coincide with regions that stain with the blue fluorescent DNA-binding dye Hoechst 33342 (Fig. 5A, Hoechst 33342). Hoechst 33342 stains AT-rich DNA that concentrates around the centromeres [45,49]. Given the role of the centromere as a checkpoint for regulation of the mitotic phase of the cell cycle [50,51], it was hypothesized that C/EBPα localization around the centromere could play a role in C/EBPα regulation of the cell cycle [49].


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, GFP-C/EBP or, B, GFP-C/EBP deleted of amino acids 310 to 358 (ΔLZ) were expressed in GHFT1-5 cells. The ΔLZ construct is depicted in figure 1B. The transfected cells were counter-stained with the blue fluorescent, DNA-binding dye Hoechst 33342 immediately before analysis by fluorescence microscopy. Green fluorescent (left panels) and blue fluorescent (right panels) images were collected from a single cell using filter sets that selectively discriminate GFP and Hoechst 33342 fluorescence (see Materials and Methods). The subnuclear position of GFP-C/EBPα and ΔLZ were compared to the subnuclear position of the peri-centromeric chromatin stained by Hoechst 33342. Representative images are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC101385&req=5

Figure 5: A, GFP-C/EBP or, B, GFP-C/EBP deleted of amino acids 310 to 358 (ΔLZ) were expressed in GHFT1-5 cells. The ΔLZ construct is depicted in figure 1B. The transfected cells were counter-stained with the blue fluorescent, DNA-binding dye Hoechst 33342 immediately before analysis by fluorescence microscopy. Green fluorescent (left panels) and blue fluorescent (right panels) images were collected from a single cell using filter sets that selectively discriminate GFP and Hoechst 33342 fluorescence (see Materials and Methods). The subnuclear position of GFP-C/EBPα and ΔLZ were compared to the subnuclear position of the peri-centromeric chromatin stained by Hoechst 33342. Representative images are shown.
Mentions: Our prior fluorescence microscopy studies showed that C/EBPα-GFP, GFP-C/EBPα, and antibody-stained C/EBPα [45,46], concentrated at specific intranuclear domains in GHFT1-5 cells (see Fig. 5A, GFP-C/EBPα). These domains coincide with regions that stain with the blue fluorescent DNA-binding dye Hoechst 33342 (Fig. 5A, Hoechst 33342). Hoechst 33342 stains AT-rich DNA that concentrates around the centromeres [45,49]. Given the role of the centromere as a checkpoint for regulation of the mitotic phase of the cell cycle [50,51], it was hypothesized that C/EBPα localization around the centromere could play a role in C/EBPα regulation of the cell cycle [49].

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