The role of the CCAAT/enhancer-binding protein in the transcriptional regulation of the gene for phosphoenolpyruvate carboxykinase (GTP)

1990 ◽  
Vol 10 (12) ◽  
pp. 6264-6272
Author(s):  
E A Park ◽  
W J Roesler ◽  
J Liu ◽  
D J Klemm ◽  
A L Gurney ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Binding Protein ◽  
Regulatory Elements ◽  
Specific Protein ◽  
Affinity Column ◽  
Binding Domains ◽  
Nuclear Extracts ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue- and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient contransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.

scholarly journals The role of the CCAAT/enhancer-binding protein in the transcriptional regulation of the gene for phosphoenolpyruvate carboxykinase (GTP).

1990 ◽  
Vol 10 (12) ◽  
pp. 6264-6272 ◽  
Author(s):  
E A Park ◽  
W J Roesler ◽  
J Liu ◽  
D J Klemm ◽  
A L Gurney ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Binding Protein ◽  
Regulatory Elements ◽  
Specific Protein ◽  
Affinity Column ◽  
Binding Domains ◽  
Nuclear Extracts ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue- and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient contransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.

Purification of the c-fos enhancer-binding protein

1987 ◽  
Vol 7 (10) ◽  
pp. 3482-3489
Author(s):  
R Prywes ◽  
R G Roeder
Keyword(s):  
Dna Sequences ◽  
Binding Protein ◽  
Binding Activity ◽  
Affinity Column ◽  
Equilibrium Dissociation Constant ◽  
Nuclear Extracts ◽  
Enhancer Binding Protein ◽  
Low Salt ◽  
Equilibrium Dissociation ◽  
Dyad Symmetry

We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.

scholarly journals CCAAT-enhancer-binding protein α (C/EBPα) is required for the thyroid hormone but not the retinoic acid induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription

1997 ◽  
Vol 322 (1) ◽  
pp. 343-349 ◽  
Author(s):  
Edwards A. PARK ◽  
Shulan SONG ◽  
Michelle OLIVE ◽  
William J. ROESLER
Keyword(s):  
Retinoic Acid ◽  
Thyroid Hormone ◽  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Binding Protein ◽  
Dominant Negative ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by cAMP, the thyroid hormone tri-iodothyronine (T3) and retinoic acid (RA). Regulation of PEPCK transcription by T3 involves two sites in the promoter including a thyroid-hormone-response element (TRE) and a CCAAT-enhancer-binding protein (C/EBP) binding site called P3(I). Mutation of either the TRE or P3(I) eliminates the T3 response. In this study, we examined the role of C/EBPs in the induction of PEPCK transcription by T3 and RA. PEPCK-CAT vectors were transfected into HepG2 cells. Co-transfection of a dominant negative C/EBP eliminated the T3 stimulation indicating that a member of the C/EBP family is required. To determine which C/EBP isoform was required, Gal4 fusion proteins were created that contained the Gal4 DNA-binding domain ligated to the transcriptional activation domain of C/EBPα, C/EBPβ or the cAMP-responsive-element-binding protein. A Gal4 DNA-binding site was introduced into the P3(I) site of the PEPCK-CAT vector. Only co-transfection of the Gal4-C/EBPα vector was able to restore T3 responsiveness to the PEPCK-CAT vector. The T3 and RA receptors are members of the nuclear receptor superfamily and bind to repeats of the AGGTCA motif. We found that the RA receptor can bind to sequences within the PEPCK-TRE and contribute to RA responsiveness of the PEPCK gene. However, the RA induction of PEPCK transcription was found to be independent of C/EBPs, further demonstrating the specificity of the involvement of C/EBPα in the T3 effect.

scholarly journals Purification of the c-fos enhancer-binding protein.

1987 ◽  
Vol 7 (10) ◽  
pp. 3482-3489 ◽  
Author(s):  
R Prywes ◽  
R G Roeder
Keyword(s):  
Dna Sequences ◽  
Binding Protein ◽  
Binding Activity ◽  
Affinity Column ◽  
Equilibrium Dissociation Constant ◽  
Nuclear Extracts ◽  
Enhancer Binding Protein ◽  
Low Salt ◽  
Equilibrium Dissociation ◽  
Dyad Symmetry

We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.

scholarly journals Effects of Age on the Posttranscriptional Regulation of CCAAT/Enhancer Binding Protein α and CCAAT/Enhancer Binding Protein β Isoform Synthesis in Control and LPS-Treated Livers

1998 ◽  
Vol 9 (6) ◽  
pp. 1479-1494 ◽  
Author(s):  
Ching-Chyuan Hsieh ◽  
Wei Xiong ◽  
Qizhi Xie ◽  
Jeffrey P. Rabek ◽  
Sheen G. Scott ◽  
...  
Keyword(s):  
Posttranscriptional Regulation ◽  
Nuclear Translocation ◽  
Binding Protein ◽  
Binding Activity ◽  
Aged Mouse ◽  
Translational Initiation ◽  
Lps Challenge ◽  
Nuclear Extracts ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

The CCAAT/enhancer binding protein α (C/EBPα) and CCAAT/enhancer binding protein β (C/EBPβ) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPαs with molecular masses of 42, 38, 30, and 20 kDa and C/EBPβs of 35, 20, and ∼8.5 kDa. The DNA-binding activities and pool levels of p42C/EBPαand p30C/EBPαin control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPα and C/EBPβ isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42C/EBPαlevels and binding activity, whereas those of p20C/EBPαand p20C/EBPβare increased. However, translation of 42-kDa C/EBPα is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPα- and C/EBPβ-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPα and C/EBPβ isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.

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