Transactivation of cytosolic alanine aminotransferase gene promoter by p300 and c-Myb

Alanine aminotransferase (Alt) provides a molecular link between carbohydrate and amino acid metabolism. In the cell context, the predominant Alt isozyme is located in the cytosol. To gain insight into the transcriptional regulation of the cytosolic alt gene (calt), we cloned and characterized the calt promoter from gilthead sea bream (Sparus aurata). Transient transfection of sea bass larvae cells with deleted calt promoter constructs and electrophoretic mobility shift assays allowed us to identify p300 and c-Myb as new factors in the transcriptional regulation of calt expression. Transfection studies carried out with an acetylase-deficient mutant p300 (p300DY) revealed that the acetyltransferase activity of p300 is essential for the p300-mediated transcriptional activation of S. aurata calt. We had previously found up-regulation of liver cAlt2, an alternatively spliced isoform of calt, under gluconeogenic conditions and in streptozotocin (STZ)-treated S. aurata. Quantitative RT-PCR assays showed that increased p300 and c-Myb mRNA levels in the liver of starved S. aurata contribute to enhancing the transcription of cAlt2. Consistently, the administration of insulin decreased both p300 and c-Myb expression. The mRNA levels of p300 and c-Myb were also analyzed in the liver of STZ-induced diabetic S. aurata. Treatment with STZ increased the expression of p300, whereas it decreased c-Myb. Our findings suggest an involvement of p300 and c-Myb in up-regulation of cAlt2 in the liver of S. aurata under starvation. In addition, these results provide evidence for a role of p300 in diabetes.

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Sterol Regulatory Element Binding Protein-1a Transactivates 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase Gene Promoter

Endocrinology ◽

10.1210/en.2005-1506 ◽

2006 ◽

Vol 147 (7) ◽

pp. 3446-3456 ◽

Cited By ~ 14

Author(s):

Isidoro Metón ◽

Miriam Egea ◽

Ida G. Anemaet ◽

Felipe Fernández ◽

Isabel V. Baanante

Keyword(s):

Transcriptional Activation ◽

Sparus Aurata ◽

Binding Protein ◽

Regulatory Element ◽

Gene Promoter ◽

Sea Bream ◽

Proximal Promoter ◽

Mrna Levels ◽

Nutritional Regulation ◽

Sterol Regulatory Element

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a) binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription.

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Transcriptional regulation of rat Na+/H+exchanger isoform-2 (NHE-2) gene by Sp1 transcription factor

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.5.c1168 ◽

2001 ◽

Vol 280 (5) ◽

pp. C1168-C1175 ◽

Cited By ~ 25

Author(s):

Liqun Bai ◽

James F. Collins ◽

Hua Xu ◽

Fayez K. Ghishan

Keyword(s):

Transcriptional Regulation ◽

Transcriptional Activation ◽

Collecting Duct ◽

Gene Promoter ◽

Minimal Promoter ◽

Mobility Shift ◽

Sp1 Transcription Factor ◽

Medullary Collecting Duct ◽

High Level ◽

Electrophoretic Mobility Shift Assays

The rat Na+/H+ exchanger isoform-2 ( NHE-2) gene promoter lacks a TATA box and is very GC rich. A minimal promoter extending from bp −36 to +116 directs high-level expression of NHE-2 in mouse inner medullary collecting duct (mIMCD-3) cells. Four Sp1 consensus elements were found in this region. The introduction of mutations within these Sp1 consensus elements and DNA footprinting revealed that only two of them were utilized and are critical for basal transcriptional activation in mIMCD-3 cells. The use of Sp1, Sp3, and Sp4 antisera in electrophoretic mobility shift assays demonstrated that Sp1, Sp3, and Sp4 bound to this minimal promoter. We further analyzed the transcriptional regulation of NHE-2 by members of the Sp1 multigene family. In Drosophila SL2 cells, which lack endogenous Sp1, the minimal promoter cannot drive transcription. Introduction of Sp1 activated transcription over 100-fold, suggesting that Sp1 is critical for transcriptional regulation. However, neither Sp3 nor Sp4 was able to activate transcription in these cells. Furthermore, in mIMCD-3 cells, Sp1-mediated transcriptional activation was repressed by expression of Sp3 and Sp4. These data suggest that Sp1 is critical for the basal promoter function of rat NHE-2 and that Sp3 and Sp4 may repress transcriptional activation by competing with Sp1 for binding to core cis-elements.

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