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Hormonal regulation of phosphoenolpyruvate carboxykinase gene expression is mediated through modulation of an already
Y T Ip1, D K Granner, R Chalkley
1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Abstract:
We used indirect end labeling to identify a series of five hypersensitive (HS) sites in the phosphoenolpyruvate carboxykinase (PEPCK) gene in H4IIE rat hepatoma cells. These sites were found at -4800 base pairs (bp) (site A), at -1300 bp (site B), over a broad domain between -400 and -30 bp (site C), at +4650 bp (site D), and at +6200 bp (site E). Sites A to D were detected only in cells capable of expressing the PEPCK gene, whereas site E was present in all of the cells examined thus far. The HS sites were present in H4IIE cells even when transcriptional activity was reduced to a minimum by treatment with insulin. Stimulation of transcription by a cyclic AMP analog to a 40-fold increase over the insulin-repressed level did not affect the main features of the HS sites. Furthermore, increased transcription did not disrupt the nucleosomal arrangement of the coding region of the gene, nor did it affect the immediate 5' region (site C), which is always nucleosome-free. In HTC cells, a rat hepatoma line that is hormonally responsive but unable to synthesize PEPCK mRNA, the four expression-specific HS sites were totally absent. Our experimental results also showed that, although there is a general correlation between lack of DNA methylation and transcriptional competence of the PEPCK gene, the role, if any, of methylation in the regulation of PEPCK gene activity is likely to be exerted at very specific sites.
Insights
Researchers identified five hypersensitive (HS) sites in the phosphoenolpyruvate carboxykinase (PEPCK) gene. Four sites correlate with PEPCK gene expression, while one is universally present, offering insights into gene regulation.
Area of Science:
- Molecular Biology
- Gene Regulation
- Epigenetics
Background:
- The phosphoenolpyruvate carboxykinase (PEPCK) gene's regulation is crucial for cellular energy metabolism.
- Understanding the role of chromatin structure in PEPCK gene expression is essential.
Purpose of the Study:
- To identify and characterize hypersensitive (HS) sites within the PEPCK gene in rat hepatoma cells.
- To investigate the correlation between HS sites, gene expression, and DNA methylation.
Main Methods:
- Indirect end labeling was employed to map HS sites.
- Analysis was conducted in H4IIE and HTC rat hepatoma cell lines.
- Gene expression was modulated using insulin and cyclic AMP analogs.
Main Results:
- Five HS sites (A-E) were identified in the PEPCK gene.
- HS sites A-D were expression-dependent, present only in cells expressing PEPCK.
- Site E was found in all cell types, irrespective of PEPCK expression.
- HS sites remained stable despite insulin-induced repression or cyclic AMP-stimulated transcription.
- HTC cells, unable to express PEPCK, lacked the expression-specific HS sites.
- A correlation between DNA hypomethylation and PEPCK gene transcriptional competence was observed.
Conclusions:
- Expression-dependent HS sites are key regulatory elements for PEPCK gene activity.
- Chromatin accessibility, indicated by HS sites, is linked to PEPCK gene expression.
- DNA methylation may play a role in PEPCK gene regulation at specific sites.