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Isolation and characterization of the rat gene for carbamoylphosphate synthetase I
M J van den Hoff1, L P van de Zande, M A Dingemanse
1University of Amsterdam, Department of Anatomy and Embryology, The Netherlands.
Insights
The study investigated the regulation of Carbamoylphosphate synthetase I (CbmPS) gene expression in rat liver. Researchers found a specific DNA demethylation event and an enhancer region crucial for tissue-specific CbmPS expression, influenced by hormones.
Area of Science:
- Molecular Biology
- Gene Regulation
- Hepatocyte Function
Background:
- Carbamoylphosphate synthetase I (CbmPS) expression in rat hepatocytes changes significantly after birth, becoming restricted to periportal zones.
- Understanding the spatiotemporal regulation of CbmPS is crucial for comprehending liver development and function.
Purpose of the Study:
- To elucidate the molecular mechanisms governing the tissue-specific and developmental regulation of the rat Carbamoylphosphate synthetase I (CbmPS) gene.
- To identify regulatory elements within the CbmPS gene responsible for its unique expression pattern in hepatocytes.
Main Methods:
- Isolation and characterization of the 110 kb rat CbmPS gene, including its 38 exons and basal promoter.
- Analysis of DNA methylation status at a specific CCGG site (-6.3 kb) in relation to CbmPS expression.
- Transient expression assays using hepatoma cells and fibroblasts to evaluate the function of promoter and enhancer regions, assessing hormonal influences (glucocorticoids, cAMP).
Main Results:
- A CCGG sequence at -6.3 kb is selectively demethylated in adult, expressing tissues, but remains methylated prenatally.
- The region around -6.3 kb acts as an enhancer, showing constitutive activity in non-hepatic cells but hormone-dependent activity in hepatoma cells.
- Hepatocyte-specific CbmPS expression appears linked to tissue-specific sensitivity to cyclic AMP and glucocorticoids; matrix attachment regions (MAR) flank the gene.
Conclusions:
- Demethylation of the enhancer region is a consequence, not a prerequisite, for CbmPS gene expression.
- Glucocorticoids and cAMP play critical roles in regulating CbmPS expression, with tissue-specific sensitivities contributing to its hepatocyte-specific pattern.
- The identified enhancer and MARs provide insights into the complex regulatory network controlling CbmPS gene expression during liver development.
Abstract:
Carbamoylphosphate synthetase I (CbmPS) is first expressed in rat hepatocytes shortly before birth. After birth, expression of CbmPS gradually becomes confined to the hepatocytes surrounding the portal veins. To obtain insight into the spatiotemporal regulation of its expression, the rat CbmPS gene was isolated and characterized. The gene is 110 kb in length and contains 38 exons. The basal promoter comprises the first 161 nucleotides upstream of the transcription-initiation site. Determination of the state of methylation of the 5' portion of the gene identified a CCGG sequence at -6.3 kb that is selectively demethylated in adult tissues which express CbmPS. This site remains methylated before birth, however, despite recruitment of all hepatocytes for CbmPS synthesis, indicating that its demethylation is a consequence of rather than a condition for expression of CbmPS. Transient expression assays revealed that the region surrounding the CCGG site at 6.3 kb functions as an enhancer. In FTO-2B hepatoma cells and Rat-1 fibroblasts, this enhancer is constitutively active when tested in front of the basal viral thymidine kinase promoter. When tested in front of the basal CbmPS promoter in hepatoma cells, however, the activity of this enhancer is dependent on the presence of glucocorticoids. In Rat-1 fibroblasts, the presence of both glucocorticoids and cyclic AMP is required for full activity, suggesting that the hepatocyte-specific expression of CbmPS is related to tissue-specific differences in the sensitivity to cyclic AMP. Matrix-attachment regions (MAR) are present upstream and downstream of the CbmPS gene. The downstream MAR defines the 3' boundary of the gene. The upstream MAR is located midway between the basal promoter and the enhancer, and may function as a hinge point to facilitate the positioning of the enhancer in the vicinity of the basal promoter.