A Single Regulatory Module of the Carbamoylphosphate Synthetase I Gene Executes Its Hepatic Program of Expression

Abstract

A 469-base pair (bp) upstream regulatory fragment (URF) and the proximal promoter of the carbamoylphosphate synthetase I (CPS) gene were analyzed for their role in the regulation of spatial, developmental, and hormone-induced expression in vivo. The URF is essential and sufficient for hepatocyte-specific expression, periportal localization, perinatal activation and induction by glucocorticoids, and cAMP in transgenic mice. Before birth, the transgene is silent but can be induced by cAMP and glucocorticoids, indicating that these compounds are responsible for the activation of expression at birth. A 102-bp glucocorticoid response unit within the URF, containing binding sites for HNF3, C/EBP, and the glucocorticoid receptor, is the main determinant of the hepatocyte-specific and hormone-controlled activity. Additional sequences are required for a productive interaction between this minimal response unit and the core CPS promoter. These results show that the 469-bp URF, and probably only the 102-bp glucocorticoid response unit, functions as a regulatory module, in that it autonomously executes a correct spatial, developmental and hormonal program of CPS expression in the liver.In liver, many metabolic pathways are predominantly found in either the upstream, periportal region (e.g. gluconeogenesis) or the downstream, pericentral region (e.g. glycolysis) (1-3). Key enzymes of these pathways are therefore expressed in gradients along the sinusoids that connect the portal and the central vein. These zonal differences in gene expression largely avoid futile cycling of opposite metabolic processes (such as between glycolysis and gluconeogenesis) but also comprise complementary functions such as the low and high affinity detoxifying functions of the urea cycle and glutamine synthetase, respectively (1, 2, 4). Because the heterogeneous expression of genes in mammalian liver seems to relate to the function of the organ, much effort has been invested in understanding the mechanisms underlying its regulation. Nevertheless, it remains obscure what causes these differences in porto-central enzyme gradients and how critical the maintenance of these gradients is for preservation of normal liver function. For several hepatic genes it was shown that their localization within the liver is regulated at the transcriptional level (5-8). For that reason, we decided to embark upon a genetic dissection of the regulatory DNA elements that confer periportal expression upon a reporter gene.

Transcriptional Regulation of Metabolism

“…The CPS-1 promoter contains a complex regulatory module composed of multiple sites for glucocorticoid receptor, HNF3 and C/EBP family members, as well as for unknown factors that control its specific pattern of expression and regulation. The glucocorticoid response unit itself combines a glucocorticoid response element with sites for HNF3␤ and C/EBP (41). Whereas these in vitro studies performed in primary hepatocytes in culture attributed a preferential role to C/EBP␤, C/EBP␤ null mice did not present any perturbation of ureagenesis (40,147), and it seems reasonable to propose that in vivo C/EBP␣ is the functional partner of the glucocorticoid receptor.…”

Carbamoyl Phosphate Synthetase 1 deficiency in Italy: Clinical and genetic findings in a heterogeneous cohort

“…CPS1, involved in the first step of the urea cycle (Brusilow and Horwich, 2001 ), is a mitochondrial matrix enzyme expressed in the hepatocytes and epithelial cells of the intestinal mucosa (Christoffels et al, 2000;Ryall et al, 1986;Van Beers et al, 1998). The CPS1 enzyme is encoded by the CPS1 gene, mapping on 2 q35 (Summar et al, 1995) and organized in 38 exons (Funghini et al, 2003;Häberle et al, 2003).…”

Structural organization of the human carbamyl phosphate synthetase I gene (CPS1) and identification of two novel genetic lesions

“…CPS I is the enzyme involved in the first step of the Urea Cycle. It catalyses the synthesis of carbamyl phosphate from bicarbonate, ATP and ammonia (Brusilow and Horwich, 2001) and is located in the mitochondrial matrix of hepatocytes and epithelial cells of intestinal mucosa (Christoffels et al, 2000). N-acetylglutamate (NAG), synthesized from glutamate and acetyl CoA, is an allosteric cofactor of the CPS I (Brusilow and Horwich, 2001).…”