The Far-upstream Enhancer of the Carbamoyl-phosphate Synthetase I Gene Is Responsible for the Tissue Specificity and Hormone Inducibility of Its Expression

Christoffels, Vincent M.; Hoff, Maurice J.B. van den; Moorman, Antoon F.M.; Lamers, Wouter H.

doi:10.1074/jbc.270.42.24932

Cited by 36 publications

(49 citation statements)

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“…Construct 3 is identical, except that the promoter extends from Ϫ74 to ϩ138, that is, lacks sites I, II, and III. The promoter of construct 4 extends from Ϫ38 to ϩ138, that is, additionally lacks the GAG box (19,25). Constructs 5-7 are comparable with constructs 2-4, respectively, except that the URF is replaced by the 102-bp GRU fragment (20).…”

Section: Plasmidsmentioning

confidence: 75%

“…Construct 1 contains the fully active genomic rat CPS I gene promoter from Ϫ161 to ϩ138 bp, containing sites I, II, and III (19,23,24), the GAG box, the TATA motif, the transcription initiation site, and 138 bp of the 140-bp 5Ј-untranslated region, upstream of the lucϩ gene (see Fig. 1).…”

Section: Plasmidsmentioning

confidence: 99%

“…1). Construct 2 contains the 469-bp genomic rat CPS I enhancer fragment (19), in conjunction with the complete CPS promoter. Construct 3 is identical, except that the promoter extends from Ϫ74 to ϩ138, that is, lacks sites I, II, and III.…”

Section: Plasmidsmentioning

confidence: 99%

“…FTO-2B hepatoma, NIH3T3, and CHO-K1 cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Life Technologies, Inc.), supplemented with 10% fetal calf serum as described (19). Transfection of FTO-2B and CHO-K1 cells and luciferase and ␤-galactosidase activity assays were performed as described (19).…”

Section: Transient Transfectionsmentioning

confidence: 99%

“…The 12-kbp upstream DNA fragment of the CPS gene has been shown to contain all information required for the proper control of expression in vivo (7). We previously isolated a 469-bp fragment at 6 kbp upstream of the transcription initiation site which, together with a 0.3-kbp (Ϫ161 to ϩ138) proximal promoter fragment, confers hepatocyte specificity upon a reporter gene (7,18,19). This upstream regulatory fragment (URF) contains multiple recognition sites for HNF3 and C/EBP family members, the glucocorticoid receptor (GR) and several unknown factors.…”

mentioning

confidence: 99%

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

Christoffels¹,

Habets²,

Das³

et al. 2000

Journal of Biological Chemistry

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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.

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Section: Plasmidsmentioning

confidence: 75%

Section: Plasmidsmentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

Section: Transient Transfectionsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Christoffels¹,

Habets²,

Das³

et al. 2000

Journal of Biological Chemistry

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Noncoding sequence variants define a novel regulatory element in the first intron of the N ‐acetylglutamate synthase gene

et al. 2021

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N-acetylglutamate synthase deficiency is an autosomal recessive urea cycle disorder caused either by decreased expression of the NAGS gene or defective NAGS enzyme resulting in decreased production of N-acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1). NAGSD is the only urea cycle disorder that can be effectively treated with a single drug, N-carbamylglutamate (NCG), a stable NAG analog, which activates CPS1 to restore ureagenesis. We describe three patients with NAGSD due to four novel noncoding sequence variants in the NAGS regulatory regions. All three patients had hyperammonemia that resolved upon treatment with NCG. Sequence variants NM_153006.2:c.427-222G>A and NM_153006.2:c.427-218A>C reside in the 547 bp-long first intron of NAGS and define a novel NAGS regulatory element that binds retinoic X receptor α. Sequence variants NC_000017.10:g.42078967A>T (NM_153006.2:c.-3065A>T) and NC_000017.10:g.42078934C>T (NM_153006.2:c.-3098C>T) reside in the NAGS enhancer, within known HNF1 and predicted glucocorticoid receptor binding sites, respectively. Reporter gene assays in HepG2 and HuH-7 cells demonstrated that all four substitutions could result in reduced expression

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Datamining approaches for examining the low prevalence of N‐acetylglutamate synthase deficiency and understanding transcriptional regulation of urea cycle genes

Caldovic,

Ahn,

Andricovic

et al. 2023

J of Inher Metab Disea

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Ammonia, which is toxic to the brain, is converted into non‐toxic urea, through a pathway of six enzymatically catalyzed steps known as the urea cycle. In this pathway, N‐acetylglutamate synthase (NAGS, EC 2.3.1.1) catalyzes the formation of N‐acetylglutamate (NAG) from glutamate and acetyl coenzyme A. NAGS deficiency (NAGSD) is the rarest of the urea cycle disorders, yet is unique in that ureagenesis can be restored with the drug N‐carbamylglutamate (NCG). We investigated whether the rarity of NAGSD could be due to low sequence variation in the NAGS genomic region, high NAGS tolerance for amino acid replacements, and alternative sources of NAG and NCG in the body. We also evaluated whether the small genomic footprint of the NAGS catalytic domain might play a role. The small number of patients diagnosed with NAGSD could result from the absence of specific disease biomarkers and/or short NAGS catalytic domain. We screened for sequence variants in NAGS regulatory regions in patients suspected of having NAGSD and found a novel NAGS regulatory element in the first intron of the NAGS gene. We applied the same datamining approach to identify regulatory elements in the remaining urea cycle genes. In addition to the known promoters and enhancers of each gene, we identified several novel regulatory elements in their upstream regions and first introns. The identification of cis‐regulatory elements of urea cycle genes and their associated transcription factors holds promise for uncovering shared mechanisms governing urea cycle gene expression and potentially lead to new treatments for urea cycle disorders.This article is protected by copyright. All rights reserved.

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The Far-upstream Enhancer of the Carbamoyl-phosphate Synthetase I Gene Is Responsible for the Tissue Specificity and Hormone Inducibility of Its Expression

Cited by 36 publications

References 50 publications

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

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

Noncoding sequence variants define a novel regulatory element in the first intron of the N ‐acetylglutamate synthase gene

Datamining approaches for examining the low prevalence of N‐acetylglutamate synthase deficiency and understanding transcriptional regulation of urea cycle genes

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