Analysis by cell-free transcription of the liver-specific pyruvate kinase gene promoter.

Abstract

A DNA fragment spanning nucleotides -183 to -4 with respect to the cap site of the rat L-type pyruvate kinase (L-PK) gene contains at least four binding sites for putative transcriptional factors: hepatocyte nuclear factor 1 (HNF1), liver factor Al (LF-A1), nuclear factor 1 (NFl), and major late transcription factor (MLTF). This fragment was used to direct transcription of a reporter sequence (a G-free cassette) in cell extracts. This L-PK promoter was active in liver nuclear extracts, but not in extracts from nonhepatic tissues. A reduction of 50% of the activity was obtained with a deleted L-PK promoter containing only the HNFl-binding site. In contrast, deletion of the HNFl-binding site inactivated the promoter by more than 90%. These results were confirmed by titration experiments with synthetic oligonucleotides. Titration of HNF1 resulted in an 85% decrease of transcriptional activity, while titration of LF-A1 resulted in only a 40% decrease. The influence of NF1 and MLTF seemed to be marginal in this system. The proximal 5'-flanking sequence of the L-PK gene therefore appears to function in vitro as an efficient liver-specific promoter which requires the binding of the liver factor HNF1 and which is also stimulated by the binding of another liver-specific factor, LF-A1.

Transcriptional control of genes that regulate glycolysis and gluconeogenesis in adult liver

“…The binding of HNF-4 apparently stabilizes NF-I binding [80]. The promoter corresponds to a liver-specific DNAase I-hypersensitive site HSS-1 [85] and suffices to confer tissue-specificity in vitro as well as in transfection and in transgenic mice [79,81,86,87]. The most potent transcriptional stimulators are the liver-specific factors HNF-I and HNF-4.…”

“…The most potent transcriptional stimulators are the liver-specific factors HNF-I and HNF-4. In transfection NF-I and USF in basal transcription is unclear [79,86,88,89]. Yamada et al [81] transfected hepatocytes in culture and proposed an activating function for USF, while Vaulont et al [80] could not find any function for USF in vitro.…”

Characterization of the rat pulmonary surfactant protein A promoter

“…As previously described with other cell-free extracts obtained from non-hepatic tissues [25], the liver pyruvatekinase promoter was also specifically transcribed and not active with fetal lung extracts. The absence of hepatic nuclear factor l a in lung [49] and the low contents in hepatic nuclear factor 1p [SO], a family of factors essential for an efficient in vitro transcription of the liver pyruvatekinase promoter [25], might explain this result. The aldolase B promoter was able to direct a weak transcription with fetal lung extracts.…”

“…Liver pyruvate kinase and aldolase B are two glycolytic enzymes highly expressed in liver. These two constructs are able to direct an efficient RNA polymerase 11-dependent in vitro transcription of the G-free reporter cassette with liver nuclear extract [25,26]. The 5' flanking region of the major late adenovirus promoter, along with a shortened G-free cassette of 270 bp, were subcloned in Bluescript.…”

Novel Mutations and a Mutational Hotspot in the M0DY3 Gene

“…The promoters of the glucose transporter (GLUT2) and glucokinase, required in islet cells for efficient insulin secretion, both contain HNF-la binding sites (25). Several glycolytic and gluconeogenic enzymes are also known to be regulated by or contain binding sites for HNF-la, including glucokinase (25), pyruvate kinase (26), and phosphoenolpyruvate carboxykinase (27). Transcriptional activation of glycolytic genes in hepatocytes is dependent on the action of glucose and insulin (28), so HNF-la might also directly influence liver glucose metabolism.…”