Elements responsible for hormonal control and tissue specificity of L-type pyruvate kinase gene expression in transgenic mice.

Abstract

L-tpe pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway specifically expressed in the liver and, to a lesser degree, in the small intestine and kidney. One important characteristic of L-PK gene expression is its strong activation by glucose and insulin and its complete inhibition by fasting or glucagon treatment.Having previously established that the entire rat L-PK gene plus 3.2 kbp of 5'-flanking region functions in mice in a tissue-specific and hormonally regulated manner, various deletions of these 3.2 kbp of 5'-flanking regions were tested in transgenic animals to map the cis-acting elements involved in transcriptional gene regulation.Our experiments indicate that the proximal region between -183 and +11 confers tissue specificity and contains all the information necessary for dietary and hormonal control of L-PK gene expression in vivo. We found, however, that the transcriptional activity generated by this proximal promoter fragment can be modulated by distal sequences in a tissue-specific manner. (i) Sequences between bp -183 and -392 seem to play a dual role in the liver and small intestine; they induce L-PK expression in the liver but repress it in the small intestine. (ii) Sequences from bp -392 up to -1170 do not seem to have any additional effect on promoter activity. (iii) Between bp -1170 and -2080, we found a putative extinguisher whose transcriptional inhibitory effect is much more marked in the small intestine than in the liver. (iv) Finally, between bp -2080 and -3200, we identified an activating sequence required for full expression of the gene in the liver.

Determinants of the brain‐specific expression of the rat aldolase C gene: ex vivo and in vivo analysis

“…With brain extracts, the A footprint (-197 to -171) was shifted to the 5' side and began on the 3' side at the same nucleotide as a faint footprint resulting from protection by recombinant Krox2O/Krox24 proteins. In gel retardation experiments, oligonucleotide NA', however, formed a single very faint complex with brain nuclear pro- CAT activity was determined with 50 pg tissue extracts previously heated for 20 min at 60°C to destroy tissue deacetylase activity (Cuif et al, 1992). B, brain; I, intestine; M, muscle; H, heart; K, kidney; S, spleen; T, testes; L, lung; Li, liver.…”

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

“…Experiments with transgenic mice indicated that this region confers tissue-specificity to L promoter activity [77][78][79]. The promoter (-183 to +11) contains binding sites for HNF-1, NF-I, HNF-4, upstream stimulating factor (USF)-related proteins and L5-binding factors (Figure 1) [80][81][82][83].…”

“…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.…”

“…This extinguishing activity could not be observed by transfection of hepatocytes in culture [88]. An activating region located around -3 kb is required for full promoter activity [79,88]. This region corresponds to a liver-specific DNAase I-hypersensitive site (HSS-2) [85] [93].…”