title: Identification and characterization of hepatocyte-specific regulatory regions of the rat pyruvate kinase L gene. The synergistic effect of multiple elements.
abstract: The rat pyruvate kinase L (PKL) gene produces the L- and R-type isozymes by alternative transcription that is regulated in a tissue-specific manner. To investigate which DNA elements are involved in hepatocyte-specific expression of the L-type isozyme, we performed transient DNA transfer experiments with PKL/chloramphenicol acetyltransferase fusion genes. We found three positive regulatory regions required for expression of the L-type isozyme in adult rat hepatocytes by functional analyses of a series of 5' and internal deletion constructs of the fusion genes. These regions, designated as PKL-I, PKL-II, and PKL-III, were located between nucleotides -76 and -94, -126 and -149, and -150 and -170, respectively. PKL-I showed enhancer-like activity alone, whereas PKL-II and PKL-III did not have any independent effect. Combinations of L-I + L-II and L-II + L-III, but not of L-I + L-III, showed synergistic enhancer activities when oriented in the same direction. The inclusion of all three elements oriented in the same direction had the maximum synergistic effect, indicating that these elements function as a unit. This unit enhanced expression from heterologous as well as homologous promoters in a manner that was independent of its orientation and position relative to the cap site. The activity of the unit was not detected in HeLa cells or K562 erythroleukemia cells, suggesting that this unit possessed cell-type specificity. PKL-I consists of a palindrome sequence 5'-CTGGTTATACTTTAACCAG-3', which contain a sequence homologous to the LF-B1-binding site. PKL-II contains the sequence 5'-TTCCTGGACTCTGGCCCCCAGTGT-3', which is similar to that of the LF-A1-binding site. PKL-III contains a palindrome sequence 5'-CCACGGGGCACTCCCGTGG-3', which include a sequence homologous to the binding site of the adenovirus major late transcription factor. Gel retardation assay indicated that the different trans-acting factors interacted with three elements and that the transacting protein bound to PKL-I was in fact LF-B1. However, the trans-acting factors bound to PKL-II and PKL-III were different from LF-A1 and major late transcription factor, respectively. Thus, we conclude that three cis-acting elements are very important for specific expression of the PKL gene in hepatocytes and that LF-B1 and two unknown factors bound to these elements interact with each other to cause a synergistic effect.
authors: Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Japan.
authors: K
authors: K
authors: Yamada
authors: T
authors: T
authors: Noguchi
authors: T
authors: T
authors: Matsuda
authors: M
authors: M
authors: Takenaka
authors: P
authors: P
authors: Monaci
authors: A
authors: A
authors: Nicosia
authors: T
authors: T
authors: Tanaka
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004274:DNA, Recombinant; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011770:Pyruvate Kinase / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D011770:Pyruvate Kinase