Nuclear factor 1 family members interact with hepatocyte nuclear factor 1alpha to synergistically activate L-type pyruvate kinase gene transcription.
Transcription of hepatic L-type pyruvate kinase (L-PK) gene is cell type-specific and is under the control of various nutritional conditions. The L-PK gene contains multiple cis-regulatory elements located within a 170-bp upstream region necessary for these regulations. These elements can synergistically stimulate L-PK gene transcription, although their mechanisms are largely unknown. Because nuclear factor (NF) 1 family members bind to specific cis-regulatory elements known as L-IIA and L-IIB and hepatocyte nuclear factor (HNF) 1alpha binds to the adjacent element L-I, we examined the functional and physical interactions between these two transcription factors. Reporter gene assay showed that these two factors synergistically activated the L-PK promoter containing the 5'-flanking region up to -189. Although two NF1-binding sites are required for the maximum synergistic effect of NF1 family members with HNF1alpha, significant functional interaction between the two factors was observed in the L-PK promoter containing two mutated NF1-binding sites and also in the promoter containing only the HNF1alpha-binding site, raising the possibility that NF1 proteins function as HNF1alpha co-activators. Chromatin immunoprecipitation assay revealed that both NF1 proteins and HNF1alpha bound to the promoter region of the L-PK gene in vivo. In vitro binding assay confirmed that NF1 proteins directly interacted mainly with the homeodomain of HNF1alpha via their DNA-binding domains. This interaction enhanced HNF1alpha binding to the L-I element and was also observed in rat liver by co-immunoprecipitation assay. Thus, we conclude that cooperative interaction between NF1 family members and HNF1alpha plays an important role in hepatic L-PK transcription.
Satoh
Shin-ichi
S
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.
Noaki
Takashi
T
Ishigure
Tatsuya
T
Osada
Shigehiro
S
Imagawa
Masayoshi
M
Miura
Naoyuki
N
Yamada
Kazuya
K
Noguchi
Tamio
T
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008099:Liver / Q000378:metabolism; D008297:Male; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051839:NFI Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011770:Pyruvate Kinase / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D051538:Hepatocyte Nuclear Factor 1-alpha; D051839:NFI Transcription Factors; D009841:Oligonucleotides; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase; D011770:Pyruvate Kinase