CCAAT-enhancer binding protein-α (C/EBPα) and hepatocyte nuclear factor 4α (HNF4α) regulate expression of the human fructose-1,6-bisphosphatase 1 (FBP1) gene in human hepatocellular carcinoma HepG2 cells

• Published in: PloS one 2018-03, Vol.13 (3), p.e0194252-e0194252
• Main Authors: Wattanavanitchakorn, Siriluck, Rojvirat, Pinnara, Chavalit, Tanit, MacDonald, Michael J, Jitrapakdee, Sarawut
• Format: Article
• Language: English
• Subjects: Binding sites; Biochemistry; Biology and Life Sciences; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - pathology; CCAAT-enhancer binding protein; CCAAT-Enhancer-Binding Proteins - genetics; CCAAT-Enhancer-Binding Proteins - metabolism; Cell culture; Cell Nucleus - genetics; Cell Nucleus - metabolism; Cloning; Deoxyribonucleic acid; Diabetes; DNA; DNA Helicases - genetics; DNA Helicases - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Down-Regulation; Electrophoretic mobility; Electrophoretic Mobility Shift Assay; Fasting; FBP1 gene; Fructose; Gene expression; Gene Expression Regulation, Neoplastic; Gluconeogenesis; Glucose; Hep G2 Cells; Hepatocellular carcinoma; Hepatocyte Nuclear Factor 4 - genetics; Hepatocyte Nuclear Factor 4 - metabolism; Hepatoma; Homeostasis; Humans; Kinases; Liver; Liver - pathology; Liver cancer; Liver Neoplasms - genetics; Liver Neoplasms - pathology; Medicine and Health Sciences; Metabolism; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Nucleotides; Promoter Regions, Genetic - genetics; Proteins; Research and Analysis Methods; RNA Interference; RNA, Small Interfering - metabolism; Rodents; siRNA; Transcription activation; Transcription factors; Up-Regulation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0194252

Fructose-1,6-bisphosphatase (FBP1) plays an essential role in gluconeogenesis. Here we report that the human FBP1 gene is regulated by two liver-enriched transcription factors, CCAAT-enhancer binding protein-α (C/EBPα) and hepatocyte nuclear factor 4α (HNF4α) in human hepatoma HepG2 cells. C/EBPα regulates transcription of FBP1 gene via binding to the two overlapping C/EBPα sites located at nucleotide -228/-208 while HNF4α regulates FBP1 gene through binding to the classical H4-SBM site and direct repeat 3 (DR3) located at nucleotides -566/-554 and -212/-198, respectively. Mutations of these transcription factor binding sites result in marked decrease of C/EBPα- or HNF4α-mediated transcription activation of FBP1 promoter-luciferase reporter expression. Electrophoretic mobility shift assays of -228/-208 C/EBPα or -566/-554 and -212/-198 HNF4α sites with nuclear extract of HepG2 cells overexpressing C/EBPα or HNF4α confirms binding of these two transcription factors to these sites. Finally, we showed that siRNA-mediated suppression of C/EBPα or HNF4α expression in HepG2 cells lowers expression of FBP1 in parallel with down-regulation of expression of other gluconeogenic enzymes. Our results suggest that an overall gluconeogenic program is regulated by these two transcription factors, enabling transcription to occur in a liver-specific manner.