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

Siriluck Wattanavanitchakorn; Pinnara Rojvirat; Tanit Chavalit; Michael J MacDonald; Sarawut Jitrapakdee

doi:10.1371/journal.pone.0194252

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

PLoS One. 2018 Mar 22;13(3):e0194252. doi: 10.1371/journal.pone.0194252. eCollection 2018.

Authors

Siriluck Wattanavanitchakorn¹, Pinnara Rojvirat², Tanit Chavalit¹, Michael J MacDonald³, Sarawut Jitrapakdee¹

Affiliations

¹ Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
² Division of Interdisciplinary, Mahidol University, Kanjanaburi, Thailand.
³ Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States of America.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CCAAT-Enhancer-Binding Proteins / genetics
CCAAT-Enhancer-Binding Proteins / metabolism*
Carcinoma, Hepatocellular / genetics*
Carcinoma, Hepatocellular / pathology
Cell Nucleus / genetics
Cell Nucleus / metabolism
DNA Helicases / genetics
DNA Helicases / metabolism
DNA-Binding Proteins / genetics
DNA-Binding Proteins / metabolism
Down-Regulation
Electrophoretic Mobility Shift Assay
Gene Expression Regulation, Neoplastic*
Hep G2 Cells
Hepatocyte Nuclear Factor 4 / genetics
Hepatocyte Nuclear Factor 4 / metabolism*
Humans
Liver / pathology
Liver Neoplasms / genetics*
Liver Neoplasms / pathology
Mutagenesis, Site-Directed
Promoter Regions, Genetic / genetics
RNA Interference
RNA, Small Interfering / metabolism
RNA-Binding Proteins
Up-Regulation

Substances

CCAAT-Enhancer-Binding Proteins
CEBPA protein, human
DNA-Binding Proteins
FUBP1 protein, human
HNF4A protein, human
Hepatocyte Nuclear Factor 4
RNA, Small Interfering
RNA-Binding Proteins
DNA Helicases

Grants and funding

This work was supported by an international research network (grant IRN 59W0003), the Thailand Research Fund. S.W. was supported by an RGJ-PHD scholarship (4CMU56010), the Thailand Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.