title: Cell type-specific target selection by combinatorial binding of Smad2/3 proteins and hepatocyte nuclear factor 4alpha in HepG2 cells.
abstract: Specific regulation of target genes by transforming growth factor-β (TGF-β) in a given cellular context is determined in part by transcription factors and cofactors that interact with the Smad complex. In this study, we determined Smad2 and Smad3 (Smad2/3) binding regions in the promoters of known genes in HepG2 hepatoblastoma cells, and we compared them with those in HaCaT epidermal keratinocytes to elucidate the mechanisms of cell type- and context-dependent regulation of transcription induced by TGF-β. Our results show that 81% of the Smad2/3 binding regions in HepG2 cells were not shared with those found in HaCaT cells. Hepatocyte nuclear factor 4α (HNF4α) is expressed in HepG2 cells but not in HaCaT cells, and the HNF4α-binding motif was identified as an enriched motif in the HepG2-specific Smad2/3 binding regions. Chromatin immunoprecipitation sequencing analysis of HNF4α binding regions under TGF-β stimulation revealed that 32.5% of the Smad2/3 binding regions overlapped HNF4α bindings. MIXL1 was identified as a new combinatorial target of HNF4α and Smad2/3, and both the HNF4α protein and its binding motif were required for the induction of MIXL1 by TGF-β in HepG2 cells. These findings generalize the importance of binding of HNF4α on Smad2/3 binding genomic regions for HepG2-specific regulation of transcription by TGF-β and suggest that certain transcription factors expressed in a cell type-specific manner play important roles in the transcription regulated by the TGF-β-Smad signaling pathway.
authors.affiliation: Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Anna
authors.initials: A
authors.lastname: Mizutani
authors.forename: Daizo
authors.initials: D
authors.lastname: Koinuma
authors.forename: Shuichi
authors.initials: S
authors.lastname: Tsutsumi
authors.forename: Naoko
authors.initials: N
authors.lastname: Kamimura
authors.forename: Masato
authors.initials: M
authors.lastname: Morikawa
authors.forename: Hiroshi I
authors.initials: HI
authors.lastname: Suzuki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imamura
authors.forename: Kohei
authors.initials: K
authors.lastname: Miyazono
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Aburatani
mesh_terms: D020816:Amino Acid Motifs; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009928:Organ Specificity / Q000502:physiology; D011485:Protein Binding; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D018398:Homeodomain Proteins; C499230:MIXL1 protein, human; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta