Recent technological advances, including ChIP-chip and ChIP-seq, provide a functional platform for comprehensive understanding of transcriptional regulation. This study revealed that Smad2/3 binding regions specifically observed in HepG2 cells were enriched in HNF4α binding regions. HNF4α was also expressed in Hep3B cells, and HNF4α-binding motif was identified in Smad2/3 binding regions in Hep3B cells by CEAS analysis (data not shown), which suggests that the functional relation between Smad2/3 and HNF4α is commonly observed in hepatocyte-derived cells. Based on the findings on the HNF4α-Smad interaction (18
), physical interaction between HNF4α and Smads is important, at least in part, for TGF-β-induced Smad2/3 binding and transcriptional activation in HepG2 cells. It is also possible that HNF4α has additional indirect interactive functions for TGF-β signaling. Many regulatory mechanisms control the expression of a proper set of genes in various cells and tissues. At the genome level, CpG methylation plays a central role to avoid unintended expression of genes that are not suitable for the given tissue (33
). Modification of the histone tail is also well known to lead to the formation of either euchromatin or heterochromatin. These modifications of the genome or histones allow transcription factors and cofactors to access the cell- and tissue-specific genomic loci to exert their actions. Modifications of the genome and histones are sometimes induced by trans
factors during differentiation of the cells and tissues (34
). HNF4α physically interacts with the histone acetyltransferase complex and chromatin remodeling complex (29
), and it is thus possible that HNF4α induces such epigenomic changes in the liver and indirectly provides Smad2/3 to access to hepatocyte-specific binding regions.
Identification of Smad binding regions downstream of the TGF-β/activin signaling by ChIP-chip analysis has been performed using several cell lines. Recently, Fei et al.
) reported promoter analysis of Smad2 binding regions in mouse embryonic stem cells by ChIP-chip. We and Qin et al.
) analyzed Smad4 binding regions under TGF-β stimulation using HaCaT and ovarian surface epithelial cells, respectively. It has been reported that transcription factor binding regions in the same target gene loci differ among the five vertebrate species (38
); it is thus difficult to compare the ChIP-chip or ChIP-seq data obtained from mouse and human. Differences in the ChIP efficiencies of the antibodies also make the comparison of the data difficult (12
). Importantly, we used the same antibody and sample preparation procedures for HaCaT cells and HepG2 cells. Our present analysis thus revealed for the first time that Smad binding regions greatly differ among cell lines. Analysis of HaCaT-specific trans
factors will facilitate our understanding of cell type-specific TGF-β-induced transcription in the future. However, comparison of the number of binding regions in different cell types is still difficult. We found a greater number of Smad2/3 binding regions in HepG2 cells than in HaCaT cells. Because the phosphorylation of Smad3 was weaker and the percent input value of the Smad2/3 ChIP sample was smaller in HepG2 than HaCaT cells, we cannot conclude that HepG2 cells have more Smad2/3 binding regions than HaCaT. It should also be noted that we cannot fully exclude that the antibody recognizes unknown genome-bound molecules in addition to Smad2/3.
Comparison of ChIP-chip and ChIP-seq data of the same transcription factor has been reported (39
). In general, ChIP-seq is reported to be more sensitive and specific than ChIP-chip. Oligonucleotide-based array analysis has a potential risk of cross-hybridization and false discovery. Conversely, ChIP-seq also has difficulty in identifying GC-rich sequences (10
). We primarily focused on the comparison of our previously reported Smad2/3 binding regions to those of different cell types by the same platform. However, based on the known problems as described above, comparison of the Smad2/3-HNF4α binding regions will be more accurately performed by the ChIP-seq in the future.
Interaction of several transcription factors at the same enhancer positions has been recognized, and the complex is called “enhanceosome.” Structure of such complex and their binding DNA motifs have been analyzed in the interferon-β promoter as reviewed by Panne (40
). In enhanceosome, each transcription factor physically interacts with others to provide its adequate surface that can bind to the series of their corresponding DNA motifs. Several reports have identified HNF4α binding regions by ChIP-chip and ChIP-seq analyses (29
). Many transcription factors, e.g.
FOXA2, GABP, HNF1α, HNF4γ, HNF6, cohesin, and CDX2, were identified to co-localize with HNF4α through these analyses. Other reports also revealed interaction of FOXO1 or retinoic acid receptor/retinoid X receptor with HNF4α on specific promoters (44
). These findings clearly revealed steady-state binding regions of HNF4α on the genome and suggested that transcription factors that co-localize or interact with HNF4α may form enhanceosome with HNF4α. Changes in the HNF4α binding regions were found during differentiation of an intestinal epithelial cell line CaCo2 (43
); however, to our knowledge, the effect of single extracellular stimulation on genome-wide HNF4α binding regions has not yet been elucidated. Our present analysis provides the data of HNF4α binding regions following TGF-β stimulation, which were compared with the Smad2/3 binding regions in HaCaT cells that lack the expression of HNF4α. We have found that large proportions of HNF4α binding regions in HepG2 cells were unchanged by TGF-β stimulation. However, some changes in HNF4α binding regions were observed with regard to their positions and their strength, suggesting that TGF-β might regulate a subset of HNF4α binding regions. de Boussac et al.
) reported that hepatocyte growth factor inhibited HNF4α binding to the ABCC6
promoter, which together suggest the importance of changes in the HNF4α binding positions by external stimuli. We also found that the effect of HNF4α on the TGF-β-induced gene expression after 24 h of TGF-β stimulation was different from that after 1.5 h of TGF-β stimulation. Studies on the changes in the genome-wide HNF4α and Smad2/3 binding after TGF-β stimulation at several time points and ChIP-seq analysis of HNF4α with other interactive factors in relation to their binding DNA sequences will reveal new mechanisms of the regulation of HNF4α-induced transcription in the context of the enhanceosome.
MIXL1 is an ortholog of Xenopus
Mix.1, a transcription factor rapidly induced by activin during the early stage of Xenopus
). There are six known homologs that have been identified in Xenopus
to engage in the formation of mesoderm and endoderm (48
). However, only one ortholog of Mix.1 is known in human and mouse (50
). MIXL1 is required for the development of the chordamesoderm, heart, and gut in mouse (51
). Forced expression of MIXL1 in embryonic stem cells resulted in the differentiation of the cells to endoderm (52
). TGF-β is reported to induce Mix.2
promoter activity by formation of a Smad2/Smad4/FAST-1 (FoxH1) complex (53
). In mouse, Smads and FAST-1 interact to up-regulate the transcriptional activity of the MIXL1
). However, FAST-1 is not expressed in HepG2 cells (56
). Our finding of TGF-β-induced MIXL1
expression in HepG2 cells suggests a previously unrecognized regulatory mechanism of its expression by HNF4α in the absence of FAST-1. During development, HNF4α is expressed in the visceral endoderm during the gastrulation stage and plays a role in the differentiation of the embryonic mesoderm (57
). MIXL1 is also expressed in the visceral endoderm and induces migration of the embryonic endoderm. HNF4α-null mice embryo showed impaired development of mature visceral endoderm, indicating that HNF4α acts upstream of MIXL1, at least in the visceral endoderm. Notably, both HNF4α and MIXL1 positively regulate the E-cadherin expression (52
), and the HNF4α expression was repressed in a model of progression of hepatocellular carcinoma (59
). Functional analysis of MIXL1 in liver fibrosis and hepatocellular carcinoma in relation to TGF-β signaling might reveal the roles of MIXL1 in the adult liver in the future.