DDBJ/EMBL/GenBank accession no. AF015275
ABSTRACT
The transcription factor hepatocyte nuclear factor 4[alpha] (HNF4[alpha]) is a tissue specific transcription factor mainly expressed in the liver, kidney, intestine and the endocrine pancreas, but is also an essential regulator for early embryonic events. Based on its protein structure HNF4[alpha] is classified as an orphan member of the nuclear receptor superfamily. Comparing HNF4[alpha] transcription factors in the differentiated and dedifferentiated murine hepatocyte cell line MHSV-12 we identified in dedifferentiated cells the novel splice variant HNF4[alpha]7. This variant is characterized by an alternative first exon and has a lower transactivation potential in transient transfection assays using HNF4 dependent reporter genes. HNF4[alpha]7 mRNA and the corresponding protein are expressed in the undifferentiated pluripotent embryonal carcinoma cell line F9, whereas HNF4[alpha]1 only appears after differentiation of F9 cells to visceral endoderm. HNF4[alpha]7 mRNA is also found in totipotent embryonic stem cells. However, the function of HNF4[alpha]7 seems not to be restricted to embryonic cells as the HNF4[alpha]7 mRNA is also present in adult tissues, most notably the stomach. All these features suggest that the presence of distinct splice variants of HNF4[alpha] modulates the activity of HNF4[alpha] in a cell type specific way.
Hepatocyte nuclear factor 4 (HNF4) constitutes a transcription factor family (1 ,2 ) whose first member HNF4[alpha] has been identified as a factor interacting with promoter elements mediating liver specific transcription (3 ). Based on the zinc finger motif of the DNA binding domain and on a potential ligand binding domain HNF4 is classified as a member of the nuclear orphan receptor superfamily (4 ). HNF4[alpha] turned out to be present also in non-hepatic cells such as kidney, intestine, stomach and pancreas (5 -7 ). The importance of HNF4[alpha] in gene control in tissues distinct from the liver has been documented by the fact that a human inherited disease is based on the expression of a mutated HNF4[alpha] gene in the endocrine pancreas leading to maturity-onset diabetes of the young (MODY1; 8 ). In addition to the role of HNF4[alpha] as a tissue specific transcription factor it functions also in early embryogenesis: in the Xenopus egg we identified the HNF4[alpha] protein as a maternal transcription factor that contributes to the zygotic activation of the gene encoding HNF1[alpha] (9 ). In mouse embryos HNF4[alpha] transcripts have been identified in the primary endoderm as early as day 4.5 and later in the liver diverticulum, the gut and nephrogenic tissues starting from day 8.5 (6 ,10 ). The early embryonic requirement of HNF4[alpha] is well established in the mouse, as the knock-out of the HNF4[alpha] gene leads to a disturbance of gastrulation and thus to early embryonic lethality (11 ). The embryonic role of HNF4 seems to be evolutionary conserved, as maternal HNF4 mRNA has been identified in the Drosophila egg. Furthermore, a large chromosomal deletion spanning the HNF4 locus results in defects in the gut and malpighian tubules of the developing Drosophila and these malformations are interpreted that HNF4 plays similar functions in invertebrates and vertebrates (7 ).
In addition to HNF4[alpha] we have recently identified two other members of the HNF4 family, i.e. HNF4[beta] in Xenopus (2 ) and HNF4[gamma] in humans (1 ). Both these new members of the HNF4 family seem to have distinct but overlapping functions compared to HNF4[alpha], since both gene products show a differential expression pattern and a distinct transactivation potential of HNF4 dependent reporter genes.
Further complexity of gene control by HNF4[alpha] transcription factors is apparently given by the differential splicing of the 10 initially identified exons of the HNF4[alpha] gene (6 ,8 ). Thus far, six distinct splice variants of HNF4[alpha] have been identified in human and murine cDNA samples. We use the nomenclature most recently used for the human splice variants (12 ): HNF4[alpha]1 represents the initially identified transcript (3 ) and HNF4[alpha]2 through HNF4[alpha]6 are the splice variants identified subsequently. HNF4[alpha]2 is a splice variant that contains a larger exon 9 resulting in an insertion of 10 additional amino acids in the Pro-rich C-terminal part and has been identified in the rat (13 ), the mouse (14 ) and human (1 ,15 ). HNF4[alpha]3 initially refered to as HNF-4C (16 ) has been identified in human liver cDNA and is characterized by a shorter and distinct C-terminus, whereas HNF4[alpha]4 found in human kidney and liver RNA contains in the N-terminal region the two additional exons 1B and 1C (1 ,12 ). The fifth splice variant HNF4[alpha]5 carries the exon 1B and 1C found in HNF4[alpha]4 together with the larger exon 9 found in HNF4[alpha]2, whereas HNF4[alpha]6 combines exon 1B and exon 1C with the C-terminal truncation found in HNF4[alpha]3 (12 ). We speculate that all these splice variants have unique properties allowing a modulation of the HNF4[alpha] activity. Indeed, we have shown in transient transfection assays using HNF4 dependent reporter genes, that HNF4[alpha]4 has a lower transactivation potential compared to HNF4[alpha]2 (1 ). However, it should be stressed that all these HNF4[alpha] variants have only been identified as mRNAs and the existence of the corresponding proteins has not been proven.
The broad spectrum of tissues expressing HNF4[alpha] argues for a function of HNF4[alpha] in the establishment of the differentiation of various cell types, but most data concerning the role of HNF4[alpha] have been investigated in hepatic cells. Most notably, the presence of HNF4[alpha] expression has been correlated with the differentiated phenotype of hepatocytes (reviewed in 17 ) . In fact, it has been shown that the introduction of HNF4[alpha] in dedifferentiated hepatocytes can lead to a redifferentiation as shown by the activation of several liver specific functions (18 -20 ), whereas the forced expression of a dominant negative mutant down-regulates several liver specific genes in differentiated hepatoma cells (21 ,22 ).
In the present report we analyzed in the murine cell line MHSV-12 the highly differentiated hepatic phenotype that is reversibly decreased in serum-containing culture medium (23 ). We reasoned that this change in hepatic differentiation might correlate with an altered expression of HNF4[alpha]. Using these cells we succeeded to define a novel HNF4[alpha]7 splice variant that is expressed in a tissue specific manner and most notably found as the major component in embryonic stem cells.
The sequence of the splice variant HNF4[alpha]7 was deposited in GenBank (accession number AF015275).
All culture media contained penicillin (100 U/ml) and streptomycin (100 U/ml). The MHSV-12 cell clone was maintained in the chemically defined MX83 medium supplemented with 1 µg/ml transferrin, 10 µg/ml insulin, 1 µg/ml hydrocortisone and 100 ng/ml EGF as described (23 ). For splitting, the cells were trypsinized and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated calf serum for one day before adding the MX83 medium. F9 cells were propagated on gelatine coated cell culture dishes in DMEM with 10% heat-inactivated calf serum. To induce visceral endoderm differentiation the F9 cells were seeded into bacterial dishes in the presence of 2 × 10-7 M retinoic acid (24 ,25 ). Embryonic stem cells (ES cells) (E14-1) were cultured on mitomycin-treated feeder cells in DMEM containing 15% heat inactivated fetal calf serum and supplemented with MEM non-essential amino acids (Sigma), 2 mM glutamine and 100 µM [beta]-mercaptoethanol.
For transient transfection of C2 cells the DNA-calcium phosphate coprecipitation method was used (26 ), whereas for Hela and MHSV-12 cells the lipofectin transfection protocol was applied (1 ).
High salt extracts of crude nuclear pellets of cultured cells were prepared as described (27 ) and the gel retardation assays (28 ) were performed using the HNF4 binding site of the human apolipoprotein B promoter (2 ) or the Xenopus HNF1[alpha] promoter (26 ). The monoclonal antibody recognizing the A/B-domain of HNF4[alpha]1 has been described (29 ). The polyclonal antibodies anti-A/B/C and anti-F were raised against the N-terminus of the rat HNF4[alpha]1 protein (29 ) and a peptide of the F-domain of the Xenopus HNF4[alpha] protein (2 ), respectively. These polyclonal antisera were produced in rabbits by Eurogentec.
For the 5' RACE, RNA was purified with the RNA-CleantmSystem (AGS, Heidelberg) from MHSV-12 cells grown in serum containing DMEM and 5 µg aliquots reverse transcribed with the HN7 primer into cDNA using the Superscripttm II plus kit (GIBCO/BRL). The 5'-end of the cDNA was amplified with the 5'-AmpliFINDERtm RACE kit (CLONTECH) by ligating the oligonucleotide H2N-TGGGCCTCTAGACTTAAG-5' to the 3'-end of the cDNA. This tag was used for priming with the oligonucleotide P11 (5'-ACCCGGAGATCTGAATTC-3') and the PCR product was cloned. For RT-PCR cDNA was synthesized with random primer using 5 µg RNA. To detect HNF4[alpha] transcripts the reverse primer HN7 (5'-CCTTAAGGCTTCCTTCTTCATGCCAGCCCGG-3') and the forward primers HN6 (5'-GGAATTCCGGCATGGATATGGCCGACTACAGCGC-') or HN18 (5'-GGGTACCCTTGGTCATGGTCAGTG-3') were used (see Fig. 3 A). The oligonucleotide HN7 and HN6 contain linker sequences (underlined) at the 5'-end not present in the cDNA. To quantitate GAPDH mRNA, the oligonucleotides 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3', were used as forward and reverse primers, respectively. To quantitate the HNF4[alpha] transcripts as illustrated in Figures 6 and 7 the PCR reactions contained in addition of the 200 µM dNTP a labeled triphosphate, i.e. 1 µCi/50 µl reaction ([alpha]-32P)dCTP. This allows quantification of the PCR fragments with a phosphor imager. In addition this sensitive detection permits a lower cycle number and thus avoids reaching the saturation of the PCR (30 ). All reactions were done at 62 and 72°C for annealing and extension, respectively.
It is well established that the composition of the culture medium has a profound effect on the differentiation state of hepatocytes (23 ). In general the presence of serum leads to substantial dedifferentiation of the hepatic phenotype. To explore whether the cultivation conditions of hepatocytes has any effect on the expression of tissue specific transcription factors known to be responsible for the hepatic phenotype, we determined the presence of liver transcription factors in the immortalized murine hepatic cell line MHSV-12. This cell line has been found to express most abundantly transcripts of liver specific proteins such as albumin, aldolase A and B as well as tyrosine amino transferase, but this liver specific expression pattern is reversibly decreased in serum containing medium (23 ). Analyzing the expression of HNF1[alpha] in the MHSV-12 cell line cultured under various conditions we did not detect any change in this transcription factor (data not shown). However, probing HNF4[alpha] in gel retardation assays using various antibodies we detected some significant differences: as illustrated in Figure 1 A, in extracts of MHSV-12 cells grown in serum-free MX83 medium, that maintains a highly differentiated hepatic phenotype (23 ), HNF4[alpha] could readily be detected in gel retardation experiments (lane 1) and this DNA-protein complex could be supershifted by an antibody raised against the A/B-domain or the F-domain of HNF4[alpha] (lanes 2 and 3, respectively). In contrast, in extracts derived from MHSV-12 cells cultivated in the presence of serum, that leads to a dramatic dedifferentiation (23 ), we observed that the HNF4[alpha] protein detected in gel retardation assays (lane 4) did not react with the antibody specific for the A/B-domain of HNF4[alpha] (lane 5). However, in the same extracts the presence of HNF4[alpha] protein could be verified with the antibody specific for the F-domain (lane 6). These results imply that the properties of HNF4[alpha] change in MHSV-12 cells depending on the culture conditions.
To identify the components in the culture medium that change the properties of the HNF4[alpha] protein, we cultivated the MHSV-12 cells in MX83 medium lacking defined components and analyzed the extracts of these cells in the gel retardation assay using the A/B-domain specific antibody. As Figure 1 B illustrates, the omission of hydrocortisone in the culture medium results in cell extracts with HNF4[alpha] protein that mostly cannot be supershifted by the A/B-domain specific antibody (lanes 3 and 4). In contrast extracts derived from cultures grown in the absence of EGF (lanes 1 and 2) or insulin (lanes 5 and 6) contain HNF4[alpha] that is predominantly supershifted by the A/B-domain specific antibody. Consequently, the presence of hydrocortisone in the serum-free medium leads to the expression of the HNF4[alpha] form that is recognized by the A/B-domain specific antibody.
In a complementary approach we investigated the effect of the glucocorticoid hormone on HNF4[alpha] expression in MHSV-12 cells grown with serum. As shown in the gel retardation assay of Figure 1 C, a 24 h incubation with dexamethasone leads to a substantial amount of HNF4[alpha] protein that is recognized by the A/B-domain specific antibody (lane 5). Since this dexamethasone mediated change can be inhibited by RU486, a well known antagonist binding to the glucocorticoid receptor as a competitor of the glucocorticoid (lane 8), we assume that the effect is mediated by the nuclear hormone receptor.
The findings with the cells grown without serum as well as the data with the cultures maintained in serum, both imply that a glucocorticoid mediated event alters the properties of the HNF4[alpha] protein in such a way that it is recognized by the A/B specific antibody.
The differential recognition of HNF4[alpha] by the A/B-domain specific antibody could either reflect a protein modification or a change in the amino acid sequence in the N-terminus of the HNF4[alpha] protein. To distinguish between these two alternatives we transfected an expression vector encoding the originally described rat HNF4[alpha] protein (3 ) into MHSV-12 cells grown in the presence of serum, i.e. in the absence of hydrocortisone, that abolishes the supershift by the A/B-domain specific antibody. Analyzing increasing amounts of extracts derived from these transfected cell lines by gel retardation assays we observed a concentration dependent appearance of a HNF4[alpha] protein that can be supershifted by the A/B-domain specific antibody (marked by an arrow head in lanes 2 and 5 in Fig. 2 ), whereas in extracts of mock transfected cells no HNF4[alpha] protein could be supershifted by the A/B specific antibody (lanes 8 and 11). Using the F-domain specific antibody no qualitative difference could be seen between the HNF4[alpha] transfected cells and the control, but the supershifted band was stronger in the extract of the transfected cells (compare lanes 3 and 6 with lanes 9 and 12). These results rule out the possibility that protein modification plays a major role but rather imply that the transfected HNF4[alpha] encodes a protein that is distinct in its amino acid sequence from the protein expressed in undifferentiated MHSV-12 cells.
Comparing extracts derived from MHSV-12 cells grown without serum or in the presence of serum, we observed in western blots that HNF4[alpha] is slightly shorter in the presence of serum and thus has the size of the predicted HNF4[alpha]7 splice variant (data not shown). The presence of this splice variant was confirmed by RT-PCR using primers specific for the [alpha]7 splice variant. Figure 4 reveals the expression of the HNF4[alpha]7 splice variant in MHSV-12 cells cultured in the presence of serum (lane 1). Apparently significant amounts of the HNF4[alpha]7 variant are also found in cells grown in serum-free medium (lane 2). However, these RT-PCR reactions were performed close to saturation and, therefore, heavily overestimate the amount of HNF4[alpha]7 transcripts. In contrast the previously described HNF4[alpha]1 splice variant is exclusively detected in MHSV-12 cells grown in serum-free medium (compare lanes 4 and 5). These data confirm that the serum induced change in the differentiation state of the hepatic cells is accompanied by an alteration in the HNF4[alpha] splice variants. In differentiated cells grown under serum-free conditions HNF4[alpha]1, and to a small extent HNF4[alpha]7 transcripts, are present, whereas dedifferentiated cells grown with serum contain almost exclusively the HNF4[alpha]7 splice variant.
To address the question whether the novel splice variant has a differential transactivation potential we compared its transactivation potential in transfection experiments with the splice variant HNF4[alpha]1. Using a luciferase reporter construct containing four HNF4 binding sites in front of the tk promoter (31 ) we observed in the dedifferentiated hepatoma cell line C2 that lacks HNF4[alpha], (31 ) a 2-fold lower transactivation potential of HNF4[alpha]7 compared to HNF4[alpha]1 (Fig. 5 A). In Hela cells an even greater difference in the transactivation potential between the two splice variants was observed (Fig. 5 B). In fact using high concentrations of the HNF4[alpha]7 expression vector no transactivation of the luciferase reporter was seen. Taken together these results establish that MHSV-12 cells grown in serum, that leads to dramatic dedifferentiation of the hepatic phenotype, contain the HNF4[alpha]7 variant that has a significantly lower transactivation potential.
To explore the significance of the novel HNF4[alpha]7 splice variant we investigated by RT-PCR the presence of HNF4[alpha]1 and HNF4[alpha]7 transcripts in different mouse tissues. To allow a reliable quantification we made the PCR reaction with 32P-labeled dCTP that permits low cycling numbers and thus ensures quantitative measurements. As shown in Figure 6 both these transcripts can be identified in the liver and the stomach (lanes 1 and 3), whereas in the kidney and to a smaller extent in the intestine only the HNF4[alpha]1 transcript can be detected. In the pancreas only small amounts of HNF4[alpha]7 transcripts are present. But the significance of this finding is questionable as the GAPDH mRNA cannot be seen probably because the pancreatic RNase was not sufficiently inhibited during RNA preparation. However, traces of HNF4[alpha]7 RNA were also detected in the ovary, heart, bladder and brain, where no HNF4[alpha]1 transcripts and normal levels of GAPDH RNA were observed. Clearly, the HNF4[alpha]1 and HNF4[alpha]7 splice variants are expressed differentially in the various tissues analyzed. By quantification of the PCR products in a phosphor imager we estimate that in the stomach about a third of the HNF4[alpha] transcripts correspond to the HNF4[alpha]7 splice variant (lane 3). Furthermore, the abundance of the HNF4[alpha]7 transcripts differs by a factor of ~1000-fold between high and low expressing tissues.
Our initial experiments leading to the identification of the HNF4[alpha]7 splice variant implied some correlation of the novel splice variant with the undifferentiated phenotype of the hepatocytes. Therefore, we investigated the presence of HNF4[alpha]7 in the murine embryonal carcinoma cell line F9 that resembles the pluripotent stem cells of the inner cell mass of the early mouse embryo. This cell line can can be differentiated into distinct endodermal cell types upon retinoic acid treatment. Most interestingly these stem cells can be differentiated by retinoic acid treatment into visceral endoderm when they are cultured in suspension as aggregates and these cell types share properties of hepatocytes (24 ,32 ) . Based on RT-PCR we succeeded to show that undifferentiated F9 cells contain exclusively the HNF4[alpha]7 splice variant ( Fig. 7 A, lanes 1 and 2), whereas the differentiated cell population contains the HNF4[alpha]1 as well as the HNF4[alpha]7 splice variants (lanes 3 and 4). From three independent experiments we estimated by quantification in a phosphor imager a 15-fold increase in HNF4[alpha]7 mRNA upon differentiation into visceral endoderm, whereas the level of HNF4[alpha]1 transcripts increased from undetectable levels to a value ~60% of the amount of the HNF4[alpha]7 transcript. Since in the immortalized hepatic MHSV-12 cells grown in the presence of serum, the balance between HNF4[alpha]1 and HNF4[alpha]7 is altered by the addition of the glucocorticoid dexamethasone (Fig. 1 ), we wondered whether a similar effect is seen in F9 cells. As Figure 7 A demonstrates the expression pattern of HNF4[alpha]1 and HNF4[alpha]7 is not changed upon dexamethasone treatment in either undifferentiated or differentiated F9 cells.
To determine whether the presence of the HNF4[alpha]7 transcript in the embryonal cell line F9 also reflects the expression of the corresponding protein, we prepared nuclear extracts from F9 cells. In gel retardation assays with extracts from undifferentiated F9 cells (Fig. 7 B) we could detect a supershift with the F-domain specific antibody (lane 3) and the A/B/C-domain specific antibody (lane 4) but no reaction with the A/B-domain specific antibody (lane 2). In contrast in F9 cells differentiated into visceral endoderm a supershift was seen with each of the three antibodies (lanes 6, 7 and 8). This result is consistent with the RT-PCR analysis and implies that undifferentiated F9 cells contain HNF4[alpha]7 protein, whereas HNF4[alpha]1 as well as HNF4[alpha]7 proteins are present in differentiated F9 cells.
To further investigate the early embryonic expression of HNF4[alpha]7 we searched for the corresponding transcript in the totipotent embryonic stem cell line E14-1. The RT-PCR analysis given in Figure 7 C demonstrates the presence of HNF4[alpha]7 mRNA in this embryonal stem cell (lane 2), whereas the RNA sample derived from the feeder cells used for the ES cell culture lack any HNF4[alpha]7 transcripts (lane 3). Probing the same ES cell RNA for the HNF4[alpha]1 mRNA we did not detect any transcript (data not shown).
From the analysis of both the F9 cells and the ES cells we conclude that the presence of HNF4[alpha]7 mRNA is a characteristic of undifferentiated embryonic cells.
The novel splice variant HNF4[alpha]7 is expressed as the exclusive HNF4[alpha] transcript in the undifferentiated embryonal carcinoma cell line F9 and the ES cell line E14-1. The transcript is also found in the hepatocyte cell line MHSV-12 that has been established by immortalization of embryonic hepatocytes. In this immortalized hepatic cell culture HNF4[alpha]7 predominates provided these cells are cultured under conditions that lead to dedifferentiation. In this context it is important to stress that the MHSV-12 cell line dedifferentiated by serum addition has far more hepatic characters than the dedifferentiated hepatoma cell lines C2 and H5 that both lack any HNF4[alpha] and HNF1[alpha] transcription factors (18 ,32 ).
Recently, the expression of HNF4[alpha] has been investigated in the ES cell line D3 (33 ). Under differentiating conditions allowing the formation of embryoid bodies the appearance of HNF4[alpha] RNA has been observed, but no signal was detected in the undifferentiated embryonic stem cells. The lack of HNF4[alpha] transcripts in undifferentiated ES cells probably reflects the insensitivity of the Northern blots compared to our RT-PCR assay as these authors used a full length cDNA probe that would hybridize also to the HNF4[alpha]7 splice variant.
The presence of HNF4[alpha]7 in various embryonic cell types suggests that this novel HNF4[alpha] splice variant has some function during early embryogenesis. Early embryonic expression of the HNF4[alpha] gene starting in the primary endoderm at embryonic day 4.5 of the mouse has been documented by in situ hybridization (10 ). As the probe used in these experiments did not differentiate between the [alpha]1 and [alpha]7 splice variant, the identity of this signal in respect to the splice variants is not clear. However, another in situ hybridization used a genomic fragment of the exon 1 of the original HNF4[alpha] transcript (6 ) and thus, in retrospect, was a HNF4[alpha]1 specific probe. With this probe HNF4[alpha] transcripts were first localised only on day 7 p.c. in visceral extraembryonic endoderm. This detection at a later time point in embryogenesis could well reflect the fact that this probe could not hybridize to the HNF4[alpha]7 splice variant. Similarly, the relatively late appearance of HNF4[alpha] binding activity in gel retardation assays starting at 8.5 days p.c. (25 ) might also be interpreted as a lack of reactivity of the antibody with the HNF4[alpha]7 splice variant. Therefore, by indirect evidence the earlier appearance of HNF4[alpha]7 compared to HNF4[alpha]1, we have seen in the differentiating F9 cells (Fig. 7A and B) matches the available data in the developing embryo.
Analyzing 200 unfertilized mouse eggs we could not identify HNF4[alpha]7 mRNA whereas GAPDH mRNA was readily detectable (data not shown). As unfertilized mouse eggs are about 20 times larger than the ES cells, the input RNA in the RT-PCR corresponds to ~4000 ES cells. Since in such an ES cell aliquot we could identify the HNF4[alpha]7 mRNA, we assume that HNF4[alpha]7 transcripts are absent in the egg and accumulate by zygotic transcription in early mouse embryogenesis. This is consistent with the appearance of HNF4[alpha] transcripts in in situ hybridization experiments with a probe detecting HNF4[alpha]1 and HNF4[alpha]7 only at day 4.5 p.c. (10 ). Therefore, it seems unlikely that HNF4[alpha] mRNA is a maternal component in mammals. This is in contrast to Drosophila HNF4 (7 ) and Xenopus HNF4[beta] mRNA (2 ) that are known to be present as maternal transcripts. However, the HNF4[alpha] protein itself might be a maternal component of the mammalian egg, a situation we have found for the HNF4[alpha] protein in Xenopus whose corresponding mRNA has disappeared at the egg stage (2 ,34 ).
An early function of HNF4[alpha] gene expression in mammals has been established by the disruption of the gene that results in an impaired gastrulation and early embryonic lethality (11 ). As the targeting vector used in these knock-out experiments lacked exon 2 and 3, the HNF4[alpha]1 as well as the HNF4[alpha]7 transcripts are devoid of the DNA binding domain. Therefore, it is not clear at the moment which HNF4[alpha] transcripts plays the essential function at this early embryonic stage. It should also be considered that the partial rescue of HNF4[alpha] knock-out embryos observed by supplementing the developing embryo with normal visceral endoderm (35 ) might be an effect relieving only one of the two HNF4[alpha] splice variants.
We assume that the presence of HNF4[alpha]7 in the undifferentiated embryonal carcinoma cell line F9 and in the embryonic stem cell E14-1 reflects already some first step of embryonic gene regulation. Consistent with this interpretation the presence of transcripts encoding the tissue specific transcription factor HNF1[alpha] has been reported in undifferentiated F9 cells (25 ,36 ). Since it is well established that HNF1[alpha] is a potential target gene of HNF4[alpha] (18 -20 ,26 ,37 ), we consider the possibility that HNF4[alpha]7 triggers HNF1[alpha] expression at this early stage. However, the significance of such a potential effect is questionable as the HNF1[alpha] transcripts are of aberrant size and no HNF1[alpha] protein can be detected in F9 cells (25 ).
Our analysis demonstrates that HNF4[alpha]7 is not restricted to embryonic tissues as it is also found abundantly in the stomach of the adult mouse (Fig. 6 ). In contrast, in the liver and intestine, HNF4[alpha]7 transcripts constitute only a minor fraction as compared to HNF4[alpha]1 and in the ovary, heart, bladder and brain only traces can be found. It will be most interesting to investigate whether in these tissues HNF4[alpha]7 is restricted to a subpopulation of cells, e.g. the stem cell compartment.
Our finding that the transactivation potential of the HNF4[alpha] splice variants differs in transient transfection assays and that the extent of this effect depends on the cell type used (Fig. 5 ), indicates that the two HNF4[alpha] splice variants cooperate differentially with other transcription factors. As the HNF4[alpha]1 and HNF4[alpha]7 splice variants have distinct N-termini, we expect that this domain determines the differential cooperation. Therefore, we exclude the reported interaction with COUP-TFI and COUP-TFII (38 ) as well as the binding to HNF1[alpha] (39 ) as the mechanisms mediating the distinct properties of of HNF4[alpha]1 and HNF4[alpha]7, since these interactions involve the C-terminus common to HNF4[alpha]1 and HNF4[alpha]7. However, the specific binding observed with the basal transcription factor TFIIB (40 ) might trigger the distinct properties of the two splice variants, as the formation of this complex requires the N-terminus of HNF4[alpha] (38 ,40 ). Alternatively, HNF4[alpha]1 and HNF4[alpha]7 may exert distinct activities due to differential heterodimerization with other members of the HNF4 family (1 ,2 ). But we would not expect differential heterodimerization with other members of the nuclear receptor superfamily, since HNF4[alpha] seems usually not to heterodimerize with these components (41 ,42 ).
Analyzing the expression of the HNF4[alpha] splice variants we revealed that HNF4[alpha]7 is under a complex regulatory network. In the hepatic cell line MHSV-12 the glucocorticoid plays a major role as the lack of this hormone seems to be a critical component for the appearance of HNF4[alpha]7. Clearly, the glucocorticoid hormone alters the balance between the HNF4[alpha]1 and HNF4[alpha]7 splice variants. As glucocorticoids are known to trigger the final stage of hepatic differentiation at birth (43 ), we have investigated the expression of these two HNF4[alpha] splice variants. Using liver extracts from 2 days before birth up to 2 days after birth we could not see any change (data not shown). In fact, as all the HNF4 binding activity observed in gel retardation assays could be supershifted by the A/B specific antibody at all stages analyzed, we have no data to support a major change in HNF4[alpha] splice variants at birth, although glucocorticoid action is an essential step at this developmental stage. A lack of glucocorticoid control was also seen in F9 cells. Neither in undifferentiated nor in differentiated F9 cells the addition of dexamethasone altered the level of the HNF4[alpha]1 and HNF4[alpha]7 mRNA (Fig. 7 A). On the other hand in these F9 cells the retinoic acid mediated differentiation to visceral endoderm leads to an increase in HNF4[alpha]7 mRNA but more significantly also to the appearance of the HNF4[alpha]1 splice variant. This change in HNF4[alpha] expression can also be seen on the protein level, as HNF4 binding activity reacting with the anti A/B antibody is only detected in the differentiated cells (Fig. 7 B).
Our data show that the splice variant HNF4[alpha]7 contains a distinct first exon. As the original exon 1 of the murine HNF4[alpha] gene has been shown to contain the initiation site of the promoter (6 ,44 ), we assume that the novel exon 1 found in HNF4[alpha]7 indicates a second promoter of the HNF4[alpha] gene. Therefore, we propose that the differential expression of the two splice variants is mainly due to the differential use of two distinct promoters of the HNF4[alpha] gene
We are grateful to K.Willecke and A.Temme for instructing us to cultivate the MHSV-12 cells, B.Rickert for providing the E14-1 cell cultures, A.Poleev for some of the murine RNA samples, T.Möröy and B.Zevnic for preparing the unfertilized mouse eggs and E.Pogge von Strandmann for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Nucleotide sequence accession number
Cell cultures and transfections
Extract preparation and gel retardation assays
5' RACE and RT-PCR
Results
A novel HNF4[alpha] protein in dedifferentiated hepatocytes
The type of H
The novel HNF4[alpha]7 protein is encoded by a differential splice variant of the HNF4[alpha] gene
The balance of the HNF4[alpha]1 and HNF4[alpha]7 splice variants changes with the MHSV-12 differentiation status
HNF4[alpha]1 and HNF4[alpha]7 have distinct transactivation potential
HNF4[alpha]1 and HNF4[alpha]7 show a distinct tissue specific expression pattern
HNF4[alpha]7 is expressed in embryonic stem cells of the mouse
Discussion
Acknowledgements
References
REFERENCES
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K. Hara, M. Horikoshi, H. Kitazato, C. Ito, M. Noda, J. Ohashi, P. Froguel, K. Tokunaga, K. Tobe, R. Nagai, et al. Hepatocyte Nuclear Factor-4{alpha} P2 Promoter Haplotypes Are Associated With Type 2 Diabetes in the Japanese Population. Diabetes, May 1, 2006; 55(5): 1260 - 1264. [Abstract] [Full Text] [PDF]
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Y. L. Muller, A. M. Infante, R. L. Hanson, L. Love-Gregory, W. Knowler, C. Bogardus, and L. J. Baier Variants in Hepatocyte Nuclear Factor 4{alpha} Are Modestly Associated With Type 2 Diabetes in Pima Indians Diabetes, October 1, 2005; 54(10): 3035 - 3039. [Abstract] [Full Text] [PDF]
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C. M. Damcott, N. Hoppman, S. H. Ott, L. J. Reinhart, J. Wang, T. I. Pollin, J. R. O'Connell, B. D. Mitchell, and A. R. Shuldiner Polymorphisms in Both Promoters of Hepatocyte Nuclear Factor 4-{alpha} Are Associated With Type 2 Diabetes in the Amish Diabetes, December 1, 2004; 53(12): 3337 - 3341. [Abstract] [Full Text] [PDF]
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L. D. Love-Gregory, J. Wasson, J. Ma, C. H. Jin, B. Glaser, B. K. Suarez, and M. A. Permutt A Common Polymorphism in the Upstream Promoter Region of the Hepatocyte Nuclear Factor-4{alpha} Gene on Chromosome 20q Is Associated With Type 2 Diabetes and Appears to Contribute to the Evidence for Linkage in an Ashkenazi Jewish Population Diabetes, April 1, 2004; 53(4): 1134 - 1140. [Abstract] [Full Text] [PDF]
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K. Silander, K. L. Mohlke, L. J. Scott, E. C. Peck, P. Hollstein, A. D. Skol, A. U. Jackson, P. Deloukas, S. Hunt, G. Stavrides, et al. Genetic Variation Near the Hepatocyte Nuclear Factor-4{alpha} Gene Predicts Susceptibility to Type 2 Diabetes Diabetes, April 1, 2004; 53(4): 1141 - 1149. [Abstract] [Full Text] [PDF]
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M. E. Torres-Padilla, F. M. Sladek, and M. C. Weiss Developmentally Regulated N-terminal Variants of the Nuclear Receptor Hepatocyte Nuclear Factor 4alpha Mediate Multiple Interactions through Coactivator and Corepressor-Histone Deacetylase Complexes J. Biol. Chem., November 15, 2002; 277(47): 44677 - 44687. [Abstract] [Full Text] [PDF]
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H. Schrem, J. Klempnauer, and J. Borlak Liver-Enriched Transcription Factors in Liver Function and Development. Part I: The Hepatocyte Nuclear Factor Network and Liver-Specific Gene Expression Pharmacol. Rev., March 1, 2002; 54(1): 129 - 158. [Abstract] [Full Text] [PDF]
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 S. F. Boj, M. Parrizas, M. A. Maestro, and J. Ferrer A transcription factor regulatory circuit in differentiated pancreatic cells PNAS, November 15, 2001; (2001) 241349398. [Abstract] [Full Text] [PDF]
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 H. Thomas, K. Jaschkowitz, M. Bulman, T. M. Frayling, S. M.S. Mitchell, S. Roosen, A. Lingott-Frieg, C. J. Tack, S. Ellard, G. U. Ryffel, et al. A distant upstream promoter of the HNF-4{alpha} gene connects the transcription factors involved in maturity-onset diabetes of the young Hum. Mol. Genet., September 1, 2001; 10(19): 2089 - 2097. [Abstract] [Full Text] [PDF]
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A. Bailly, M. E. Torres-Padilla, A. P. Tinel, and M. C. Weiss An enhancer element 6 kb upstream of the mouse HNF4{alpha}1 promoter is activated by glucocorticoids and liver-enriched transcription factors Nucleic Acids Res., September 1, 2001; 29(17): 3495 - 3505. [Abstract] [Full Text] [PDF]
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G. Peiler, B. Bockmann, H. Nakhei, and G. U. Ryffel Inhibitor of the Tissue-Specific Transcription Factor HNF4, a Potential Regulator in Early Xenopus Development Mol. Cell. Biol., December 1, 2000; 20(23): 8676 - 8683. [Abstract] [Full Text] [PDF]
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J. Lausen, H. Thomas, I. Lemm, M. Bulman, M. Borgschulze, A. Lingott, A. T. Hattersley, and G. U. Ryffel Naturally occurring mutations in the human HNF4{alpha} gene impair the function of the transcription factor to a varying degree Nucleic Acids Res., January 15, 2000; 28(2): 430 - 437. [Abstract] [Full Text] [PDF]
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F. M. Sladek, M. D. Ruse Jr., L. Nepomuceno, S.-M. Huang, and M. R. Stallcup Modulation of Transcriptional Activation and Coactivator Interaction by a Splicing Variation in the F Domain of Nuclear Receptor Hepatocyte Nuclear Factor 4alpha 1 Mol. Cell. Biol., October 1, 1999; 19(10): 6509 - 6522. [Abstract] [Full Text] [PDF]
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H. Dell and M. Hadzopoulou-Cladaras CREB-binding Protein Is a Transcriptional Coactivator for Hepatocyte Nuclear Factor-4 and Enhances Apolipoprotein Gene Expression J. Biol. Chem., March 26, 1999; 274(13): 9013 - 9021. [Abstract] [Full Text] [PDF]
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S. F. Boj, M. Parrizas, M. A. Maestro, and J. Ferrer From the Cover: A transcription factor regulatory circuit in differentiated pancreatic cells PNAS, December 4, 2001; 98(25): 14481 - 14486. [Abstract] [Full Text] [PDF]
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