Identification of target genes of the transcription factor HNF1β and HNF1α in a human embryonic kidney cell line

Biochimica et Biophysica Acta 1731 (2005) 179 – 190 http://www.elsevier.com/locate/bba

Identification of target genes of the transcription factor HNF1h and HNF1a in a human embryonic kidney cell line Sabine Senkel, Bele´n Lucas, Ludger Klein-Hitpass, Gerhart U. Ryffel * Institut fu¨r Zellbiologie (Tumorforschung), Universita¨tsklinikum Essen, D-45122 Essen, Germany Received 2 June 2005; received in revised form 14 September 2005; accepted 10 October 2005 Available online 2 November 2005

Abstract Hepatocyte nuclear factor 1beta (HNF1h, TCF2) is a tissue-specific transcription factor whose mutation in humans leads to renal cysts, genital malformations, pancreas atrophy and maturity onset diabetes of the young (MODY5). Furthermore, HNF1h overexpression has been observed in clear cell cancer of the ovary. To identify potential HNF1h target genes whose activity may be deregulated in human patients, we established a human embryonic kidney cell line (HEK293) expressing HNF1h conditionally. Using Flp recombinase, we introduced wild type or mutated HNF1h at a defined chromosomal position allowing a most reproducible induction of the HNF1h derivatives upon tetracycline addition. By oligonucleotide microarrays we identified 25 HNF1h-regulated genes. By an identical approach, we identified that the related transcription factor HNF1a (TCF1) affects only nine genes in HEK293 cells and thus is a less efficient factor in these kidney cells. The HNF1h target genes dipeptidyl peptidase 4 (DPP4), angiotensin converting enzyme 2 (ACE2) and osteopontin (SPP1) are most likely direct target genes, as they contain functional HNF1 binding sites in their promoter region. Since nine of the potential HNF1h target genes are deregulated in clear cell carcinoma of the ovary, we propose that HNF1h overexpression in the ovarian cancer participates in the altered expression pattern. D 2005 Elsevier B.V. All rights reserved. Keywords: Kidney cell; Clear cell ovarian cancer; Gene expression profile; HNF1a; HNF1h; HNF4a

1. Introduction The tissue-specific transcription factor HNF1h (TCF2) is expressed in epithelial cells of kidney, urogenital tract, liver, lung, gut and pancreas [1]. It constitutes together with its highly related factor HNF1a (TCF1) a unique subgroup of the homeodomain transcription factors that is characterized by an additional 21-amino acid loop between the helix 2 and helix 3 of the homeodomain [2,3]. HNF1h and HNF1a contain highly identical DNA binding domains and recognize the same DNA sequence. This HNF1 binding site has been found in the promoter of many genes and a consensus sequence has been identified [4]. However, this sequence has considerable flexibility and therefore in silico analysis is not sufficient to define a functional HNF1 binding site. HNF1h is more abundant than HNF1a in kidney cells and is the exclusive HNF1 transcription factor in the lung. This distinct tissue-specific distribution of HNF1h compared to * Corresponding author. Tel.: +49 201 723 3110; fax: +49 201 723 5905. E-mail address: [email protected] (G.U. Ryffel). 0167-4781/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2005.10.003

HNF1a could possibly just reflect the need to regulate HNF1 differentially. However, it could also be essential because the two transcription factors are functionally different. Consistent with this assumption, it has been observed that HNF1a is a stronger transactivator on reporter genes than HNF1h [2,3]. On the other hand the function of HNF1a and HNF1h appear equivalent in early murine embryogenesis, as HNF1a can rescue the defective differentiation program in HNF1h deficient embryonic cells [5]. In contrast, we have observed significant differences between the two factors in nephrogenesis, as the introduction of HNF1h interferes with kidney development in the frog Xenopus, whereas HNF1a has no effect [6]. This functional difference can be traced back to three nephrogenic domains of the HNF1h protein [7]. In humans, mutations in the HNF1h gene are associated with a broad spectrum of clinically relevant abnormalities including maturity-onset diabetes of the young (MODY5), renal and genital malformations as well as atrophy of the pancreas [8 –18]. Furthermore, overexpression of HNF1h has been reported in clear cell ovarian cancer, a specific subtype of ovarian cancer [19,20].

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HNF1 target genes have initially been identified by the presence of HNF1 binding sites in the promoter of genes that are specifically active in hepatocytes. In this approach, the regulation by HNF1 is proven in transient transfection assays using a promoter reporter construct and cotransfecting expression vectors encoding HNF1a or HNF1h. This approach does not take into account higher order regulatory control such as chromatin structure and thus lacks an important feature of tissue-specific gene control. Therefore, it is more meaningful to introduce HNF1h into cell lines and monitor the activity of the endogenous genes. Using this approach, we have recently identified 292 HNF1h target genes in the insulinoma cell line INS-1 [21]. Alternatively, the knock-down of HNF1h has been performed by RNAi expression in mouse hepatoma cells leading to a set of 243 decreased genes [22]. HNF1h target genes have also been identified by gene expression profiling in mice with tissue restricted knock-out of the HNF1h gene. Selective inactivation of HNF1h in h-cells of the pancreas revealed specific changes in gene activities in pancreatic h-cells [23]. A more detailed analysis has been made in kidney cells. Using renal-specific inactivation of the HNF1h gene, expression analysis combined with chromatin immunoprecipitation has shown a direct transcriptional hierarchy between HNF1h and the Umod, Pkhd1 and Pkd2 genes that are involved in cystic disease [24]. A similar approach based on overexpression of a dominant negative mutant of HNF1h in the kidney identified the Pkhd1 gene as a HNF1h target [25]. Based on both these experiments, a regulatory network of HNF1h has been described whose dysfunction leads to polycystic kidney disease. Clearly, such approaches in genetically modified mice are most powerful, but the analysis of the whole set of target genes using microarrays is limited, as the analysis of entire organs involves the simultaneous analysis of several different cell types. Therefore, we decided to restrict our analysis to a specific cell type using a cell culture system to define the genes whose activities are altered by HNF1h. As cell line we have chosen the human embryonic kidney cell line HEK293 that represents a cell type along the differentiation of the condensed mesenchyme to the S-shaped body in nephrogenesis [26]. This embryonic cell line expresses Wilm’s tumor gene, Wnt4 and Pax2 typical for the condensed metanephric mesenchyme, but does not express HNF1h and HNF1a and lacks markers of the differentiated kidney (see below). Furthermore, we wanted to compare the activities of HNF1h with HNF1a to assess functional differences between these two closely related factors. We used Flp recombinase mediated site directed integration to avoid integration site dependent activities of the transgenes [21,27]. Furthermore, we used tetracycline induction of the transgene for temporal control of gene activity. 2. Materials and methods 2.1. Establishment of HEK293 cells expressing HNF1 derivatives The HEK293 cell lines carrying the inducible transgenes were generated essentially as described in the Flp-Ini T-RExi Core Kit Manual using the

cell clone Flp-Ini T-RExi -293 from Invitrogen. Co-transfection of the Flp expression vector pCSFLPe [28] together with the pcDNA5/FRT/TO vector containing the gene of interest was done with lipofectamine and cells were selected with hygromycin (75 Ag/ml). The pcDNA5/FRT/TO vector containing the myc tagged human HNF1h wild type sequence or the corresponding MODY5 mutant A263insGG have been described [21]. The pcDNA5/FRT/TO vector containing the myc tagged human HNF1h was generated from the Rc/ CMV plasmid previously described [29].

2.2. Western blots, immunofluorescence and gel retardation assays For Western blots, the anti-myc tag antibody 9E10 was combined with the peroxidase coupled sheep anti-mouse immunoglobulin antibody (Amersham, NXA931) using the ECL system (Amersham). For immunofluorescence Cy3conjugated rat anti-mouse (Dianova, #415-166-166) antibody were used. Gel retardation assays were done as described [6].

2.3. Oligonucleotide microarray analysis Microarray analysis was performed with the Affymetrix GeneChip platform using the HG-U133A microarray chip and RNA prepared as described [21]. Target identification was restricted to probe sets which received at least one Present detection call in the induced versus uninduced sample pair. Probe sets exhibiting a significant increase (change P-value < 0.0045) or decrease (change P-value > 0.9955) were identified by filtering using the Affymetrix Data Mining Tool 3.0. The microarray data have been deposited in Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through the GEO series accession number GSE3308.

2.4. Real-time RT-PCR Real-time RT-PCR was performed using TaqMan\ Gene Expression Assays (Applied Biosystems) as described by the manufacturer employing the GeneAmp 5700 Sequence Detection System. The assays used were: Hs00202061_m1, Hs00175210_m1, Hs00222343_m1 and Hs00167093_m1 for TKTKL1, DPP4, ACE2 and osteopontin (SSP1), respectively. TKTL1 was also assayed with SYBR Green using 5V-AATCCGGACAACGACCGAT- 3V and 5V-GCCATTCCACATGCAACTCC-3V as primers. The amount of GAPDH was used for standardization. cDNA was generated with random primers using the Omniscript RT Kit (Qiagen) using 1 Ag total RNA in a 20-Al reaction volume.

2.5. Luciferase assays on ACE2 and osteopontin promoter constructs To generate promoter luciferase constructs the plasmid pGL3-Basic (Promega) was used. For the osteopontin gene the promoter fragment was amplified from genomic HEK293 DNA using 5V-CCCAGAGCTCTGCTATCC-3V and 5V-CGCCTCGAGGGTCGGCGTTTGGCTGAG-3V as primers. The SacI/XhoI fragment (restriction sites underlined) of the PCR product was cloned into SacI and XhoI digested pGL3-Basic vector. The 5V deletion constructs 783 or 527 were made by BlnI or PinAI digestion. All the other 5V deletion constructs were made by PCR using a primer targeting the desired position and containing a XbaI restriction site for cloning into the NheI site of pGL3-Basic. For the ACE2 gene the promoter fragment was amplified from genomic HEK293 DNA using 5V-GACACTGAGCTCGCTTCTG-3V and 5VGGAAGATCTCGTCCCCTGTGAGCCAAG- 3V as primers. The SacI/BglII fragment (restriction sites underlined) of the PCR product was cloned into SacI and BglII digested pGL3-Basic vector. The 5V deletion constructs 285 or 202 were made by XbaI or AgeI digestion. All the other 5V deletion constructs were made by PCR using a primer targeting the desired position and containing a XbaI restriction site for cloning into the NheI site of pGL3-Basic. All PCR fragments were verified by sequencing. Transfection and luciferase activity measurements were done as described [30]. For each promoter construct, at least two independent plasmid preparations were used and in each transfection the full-length promoter construct was included for comparison. The activity of the pGL3-Basic vector was not changed by cotransfection of the HNF1h expression vector.

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3. Results 3.1. Cell lines expressing HNF1b conditionally To establish cell lines expressing HNF1h conditionally we have chosen the cell clone Flp-In T-REx-293 (Invitrogen) that has been derived from the human embryonic kidney cell line HEK293. It contains a stable integrated gene encoding a constitutively expressed Tet repressor as well as a target site (FRT/lacZ-Zeocin) to integrate a gene of interest by site directed Flp recombination (Fig. 1A). To introduce HNF1h at this site, we cloned HNF1h into the plasmid pcDNA5/FRT/

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TO (Fig. 1A) downstream of the Tet operator (2xTetO2) that allows tetracycline-regulated expression. As a control we also used the MODY5 mutant A263insGG that due to the frameshift mutation encodes a truncated HNF1h protein lacking the transactivation domain and part of the region involved in DNA binding [31]. Both constructs contain a myc tag at the N-terminus to allow proper detection with a myc-specific antibody. By Flp recombinase mediated integration hygromycin resistant cell lines were established and proper recombination of the HNF1h cassette at the FRT integration site was verified by the loss of lacZ-Zeocin expression.

Fig. 1. Conditional expression of HNF1h in the human embryonic kidney cell line HEK293. (A) Flp recombinase mediated site directed integration of the plasmid pcDNA5/FRT/TO containing the myc tagged HNF1h gene is illustrated. The Flp-Ini T-RExi 293 cell line containing the chromosomal FRT site as well as a gene constitutively expressing the tetracycline repressor is available from Invitrogen. Further details are given in the text and at http://www.invitrogen.com/content/ sfs/manuals/flpinsystem_man.pdf. (B) Western blot with a myc tag-specific antibody on extracts from untreated cells ( ) or cells treated for 14 h with 1 Ag/ml tetracycline. The cell lines used contain HNF1h (cell line beta#2a, lanes 1 and 2), the mutant A263insGG (cell line A263#5b, lanes 3 and 4) or the empty vector pcDNA5/FRT/TO (cell line#9b, lanes 5 and 6). (C) Gel retardation assay with an HNF1 binding site using extracts from cell lines containing HNF1h (beta#2a) or the mutant A263insGG (A263#6a). The cells were untreated ( ) or stimulated for 14 h with 1 Ag/ml tetracycline. The addition of a myc-tag-specific antibody (ab) is indicated. (D) The cell line beta#1a and beta#2a containing HNF1h were transfected with either a vector lacking HNF1 binding sites (pGL3-Basic) or with the HNF1-dependent reporter syn04tkluc containing 4 HNF1 binding sites [34]. The cells were treated with tetracycline as indicated and luciferase activity was quantified 16 h after transfection. The bars refer to the determination in duplicate.

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To characterize the induction system, we monitored the tetracycline induction using the myc-tag-specific antibody for immunostaining. Whereas in untreated cells less than 5% of the cells expressed HNF1h or the mutant A263insGG, more than 95% were positive after tetracycline induction for 24 h (data not shown). To evaluate the induction of the HNF1h or the mutant A263insGG protein, the cell lines were treated with tetracycline for 14 h. Fig. 1B illustrates the induction of the full length HNF1h (lane 2) and of the truncated A263insGG protein (lane 4) in a Western blot. To verify the DNA binding properties of the induced HNF1h proteins we performed a gel retardation assay (Fig. 1C). As expected, the induced wild type HNF1h protein generated a complex that was retarded by the addition of an HNF1h-specific antibody (lanes 3 and 4), whereas the protein induced in the cell line containing A263insGG was unable to bind DNA as it lacks part of the DNA binding domain (lanes 7 and 8). The gel retardation experiment also documents that the Flp-In T-REx-293 cells lack endogenous HNF1h protein, as the cell line containing the A263insGG mutant lacks any binding activity (lanes 5 to 8). Comparing the expression level of HNF1h protein, we deduce a more than 10-fold lower level in HEK293 cells compared to the renal cell carcinoma cell lines SK-RC-28 and KT-CL-13 [32]. The absence of HNF1h transcripts in the Flp-In T-REx293 cells was further quantified in microarray experiments by comparing the expression level to the renal adenocarcinoma cell line ACHN [33]. This cell line has a detectable call that is 7-fold above the absent call found in Flp-In T-REx-293 cells. In a transfection experiment using the HNF1-dependent reporter syn04tkluc [34] we could show a dramatic increase in reporter activity upon tetracycline addition (Fig. 1D) that was absent on reporters lacking HNF1 binding sites (pGL3-Basic). Comparing the two independent cell lines expressing wild type HNF1h conditionally (beta#1a and beta#2a), a similar fold induction was observed (Fig. 1D). This is consistent with the identical level of HNF1h induced as measured in Western blots and band shift assays (data not shown). Based on these data, we conclude that we have established for wildtype HNF1h as well as the MODY mutant A263insGG two independent cell lines with most similar induction efficiency. 3.2. Identification of HNF1b target genes by microarray analysis To identify alterations in gene expression mediated by HNF1h, we performed gene expression profiling. We used the two independent cell lines either containing wild-type HNF1h (cell line beta#1a and beta#2a) or encoding the mutant A263insGG (cell line A263ins#5 and A263ins#6). RNA was prepared from untreated cells or cells treated for 24 h with tetracycline and processed for microarray analysis. As the parallel cell lines showed most similar changes, we scored in the cell lines beta#1a and beta#2a all changes that were simultaneously increased (change P-value < 0.0045) or decreased (change P-value > 0.9955) upon tetracycline induction. Using these selection criteria, we obtained 23 induced and 7 repressed probe sets (Table 1). None of these probes regulated

by wildtype HNF1h showed a significant change in the mutant lines A263ins#5 and A263ins#6. More importantly, using the same selection criteria for the microarray data of the cell lines A263ins#5 and A263ins#6 encoding the mutant A263insGG, no probe sets were scored to be regulated. This finding strongly supports the validity of our scoring criteria. Furthermore, for three genes (CD24, NEFL and Sox9) 2 or even 3 independent probe sets revealed the change in expression (Table 1). We assume that these subtle changes can be detected because the Flp-In system generates most reproducible cell lines. As Table 1 documents, fold-induction was up to 35-fold, but could be as low as 1.2-fold. In contrast, the decrease was much lower with 0.5-fold at most. As some probe sets detect the same gene, we conclude that the activity of 25 genes is affected by HNF1h in HEK293 cells with 19 up-regulated and 6 downregulated. The expression of HNF1h failed to induce significant expression of HNF1a in HEK293 cells (absent call under all conditions). Therefore, the cross regulation between these transcription factors observed in murine embryoid bodies [5] does not exist in HEK293 cells. 3.3. Validation of HNF1b-regulated genes To validate the microarray data with an independent assay we performed real-time RT-PCR for the four transcripts most activated by HNF1h expression. Fig. 2 shows that the transcripts for dipeptidyl peptidase 4 (DPP4), angiotensin converting enzyme 2 (ACE2) and osteopontin (SPP1) were increased upon HNF1h induction. However, the fold induction was considerably different in comparison to the microarray, possibly reflecting the fact that in the uninduced state the level was close to background in the microarray analysis. In contrast, the transketolase like 1 (TKTL1) transcripts remained unchanged in real-time RT-PCR assay. As this was also observed with another probe specific for TKTL1, we assume that the transcript induced by HNF1h cannot correspond to TKTL1 mRNA. 3.4. HNF1b affects more genes than HNF1a in HEK293 cells To compare the ability to affect gene expression between the two related transcription factors HNF1h and HNF1a we established cell lines expressing conditionally HNF1a applying the same protocol as used for the HNF1h cell lines. The two HNF1a cell lines (alpha#1 and alpha#4) expressed HNF1a in a tetracycline dependent fashion and the amount induced was identical to the one found in the HNF1h expressing cell lines (data not shown). To identify the genes regulated by HNF1a, the gene expression profile was compared between control cells and cells induced with tetracycline for 24 h using the same protocol as for the identification of the HNF1h-regulated genes. Based on this analysis we identified nine genes regulated by HNF1a (Table 2) and that is less than 40% compared to the 25 genes regulated by HNF1h (Table 1). The Venn diagram given in Fig. 3 compares the genes affected by the HNF1 proteins to the genes regulated by the unrelated transcription factor HNF4a [35]. Clearly, HNF1h as well as

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Table 1 Potential HNF1h target genes in HEK293 cells Gene symbol

Gene title

Probe set

beta#1a

beta#2a

Fold

P-value

Fold

P-value

TKTL1a DPP4 ACE2 SPP1 ACE2 SAH RBPMS PCBD CD24 CD24 NID2 CD24 – CAV2 NEFL MOXD1 TMSB4X NEFL LAMB1 GLA RHOB HEY1 AIM1 DDIT4 PAK1IP1 SOX9 SOX9 GPR49 KCTD12 NRP1

Transketolase-like 1 Dipeptidylpeptidase 4 Angiotensin I converting enzyme 2 Secreted phosphoprotein 1 (osteopontin) Angiotensin I converting enzyme 2 SA hypertension-associated homolog (rat) RNA binding protein with multiple splicing 6-pyruvoyl-tetrahydropterin synthase (DCoH) CD24 antigen CD24 antigen Nidogen 2 CD24 antigen MRNA; cDNA DKZZp564B222 Caveolin 2 Neurofilament, light polypeptide 68 kDa Monooxygenase, DBH-like 1 Thymosin, beta 4, X linked Neurofilament, light polypeptide 68 kDa Laminin, beta 1 Galactosidase, alpha Ras homolog gene family, member B Hairy/enhancer-of-split related with YRPW motif 1 Absent in melanoma 1 DNA-damage-inducible transcript 4 PAK1 interacting protein 1 SRY (sex determining region Y)-box 9 SRY (sex determining region Y)-box 9 G protein-coupled receptor 49 Potassium channel tetramerisation domain containing 12 Neuropilin 1

214183_s_at 211478_s _at 219962_at 209875_s_at 222257_s_at 205942_s_at 207836_s_at 203557_s_at 208651_x_at 216379_x_at 204114_at 266_s_at 213429_at 203324_s_at 221805_at 209708_at 216438_s_at 221916_at 201505_at 214430_at 212099_at 44783_s_at 212543_at 202887_s_at 218886_at 202936_s_at 202935_s_at 213880_at 212188_at 210510_s_at

24.3 10.3 10.1 9.8 5.0 2.6 2.1 1.9 1.8 1.8 1.7 1.7 1.6 1.6 1.6 1.5 1.4 1.4 1.4 1.4 1.4 1.3 1.2 0.8 0.8 0.8 0.7 0.7 0.6 0.5

0.000389 0.000618 0.000147 0.002250 0.000618 0.000030 0.002490 0.000020 0.000167 0.000068 0.000241 0.000014 0.000774 0.001486 0.000189 0.000101 0.000189 0.000052 0.001077 0.000020 0.000114 0.001664 0.002032 0.999811 0.996301 0.999833 0.998349 0.999980 0.999980 0.999308

26.9 1.9 5.8 34.5 4.3 1.7 1.5 1.6 1.5 1.4 1.6 2.0 1.7 1.5 1.5 1.3 1.4 1.4 1.8 1.4 1.3 1.2 1.4 0.8 0.7 0.7 0.8 0.7 0.6 0.6

0.002753 0.002250 0.000078 0.000101 0.000346 0.000046 0.002753 0.000020 0.000130 0.000020 0.000088 0.000253 0.000214 0.000023 0.000020 0.000552 0.000020 0.000130 0.000035 0.000023 0.000241 0.000124 0.001651 0.997750 0.996301 0.996301 0.999853 0.999811 0.999922 0.997510

List of probes giving significant changes comparing RNA samples of untreated and 24 h tetracycline treated cells in the beta#1a and beta#2a cell lines. The official gene symbols and the gene titles are given with more common aliases in brackets. indicates that no official gene symbol is annotated to this probe set. The probe sets refer to the HG-U133A microarray from Affymetrix. Angiotensin converting enzyme 2 (ACE2), NEFL and Sox9 are listed twice and CD24 three-times, as independent probe sets for the same gene gave a significant change. a Based on real-time RT-PCR, the assignment of this probe set to TKTL1 is questionable.

HNF1a have about a 10-fold lower potential to alter gene expression in HEK293 cells and there is hardly any overlap in the expression profiles. In contrast, there was substantial overlap in the genes regulated by the HNF1 proteins. In fact, the four genes up-regulated by HNF1a were also up-regulated by HNF1h, but the five genes down-regulated by HNF1a are unique. 3.5. HNF1b activates the osteopontin and ACE2 promoter It has previously been reported that the human DPP4 promoter is activated by HNF1h in transfection assays using reporter constructs [36], but no data are available on the osteopontin and ACE2 gene promoters. Whereas the ACE2 promoter has not been analyzed so far, the osteopontin promoter from human [37,38] and rodent [39] genes has been characterized extensively. Using an in silico approach we searched for HNF1 binding sites upstream of the transcription start site of the human osteopontin and ACE2 promoters. Using three different programs to search for HNF1 binding sites [40 – 42], we obtained a distinct distribution pattern of HNF1 binding sites dependent on the program used.

In the case of the osteopontin promoter we found with the three programs a total of seven potential HNF1 binding sites within 1.2 kb upstream of the transcription start site (Fig. 4A). To analyze whether these sites confer HNF1h inducibility, we constructed the luciferase reporter OPN ( 1206/+87)-luc containing the region from 1206 bp to +87 bp as illustrated in Fig. 4A. Transfection of this reporter construct into HEK293 cells gave a high activity (Fig. 4C) that could be stimulated about 4-fold by cotransfection with an expression vector encoding HNF1h (Fig. 4D) suggesting the presence of functional HNF1h binding sites in the osteopontin promoter. By generating 5V-deletions within this osteopontin promoter fragment, we observed a gradual loss of induction by HNF1h. The deletion to 763 bp gave a small decrease suggesting the presence of functional HNF1h sites in the distal promoter area. Upon further 5V deletion a clear decrease in activation was seen between 234 bp and 213 bp, a loss of activation was observed at 199 bp and the promoter fragment extending to 139 bp was even inhibited by HNF1h (Fig. 4D). These transient transfection assays show that the HNF1 binding sites upstream of 234 are largely dispensable and the region between 234 and

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Fig. 2. Comparison between the induction assayed by microarrays or by realtime RT-PCR. The open bars give the fold induction as assayed by the oligonucleotide microarrays (data from Table 1). The closed bars give the fold induction determined by real-time RT-PCR using TaqMan\ probes (Applied Biosystems, Assay-On-Demand) for TKTL1, DPP4, ACE2 and osteopontin. For the microarray data, the average of the two independent cell lines is given and for the ACE2 gene the value refer in addition to the average of the two independent probe sets (see Table 1). Microarrays and real-time RT-PCR measurements were made on the two independent cell line beta#1a and beta#2a.

213 lacking an apparent HNF1 binding site is most critical for the extent of transactivation in the transfection assays. We assume that this complex pattern of regulation results from the activity of other factors (Fig. 4B) known to act on the osteopontin promoter [37,38]. Consistent with this assumption, we observed some variation in the basal promoter activity (Fig. 4C) with a prominent activity loss between 139 and 51, the region reported to contain binding sites for at least eight distinct transcription factors. Analyzing in silico the human ACE2 promoter, we found a total of eight potential HNF1 binding sites within 1.1 kb of the transcription start site (Fig. 5A) and using the

Fig. 3. Overlap of the genes regulated by HNF1h, HNF1a and HNF4a. The number of regulated genes is given in a Venn diagram. The total number of genes regulated by a given transcription factor is given in brackets. The size of the circles and the overlapping regions are not to scale, but can be deduced from the gene numbers given. The values for HNF4a are taken from our recent analysis [35] by scoring all changes that were simultaneously increased (change P-value < 0.0045) or decreased (change P-value > 0.9955) upon tetracycline induction in the cell lines wt#1 and wt#4.

luciferase reporter ACE2( 1119/+165)-luc we obtained a 5fold increase of the activity of the promoter-less construct (Fig. 5C) and this level could be increased 6-fold by cotransfection of a HNF1h expression vector (Fig. 5D). To define the minimal promoter requirement for activation by HNF1h, we analyzed a series of 5V-deletions of the ACE2 promoter constructs. The basal activity of most of these promoter fragments resulted in an increased activity indicating the removal of negative regulatory elements (Fig. 5C). Analyzing regulation by HNF1h (Fig. 5D) a deletion up to position 285 reduced the fold activation to a factor of three, implying that the HNF1 binding sites at 818 and 812 (Fig. 5A) contribute to the response to HNF1h. Using various other ACE2 promoter constructs, we found that

Table 2 Potential HNF1a target genes in HEK293 cells Gene symbol a

TKTL1 SPP1 PCBD GLA KRT19 CGA REPRIMO INSIG1 NOV

Gene title

Transketolase-like 1 Secreted phosphoprotein 1 (osteopontin) 6-pyruvoyl-tetrahydropterin synthase (DCoH) Galactosidase, alpha Keratin 19 Glycoprotein hormones, alpha polypeptide Candidate mediator of the p53-dependent G2 arrest Insulin induced gene 1 Nephroblastoma overexpressed gene

Probe set 214183_s_at 209875_s_at 203557_s_at 214430_at 201650_at 204637_at 219370_at 201625_s_at 214321_at

alpha#1

alpha#4

Fold

P-value

Fold

P-value

72.5 3.6 1.8 1.5 0.7 0.7 0.7 0.6 0.5

0.000214 0.001077 0.000020 0.000023 0.996301 0.999308 0.999693 0.999382 0.999886

40.8 4.6 1.8 1.6 0.8 0.8 0.8 0.7 0.4

0.003699 0.000552 0.000167 0.000130 0.998799 0.997750 0.998799 0.998349 0.999970

List of probes giving significant changes comparing RNA samples of untreated and 24 h tetracycline-treated cells in the cell line alpha#1 and alpha#4 expressing HNF1a. The official gene symbols and the gene titles are given with more common aliases in brackets. The probe sets refer to the HG-U133A microarray from Affymetrix. a Based on real-time RT-PCR the assignment of this probe set to TKTL1 is questionable.

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Fig. 4. Transactivation of the osteopontin promoter by HNF1h. (A) The 5V-flanking region of the osteopontin gene (GeneID 6696) from the SacI site at 1206 bp to the first exon is given. Exon 1 is non-coding, as the ATG is present in exon 2 that is about 1 kb downstream. The position and orientation of potential HNF1 binding sites are given using three different programs. Program I: TfBind [41], program II: BindGene [40]; program III MatInspector [42,60]. (B) The nucleotide sequence from 270 bp to 1 bp is given with the HNF1 binding sites at 213 bp and 195 bp. The C/EBP and AML-1 binding site [38], the AP1 [61] as well as c-myc, glucocorticoid receptor (GRE), USF, Sp1 and oct1/oct2 binding sites [37] have been described previously. (C) The various osteopontin promoter fragments in front of the luciferase reporter were transfected into HEK293 cells and the activity is given relative to the pGL3-Basic vector. The numbers below the bars refer to the 5Vend of the osteopontin promoter fragment in the reporter construct. All fragments extended up to position +87 bp. (D) Osteopontin luciferase reporter constructs were transfected into HEK293 cells without or with an expression vector encoding HNF1h. The fold activation by HNF1h is given. The mean and standard deviation are of at least six determinations and significant differences (P < 0.001) are indicated with an asterisk.

252 is the most critical border as any shorter construct could not be activated by HNF1h although potential HNF1 binding sites are retained in this promoter proximal area (Fig. 5B). 4. Discussion Using a most reproducible conditional expression system for the transcription factors HNF1h and HNF1a, we found in HEK293 cells a relatively small number of genes regulated by these transcription factors, i.e., 25 for HNF1h and 9 for HNF1a. This is in contrast to our corresponding analysis of HNF1h target genes in the pancreatic h-cell line INS-1 [21] where with even a more stringent filtering 292 transcripts were regulated. This broad effect on the regulation of endogenous genes in INS-1 cells correlates with the expression of HNF1a in this cell line [21]. However, HNF4a is also not expressed in HEK293 cells [27], but upon ectopic expression regulates about 10-fold more genes in HEK293 cells (Fig. 3). Therefore, we postulate that HEK293 cells are specifically refractory to the activation of endogenous genes by HNF1h and HNF1a.

We assume that additional factors are needed to alter gene expression by HNF1h and HNF1a more substantially. These factors may include the chromatin remodeling ATPase BRG1, that allows efficient reprogramming in HEK293 cells [43]. A comparison of the number of genes affected by the two HNF1 proteins revealed that HNF1h is a more potent transcription factor than HNF1a (Fig. 3). This is an unexpected finding, as in transient reporter assays HNF1h is in general a weaker transactivator than HNF1a. Since in our assay the activation of endogenous genes is analyzed, we conclude that HNF1h is a more efficient activator in the chromosomal context. It is open at the moment, whether this reflects a general property of HNF1h or rather the fact that HNF1h activity is measured in renal cell types where HNF1h is usually more abundant than HNF1a. The genes up-regulated by HNF1a are all also upregulated by HNF1h, but these HNF1a-regulated genes include also two genes (PCBD and GLA) only weakly activated by HNF1h. This implies that the HNF1a is not generally less active in HEK293 cells, but rather has its own favorite target genes. So far, all genes directly regulated by HNF1a and HNF1h were found to be up-regulated. Our

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Fig. 5. Transactivation of the ACE2 promoter by HNF1h. (A) The 5V-flanking region of the ACE2 gene (GeneID 59272) from the SacI site at 1119 bp to the ATG codon is displayed. The position and orientation of potential HNF1 binding sites are given using three different programs (see legend of Fig. 4). (B) The nucleotide sequence from 266 bp to 107 bp is given with the potential HNF1 binding sites present in this region. (C) The various ACE2 promoter fragments in front of the luciferase reporter were transfected into HEK293 cells and the activity is given relative to the pGL3-Basic vector. The numbers below the bars refer to the 5V-end of the ACE2 promoter fragment in the reporter construct. All fragments extended up to position +103 bp. (D) ACE2 luciferase reporter constructs were transfected into HEK293 cells without or with an expression vector encoding HNF1h. The fold activation by HNF1h is given. The mean and standard deviation are of at least six determinations and significant differences (P < 0.001) are indicated with an asterisk.

analysis reveals several genes that are down-regulated and strikingly all these are distinct between HNF1a and HNF1h. We speculate that these genes are either no direct target genes, and thus underlie a more complex control, or that the downregulation is achieved by competition of the HNF1 factors with other transcription factors and that this effect differs between HNF1a and HNF1h because they have quite distinct activation domains [2,44]. Our observation that HNF1h expression induces the DPP4 mRNA qualifies DDP4 as a potential direct target of HNF1h, as a functional HNF1 binding site has been identified in the promoter in transient transfection experiments [36,45,46]. Based on the same criteria, the ACE2 and osteopontin genes are most likely direct HNF1h targets as well, since we could localize functional HNF1 binding sites in the promoter area

(Figs. 4 and 5). The in silico search for HNF1 binding sites in these two promoters gave a whole series of potential HNF1 binding sites, only a subset of which act in transfection experiments. Notably, three established programs to search for HNF1 binding sites gave different results. From our deletion analysis on the ACE2 promoter, the far upstream HNF1 binding sites ( 818 bp and 812 bp) are detected by method I, whereas the site at 248 is found by method III. In contrast, method II did not reveal these functional sites and also failed to display the crucial element at 213 bp or 199 of the osteopontin promoter. Based on this finding, we recommend applying all three methods to search for HNF1 binding sites and then to test the function using corresponding promoter constructs. Comparing the HNF1h-regulated genes with those that have previously been described only DDP4 has been reported

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187

Table 3 Potential HNF1h target genes in clear cell ovarian cancer cells (CCC) Gene symbol

TCF2 SPP1 DPP4 SAH RBPMS CD24 NID2 LAMB1 RHOB SOX9 TMSB4X PCBD CAV2 NEFL GLA AIM1

Gene title

HNF1h Secreted phosphoprotein 1/osteopontin Dipeptidylpeptidase 4 SA hypertension-associated homolog (rat) RNA binding protein with multiple splicing CD24 antigen Nidogen 2 Laminin, beta 1 Ras homolog gene family, member B SRY (sex determining region Y)-box 9 Thymosin, beta 4, X linked 6-pyruvoyl-tetrahydropterin synthase/DCoH) Caveolin 2 Neurofilament, light polypeptide 68 kDa Galactosidase, alpha Absent in melanoma 1

Fold change HEK293a

CCCb

CCC cell linesc

induced 22.2 6.1 2.2 1.8 1.7 1.65 1.6 1.35 0.75 1.4 1.75 1.55 1.5 1.4 1.3

7.4 2.6 2.2 2.1d 2.5 1.7 8.6 7.6 2.2 0.36 0.59 NC NC NC NC NC

8.6 14.3

11.5d

0.26

The HNF1h-regulated genes identified in HEK293 cells (Table 1) that were also analyzed in the ovarian carcinomas [19] are listed. a The fold induction by HNF1h in HEK293 cells is given as average of the two cell lines analyzed in Table 1. In cases where several probe sets detect the same gene the average is given. The introduced HNF1h transcript is not measured in the microarray, as the probe set targets the 3V untranslated region absent in the expression vector. b The fold change in CCC (8 samples) versus the combined values of the mucinous, endometrioid and serous ovary cancer (96 samples) were calculated from the data available at http://www.dot.ped.med.umich.edu:2000/pub/Ovary/index.html. NC indicates no significant change (P > 0.01). Ovary cancers with mixed histology were ignored. c The fold change of clear cell carcinoma (CCC) cell lines versus non-CCC cell lines of the ovary are listed as published [20]. d Average of two probe sets given in [19,20], respectively.

to be regulated by HNF1a and HNF1h. The overall lack of overlap with genes known to be regulated by HNF1h is partly due to the fact that the target genes have been identified in other cell types, including hepatocytes and pancreatic h-cells. Furthermore, HEK293 cells represent an embryonic kidney cell type and are distinct from the various cell types forming the differentiated kidney [26]. As the known target genes of HNF1h whose dysfunction leads to polycystic kidney disease [24,25] are not regulated by HNF1h in HEK293 cells, we assume that HNF1h is not sufficient to activates these genes in a relatively undifferentiated cell type. In this context it is interesting that HNF1h activates the CD24 gene, a cell surface protein that has recently been identified as a marker of the renal progenitor population in the uninduced metanephric mesenchyme [47]. Several genes we have identified to be regulated by HNF1h may be relevant for the clinical phenotype of patients with a mutated HNF1h gene. DPP4 is the dipeptidyl peptidase IV whose enzymatic activity is implicated in the regulation of the biological activity of hormones (e.g., glucagon-like peptide 1, GLP1) and chemokines. By inactivating GLP1, DPP4 seems to play a critical role in glucose homeostasis and has been put forward as a potential target for type II diabetes therapy [48]. The expression in the embryonic kidney cell line HEK293 may not be too surprising, as DPP4 is highly expressed in the brush border membranes of the kidney proximal tubules [49]. ACE2 is the angiotensin converting enzyme 2 that is a membrane-bound carboxy-monopeptidase cleaving angiotensin but also other regulatory peptides. Its action on angiotensin I and II implies a crucial function in vasoconstriction as well as vasodilatation [50,51]. It has been reported to be expressed in

renal proximal tubular cells [52] and an alteration in ACE2 expression has been observed in human kidney disease. However, it is not clear how these changes are related to pathophysiological processes in the kidney [53]. Osteopontin is a secreted protein initially identified in bones but later also found in the kidney especially in the thick ascending limbs of the loop of Henle. It has been implicated in renal stone formation both as an inhibitor or promoter and is found in kidney stones [54]. Osteopontin might be a critical parameter for kidney morphogenesis, as the addition of antiosteopontin antibodies to rat metanephric organ cultures prevents normal tubulogenesis [55]. Based on very similar effects of anti-aVh3-integrin antibodies [55] and biochemical data [56] osteopontin seems to act through integrin cell surface receptors known to play an important role in kidney development [57]. Furthermore, osteopontin is reported to have a protective effect in the kidney by enhancing the adaptation of podocytes to mechanical stress (Endlich et al., 2002). Another gene that seems noteworthy is RBPMS (RNA binding protein multiple splicing), since overexpression of a member of this family, the Xenopus hermes (the possible orthologue of RBPMS2 in human) has been proven to prevent kidney formation in Xenopus embryos [58]. Since we have previously shown that HNF1h overexpression results in agenesis of the Xenopus kidney [6,59], it is possible that RBPMS is a HNF1h target involved in kidney formation. Recently, overexpression of HNF1h has been reported in clear cell carcinoma (CCC) of the ovary [19,20]. As ovarian tissue originates from the urogenital duct and thus is of the same origin as the kidney, we wondered whether the genes we have found to be regulated by HNF1h in the kidney cell line

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HEK293 are deregulated in CCC of the ovary. Transcripts of 15 genes of the 25 genes regulated by HNF1h in HEK293 cells were also included in the study analyzing the gene expression profiles of 114 ovarian tumor samples [19]. Whereas 5 of the 15 informative transcripts were not changed in the tumor material, eight transcripts were significantly up-regulated in ovary CCC compared to the other ovarian cancer types (Table 3). All these eight transcripts were also up-regulated by HNF1h in HEK293 cells implying that their overexpression in ovary CCC may be due to the overexpressed HNF1h transcription factor in ovary CCC (Table 3). Two transcripts were downregulated in ovary CCC and one of them, SOX9, was also down-regulated upon HNF1h overexpression in HEK293 cells. Based on these findings we propose that the genes SPP1, DPP4, SAH, RBPMS, CD24, NID2, LAMB1, RHOB and SOX9 are deregulated in ovary CCC due to the overexpression of HNF1h. This correlation was further strengthened by comparing the HNF1h-regulated genes with genes deregulated in cell lines derived from clear cell ovary cancer characterized by HNF1h overexpression [20]. As shown in Table 3, the SSP1, RBPMS and SOX9 genes were deregulated as expected for HNF1h-regulated genes in these ovary CCC cell lines. At the present time, it is open whether the overexpression of HNF1h in ovary CCC reflects the cell type the cancer has originated from or whether deregulated HNF1h constitutes a step in tumor initiation or progression. Both these alternatives are also valid for the potential HNF1h target genes in CCC of the ovary.

[7]

[8]

[9]

[10]

[11]

[12]

[13]

Acknowledgements [14]

We greatly acknowledge the technical assistance of Nadine Esser and Adriane Parchatka for microarray analysis. We thank Mike Weedon and Tim Frayling for their help to identify HNF1 binding sites and Kathleen R. Cho for giving access to her original data of molecular profiling of ovarian adenocarcinomas. We thank Heike Thomas and Christoph Waldner for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (RY5/4-5). References [1] C. Coffinier, J. Barra, C. Babinet, M. Yaniv, Expression of the vHNF1/HNF1beta homeoprotein gene during mouse organogenesis, Mech. Dev. 89 (1999) 211 – 213. [2] S. Cereghini, Liver-enriched transcription factors and hepatocyte differentiation, FASEB J. 10 (1996) 267 – 282. [3] M. Pontoglio, Hepatocyte nuclear factor 1, a transcription factor at the crossroads of glucose homeostasis, J. Am. Soc. Nephrol. 11 (Suppl. 16) (2000) S140 – S143. [4] F. Tronche, F. Ringeisen, M. Blumenfeld, M. Yaniv, M. Pontoglio, Analysis of the distribution of binding sites for a tissue-specific transcription factor in the vertebrate genome, J. Mol. Biol. 266 (1997) 231 – 245. [5] C. Haumaitre, M. Reber, S. Cereghini, Functions of HNF1 family members in differentiation of the visceral endoderm cell lineage, J. Biol. Chem. 278 (2003) 40933 – 40942. [6] W. Wild, E. Pogge v. Strandmann, A. Nastos, S. Senkel, A. Lingott-Frieg, M. Bulman, C. Bingham, S. Ellard, A.T. Hattersley, G.U. Ryffel, The mutated human gene encoding hepatocyte nuclear factor 1beta inhibits

[15]

[16]

[17]

[18]

[19]

[20]

kidney formation in developing Xenopus embryos, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 4695 – 4700. G. Wu, S. Bohn, G.U. Ryffel, The HNF1beta transcription factor has several domains involved in nephrogenesis and partially rescues Pax8/lim1-induced kidney malformations, Eur. J. Biochem. 271 (2004) 3715 – 3728. Y. Horikawa, N. Iwasaki, M. Hara, H. Furuta, Y. Hinokio, B.N. Cockburn, T. Lindner, K. Yamagata, M. Ogata, O. Tomonaga, H. Kuroki, T. Kasahara, Y. Iwamoto, G.I. Bell, Mutation in hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY, Nat. Genet. 17 (1997) 384 – 385. T.H. Lindner, P.R. Njolstad, Y. Horikawa, L. Bostad, G.I. Bell, O. Sovik, A novel syndrome of diabetes mellitus, renal dysfunction and genital malformation associated with a partial deletion of the pseudo-POU domain of hepatocyte nuclear factor-1beta, Hum. Mol. Genet. 8 (1999) 2001 – 2008. C. Bingham, S. Ellard, L. Allen, M. Bulman, M. Shepherd, T. Frayling, P.J. Berry, P.M. Clark, T. Lindner, G.I. Bell, G.U. Ryffel, A.J. Nicholls, A.T. Hattersley, Abnormal nephron development associated with a frameshift mutation in the transcription factor hepatocyte nuclear factor1beta, Kidney Int. 57 (2000) 898 – 907. C. Bingham, M.P. Bulman, S. Ellard, L.I. Allen, G.W. Lipkin, W.G. Hoff, A.S. Woolf, G. Rizzoni, G. Novelli, A.J. Nicholls, A.T. Hattersley, Mutations in the hepatocyte nuclear factor-1beta gene are associated with familial hypoplastic glomerulocystic kidney disease, Am. J. Hum. Genet. 68 (2001) 219 – 224. A. Montoli, G. Colussi, O. Massa, R. Caccia, G. Rizzoni, G. Civati, F. Barbetti, Renal cysts and diabetes syndrome linked to mutations of the hepatocyte nuclear factor-1 beta gene: description of a new family with associated liver involvement, Am. J. Kidney Dis. 40 (2002) 397 – 402. C. Bingham, S. Ellard, T.R. Cole, K.E. Jones, L.I. Allen, J.A. Goodship, T.H. Goodship, D. Bakalinova-Pugh, G.I. Russell, A.S. Woolf, A.J. Nicholls, A.T. Hattersley, Solitary functioning kidney and diverse genital tract malformations associated with hepatocyte nuclear factor-1beta mutations, Kidney Int. 61 (2002) 1243 – 1251. C. Bingham, S. Ellard, W.G. Van’t Hoff, H.A. Simmonds, A.M. Marinaki, M.K. Badman, P.H. Winocour, A. Stride, C.R. Lockwood, A.J. Nicholls, K.R. Owen, G. Spyer, E.R. Pearson, A.T. Hattersley, Atypical familial juvenile hyperuricemic nephropathy associated with a hepatocyte nuclear factor-1beta gene mutation, Kidney Int. 63 (2003) 1645 – 1651. L.W. Harries, S. Ellard, R.W. Jones, A.T. Hattersley, C. Bingham, Abnormal splicing of hepatocyte nuclear factor-1 beta in the renal cysts and diabetes syndrome, Diabetologia 47 (2004) 937 – 942. J.V. Sagen, L. Bostad, P.R. Njolstad, O. Sovik, Enlarged nephrons and severe nondiabetic nephropathy in hepatocyte nuclear factor-1beta (HNF-1beta) mutation carriers, Kidney Int. 64 (2003) 793 – 800. C. Bellanne-Chantelot, D. Chauveau, J.F. Gautier, D. Dubois-Laforgue, S. Clauin, S. Beaufils, J.M. Wilhelm, C. Boitard, L.H. Noel, G. Velho, J. Timsit, Clinical spectrum associated with hepatocyte nuclear factor-1beta mutations, Ann. Intern. Med. 140 (2004) 510 – 517. E. Barbacci, A. Chalkiadaki, C. Masdeu, C. Haumaitre, L. Lokmane, C. Loirat, S. Cloarec, I. Talianidis, C. Bellanne-Chantelot, S. Cereghini, HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment, Hum. Mol. Genet. 13 (2004) 3139 – 3149. D.R. Schwartz, S.L. Kardia, K.A. Shedden, R. Kuick, G. Michailidis, J.M. Taylor, D.E. Misek, R. Wu, Y. Zhai, D.M. Darrah, H. Reed, L.H. Ellenson, T.J. Giordano, E.R. Fearon, S.M. Hanash, K.R. Cho, Gene expression in ovarian cancer reflects both morphology and biological behavior, distinguishing clear cell from other poor-prognosis ovarian carcinomas, Cancer Res. 62 (2002) 4722 – 4729. A. Tsuchiya, M. Sakamoto, J. Yasuda, M. Chuma, T. Ohta, M. Ohki, T. Yasugi, Y. Taketani, S. Hirohashi, Express ion profiling in ovarian clear cell carcinoma: identification of hepatocyte nuclear factor-1beta as a molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma, Am. J. Pathol. 163 (2003) 2503 – 2512.

S. Senkel et al. / Biochimica et Biophysica Acta 1731 (2005) 179 – 190 [21] H. Thomas, S. Senkel, S. Erdmann, T. Arndt, G. Turan, L. Klein-Hitpass, G.U. Ryffel, Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4alpha and HNF1beta in a pancreatic beta-cell line, Nucleic Acids Res. 32 (2004) e150. [22] T. Tanaka, Y. Tomaru, Y. Nomura, H. Miura, M. Suzuki, Y. Hayashizaki, Comprehensive search for HNF-1beta-regulated genes in mouse hepatoma cells perturbed by transcription regulatory factor-targeted RNAi, Nucleic Acids Res. 32 (2004) 2740 – 2750. [23] L. Wang, C. Coffinier, M.K. Thomas, L. Gresh, G. Eddu, T. Manor, L.L. Levitsky, M. Yaniv, D.B. Rhoads, Selective deletion of the Hnf1beta (MODY5) gene in beta-cells leads to altered gene expression and defective insulin release, Endocrinology 145 (2004) 3941 – 3949. [24] L. Gresh, E. Fischer, A. Reimann, M. Tanguy, S. Garbay, X. Shao, T. Hiesberger, L. Fiette, P. Igarashi, M. Yaniv, M. Pontoglio, A transcriptional network in polycystic kidney disease, EMBO J. 23 (2004) 1657 – 1668. [25] T. Hiesberger, Y. Bai, X. Shao, B.T. McNally, A.M. Sinclair, X. Tian, S. Somlo, P. Igarashi, Mutation of hepatocyte nuclear factor-1beta inhibits Pkhd1 gene expression and produces renal cysts in mice, J. Clin. Invest. 113 (2004) 814 – 825. [26] E. Torban, P.R. Goodyer, Effects of PAX2 expression in a human fetal kidney (HEK293) cell line, Biochim. Biophys. Acta 1401 (1998) 53 – 62. [27] B. Lucas, K. Grigo, S. Erdmann, J. Lausen, L. Klein-Hitpass, G.U. Ryffel, HNF4alpha reduces proliferation of kidney cells and affects genes deregulated in renal cell carcinoma, Oncogene (2005). [28] D. Werdien, G. Peiler, G.U. Ryffel, FLP and Cre recombinase function in Xenopus embryos, Nucleic Acids Res. 29 (2001) E53. [29] H. Thomas, B. Badenberg, M. Bulman, I. Lemm, J. Lausen, L. Kind, S. Roosen, S. Ellard, A.T. Hattersley, G.U. Ryffel, Evidence for haploinsufficiency of the human HNF1alpha gene revealed by functional characterization of MODY3-associated mutations, Biol. Chem. 383 (2002) 1691 – 1700. [30] T. Drewes, S. Senkel, B. Holewa, G.U. Ryffel, Human hepatocyte nuclear factor 4 isoforms are encoded by distinct and differentially expressed genes, Mol. Cell. Biol. 16 (1996) 925 – 931. [31] H. Nishigori, S. Yamada, T. Kohama, H. Tomura, K. Sho, Y. Horikawa, G.I. Bell, T. Takeuchi, J. Takeda, Frameshift mutation, A263fsinsGG, in the hepatocyte nuclear factor-1beta gene associated with diabetes and renal dysfunction, Diabetes 47 (1998) 1354 – 1355. [32] I. Lemm, A. Lingott, E. Strandmann, C. Zoidl, M.P. Bulman, A.T. Hattersley, W.A. Schulz, T. Ebert, G.U. Ryffel, Loss of HNF1alpha function in human renal cell carcinoma: frequent mutations in the VHL gene but not the HNF1alpha gene, Mol. Carcinog. 24 (1999) 305 – 314. [33] T. Ozeki, Y. Takahashi, K. Nakayama, T. Kamataki, Hepatocyte nuclear factor (HNF)-4alpha/gamma, HNF-1alpha, and vHNF-1 regulate the cell-specific expression of the human dihydrodiol dehydrogenase (DD)4/AKR1C4 gene, Arch. Biochem. Biophys. 405 (2002) 185. [34] T. Drewes, A. Clairmont, L. Klein-Hitpass, G.U. Ryffel, Estrogeninducible derivatives of hepatocyte nuclear factor-4, hepatocyte nuclear factor-3 and liver factor B1 are differently affected by pure and partial antiestrogens, Eur. J. Biochem. 225 (1994) 441 – 448. [35] B. Lucas, K. Grigo, S. Erdmann, J. Lausen, L. Klein-Hitpass, G.U. Ryffel, HNF4alpha reduces proliferation of kidney cells and affects genes deregulated in renal cell carcinoma, Oncogene 24 (2005) 6418 – 6431. [36] R.H. Erickson, R.S. Lai, Y.S. Kim, Role of hepatocyte nuclear factor 1alpha and 1beta in the transcriptional regulation of human dipeptidyl peptidase IV during differentiation of Caco-2 cells, Biochem. Biophys. Res. Commun. 270 (2000) 235 – 239. [37] D. Wang, S. Yamamoto, N. Hijiya, E.N. Benveniste, C.L. Gladson, Transcriptional regulation of the human osteopontin promoter: functional analysis and DNA – protein interactions, Oncogene 19 (2000) 5801 – 5809. [38] Y.N. Liu, B.B. Kang, J.H. Chen, Transcriptional regulation of human osteopontin promoter by C/EBPalpha and AML-1 in metastatic cancer cells, Oncogene 23 (2004) 278 – 288. [39] Y. Guo, R. Costa, H. Ramsey, T. Starnes, G. Vance, K. Robertson, M.

[40]

[41] [42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53] [54]

[55]

[56]

[57]

[58]

[59]

189

Kelley, R. Reinbold, H. Scholer, R. Hromas, The embryonic stem cell transcription factors Oct-4 and FoxD3 interact to regulate endodermalspecific promoter expression, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 3663 – 3667. C.R. Lockwood, C. Bingham, T.M. Frayling, In silico searching of human and mouse genome data identifies known and unknown HNF1 binding sites upstream of beta-cell genes, Mol. Genet. Metab. 78 (2003) 145 – 151. T. Tsunoda, T. Takagi, Estimating transcription factor bindability on DNA, Bioinformatics 15 (1999) 622 – 630. K. Quandt, K. Frech, H. Karas, E. Wingender, T. Werner, MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data, Nucleic Acids Res. 23 (1995) 4878 – 4884. C. Hansis, G. Barreto, N. Maltry, C. Niehrs, Nuclear reprogramming of human somatic cells by Xenopus egg extract requires BRG1, Curr. Biol. 14 (2004) 1475 – 1480. G.U. Ryffel, Mutations in the human genes encoding the transcription factors of the hepatocyte nuclear factor (HNF)1 and HNF4 families: functional and pathological consequences, J. Mol. Endocrinol. 27 (2001) 11 – 29. R.H. Erickson, J.R. Gum, C.D. Lotterman, J.W. Hicks, R.S. Lai, Y.S. Kim, Regulation of the gene for human dipeptidyl peptidase IV by hepatocyte nuclear factor 1 alpha, Biochem. J. 338 (Pt. 1) (1999) 91 – 97. R.H. Erickson, R.S. Lai, C.D. Lotterman, Y.S. Kim, Identification of upstream stimulatory factor as an activator of the human dipeptidyl peptidase IV gene in Caco-2 cells, Gene 258 (2000) 77 – 84. G.A. Challen, G. Martinez, M.J. Davis, D.F. Taylor, M. Crowe, R.D. Teasdale, S.M. Grimmond, M.H. Little, Identifying the molecular phenotype of renal progenitor cells, J. Am. Soc. Nephrol. 15 (2004) 2344 – 2357. D. Marguet, L. Baggio, T. Kobayashi, A.M. Bernard, M. Pierres, P.F. Nielsen, U. Ribel, T. Watanabe, D.J. Drucker, N. Wagtmann, Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 6874 – 6879. A.C. Girardi, B.C. Degray, T. Nagy, D. Biemesderfer, P.S. Aronson, Association of Na(+) – H(+) exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule, J. Biol. Chem. 276 (2001) 46671 – 46677. R.M. Carey, H.M. Siragy, The intrarenal renin – angiotensin system and diabetic nephropathy, Trends Endocrinol. Metab. 14 (2003) 274 – 281. L.M. Burrell, C.I. Johnston, C. Tikellis, M.E. Cooper, ACE2, a new regulator of the renin – angiotensin system, Trends Endocrinol. Metab. 15 (2004) 166 – 169. D. Harmer, M. Gilbert, R. Borman, K.L. Clark, Quantitative mRNA expression profiling of ACE 2, a novel homologue of angiotensin converting enzyme, FEBS Lett. 532 (2002) 107 – 110. A. Lely, I. Hamming, H. van Goor, G. Navis, Renal ACE2 expression in human kidney disease, J. Pathol. 204 (2004) 587 – 593. Y. Xie, M. Sakatsume, S. Nishi, I. Narita, M. Arakawa, F. Gejyo, Expression, roles, receptors, and regulation of osteopontin in the kidney, Kidney Int. 60 (2001) 1645 – 1657. S.A. Rogers, B.J. Padanilam, K.A. Hruska, C.M. Giachelli, M.R. Hammerman, Metanephric osteopontin regulates nephrogenesis in vitro, Am. J. Physiol. 272 (1997) F469 – F476. S. Denda, L.F. Reichardt, U. Muller, Identification of osteopontin as a novel ligand for the integrin alpha8 beta1 and potential roles for this integrin – ligand interaction in kidney morphogenesis, Mol. Biol. Cell 9 (1998) 1425 – 1435. J.A. Kreidberg, J.M. Symons, Integrins in kidney development, function, and disease, Am. J. Physiol.: Renal. Physiol. 279 (2000) F233 – F242. W.V. Gerber, S.A. Vokes, N.R. Zearfoss, P.A. Krieg, A role for the RNAbinding protein, hermes, in the regulation of heart development, Dev. Biol. 247 (2002) 116 – 126. S. Bohn, H. Thomas, G. Turan, S. Ellard, C. Bingham, A.T.

190

S. Senkel et al. / Biochimica et Biophysica Acta 1731 (2005) 179 – 190

Hattersley, G.U. Ryffel, Distinct molecular and morphogenetic properties of mutations in the human HNF1beta gene that lead to defective kidney development, J. Am. Soc. Nephrol. 14 (2003) 2033 – 2041. [60] K. Cartharius, K. Frech, K. Grote, B. Klocke, M. Haltmeier, A. Klingenhoff, M. Frisch, M. Bayerlein, T. Werner, MatInspector and

beyond: promoter analysis based on transcription factor binding sites, Bioinformatics (2005). [61] Z. Mi, H. Guo, P.Y. Wai, C. Gao, J. Wei, P.C. Kuo, Differential osteopontin expression in phenotypically distinct subclones of murine breast cancer cells mediates metastatic behavior, J. Biol. Chem. 279 (2004) 46659 – 46667.