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The enzyme was first shown to be able to convert estrone to estradiol in vitro. Later involvement of this enzyme in postsqualene cholesterol biosynthesis was postulated conversion of zymosterone to zymosterol and could be proven in vitro. In this work, we performed a detailed analysis of the transcriptional regulation of both the human and murine genes. Despite relatively low sequence similarity, both promoters contain similar contexts of transcription factor-binding sites.
The participation of these sites in transcriptional regulation of HSD17B7 was proven by electro-mobility shift assay and site-directed mutagenesis of the corresponding binding sites. The results of our study provide unequivocal evidence for a role of HSD17B7 in cholesterol biosynthesis. A common feature of this group is the ability to interconvert keto- and hydroxy-groups on position C17 of the steroid backbone, thereby controlling the biological action of hormones.
Besides its ability to bind the short form of the prolactin receptor, the biological function was unclear. The enzyme was shown to convert estrone to biologically active estradiol with the highest expression levels detected in ovaries of pregnant mice. Consequently, an important function during pregnancy was postulated. With the identification of the human homolog shortly after, in silico analysis showed high levels of expression in brain and fetal liver Krazeisen et al.
By phylogenetic analysis, a significant similarity with yeast 3-keto sterol reductase ERG27 was detected Breitling et al. This protein catalyzes an important step in yeast ergosterol biosynthesis conversion of zymosterone to zymosterol , the yeast equivalent of vertebrate cholesterol biosynthesis Gachotte et al.
Furthermore, co-expression of HSD17b7 with Hmgcr, the rate-limiting enzyme of cholesterol biosynthesis, and other cholesterogenic enzymes in the developing mouse embryo has been shown Laubner et al.
Highest expression levels were found in neuronal tissues and some apoptotic regions. Therefore, several lines of evidence point to a dual functionality of HSD17B7 both human and murine , being involved in both steroidogenesis and cholesterol biosynthesis.
In order to gain further insight into its in vivo functionality and due to possible differences between the human and murine enzymes, we performed a detailed analysis of the transcriptional regulation of the human and murine homologs of HSD17B7.
The complex and controversial data known so far prompted us to adopt this comparative and open approach. Transcription of genes involved in steroidogenesis is in most described cases mediated by the transcription factors TFs of the steroidogenic factor 1 SF1 family SF1 and liver receptor homolog 1, LRH-1; for review, see Val et al. To date, little is known about the transcriptional regulation of both the human and murine HSD17B7 genes.
The first evidence for cholesterol-dependent regulation of the murine gene was reported by Horton et al. The authors showed that among many other genes, HSD17B7 displayed significantly higher expression levels in the transgenic mice compared with wild-type ones. In a recent study addressing transcriptional regulation of rat HSD17b7 in luteal cell line and in dependence on luteinizing hormone LH , two binding sites for SP1 and one for NF-Y were identified Risk et al.
In our preliminary studies, we revealed the existence of several adjacent transcription start sites and the absence of a conserved TATA box for both the human and murine genes. In the present study, we proved the involvement of bioinformatically identified TFs and examined their influence on transcriptional regulation of human and murine HSD17B7 under basal and inducedconditions.
We now present data which expand the knowledge on both the number and significance of other TFs and provide novel and unequivocal evidence for in vivo activity of HSD17B7 and its role in cholesterol biosynthesis.
For bioinformatic analyses, the Genomatix software package Genomatix Software, München, Germany was used. Identification of TF-binding sites was performed by MatInspector for the identification of a conserved pattern of TF-binding sites across species the FrameWorker genomatix software software was used Cartharius et al.
Both analyses were conducted with the recommended default settings at high threshold values. All constructs were confirmed by sequencing. Sequences of nucleotides for amplification of promoter constructs and expression analysis. All mutations were confirmed by sequencing. For analyses of transcriptional activity the Dual Luciferase Assay Promega was used. The cells were seeded in well plates and grown in medium described above.
In order to obtain a higher concentration of nuclear proteins, only half the amount of recommended Nuclear Extraction Reagent was used. After 2-min incubation at room temperature, approximately 25 fmol biotin-labeled double-stranded probe containing the corresponding binding site was added.
The reaction mixtures were incubated for 30 min at room temperature. For competition experiments, a fold molar excess of unlabeled wild-type or mutated probe was added to the reaction mixtures before adding the labeled probe. Reaction mixtures were analyzed by native PAGE. Subsequent signal detection was carried out according to the instruction manual. Further details are given by Ebert et al.
Expression was tested by means of a standard PCR with Taq polymerase. Template used for PCR was applied either plain as received from first-strand cDNA synthesis or diluted five- or tenfold. Our sequence analysis revealed MatInspector analysis of this sequence provided no evidence for additional or missing putative binding sites and the existing differences affect none of the TF-binding sites as identified in our study.
In this work, only the promoter of HSD17B7 residing on chromosome 1 was analyzed. Due to these observations and the previously described involvement of HSD17B7 in postsqualene cholesterol biosynthesis Marijanovic et al. As shown in Fig. The inducing effect of delipidized serum is remarkably stronger for the human promoter human: Results of dual luciferase assays performed with wild-type HSD17B7 promoter fragments. A Luciferase activity of human promoter constructs in HepG2 cells.
B Luciferase activity of murine promoter constructs in Hepa 1—6 cells. Cells were grown under the conditions indicated for 24 h. The same effects were observed with undiluted and diluted templates five- or tenfold, not shown.
In HeLa cells, the effect of induction by cholesterol depletion is negligible. Activity of HSD17B7 promoter in different human cell lines. For analysis of differential expression, the construct showing strongest luciferase activity in promoter fine-mapping experiment was transfected also in HEK and HeLa cells.
Strongest induction of promoter activity can be seen in HepG2 cells, which is indicative for liver-specific induction by cholesterol depletion.
In order to identify a conserved pattern of transcriptional regulation in different species, we used the FrameWorker software. Despite relatively low overall sequence similarity not shown , the promoters of all the three species examined displayed significant similarity of TF-binding motifs Fig. Putative TF-binding sites are underlined. Arrows indicate the position of transcription start points. In depth MatInspector analysis revealed more common and also distinct putative TF-binding sites.
The software identified a putative HNF4-binding site for both promoters. In contrast to the murine promoter, the human contains two putative SP1-binding sites Fig. The human promoter is lacking both binding sites within 1. In order to determine the biological relevance of the bioinformatically identified putative TF-binding sites, we tested the impact of mutations within these sites. In most cases, the effect of mutation of each TF-binding site is at least comparable for the human and murine promoters, concerning basal or induced promoter activity.
Constructs containing double mutations of these sites show an almost complete loss of induction. Interestingly, mutations of SREBP-binding sites also lead to a decreased basal promoter activity, whereas these constructs still exhibit remarkable increases in luciferase activity under inducing conditions.
Mutations of CREB sites had no significant effect, either under basal or induced conditions. Constructs containing the indicated mutations were transfected in HepG2 cells and grown under basal or induced conditions respectively. Cells were lysed and luciferase activity was measured and compared with activity of the wild-type construct 30 h after transfection. Constructs containing the indicated mutations were transfected in Hepa 1—6 cells and grown under basal or induced conditions respectively.
For mutations of HNF4-binding sites, the results are species specific. While we were not able to show any effect by mutation of the corresponding binding site in the murine promoter, a weak but significant change in response to cholesterol depletion can be seen for the human fragment compared with the wild type.
The factor binding to the proximal NF-Y-binding site obviously acts as a silencer, as mutation of this site leads to a strong increase in promoter activity under basal conditions. Thereby, those factors were shown to be responsible for the effects shown in mutagenesis analysis. All binding sites showing different luciferase activity due to mutagenesis were included in these experiments.
Nuclear extracts of HepG2 human and Hepa 1—6 murine were incubated with biotin-labeled probes containing putative TF sites as indicated in Table 2. As indicated for each figure, unlabeled wild type or mutated competitors were used in fold molar excess.
To confirm the identity of proteins, we added antibodies specific to the corresponding TFs. As can be seen in Fig. The addition of specific antibodies leads to the supershifted bands in both species. Due to the usage of recombinant SP1 protein, no supershift experiment was required. In contrast to the murine promoter, we could prove binding of HNF4 to the corresponding binding site in the human promoter. The addition of HNF4-specific antibody leads to a supershifted band Fig.
Finally, binding of NF-Y to the predicted distal-binding sites in both promoters is shown in Fig. In both the species, the addition of NF-Y-specific antibodies leads to a supershifted band. In our study, we identified the TFs responsible for basal-and cholesterol-dependent regulation of both the human and murine HSD17B7. Although we did not examine the corresponding promoter region of the rat experimentally, the results of the bioinformatic analysis point to a very similar regulation.
Furthermore, the similarity in composition and sequence of the identified TF-binding sites within the promoters, and therefore, a high probability of similar in vivo regulation, leads us to the conclusion that HSD17B7 is involved in the same metabolic pathway across the examined species, namely cholesterol biosynthesis.
On the other hand, our results do not disprove in vivo involvement of HSD17B7 in steroidogenesis. In a recent study addressing transcriptional regulation of rat HSD17B7 using a rat luteal cell line dependent on LH and forskolin, which led to a significant decrease in promoter activity, the authors were able to identify and verify two binding sites for SP1 and one for NF-Y Risk et al.
In contrast to our findings, a conserved CREB-binding site within the rat promoter was not detected. This might be due to the use of different algorithms for the identification of TF-binding sites.
We showed that several TFs are required for both basal and induced transcription levels. This situation is a common feature for genes involved in cholesterol biosynthesis and homeostasis, as well as in fatty acid metabolism Kim et al.
Although based on limited published data, genes of postsqualene cholesterol biosynthesis and fatty acid synthesis seem to be regulated by SREBPs and both SP1 and NF-Y in a cholesterol-dependent manner Xiong et al.
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