Figures
Abstract
Background
Mutations in ATP8B1 gene were identified as a cause of low γ-glutamyltranspeptidase cholestasis with variable phenotype, ranging from Progressive Familial Intrahepatic Cholestasis to Benign Recurrent Intrahepatic Cholestasis. However, only the coding region of ATP8B1 has been described. The aim of this research was to explore the regulatory regions, promoter and 5′untranslated region, of the ATP8B1 gene.
Methodology/Principal Findings
5′Rapid Amplification of cDNA Ends using human liver and intestinal tissue was performed to identify the presence of 5′ untranslated exons. Expression levels of ATP8B1 transcripts were determined by quantitative reverse-transcription PCR and compared with the non-variable part of ATP8B1. Three putative promoters were examined in vitro using a reporter gene assay and the main promoter was stimulated with chenodeoxycholic acid. Four novel untranslated exons located up to 71 kb upstream of the previously published exon 1 and twelve different splicing variants were found both in the liver and the intestine. Multiple transcription start sites were identified within exon −3 and the proximal promoter upstream of this transcription start site cluster was proven to be an essential regulatory element responsible for 70% of total ATP8B1 transcriptional activity. In vitro analysis demonstrated that the main promoter drives constitutive ATP8B1 gene expression independent of bile acids.
Citation: Cebecauerová D, Strautnieks SS, Byrne JA, Jirsa M, Thompson RJ (2012) ATP8B1 Gene Expression Is Driven by a Housekeeping-Like Promoter Independent of Bile Acids and Farnesoid X Receptor. PLoS ONE 7(12): e51650. https://doi.org/10.1371/journal.pone.0051650
Editor: Andrea Dardis, University Hospital S. Maria della Misericordia, Italy
Received: September 22, 2012; Accepted: November 2, 2012; Published: December 10, 2012
Copyright: © 2012 Cebecauerová et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Supported by EASL Sheila Sherlock Post-doc Fellowship (DC) and Institutional grant IKEM MZO 00023001 (MJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Mutations in ATP8B1 (18q21-q22) cause variable cholestatic phenotypes ranging from progressive to benign recurrent forms (Progressive Familial Intrahepatic Cholestasis type 1, PFIC1, formerly Byler disease, and Benign Recurrent Intrahepatic Cholestasis type 1, BRIC1; OMIM 211600, 243300) [1], [2]. ATP8B1 deficient patients suffer from intrahepatic cholestasis, often accompanied with extrahepatic symptoms including diarrhoea, pancreatitis and hearing problems. Milder phenotype presents with recurrent attacks of cholestasis typically without permanent liver damage [3]. Serum γ-glutamyltranspeptidase (γ-GT) activity and cholesterol concentrations are normal.
The ATP8B1/FIC1 (Familial Intrahepatic Cholestasis 1) protein, a member of the P4-type ATPases subfamily, is widely expressed in epithelial tissues [4], [5], [6] and is considered a phosphatidylserine flippase, translocating phosphatidylserine from the outer to the inner leaflet of the plasma membrane [6], [7]. The ATP8B1 disease mechanism is, however, poorly understood. In vivo experiments using “Byler“ Atp8b1G308V/G308V mice or ATP8B1 deficient hepatocytes demonstrated defective membrane order due to the impaired flippase activity of ATP8B1 [8], [9]. A more recent study [10] challenged the proposed mechanism and on ATP8B1-depleted Caco-2 cells demonstrated an unimpaired flippase activity, with profound disorganization of apical actin cytoskeleton and loss in microvilli. Since ATP8B1 deficiency is primarily characterised by cholestasis, some studies attempted to attribute the phenotype to a defective farnesoid X receptor (FXR) signalling pathway [11], [12], [13]. Others [14] suggested that impaired FXR activity is secondary to cholestasis and, as such, is not responsible for the PFIC1/BRIC1 phenotype. Unperturbed activity of FXR and its target genes was observed in ATP8B1-depleted Caco-2 cells using small hairpin RNA and small interfering RNA respectively [9], [10], which suggests an unimpaired FXR signalling pathway in PFIC1/BRIC1 patients.
In a large study by Klomp et al. [15] ATP8B1 mutations were detected in fewer than 50% of the families screened, irrespective of severity of their disease. It may be partly because only the coding region of the gene was analyzed. Even though the ATP8B1 gene is important biologically and clinically, our knowledge of its regulatory regions remains limited. Our aim was to characterise the transcriptional control of the ATP8B1 gene by identifying its promoter and 5′untranslated (5′UTR) regions, and to search for putative regulatory sites in any newly discovered parts of the gene.
Results
The 5′UTR of ATP8B1 Comprises Four Novel Exons and Spans a 71 kb Genomic Region
5′RACE using RNA from a number of different human tissues including liver, small intestine, large intestine and pancreas, revealed four novel untranslated exons located 30, 70, 71 and 72 kb upstream of the known exon +1 (Ex +1). These new exons have been designated exons −1 to −4 (Ex −1 to Ex −4) and their lengths and positions are summarized in Fig. 1A. Six different splicing variants comprising different combinations of the novel untranslated exons were detected (Fig. 1B). Due to the existence of two donor splice sites (tandem acceptors) at the 5′end of Ex +1, two different ways of splicing the untranslated exons to Ex +1 are possible (Fig. 1C). Indeed, two different variants for each splicing event including Ex +1, differing from each other by only 3 bases (CAG), were observed. This subtle change represents a further source of 5′UTR variability which generates, in total, twelve ATP8B1 mRNA isoforms (Fig. 1D).
(A) Length and position of novel untranslated exons of ATP8B1 gene. (B) Six identified alternatively spliced variants are indicated by diagonal lines. (C) The existence of two acceptor splice sites CAGCAG (tandem acceptors) at the 5′ boundary of the first translated exon (Ex +1) of ATP8B1 allows the generation of two different splice forms for each combination of upstream exons with Ex +1. Thus generated splice forms differ from each other by only three nucleotides CAG and give rise to twelve ATP8B1 isoforms in total (D).
Using commercially available First Choice® RACE-Ready cDNA from human liver and intestinal tissue, several putative transcription start sites were identified: One each at the beginning of the novel UTR exons depicted in Fig. 2A, except Ex −1, and several alternative transcription start sites within Ex −3. A transcription start site cluster was located in the region between nucleotides 135 and 115 upstream from the 3′end of Ex −3 (Fig. 2B). This region contains a putative initiator sequence (Inr) [16], [17]. RACE-Ready experiments also identified two other novel exons in the vicinity of Ex −3 and Ex −2, designated Ex-3b (Chr18: 55470138-55470074; GRCh37/hg19) and Ex −2b (Chr 18: 55468948-55468914; GRCh37/hg19) (Fig. 2A). Ex −4, which was identified by classical RACE above, was not detected using RACE-Ready cDNA. The transcription start site (TSS) at the beginning of Ex −1 was only found by RACE, while using RACE-Ready cDNA Ex −1 was a part of transcripts initiating further upstream of Ex −1.
(A) Transcription of ATP8B1 gene originates from multiple TSSs (indicated by arrows); two additional TSS were identified at the beginning of rarely used exons, adjacent to Ex −3 and −2 respectively (dashed boxes). Sixteen different TSSs were found within the 509 bp-long Ex −3 with the main cluster located between nucleotides 135 and 115 upstream from the 3′end of Ex −3 (B).
Consistent Expression Pattern of the Novel ATP8B1 mRNA Isoforms in the Liver and Intestine
To define the biological relevance of the different ATP8B1 transcripts we used 7 normal human livers and 3 normal human intestinal samples in qRT-PCR experiments with individually-designed probe/primer sets covering all identified alternative exon-exon boundaries found by 5′RACE (Tab. S1). The results are presented in Fig. 3, where the diagrams represent probe/primer sets and Latin numbers relevant splicing variant(s) detected. The results show the abundance of each 5′UTR splicing variant relative to the protein coding region, represented by the Ex +1/+2 boundary. qRT-PCR demonstrated the prevalent expression of transcripts containing Ex −3 directly spliced to Ex +1; these splicing variants, Ex −3/Ex +1 and Ex −3/CAG/Ex +1 (Fig. 3) comprise almost 2/3 of total ATP8B1 expression, whilst the alternative splicing event, Ex −3/Ex −1 that in fact comprises two mRNA isoforms: Ex−3/Ex −1/Ex +1 and Ex−3/Ex −1/CAG/Ex +1, comprises less than 10% of the total transcripts. Splicing variants Ex −1/Ex +1 and Ex −1/CAG/Ex +1 account for almost 26% but splicing events including Ex −2 account for less than 4% of the total transcripts. The expression level of Ex −4/Ex +1 (Fig. 3, variants I and II), found by classical 5′RACE but not RACE-Ready cDNA, varied significantly amongst the samples, ranging from complete absence to 3.4% of total gene expression. The expression levels of the two small rarely detected exons, located 91 bp and 215 bp downstream from Ex −3 and Ex −2 respectively (Ex−3b and Ex −2b, Fig. 2A), and identified only using RACE-ready cDNA, were below 1% in all experiments (data not shown). Additional qRT-PCR experiments on a limited (n = 3) number of human intestinal samples did not exhibit any significant difference compared to the expression pattern detected in liver, with Ex −3 proving to be the most prevalent 5′UTR exon expressed in both, liver and intestinal tissues (data not shown).
Diagram of individually designed probes (for probe and primer sequences see Table S1) used to evaluate the expression levels of twelve identified splicing variants of ATP8B1 5′UTR. Tested splicing variants are indicated by Latin numbers on the left, average expression levels for each transcript from normal liver tissues (n = 7) are shown on the right. The expression levels are presented as a relative value normalised to the expression of the protein coding region represented by Ex +1/Ex +2 boundary.
Searching of the NCBI EST database (National Centre for Biotechnology and Information, http://www.ncbi.nlm.nih.gov/) revealed one ATP8B1 transcript with Ex −3 spliced to Ex +1 without the CAG triplet (GenBank accession: DR005588.1, GRCh37/hg19). This transcript does not include the protein coding Ex +2, and thus Ex +1 must be spliced directly to Ex +3. The resultant predicted protein sequence would have a premature stop codon (TAA) at position 62, which would not give a viable ATP8B1 protein. We searched for the existence of Ex +1/Ex +3 splicing event by PCR using cDNA templates prepared from liver and intestinal RNA. qRT-PCR using the specifically-designed probe for the Ex +1/Ex +3 boundary demonstrated 20-fold lower expression of this transcript in normal human liver and intestinal tissue compared to the Ex +1/Ex +2 splicing variant (data not shown). The biological significance of the low abundance transcript therefore remains unclear.
Also, our results did not confirm the existence of previously identified 909 bp-long ATP8B1 5′exon [15].
In Silico Identification of the Putative Promoters in ATP8B1 Gene
On the basis of the 5′RACE and qRT-PCR results, we predicted the major promoter region of ATP8B1 to be located upstream of the cluster of TSSs within Ex −3 (P3, Fig. 4) and two weaker promoters to be upstream of Ex −2 and −1 (P2 and P1, Fig. 4). In agreement with our hypothesis, in silico analysis employing three independent promoter prediction programs located putative promoter regions within a CpG island 70–72 kb upstream of the protein coding Ex +1 corresponding to the chromosomal location of the novel Ex −3 (Fig. S1). Computer analysis did not predict the presence of promoter upstream of Ex −1.
HepG2 cells were transiently transfected with luciferase reporter gene constructs containing 12 different fragments of putative ATP8B1 promoters. Nine luciferase constructs (Prom 1 to Prom 9) were designed to comprise the putative dominant promoter P3, two constructs covered promoter P1 (Proms 11 and 12) and one covered promoter P2 (Prom 10). The position of the tested fragments are indicated by horizontal double arrow lines. The number in brackets next to the construct name represents its size (bp). Prom 3 and Prom 4 were designed to include/exclude a putative FXR/RXR binding site indicated by black oval. Antisense construct encodes the same region as Prom 5, but in antisense orientation. Putative promoters (P1–P3) are depicted as horizontal thick arrows. Transcriptional activity for each construct was measured in relative light units per second (RLU/s) and corrected for the transfection efficiency using the internal control Renilla pRL-TK expression plasmid. The data shown are calculated from 3–5 independent experiments and related to the pGL3 Basic activity.
Experimental Validation of the Major Promoter of the ATP8B1 Gene
In order to identify whether the major promoter of ATP8B1 resides upstream of Ex −3, twelve promoter constructs (Prom 1–12, Fig. 4) utilising the luciferase reporter gene system were prepared: Six comprised the sequences upstream of the TSS cluster in Ex −3, whilst three focused on the region upstream of Ex −4 and three represented the regions upstream of Ex −2 and Ex −1. The promoter sequences cloned ranged from 434 bp to 3379 bp in length.
The luciferase assay results, summarised in graph on Fig. 4, demonstrate the highest relative promoter activity for the short 434 bp construct (Prom 1) which is situated immediately upstream of the major TSS cluster in Ex −3. Assessment of the longer promoter constructs ranging from 747 bp up to 3379 bp (Proms 2–6) exhibited approximately 65% reduced promoter activity compared to the Prom 1 construct. Only minimal differences in the reporter assay were observed among Proms 2–6 constructs relative to each other. Removal of the 434 bp region upstream of Ex −3 resulted in a complete absence of luciferase activity in all constructs tested (Proms 7–9; Fig. 4), thus emphasizing the importance of the proximal P3 promoter in driving luciferase activity.
A putative TSS in Ex-1, not predicted in silico, was detected only by classical 5′RACE. Nevertheless, the 40 kb distance between the main promoter and Ex −1 in combination with the qRT-PCR results suggested the presence of an alternative regulatory region in the vicinity of Ex −1. Constructs, which included the putative promoter upstream of Ex −1 and also upstream of Ex −2 (Proms 10–12), showed only basal transcription activity which was less than 10% that of the Prom 1 construct upstream of Ex −3 (Fig. 4).
The 5′UTR of ATP8B1 is Highly Conserved between Human, Mouse and Rat
The sequences of mouse and rat Atp8b1 5′UTR were obtained from Ensembl database (http://www.ensembl.org/index.html) and aligned with nucleotide sequences of newly identified human UTR exons using the ClustalW (http://www.ebi.ac.uk/clustalw) program. A high degree of conservation was found in the region corresponding to Ex −3 and Ex −4: 83% and 82%, respectively, for a human-mouse alignment and 66% and 69%, respectively, for a human-rat alignment (Fig. 5).
(A) Sequence alignment using ClustalW algorithm (http://www.ebi.ac.uk/Tools/clustalw/). High level of conservation among mouse (M), rat (R) and human (H) genome was detected for Ex −3 and Ex −4 (conserved nucleotides indicated by stars). Putative Sp-1, Ap-2, NFκB transcription factor binding sites were predicted in exonic/promoter region P3. Putative CREB and HNF-4 binding sites were identified within a distal part of promoter P3 corresponding to Ex −4 sequence. Initiator element sequence encompasses the main TSS cluster in Ex −3. Exonic regions are underlined, transcription start sites are indicated by arrows and bold letters and putative transcription factor binding sites by grey boxes. Two upstream ATG are in bold. (B) Sequence of Ex −2 and (C) Ex −1. Exonic region is highlighted in bold. Alu consensus sequences are underlined.
Transcription Factor Binding Sites Present in the ATP8B1 Promoter Region
No consensus TATA or CAAT boxes were found in the proximal P3 promoter of the ATP8B1 gene. On the other hand, several putative binding sites for non-specific, general transcription factors (Sp1, AP-2, NFκB) were identified in that DNA sequence. No liver- or intestine-specific transcription factor binding sites were found in the vicinity of the TSS cluster of Ex −3 (Fig. 5). A putative FXR binding site was identified using only the MatInspector computer analysis software, and this was 807 bp upstream of Ex −4. However, this site, GAGTGAcTGACCA, does not correspond to any known consensus FXR binding sequence and the sequence is not conserved between human, mouse and rat.
Influence of Bile Acids on Promoter P3 Activity
To investigate the effect of bile acids on P3 promoter activity, HepG2 cells were transfected with the Prom 3, Prom 4, and Prom 6 ATP8B1 luciferase constructs, which comprise the main promoter P3, and then incubated in the presence or absence of CDCA and TCA for 24 hours. Prior to the commencement of the luciferase experiments, we evaluated the expression levels of endogenous ATP8B1, ABCB11, SHP and CYP7A1 by qRT-PCR before and after CDCA treatment to assess the normal response of HepG2 cells to bile acid stimulation. While ATP8B1 levels remained constant, ABCB11 and SHP mRNAs were up-regulated and CYP7A1 mRNA down-regulated in the presence of 50 and 100 µM CDCA respectively (data not shown), proving HepG2 as a suitable model to assess the response of the ATP8B1 promoter constructs to bile acids.
In agreement with the unchanged mRNA expression of ATP8B1 under CDCA stimulation, none of the luciferase constructs tested demonstrated a significant change in promoter activity in HepG2 cells after CDCA (Fig. 6A) or TCA (not shown) treatment. Since HepG2 cells do not express the NTCP, all experiments were repeated in a HepG2 cell line stably expressing rat Ntcp (rNtcp-HepG2 cells), that were in addition co-transfected with vectors expressing human FXR and RXRα. To minimise the effect of bile acids present in fetal calf serum, the cells were cultured in medium containing charcoal-stripped fetal bovine serum with minimal content of bile acids [13]. In agreement with the previous experiments, no significant change in ATP8B1 promoter activity was observed (Fig. 6B). These results indicate no direct link between FXR, bile acids and the ATP8B1 major promoter.
No significant change in activity was detected after stimulation with CDCA. (A) HepG2 cells were transiently transfected with three previously characterised (Fig. 4) ATP8B1 promoter gene constructs (Prom 3, 4 and 6) and stimulated with 0, 10, 50 and 100 µM CDCA for 24 hours. All constructs comprise proximal 434 bp-promoter P3, Prom 4 includes putative FXR binding site identified by MatInspector computer analysis software, and Prom 6 represents the largest construct containing 3379 bp of 5′flanking region. (B) HepG2 cells stably expressing rat sodium-taurocholate co-transporting polypeptide (rNtcp) were transiently transfected with constructs Prom 3, 4 and 6 together with 50 ng of pCI_hRXRα and 50 ng pCI_hFXR plasmids and treated with 0, 10 and 25 µM CDCA for 24 hours.
Discussion
In this study we demonstrate that ATP8B1 expression is regulated by a highly structured 5′UTR which spans 71,964 kb and comprises four untranslated exons located a considerable distance upstream of the first protein coding exon. These exons are alternatively spliced. The main TSS cluster was located within nucleotides 135 to 115 upstream from the 3′end of Ex −3. This TSS cluster contains a putative initiator element (Inr) [17], [18] and is encompassed by Sp1 binding sites (Fig. 5). The previously published TSS [1], [15] is not significant. Transcription of ATP8B1 is driven by three newly identified promoters (P1, P2 and P3; Fig. 4) In liver, the promoters P1 and P2 play only minor role under physiological conditions. Promoter P3 was identified as the essential regulatory element responsible for 70% of total ATP8B1 gene expression. The 434 bp part of P3 (Prom 1 construct in Fig. 4) upstream of the main TSS cluster promiscuously serves both as exonic and promoter region and represents the main driving force of ATP8B1 gene expression. The importance of this region was further confirmed by sporadic use of TSSs located further upstream of the main TSS cluster in Ex −3.
The dominant ATP8B1 promoter P3 displays typical features for promoters of housekeeping genes: TATA-less, GC-rich sequence with multiple TSSs [19] in which only non-specific putative transcription factor binding sites for Sp1, AP-2 and NFκB were identified. (Fig. 5). These data are in agreement with the ubiquitous expression of ATP8B1 (FIC1) and its putative complex role in maintenance of apical membrane structure [8], [10].
Genes regulated by alternative promoters are common in humans. Multiple promoters can be utilised according to environmental conditions or to a particular developmental stage to ensure the tissues-specific or spatio-temporal expression of the appropriate gene isoform [20]. Various mRNA isoforms may also interact to achieve a transcriptional repression of an alternative transcript [20], [21]. It has been shown that of several alternative mRNA isoforms, one can be ubiquitously expressed among various cell types, whereas the remaining ones may be limited to a small number of tissues [22], [23], [24], [25]. This might be the case of ATP8B1 alternative transcripts. Even though our study does not support such tissue specific regulation of ATP8B1 at the transcriptional level in the tested samples, further research could characterise the role of all three promoters in different organs under varying conditions and address the involvement of post-transcriptional control mechanisms.
In a view of the complex structure of the ATP8B1 gene and its highly variable mRNA levels across cell types, RNA stability and post-transcriptional control appears to be more important in ATP8B1 regulation than previously expected. Our data demonstrate significant differences between the activity observed for the promoter upstream of Ex −1 (P1) versus the promoter upstream of Ex −3 (P3). However, this difference was not replicated at the level of abundance of the mRNA transcripts associated with these promoters: Whereas the reporter gene activity mediated by promoter P1 was 15-fold lower than that of the principle promoter P3 (Fig. 4), the mRNA levels of corresponding transcripts displayed only a 3-fold difference (Fig. 3). The observed discrepancy suggests possible differential efficiencies in post-transcriptional processing of the corresponding pre-mRNA ATP8B1 forms.
5′UTRs are known as key mediators of post-transcriptional control. The mechanisms of UTR-mediated regulation comprise, among others, stable secondary structures including those formed by repetitive sequences such as Alu and upstream open reading frames [26]. Alternatively spliced Ex −1 of ATP8B1 is apparently an exonized Alu [27] element with the promoter P1 containing complementary Alu sequence. Alu sequences embedded in 5′UTR have been shown to modulate both transcription and translation [28], [29].
Other potent modulators of transcriptional and translational efficiency are Upstream Open Reading Frames (uORFs) which can affect gene expression by inhibition of mRNA stability and translational repression [30], [31]. Recently demonstrated uORF-mediated ability to trigger the nonsense-mediated mRNA decay [32], [33] or to inhibit the downstream ORF by upstream located uAUG [34] represents processes that could be potentially involved in posttranscriptional regulation of ATP8B1. Indeed we identified ATP8B1 transcripts that differ in their leader sequences and in the presence of putative upstream start codons AUG (uAUG) (Fig. 5). Whereas no uAUG was found within the transcript containing Ex −1, two uAUGs and two uORFs were identified within the prevalent transcript containing Ex −3. Further factors known to influence regulation of gene expression [35], [36], [37] are heterogeneity, a high GC content and the unusually long length of 5′UTR. Their potential contribution to regulation of ATP8B1 expression is discussed in Fig. 7, Fig. S2 and their legends.
The comparison of the 5′UTR length and RNA secondary structure free energy and percentage of minimal free energy (MFE) for all identified ATP8B1 5′UTR isoforms, schematically depicted on the left. Prediction for the most frequent isoforms initiating at Ex −3 was calculated using TSS at position −125 from 3′end of Ex −3. Data in brackets (row 3 and 4) represent data for TSS at position −509. Putative secondary RNA structures predicted using RNAfold web tool (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) are summarised in Fig. S2.
Since ATP8B1 deficiency is associated with cholestatic liver disease, most studies have focused on the role of the gene in cholestasis. Zollner and colleagues [38] found no changes in ATP8B1 mRNA level in cholestatic patients. In our experiments, stimulation of HepG2 cells with CDCA or TCA respectively showed no change in ATP8B1 mRNA levels and CDCA or TCA treatment of HepG2 or rNTCP-HepG2 cells expressing various ATP8B1 promoter constructs did not significantly alter the luciferase activity compared with untreated cells. This observation is in agreement with the gene’s ubiquitous expression and suggests that bile acid independent mechanisms regulate ATP8B1 expression across different cell types.
In conclusion our study provides fundamental data about the complexity of ATP8B1 gene regulation. Newly identified ATP8B1 mRNA isoforms differ in their 5′UTRs and both transcriptional and post-transcriptional efficiency. The basal expression of ATP8B1 gene in the liver and the intestine is driven by a promoter with house-keeping like properties. Regulatory parts of ATP8B1 characterised in this study represent a feasible region for mutational search in patients with features suggestive of ATP8B1 deficiency, in whom no mutations have been identified within the coding region.
Materials and Methods
5′Rapid Amplification of cDNA Ends (5′RACE)
The 5′ends of the ATP8B1 gene were mapped using the 5′/3′ RACE kit, 2nd Generation (Roche, Switzerland) according to manufacturer’s instructions. Total RNA was isolated using RNA-Bee (Tel-test, Inc., Friendswood, USA) from 50 mg of human liver or intestinal tissue, or 5×106 of HepG2 cells. All procedures were conducted with written informed consent under an institutional-review-board approved protocol or using anonymised bank samples, previously collected with consent for research. To confirm any newly identified transcriptional origins, liver and intestinal RACE-ready cDNA (Ambion, Austin, USA) was used. ATP8B1 gene-specific primers for RACE were designed to span the junctions of exons 3/4, 2/3 and 1/2. Resultant PCR products were cloned into the pDrive Cloning Vector (Qiagen, Hilden, Germany) and sequenced in both directions using ABI Big Dye (Version 3.1) on a 3100 automated DNA Sequencer (Applied Biosystems, Foster City, USA) using vector specific primers.
Quantitative Real–time PCR
Twelve sets of individually designed TaqMan® MGB probes labelled with Fam and non-fluorescent quencher and primers were generated using Primer Express® Software Version 2.0 (Applied Biosytems, Warrington, UK), to cover all variants of alternative splicing of the untranslated exons (Table S1, No.1-12). 100 ng of DNase-treated total RNA from normal human liver and intestinal samples was used as a template in a 20 µl reverse transcription reaction using Transcriptor (Roche, West Sussex, UK) and mix of random hexamer or gene specific primers (Invitrogen, Paisley, UK, Sigma-Aldrich, Dorset, UK). 1 µl of first strand cDNA was then assessed in triplicate for levels of the different ATP8B1 transcripts on an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Expression levels of the studied transcripts and the overall expression of the ATP8B1 coding sequence represented by Ex +1/Ex +2 boundary, were corrected to the level of 18S rRNA (delta Ct) (TaqMan® MGB probe, Applied Biosytems, Warrington, UK). A PCR of non-reverse transcribed RNA was performed as a negative control to check for any genomic DNA contamination. Delta delta Ct values were calculated using ABI SDS software with RQ study application (Version 1.2.3, Applied Biosystems) and the data analysed using Microsoft Excel.
Promoter and First Exon Analysis in Silico
Three independent algorithms for promoter prediction (http://genome.ucsc.edu/, http://www.genomatix.de, http://bimas.dcrt.nih.gov/molbio/proscan/) were used to analyse the 5′UTR of the ATP8B1 gene. The University of California Santa Cruz Genome Bioinformatics server was also used to predict the gene’s first exon. The data obtained were compared with the EST database and the 5′RACE experimental results.
Luciferase and Expression Plasmid Construction
Twelve fragments of the 5′UTR (Prom 1–12 in Fig. 4) of the ATP8B1 gene were PCR amplified using human genomic DNA as a template, Pfx polymerase (Invitrogen) and specific primers containing XhoI restriction sites. PCR products were cloned (Invitrogen Zero blunt kit or Qiagen Cloning kit), sequenced, digested with XhoI, and ligated into XhoI-digested luciferase reporter gene vector pGL3-Basic (Promega, Southampton, UK) using Quick Ligation Kit (New England Biolabs, Hitchin, UK). Resultant constructs were checked for the correct sequence with various restriction enzymes and by direct sequencing prior to transfection. Stocks were prepared using an Endotoxin-free Maxiprep kit (Qiagen, West Sussex, UK). Human retionid X receptor α (hRXRα) and farnesoid X receptor (hFXR) cDNAs were PCR amplified, cloned into Invitrogen’s TOPO TA- cloning kit, sequenced and then sub-cloned to the mammalian expression vector pCI (Promega).
Cell Culture and Transfection
HepG2 cell lines were purchased from the ATCC (Teddington, UK). Cells were maintained in Dulbecco’s modified Eagle’s medium (PAA, Farnbourough, UK) supplemented with 5% or 10% fetal calf serum (PAA) and glutamine. rNTCP-HepG2 cells, kindly provided by Ulrich Beuers (Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands) and Christopher Rust (Department of Medicine 2– Grosshadern, University of Munich, Munich, Germany), were maintained in Dulbecco’s modified Eagle’s medium containing 5% fetal calf serum and 1 mg/ml G418 (Invitrogen). For transfection, cells were seeded in 24-well plates (TPP) in medium containing 5% fetal calf serum or 5% charcoal-stripped bovine calf serum (GibcoBRL). For transient transfection, 1.5 µl of FuGene HD (Roche) and 500 ng of plasmid DNA were used per well. Plasmid DNA comprised 450 ng of the appropriate LucATP8B1 promoter construct and 50 ng of the Renilla pRL-TK internal control plasmid (Promega). For some stimulation experiments, 50 ng of pCI_hFXR and 50 ng of pCI_hRXR contructs were co-transfected with the luciferase constructs. Twenty-four hours after transfection, cells were treated with 0 to 100 µM dimethyl sulfoxide, chenodeoxycholic acid, CDCA or taurocholic acid, TCA (Sigma Aldrich), respectively.
Reporter Gene Assay
Cells were harvested 48 hours after transfection (24 hours after stimulation with bile acids) and then Firefly and Renilla luciferase activities in cell lysates were determined using the Dual luciferase system (Promega) on a Glomax luminometer (Promega). All reporter gene assays were performed in triplicate and results are presented as the average value from at least three independent experiments corrected for the transfection efficiency using Renilla luciferase activities. The data from individual experiments were related to the activity of the control expression plasmid pGL3 Basic (Promega).
Supporting Information
Figure S1.
In silico analysis of first exon and promoter region for ATB8B1 gene compared to the 5′RACE results. Four putative first exons were predicted by “First EF” computer prediction software (horizontal double-arrow lines) [1]. Predicted exons correspond to the chromosomal location of the novel exons −2, −3 and −4 identified in the 5′RACE experiments. Three independent computer algorithms localised putative promoters (dashed horizontal double-arrow lines) in a CpG island 70–72 kb upstream of Ex +1.
https://doi.org/10.1371/journal.pone.0051650.s001
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Figure S2.
Putative secondary structures of ATP8B1 5′UTR isoforms depicted in Fig. 7 of the main text. Drawings of the minimum free energy (MFE) structures for each splicing variant of ATP8B1 5′UTR suggest their possible different role in regulation of gene expression. One of the regulatory mechanism, formation of stable secondary structures, was shown to impede the progress of the scanning ribosome [2]. Such scanning is influenced by the size and the position of the secondary structure(s) towards the 5′cap of the mRNA species: that is, an alternative transcript with a shorter version of the 5′UTR is frequently translated more efficiently than the one with a longer 5′ region [3], [4], [5]. Likewise, a stem-loop structure located a considerable distance from the 5′cap will require a higher free energy compared to one situated closer to it to affect the access of a pre-initiation complex to the mRNA. [2], [6].
https://doi.org/10.1371/journal.pone.0051650.s002
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Table S1.
Individually designed TaqMan® MGB probes labelled with Fam and non-fluorescent quencher and primers were generated using Primer Express® Software Version 2.0 (Applied Biosytems), to cover all variants of alternative splicing of the untranslated exons. The abundance of each splicing variant was compared relatively to a non-variable coding region of ATP8B1 represented by Ex+1/+2 boundary. Probe/primer set for Ex+1/+3 boundary (No.14) was used to test the biological significance of transcript excluding protein coding Ex +2, found in EST database (GenBank accession: DR005588.1). All probe sets were designed across exon/exon boundaries to eliminate the possibility of genomic contamination. Primers used in various amplifications are indicated by upper index (a, b, c). The amplification efficiency was tested for each probe/primer set on control templates (obtained by cloning the appropriate cDNA region) using different concentrations of positive and negative controls. As each probe set worked with a slightly different efficiency, the concentration of probes was adjusted for each positive control to reach a cycle threshold (Ct) value difference not greater than 1.
https://doi.org/10.1371/journal.pone.0051650.s003
(DOC)
Acknowledgments
The authors thank to Jonathan Gilthorpe and Vladimír Kořínek for helpful discussion and technical support with reporter gene assay and Marek Cebecauer for critical reading of the manuscript. Supported by EASL Sheila Sherlock Post-doc Fellowship (D.C.) and Institutional grant IKEM MZO 00023001 (M.J.)
Author Contributions
Conceived and designed the experiments: DC SSS JAB RJT. Performed the experiments: DC SSS. Analyzed the data: DC JAB MJ RJT. Contributed reagents/materials/analysis tools: DC SSS JAB. Wrote the paper: DC MJ RJT.
References
- 1. Bull LN, van Eijk MJ, Pawlikowska L, DeYoung JA, Juijn JA, et al. (1998) A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat Genet 18: 219–224.
- 2. Houwen RH, Baharloo S, Blankenship K, Raeymaekers P, Juyn J, et al. (1994) Genome screening by searching for shared segments: mapping a gene for benign recurrent intrahepatic cholestasis. Nat Genet 8: 380–386.
- 3. van Mil SW, Klomp LW, Bull LN, Houwen RH (2001) FIC1 disease: a spectrum of intrahepatic cholestatic disorders. Semin Liver Dis 21: 535–544.
- 4. van Mil SW, van Oort MM, van den Berg IE, Berger R, Houwen RH, et al. (2004) Fic1 is expressed at apical membranes of different epithelial cells in the digestive tract and is induced in the small intestine during postnatal development of mice. Pediatr Res 56: 981–987.
- 5. Stapelbroek JM, Peters TA, van Beurden DH, Curfs JH, Joosten A, et al. (2009) ATP8B1 is essential for maintaining normal hearing. Proc Natl Acad Sci U S A 106: 9709–9714.
- 6. Ujhazy P, Ortiz D, Misra S, Li S, Moseley J, et al. (2001) Familial intrahepatic cholestasis 1: studies of localization and function. Hepatology 34: 768–775.
- 7. Paulusma CC, Folmer DE, Ho-Mok KS, de Waart DR, Hilarius PM, et al. (2008) ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity. Hepatology 47: 268–278.
- 8. Paulusma CC, Groen A, Kunne C, Ho-Mok KS, Spijkerboer AL, et al. (2006) Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport. Hepatology 44: 195–204.
- 9. Cai SY, Gautam S, Nguyen T, Soroka CJ, Rahner C, et al. (2009) ATP8B1 deficiency disrupts the bile canalicular membrane bilayer structure in hepatocytes, but FXR expression and activity are maintained. Gastroenterology 136: 1060–1069.
- 10. Verhulst PM, van der Velden LM, Oorschot V, van Faassen EE, Klumperman J, et al. (2010) A flippase-independent function of ATP8B1, the protein affected in familial intrahepatic cholestasis type 1, is required for apical protein expression and microvillus formation in polarized epithelial cells. Hepatology 51: 2049–2060.
- 11. Chen F, Ananthanarayanan M, Emre S, Neimark E, Bull LN, et al. (2004) Progressive familial intrahepatic cholestasis, type 1, is associated with decreased farnesoid X receptor activity. Gastroenterology 126: 756–764.
- 12. Alvarez L, Jara P, Sanchez-Sabate E, Hierro L, Larrauri J, et al. (2004) Reduced hepatic expression of farnesoid X receptor in hereditary cholestasis associated to mutation in ATP8B1. Hum Mol Genet 13: 2451–2460.
- 13. Frankenberg T, Miloh T, Chen FY, Ananthanarayanan M, Sun AQ, et al. (2008) The membrane protein ATPase class I type 8B member 1 signals through protein kinase C zeta to activate the farnesoid X receptor. Hepatology 48: 1896–1905.
- 14. Demeilliers C, Jacquemin E, Barbu V, Mergey M, Paye F, et al. (2006) Altered hepatobiliary gene expressions in PFIC1: ATP8B1 gene defect is associated with CFTR downregulation. Hepatology 43: 1125–1134.
- 15. Klomp LW, Vargas JC, van Mil SW, Pawlikowska L, Strautnieks SS, et al. (2004) Characterization of mutations in ATP8B1 associated with hereditary cholestasis. Hepatology 40: 27–38.
- 16. Smale ST, Baltimore D (1989) The “initiator” as a transcription control element. Cell 57: 103–113.
- 17. Javahery R, Khachi A, Lo K, Zenzie-Gregory B, Smale ST (1994) DNA sequence requirements for transcriptional initiator activity in mammalian cells. Mol Cell Biol 14: 116–127.
- 18. Kaufmann J, Smale ST (1994) Direct recognition of initiator elements by a component of the transcription factor IID complex. Genes Dev 8: 821–829.
- 19. Zhu J, He F, Hu S, Yu J (2008) On the nature of human housekeeping genes. Trends Genet 24: 481–484.
- 20. Davuluri RV, Suzuki Y, Sugano S, Plass C, Huang TH (2008) The functional consequences of alternative promoter use in mammalian genomes. Trends Genet 24: 167–177.
- 21. Martianov I, Ramadass A, Serra Barros A, Chow N, Akoulitchev A (2007) Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript. Nature 445: 666–670.
- 22. Barker DF, Husain A, Neale JR, Martini BD, Zhang X, et al. (2006) Functional properties of an alternative, tissue-specific promoter for human arylamine N-acetyltransferase 1. Pharmacogenet Genomics 16: 515–525.
- 23. Fusco F, Mercadante V, Miano MG, Ursini MV (2006) Multiple regulatory regions and tissue-specific transcription initiation mediate the expression of NEMO/IKKgamma gene. Gene 383: 99–107.
- 24. Husain A, Zhang X, Doll MA, States JC, Barker DF, et al. (2007) Functional analysis of the human N-acetyltransferase 1 major promoter: quantitation of tissue expression and identification of critical sequence elements. Drug Metab Dispos 35: 1649–1656.
- 25. Shibahara S, Takeda K, Yasumoto K, Udono T, Watanabe K, et al. (2001) Microphthalmia-associated transcription factor (MITF): multiplicity in structure, function, and regulation. J Investig Dermatol Symp Proc 6: 99–104.
- 26. Mignone F, Gissi C, Liuni S, Pesole G (2002) Untranslated regions of mRNAs. Genome Biol 3: REVIEWS0004.
- 27. Sorek R, Lev-Maor G, Reznik M, Dagan T, Belinky F, et al. (2004) Minimal conditions for exonization of intronic sequences: 5′ splice site formation in alu exons. Mol Cell 14: 221–231.
- 28. Sobczak K, Krzyzosiak WJ (2002) Structural determinants of BRCA1 translational regulation. J Biol Chem 277: 17349–17358.
- 29. Landry JR, Medstrand P, Mager DL (2001) Repetitive elements in the 5′ untranslated region of a human zinc-finger gene modulate transcription and translation efficiency. Genomics 76: 110–116.
- 30. Diba F, Watson CS, Gametchu B (2001) 5′UTR sequences of the glucocorticoid receptor 1A transcript encode a peptide associated with translational regulation of the glucocorticoid receptor. J Cell Biochem 81: 149–161.
- 31. Lammich S, Buell D, Zilow S, Ludwig AK, Nuscher B, et al. (2010) Expression of the anti-amyloidogenic secretase ADAM10 is suppressed by its 5′-untranslated region. J Biol Chem 285: 15753–15760.
- 32. Hood HM, Neafsey DE, Galagan J, Sachs MS (2009) Evolutionary roles of upstream open reading frames in mediating gene regulation in fungi. Annu Rev Microbiol 63: 385–409.
- 33. Zhao C, Datta S, Mandal P, Xu S, Hamilton T (2010) Stress-sensitive regulation of IFRD1 mRNA decay is mediated by an upstream open reading frame. J Biol Chem 285: 8552–8562.
- 34. Zhang Y, Zhao T, Li W, Vore M (2010) The 5′-untranslated region of multidrug resistance associated protein 2 (MRP2; ABCC2) regulates downstream open reading frame expression through translational regulation. Mol Pharmacol 77: 237–246.
- 35. Arrick BA, Grendell RL, Griffin LA (1994) Enhanced translational efficiency of a novel transforming growth factor beta 3 mRNA in human breast cancer cells. Mol Cell Biol 14: 619–628.
- 36. Pickering BM, Willis AE (2005) The implications of structured 5′ untranslated regions on translation and disease. Semin Cell Dev Biol 16: 39–47.
- 37. Hughes TA (2006) Regulation of gene expression by alternative untranslated regions. Trends Genet 22: 119–122.
- 38. Zollner G, Fickert P, Zenz R, Fuchsbichler A, Stumptner C, et al. (2001) Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases. Hepatology 33: 633–646.