Human Molecular Genetics, 2001, Vol. 10, No. 14 1455-1464
© 2001 Oxford University Press
An element in intron 1 of the CFTR gene augments intestinal expression in vivo
Paediatric Molecular Genetics, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK and 1Section of Cell and Molecular Biology, Division of Biomedical Sciences, Imperial College School of Medicine, London SW7 2AZ, UK
Received February 20, 2001; Revised and Accepted May 18, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The elements controlling the complex developmental and tissue-specific expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene lie outside the basal promoter region and have not been characterized. We previously identified a tissue-specific DNase I hypersensitive site (DHS) in intron 1 (185 + 10 kb) of the CFTR gene. Here we show that removal of the core element abolishes the activity of this DHS in transient transfection assays of reporter/enhancer gene constructs. We then compared expression from a 310 kb yeast artificial chromosome (YAC) that contains the entire CFTR gene with expression from the same YAC from which the DHS element had been deleted. Stable transfection of a human colon carcinoma cell line showed that transcription from the deleted YAC was reduced by
60%. In transgenic mice, deletion of the intron 1 DHS had no effect on expression in the lung, but reduced expression in the intestine by 60%. Thus, the regulatory element associated with the intron 1 DHS is tissue-specific and is required for normal CFTR expression levels in the intestinal epithelium in vivo. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The cystic fibrosis transmembrane conductance regulator (CFTR) gene (1,2) exhibits a complex pattern of expression that shows temporal and spatial regulation but is poorly understood (3–7). Extensive evaluation of the CFTR promoter failed to reveal the regulatory elements responsible for tissue-specific CFTR expression (8–12). To identify elements involved in CFTR expression
400 kb of genomic DNA was screened for DNase I hypersensitive sites (DHS). DHS were identified at –79.5 and –20.9 kb relative to the translational start site (13), within introns 1, 2, 3, 10, 16, 17a, 18, 20 and 21 of the CFTR gene (14), and in two clusters 3' to the gene at 4574 + 5.4 to + 7.4 and + 15.6 kb (15). The DHS in the first intron of the CFTR gene at 185 + 10 kb (16) showed complete correlation with CFTR expression in cell lines. The 185 + 10 kb region contained an element that augmented CFTR promoter activity in transient transfections only of CFTR-expressing cell lines (16). Further, CFTR-expressing cells contained proteins which bound to this element in vitro, which were absent from cells that did not express CFTR (16). The properties of the 185 + 10 kb element warranted further evaluation in vivo. We first used transient transfection of enhancer/reporter gene constructs in which the CFTR basal promoter drives luciferase expression. The 185 + 10 kb region increased CFTR promoter activity and deletion of the core element abolished this effect. Further analysis was carried out with a yeast artificial chromosome (YAC) containing the entire CFTR gene (yCFTRtag). When this YAC was introduced into the human colon carcinoma cell line Caco2, which expresses CFTR, the YAC-derived transcript showed copy number-dependent expression and was expressed in a 1:1 ratio with the endogenous gene (17). yCFTR also corrected the CF phenotype when introduced onto the cftrm1CAM/cftrm1CAM null CF mouse background (18), indicating that the majority of CFTR regulatory elements were present in this YAC. When a 209 bp fragment spanning the intron 1 DHS element was removed from yCFTRtag by homologous recombination, CFTR mRNA expression levels were reduced by 60% both in human colon carcinoma cells and in the small intestine of transgenic mice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Removal of the core segment of the DHS region abolishes its activity in transient assays
The 185 + 10 kb DHS in intron 1 augments the activity of the CFTR promoter in transient transfection of the Caco2 colon carcinoma cell line which endogenously expresses CFTR (16). Previously, a 787 bp fragment containing the CFTR basal promoter was cloned into the pGL2B vector to give pGL2B-245, and a 755 bp fragment spanning the putative regulatory element (BS0.7) was then cloned downstream of the basal promoter to give pGL2B-245/BS0.7 (16). In order to better define the enhancer element a 32 bp segment that bound proteins in DNase I footprinting and EMSA experiments (16 and unpublished data) was removed from BS0.7 by cloning. This modified fragment, BS0.7
32, was inserted into the enhancer site to give pGL2B-245/BS0.732. The three plasmids were transfected into the Caco2 cell line (expresses CFTR) and the mammary carcinoma cell line MCF-7 (does not express CFTR). The vector pCMV/ß was co-transfected to allow normalization of transfection efficiency. Both luciferase and ß-galactosidase activities were assayed and the luciferase activities for each construct, corrected for transfection efficiency, are expressed as a ratio of the luciferase activity obtained with the pGL2B-245 vector (Fig. 1). As expected (16) the pGL2B-245/BS0.7 construct caused a 3.3-fold increase in luciferase activity ( SD 1.02) in comparison to the promoter-only plasmid in Caco2 cells. This increase was determined to be statistically significant by using a non-paired t-test assuming unequal variance (P < 0.01). The pGL2B-245/BS0.732 construct showed no enhancement of luciferase activity, suggesting that the 32 bp deletion contained sequences that are important for the function of this regulatory element.
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Though the pGL2B-245/BS0.7
32 construct shows a slight decrease in reporter expression when compared with pGL2B-245 (P < 0.02), this is unlikely to be functionally significant. Neither pGL2B-245/BS0.7 nor pGL2B-245/BS0.732 showed any increase of luciferase activity in MCF-7 cells, which do not express CFTR, confirming the cell-specificity of this function (P > 0.6 and P > 0.06, respectively).
Generation of a CFTR gene YAC that lacks the 185 + 10 kb DHS
The 37AB12 YAC encompasses the whole of the CFTR gene, including 58 kb of 5' sequence and 50 kb of 3' sequence (19). This YAC has been retrofitted with a neomycin resistance gene [37AB12-pLNA or yCFTR (20)] and has been used previously to make two transgenic mouse lines called T30 and T57 (18).
yCFTR was then adapted to incorporate a modified restriction enzyme site (BclI to ClaI) in the 3'-untranslated region (3'-UTR) (yCFTRtag) to allow detection of YAC-derived CFTR transcripts (17) (Fig. 2A). A derivative of yCFTRtag that lacked 209 bp of the regulatory element at 185 + 10 kb (yCFTRtag185 + 10 kb) was generated by homologous recombination in a yeast host. This region was chosen as it encompassed the entire region shown to be important in previous transient transfection assays (16) and contained suitable restriction enzymes for YAC manipulation. Two regions of homology flanking the region to be deleted, 1AIR/TSR4 and TSR12/13, were cloned in a shuttle vector and used to delete the 209 bp using the pop-in/pop-out technique (Fig. 2B) (21). The deleted YAC (yCFTRtag185 + 10 kb) was checked for total size by pulsed-field gel electrophoresis (PFGE), for all the non-rearranged exon fragments by digestion with HindIII and probing with the CFTR cDNA, and for the correct deletion by digestion with NcoI and hybridization (data not shown).
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The yCFTRtag
185 + 10 kb YAC expresses 40% of the CFTR mRNA levels derived from yCFTRtag in Caco2 cellsIn previous experiments we have introduced the full-length YAC (yCFTRtag) into Caco2 cells by spheroplast fusion. The ratio of YAC-derived CFTR genes to genomic copies was determined by PCR using primers on either side of the BclI/ClaI site, which has been modified in the yCFTRtag. The primers, GV4579 and GV5508, are homologous to both DNA templates and therefore amplify both products equally efficiently, as shown previously (17). The PCR products were radiolabelled using [
-33P]dATP and digested with BclI (cuts the endogenous gene), ClaI (cuts the YAC gene) or both enzymes, run out on a gel and quantified using a phosphorimager. The relative level of mRNA being expressed from the YAC versus the genomic CFTR genes was similarly determined by RT–PCR using the primers GV4321 and GV5340 which selectively amplify cDNA rather than genomic DNA as the 1.5 kb intron 23 lies between them. Quantitation was then carried out as described for YAC:genomic DNA ratio determination. The CFTR gene on the YAC was found to be expressed in a copy-number-dependent manner and at a 1:1 ratio with expression of the endogenous gene in Caco2 cells (17).
The modified YAC (yCFTRtag185 + 10 kb) was similarly introduced into the human colon carcinoma cell line Caco2 by spheroplast fusion and four clones were isolated (int1/7.1, int1/7.2, int1/10.1, int1/10.2) after selection with G418. The presence of the yCFTRtag185 + 10 kb was confirmed by PCR amplification across the deletion site using primers TSR3 and TSR13 (data not shown). These primers generate a 672 bp product from the genomic copies of the CFTR gene in Caco2 cells and a 463 bp product from yCFTRtag185 + 10 kb. The four int1 cell lines all showed 463 and 672 bp products, whereas only the latter was seen in the parental Caco2 cell line DNA.
The copy number of the YAC relative to the endogenous CFTR genes was evaluated by PCR in the four int1 cell lines as described above (Table 1). The ratios in the four cell lines were: int1/7.1 = 6.16 ± SD 1.0; int1/7.2 = 5.26 ± SD 0.4; int1/10.1 = 0.85 ± SD 0.1; and int1/10.2 = 2.80 ± SD 0.7. The levels of CFTR mRNA expression derived from the YAC relative to that from the endogenous genes were also determined by RT–PCR as described above. The ratios obtained were: int1/7.1 = 2.60 ± SD 0.1; int1/7.2 = 2.17 ± SD 0.3; int1/10.1 = 0.46 ± SD 0.2; and int1/10.2 = 0.62 ± SD 0.2. Thus the CFTR expression from each CFTR gene on the yCFTRtag185 + 10 kb relative to each genomic gene was: int1/7.1 = 0.43 ± SEM 0.04; int1/7.2 = 0.41 ± SEM 0.04; int1/10.1 = 0.52 ± SEM 0.11; and int1/10.2 = 0.23 ± SEM 0.06. Relative CFTR expression levels from the four intron 1 cell lines are statistically significantly different from the full-length YAC (CYAC1) described in Vassaux et al. (17) with P < 0.0001 in three cases (lines int1/7.1, int1/7.2 and int1/10.2) and P < 0.09 for line int1/10.1.
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Overall, each CFTR gene on the yCFTRtag
185 + 10 kb YAC generates between 23 and 52% (mean 40%) of the expression levels of the endogenous CFTR gene. As for the undeleted YAC (17), the relative levels of expression in each cell line are quite uniform even though the relative copy number varies from 1 to 6, indicating that the CFTR expression from the YAC is largely copy number dependent and position independent. Since the CFTR gene on the undeleted yCFTRtag is expressed at a 1:1 ratio to each endogenous gene in Caco2 (17), these ratios indicate that in this cell line the intron 1 DHS (185 + 10 kb) accounts for 60% of CFTR expression.
Transgenic mice carrying yCFTRtag185 + 10 kb show a 60% reduction in intestinal expression of the YAC-derived CFTR mRNA
Three lines of transgenic mice i39, i59 and i93 were generated carrying the yCFTRtag185 + 10 kb YAC to allow for possible position effects. These mice were analysed in parallel with the T30 and T57 lines that carry the full-length yCFTR and have been described previously (18). The T30 and T57 mice provide control values of CFTR expression from the intact yCFTR in mice. The presence of the yCFTRtag185 + 10 kb in the i39, i59 and i93 mice was verified using PCR as described for the Caco2 cell lines above (Fig. 3). A PCR product of 672 bp was observed for both the T30 and T57 mice, confirming that these mice carry the wild-type YAC, whereas the i39, i59 and i93 mice generated fragments of 463 bp, confirming the presence of yCFTRtag185 + 10 kb.
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YAC copy number of transgenic mice
The YAC copy number of the
i39, i59 and i93 yCFTRtag185 + 10 kb transgenic lines and the T30 and T57 lines carrying yCFTR was estimated by Southern blotting. A series of increasing amounts of HindIII-digested human genomic DNA was loaded onto an agarose gel with HindIII-digested genomic DNA from each transgenic mouse line. This enabled the human DNA lane containing equivalent amounts of genomic DNA to each mouse lane to be identified by measuring the overall ethidium bromide staining in each lane of the gel using NIH Image 1.62 (Fig. 4A). Southern blots were probed sequentially with [32P]dCTP-radiolabelled DNA probes for exons 4, 9 and the 3'-UTR to evaluate the YAC copy number in each line (Fig. 4B–D). Hybridization with these probes showed mouse lines i39, i59 and i93 to have YAC copy numbers of three, one and two, respectively. The YAC copy numbers of lines T30 and T57 were verified simultaneously and confirmed to be two and one respectively (18). HindIII-digested non-transgenic mouse DNA gave no hybridization with any probe (Fig. 4B–D, WT lane), confirming the specificity of the probe hybridizations.
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CFTR expression from transgenic mouse tissues
To determine whether the removal of the 185 + 10 kb DHS affects CFTR transcription levels in specific tissues, the levels of transcript were measured by comparative RT–PCR. The primers HMEx2/HMEx6a-b amplify the same regions of the murine and human cDNAs at approximately equal efficiency (Fig. 5A). The PCR was shown to be in the linear range for the total amount of cDNA used and the ratios of human:mouse cDNA product stayed constant during the course of the PCR reaction (data not shown). The RT–PCR products were radiolabelled with [
-33P]dATP, digested with HindIII and NruI and resolved on 2% agarose gels that were dried down and exposed to phosphor screens. The human cDNA is cleaved with NruI to yield fragments of 390 and 237 bp, whereas the mouse cDNA produces 564 and 63 bp fragments after HindIII digestion. The ratio of human:mouse CFTR expression was determined for each cDNA synthesis reaction by measuring the sum above background (SAB) counts for the larger digestion fragment obtained for each species (Fig. 5B). The digestion fragments measured for each species are of different length and have different AT contents, so the SAB for each fragment was adjusted to account for this [SAB for human fragment divided by 221 (number of As and Ts in fragment) and SAB for mouse fragment divided by 326 (number of As and Ts in fragment)]. The human:mouse ratios were then adjusted to a copy number of two (i.e. the ratios were divided by the copy number then multiplied by two) for each transgenic line. The final percentage is the level of expression from each copy of the transgene relative to the expression from each copy of the endogenous gene. Each percentage value given below is the average of triplicate cDNA synthesis reactions from two mice from the same line (Table 2).
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Though endogenous mouse cftr transcripts were readily detectable in the mouse kidney, expression from the human yCFTR T30 and T57 transgenes was very low. Human CFTR expression was too low to measure accurately; however, it was possible to obtain figures of 8.7% for T30 and 9.0% for T57 from three independent cDNA synthesis reactions. Expression from the yCFTRtag
185 + 10 kb YAC in mouse kidney was below the level of detection for all cDNAs analysed. This suggests that there is a reduction in CFTR expression associated with the loss of the intron 1 DHS element in kidney; however, this cannot be measured accurately.
Analysis of CFTR expression in the lungs of T30 and T57 mice indicated that each transgene is expressed at 66.1% SEM 5.6 and 62.5% SEM 3.4, respectively, of the level of each murine cftr. The human CFTR gene is expressed from yCFTRtag185 + 10 kb in the transgenic mouse lung at approximately the same levels as from yCFTR, i.e. 62.3% SEM 3.1, 69.4% SEM 4.3 and 70.8% SEM 2.6 for lines i39, i59 and i93, respectively. There is no statistically significant difference between these results (using a non-paired t-test assuming unequal variance) with P > 0.47 in all cases.
Human CFTR transcript levels in the small intestine of the T30 and T57 transgenic mouse indicated that each transgene is expressed at 20.3% SEM 1.7 and 20.0% SEM 2.2, respectively, of the level of each endogenous mouse cftr gene. There is a significant reduction in human CFTR expression in the small intestine of the mice carrying yCFTRtag185 + 10 kb with levels of 9% (i.e. 7.8% SEM 1.5, 9.5% SEM 1.4 and 8.3% SEM 1.5 for lines i39, i59 and i93, respectively, an average of 8.5% for the 18 cDNAs analysed) with P < 0.001 in all cases. This value equates to 43% of the level obtained from the undeleted YAC (average of 20% relative to each endogenous gene), a reduction of 57%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Extensive evaluation of the CFTR gene promoter region previously failed to identify crucial tissue-specific and temporal control elements. Hence it is likely that these elements lie elsewhere in the gene and co-operation of multiple regulatory elements may contribute to CFTR expression in the chromatin environment in vivo. We have identified a number of DHS within and flanking the CFTR gene that may be associated with regulatory elements. Here we investigate further the in vivo function of a regulatory element located in intron 1 of the CFTR gene at 185 + 10 kb.
We previously showed by transient transfection of the CFTR-expressing cell line Caco2, that the 755 bp fragment BS0.7 encompassing the 185 + 10 kb DHS augmented reporter (luciferase) gene expression 3.4-fold when compared to CFTR promoter activity alone. This fragment was without effect in the MCF-7 cell line that does not express CFTR (16). On the basis of DNase I footprinting and EMSA experiments (16 and unpublished data) we predicted that the core element responsible for the augmented activity was within a 32 bp sequence (AC000111:29826–29857). The BS0.732 enhancer/reporter gene construct was generated that lacked these 32 bp, and when transfected into Caco2 cells the deleted fragment failed to increase luciferase expression above the levels driven by the CFTR promoter alone. This confirms that the 185 + 10 kb DHS is associated with an element that weakly increases CFTR expression in transient transfection assays in a human colon carcinoma cell line. These data are consistent with those generated by Mogayzel and Ashlock (22) using a YAC reporter gene construct containing 335 kb of CFTR 5'-flanking DNA driving luciferase gene expression. The BS0.7 fragment encompassing the 185 + 10 kb DHS was introduced downstream of the luciferase gene. When stably transfected into Chinese hamster ovary cells, clones carrying the BS0.7-containing construct showed 1.5-fold greater luciferase expression than clones carrying the unmodified YAC. These results confirm the importance of the 185 + 10 kb region once integrated into chromatin.
The key regulatory elements for the human CFTR gene are included within a 310 kb YAC yCFTR that restored a normal phenotype when introduced into a cftr–/cftr– mouse (18). Normal mice carrying yCFTR showed the 185 + 10 kb DHS in chromatin extracted from small intestine, liver and kidney, but not from lung or pancreas (23). We have now shown that deletion of the 185 + 10 kb DHS from yCFTRtag had no effect on the level of human CFTR expression in the lungs of mice transgenic from the yCFTRtag185 + 10 kb YAC in comparison to yCFTR. The absence of the 185 + 10 kb DHS in yCFTR in chromatin extracted from mouse lung tissues could be explained by absence of necessary transcription factors in murine cells. This would be consistent with the observation that removal of the 185 + 10 kb DHS did not alter the levels of human CFTR transcripts in the transgenic mouse lungs. As the DHS was not evident in mouse lung its removal would not be predicted to influence expression levels of the human transcript in the mouse lung. However, the site might still be involved in CFTR expression in the human lung.
It is of interest that the 185 + 10 kb DHS was evident in chromatin from transgenic mouse kidney (23). However, the extremely low levels of both murine and human CFTR transcripts that we detected in RNA from transgenic mouse kidney made it impossible to evaluate reliably the effect of deleting the 185 + 10 kb region from yCFTRtag. In our previous work (23) we could not detect CFTR expression in the transgenic mouse kidney by RT–PCR, though in the current experiments different PCR primer sets have been used.
We have observed a significant reduction in human CFTR expression in the small intestine of the mice carrying yCFTRtag185 + 10 kb in comparison to yCFTR. The human CFTR transcript levels from each copy of yCFTR are 20% of the levels of expression from each endogenous mouse gene, while the yCFTRtag185 + 10 kb transcripts are only 9%. The expression of yCFTRtag185 + 10 kb equates to 43% of yCFTR expression, a reduction of 57%, very similar to that observed for the Caco2 colon carcinoma cell lines carrying the same YACs. These data provide the strongest in vivo support for the hypothesis that the 185 + 10 kb DHS contains an element that is involved in tissue-specific expression of CFTR in the intestine.
Human and rodent CFTR show divergent patterns of expression in certain tissues (3,24,25) and hence might be expected to have certain unique regulatory elements. It is of interest that other distinct regulatory elements involved in intestinal expression of CFTR have been defined as DHS 5' and 3' to the rat CFTR gene (26,27). The two 3' DHS are found within regions that are conserved between humans and rodents but do not correspond to the location of the 3' DHS in the human CFTR gene (15). Transient and stable transfections showed that a 5.3 kb region 5' to the rat gene in combination with 1.3 kb containing the 3' DHS conferred intestinal-specific expression of a ß-galactosidase reporter gene in mice (27).
Further divergence between the regulation of the human and rodent CFTR genes may be illustrated by the difference in the expression levels of the yCFTRtag which we observed in the Caco2 human colon carcinoma cell line and in the transgenic mice. In Caco2 cells, CFTR expression from yCFTRtag was observed in a 1:1 ratio with the endogenous gene (17). However, in the transgenic mice carrying yCFTR the expression levels showed tissue-specific divergence in the ratio of human CFTR to mouse endogenous cftr per gene, with a maximum of about 0.7:1 in the lung and 0.2:1 in the small intestine. These differences may reflect species-specific subtleties of the CFTR regulatory mechanism and the divergence of transcription factors in the mouse which are required for the efficient expression of the human CFTR gene. This variation warrants further investigation using model systems such as the ovine CFTR gene that show greater similarity in expression patterns to the human CFTR gene (28,29).
The data presented here are the first in vivo evaluation of the regulatory element associated with the 185 + 10 kb DHS in intron 1 of the CFTR gene and they may have broader significance in the context of genes with major cis-acting regulatory elements located outside the promoter. Tissue-specific regulation of gene expression by elements located in the first intron is not uncommon. The hsp47 gene, encoding a collagen-binding heat shock protein, has a 500 bp element in the first intron that controls tissue-specific gene expression in vitro and in vivo in transgenic mice (30). An element in intron 1 of the Flk-1 gene (vascular endothelial growth factor receptor-2) functioned in concert with the 5' promoter to regulate tissue-specific gene expression in endothelial cells (31). The human angiotensin II type 2 receptor (32), protein C (33) and fast skeletal troponin 1 genes (34), all have regulatory elements in their first intron. Furthermore, erythroid-specific GATA-1 gene expression is markedly affected by elements in intron 1 (35). Expression of the acetylcholinesterase gene (AChE) is tightly controlled in skeletal muscle fibres. An enhancer element within the first 499 bp of the first intron of the rat AChE gene functioned with the 5' promoter region to regulate tissue-specific gene expression (36). The promoter of the AChE gene has similarities to the CFTR promoter in that it has no TATA-box and has a high GC content. Further elucidation of the mechanism of action of the 185 + 10 kb element of the CFTR gene may have implications for other genes with intronic regulatory elements.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture and transient transfection assays
Transient expression constructs were generated using the pGL2 Basic vector (Promega) as described previously (16). A 787 bp fragment (named 245) spanning the CFTR basal promoter (from –820 to –33 with respect to the ATG translational start codon) was cloned into NheI and BglII sites of the multiple cloning site of pGL2B in the correct orientation for driving transcription of the luciferase gene. This clone was called pGL2B-245. The full-length BS0.7 fragment of intron 1 [1AIR (5'-AAGCAAGTACGCATGATA-3')/TRS8 (5'-TAACTCATTGTACTGACGAG-3') AC000111:29163–29918] was as described previously (16). BS0.7
32 was generated by PCR amplification and cloning together of flanking DNA fragments [1AIR/ASTM6R (5'-CCAGCCAGGTCTGTCTGATTCCAAAGTAC-3') (AC000111:29163–29825) and ASTM3F (5'-TCTTAGGCAATTTACTTA-3')/TSR8 (AC000111:29858–29918)]. It encompasses the same segment of intron 1 as BS0.7 but lacks the core 32 bp (AC000111:29826–29857, 5'-AATCCTAACTCTGTCACTTATTAACAATGTGA-3') defined previously (16). Both fragments were cloned in the BamHI and SalI sites downstream of the luciferase gene in pGL2B, giving pGL2B-245/BS0.7 and pGL2B-245/BS0.732. The orientation of each fragment was determined to be 5'
3' with respect to the luciferase gene. Caco2 cells (37) and MCF-7 cells (38) were cultured in DMEM (Gibco) supplemented with 10% fetal calf serum at 37°C in 5% CO2. Cells were plated onto 30 mm dishes and transfected at 30–50% confluency. Transfections were carried out using FuGene6 (Boehringer Mannhiem) according to the manufacturer’s instructions and harvested after 24 or 48 h for MCF-7 and Caco2 cells, respectively. In all transfection experiments the transfection control was pCMV/ß (Clontech), which was used at a ratio of 1:4 with the luciferase construct. Luciferase and ß-galactosidase assays were carried out according to the manufacturer’s instructions (luciferase assay reagent, Promega; luminescent ß-gal genetic Reporter System II, Clontech). The results are expressed as relative luciferase activity compared with the pGL2B-245 construct which is given a value of 1, corrected for transfection efficiency as measured by ß-galactosidase activity. In each experiment every construct was assayed in triplicate and the transfection series was repeated at least four times. Statistical analysis was performed using non-paired t-tests assuming unequal variance (Welch) with www.graphpad.com.
Vector construction and spheroplast transformation
The YACs used in this study are derived from 37AB12 that contains the intact CFTR gene (19). This YAC has previously been retrofitted with pLNA-1 (20) (now referred to as yCFTR) and used to make the transgenic lines T30 [TgN(yCFTR)T30Clh] and T57 [TgN(yCFTR)T57Clh] (18,19). YAC yCFTR was further modified to include a ClaI restriction site in the 3'-UTR (referred to as yCFTRtag) (17). The intron 1 DHS element (185 +10 kb) was deleted from the YAC yCFTRtag by homologous recombination in the yeast host. Two regions of DNA homologous to CFTR intron 1 flanking the DHS site, and separated by 209 bp which contains the DHS itself, were generated by PCR using primers 1AIR/TSR4 AC000111:29163–29689 (1AIR: 5'-CGGGATCCAAGCAAGTACGCATGATA-3'; TSR4: 5'-TCCCCGCGGATCCAAGGGAAGATCAGGAACAAC-3') and TSR12/TSR13 AC000111:29899–30181 (TSR12: 5'-ATGAGCTCGTCAGTACAATGAG-3'; TSR13: 5'-ATGAGCTCAAACTGGAACATTG-3'). Bold letters indicate additional bases which were added to the 5' end of each primer to create new restriction enzymes sites—BamHI for 1AIR and TSR4, SacI for TSR12 and TSR13. The 1AIR/TSR4 fragment (527 bp) was cloned into the TA vector pCRII (Invitrogen). The KpnI/XhoI fragment containing the PCR product was then transferred to the vector pRS406 (Stratagene; Sikorski and Hieter, 39) (previously modified to lack a HindIII site) using the KpnI and XhoI sites. The TSR12/TSR13 fragment (282 bp) was similarly cloned into pCRII and then the SpeI/XhoI fragment cloned into the SpeI/XhoI sites of the modified pRS406 vector to give pRS406int1. pRS406int1 was linearized with HindIII (which cuts between 1AIR and TSR4) before transfection into yeast. YAC-containing yeast spheroplasts (yCFTRtag) were transfected with linearized pRS406int1 as described previously (40). Recombinants were selected on plates lacking uracil and confirmed to have pRS406int1 integrated into intron 1 in the YAC. Pop-out of the pRS406int1 vector was carried out by selection with 5'-FOA as described previously (21). A pop-out clone with a single copy of the YAC carrying the 209 bp deletion of intron 1 was obtained. This YAC, yCFTRtag185 + 10 kb, was finally retrofitted with pLUNA to introduce a neomycin resistance gene driven by a strong mammalian promoter (41). YAC integrity was verified using PFGE and Southern blotting.
Generation of Caco2 cell lines and transgenic mice
The YAC yCFTRtag185 + 10 kb was transferred into Caco2 cells by fusion as described previously (17) and four independent cell lines were grown.
The T30 [TgN(yCFTR)T30Clh] and T57 [TgN(yCFTR)T57Clh] strains of transgenic mice carrying yCFTR were described previously (18). DNA from the YAC yCFTRtag185 + 10 kb was gel-purified and microinjected as described elsewhere (42) to generate the lines i39 [TgN(yCFTR)i39Clh], i59 [TgN(yCFTR)i59Clh] and i93 [TgN(yCFTR)i93Clh]. All pro-nuclear injections were carried out in C57BL/6J x CBA/Ca F2 embryos. The transgenic lines were shown to carry the left and right arms of the YAC as well as exons 4 and 9 by PCR. Transgenic lines were bred with C57BL/6J mice and were genotyped with a PCR assay for the left arm of the YAC vector; 5'-GCTACTTGGAGCCACTATCGACTACGCGAT-3' and 5'-GTGATAAATTAAAGTCTTGCGCCTTAAACC-3'. The presence of the deletion in intron 1 was confirmed in both the cell lines and the transgenic mice. PCR was performed with the primers TSR3 (5'-CCTTAATTAAGGATCCGAGAATGTGTGATTTTCTTG-3') and TSR13 (AC000111:29510–30181); conditions were 94°C for 5 min, then 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min. A 672 bp fragment was derived from the endogenous Caco2 cells, but not from the endogenous mouse gene in the transgenics. A 463 bp fragment was generated from the modified YACs in the cell lines and the transgenics.
DNA/RNA analysis of transgene in Caco2 clones
The primer pair GV4579 (5'-GCAGCATAAAATGTTGACATG-3') and GV5508 (5'-GACTTCATGTGTGTCTACCC-3') (17) in the 3'-UTR of the CFTR gene amplifies CFTR genomic DNA and cDNA (AC000061:57465–58414). Primer pair GV4321 (5'-GTAATTCTCTGTGAACACAGGAT-3') and GV5340 (5'-CATCAAGGGAACCATCCTGTC-3') selectively amplifies CFTR cDNA due to the presence of intron 23 between the two primers (AC000061:55864–58224). Radioactive PCR was carried out as described previously (17) using genomic DNA. The ratio of YAC CFTR (digested with ClaI) to endogenous CFTR (digested with BclI) was calculated after quantification of the bands with a phosphorimager (Molecular Dynamics) to determine the YAC copy number present in each clone. RT–PCR was carried out as described below using RNA isolated from each clone using RNA Isolator (Genosys). Quantitation was carried out as for genomic DNA.
RNA preparation and RT–PCR from mouse tissues
Total cellular RNA was prepared from 150 mg of mouse tissues using RNA Isolator (Genosys). RT–PCR was carried out as described previously (43) using primers HMEx2 (AC000111 44028: 5'-CCTCTGYTGATTCWGCTGAC-3') and HMEx6a-b (AC000111 75118: 5'-GATCTCTGTACTTCAYCATC-3') (where ‘Y’ represents any pyrimidine and ‘W’ represents T or A), Superscript (Life Technologies, BRL) and Taq polymerase (Promega). The PCR reaction contained 0.5 µl of [-33P]dATP (Amersham International) per 50 µl reaction and the reaction conditions were 94°C for 5 min, then 30 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 7 min, with a final elongation at 72°C for 5 min. This cycle number was shown to lie within the exponential phase (data not shown). DNA fragments were digested with the relevant restriction enzymes in 0.8x buffer and then separated on 2% agarose gels which were dried at 80°C for 4 h before exposure to phosphorimager screen. Quantitation was carried out using ImageQuant 5.1 (Molecular Dynamics).
Preparation of genomic DNA from tissues and copy number estimation
High molecular weight DNA was prepared from mouse spleens. DNA probes for exon 4, 438 bp (AC000111:70472–70909), exon 9, 192 bp (AC000111:88347–88538) (44) and the 945 bp H set in the 3'-UTR—(AC000061:58013–58957) (43) of the CFTR gene were generated by PCR and purified by gel electrophoresis and Geneclean (Bio 101). Fifty nanograms of DNA probes were radiolabelled with 1 µl of [-32P]dCTP using the Megaprime kit (Amersham Pharmcia Biotech). Unincorporated nucleotides were removed using a NICK column (Pharmacia) prior to re-association with 30 µl of human placental DNA (0.5 mg/ml) at 65°C for 1 h before addition to the membrane.
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ACKNOWLEDGEMENTS |
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We would like to thank Ania Manson for genotyping the transgenic mice and help setting up the RT–PCR assay and Hugh Nuthall for vector construction. This work was supported by the Cystic Fibrosis Trust, UK, The Wellcome Trust and AFLM.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Fax: +44 1865 222626; Email: aharris@molbiol.ox.ac.ukPresent addresses:Georges Vassaux, ICRF Molecular Oncology Unit, ICSM at Hammersmith Hospital, Du Cane Road, London W12 ONN, UKTarra L. McDowell, MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UKAmanda McGuigan, Biological Services Unit, Hodgkin Building, King’s College, London SE1 9RT, UK
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REFERENCES |
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