Department of Microbiology, College of Natural Sciences, Pusan National University, Pusan 609-735, South Korea
Correspondence
Kyung Lib Jang
kljang{at}pusan.ac.kr
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ABSTRACT |
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INTRODUCTION |
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HERV gene expression is mostly under control of their LTRs. HERV solitary LTRs can often be found in close vicinity to functional genes (Sverdlov, 1998). In addition, the U3 region of HERV LTRs contains all the sequences required for initiation of transcription, such as promoters, enhancers and transcription factor (TF) binding sites (Domansky et al., 2000
; Knössl et al., 1999
; Schön et al., 2001
); thus it not only regulates transcription of viral genes but also influences the expression of neighbouring cellular genes. Indeed, some HERVs are transcribed, and HERV proteins as well as replication-defective virus particles have been detected in several tissues (Boller et al., 1993
; Seifarth et al., 1995
; Lyden et al., 1994
). Furthermore, HERVs might cause a range of disease processes including neoplasia, auto-immunity, encephalitis and foetal malformations (O'Reilly & Singh, 1996
; York et al., 1992
), although their biological significance awaits clarification.
Members of the HERV-W family, originally isolated from monocytes and leptomeningeal cells from patients with multiple sclerosis, are preferentially expressed in normal human placenta (Blond et al., 1999) where they might play a role in morphogenesis by mediating trophoblast fusion (Mi et al., 2000
). Computer sequence analysis suggested the presence of several putative TF binding sites in the U3 promoter region and its promoter activity varies significantly in different human cell types (Schön et al., 2001
). However, the precise transcriptional regulatory mechanism is still obscure because little is known about the action of each putative TF binding site on transcriptional initiation from the HERV-W LTR. Therefore, in the present study, we tried to identify a regulatory sequence(s) that is important for HERV-W LTR-directed transcription in order to understand transcriptional regulation of the LTR. Furthermore, we analysed nucleotide sequences in the regulatory sites of several HERV-W LTR isolates and compared their promoter activities. In addition, we investigated which regulatory site is responsible for the cell type-specific promoter activity of HERV-W LTR.
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METHODS |
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Construction of HERV-W LTR reporter plasmid.
The full-length HERV-W LTR fragment of HW6 was subcloned into the KpnI and HindIII sites of the luciferase reporter vector pGL2-basic (Promega) to create HW1847. For construction of HW1772 and HW772847, the sequence either downstream or upstream of the SacI site at nt 772, respectively, were deleted from HW1847. For construction of HW1575, the sequence downstream of the SpeI site at nt 575 was deleted from HW1847. For HW1-347, the U3R fragment from nt 1 to 347 was amplified using as primer set HW-LTR forward and HW347R (5'-ACC AGA AGC TTG CAG TTG CA-3'); the fragment was then cloned into the KpnI and HindIII sites of the luciferase vector. Five additional U3R-containing luciferase constructs, HW1 to HW5, were prepared by the same procedure. For construction of HW347847, the U5 fragment amplified using as primer set HW347F (5'-CTG CAC TCT TCT GGT CCG T-3') and HW-LTR reverse was cloned into the SmaI and SacI sites of the luciferase vector. For HW8471 and HW3471, the LTR fragments of HW1847 and HW1347, respectively, were cloned in reverse orientation into the HindIII and KpnI sites of pGL2 vector. For HW1259 and HW259347, the sequence either downstream or upstream of the NaeI site at nt 259 was deleted from HW1347. For construction of HW1143 and HW143347 the sequence either downstream or upstream of the SacI site at 143 was deleted. For HW161347, the fragment from 161 to 347 was amplified using as primer set HW161F (5'-AAA ATG CTA GCT AGC CAA AA-3') and HW347R, and was cloned into the NheI and HindIII sites of the luciferase vector. For HW191347, the fragment from 191 to 347 was amplified using as primer set mCAAT-F (5'-AAT AGC TAG CCA TCT ATT GCC TGA-3') and HW347R, and was cloned into the NheI and HindIII sites of the luciferase vector. HWdOct1, which has an internal deletion from nt 144 to 160, was constructed by ligating the two fragments from HW1143 and HW161347. For HWdC/EBP, the fragment from 1 to 178 was amplified using as primer set HW-LTR forward and HW178R (5'-TTG CTA GCA CCT CCT ATT TTT GCC T-3') and was cloned in front of the LTR fragment of HW191347. HWSp1, which has an artificially introduced Sp1 site in sequence position 220227 (5'-CGG GAG GG-3' to 5'-CGG GCG GG-3'), was constructed by PCR-directed mutagenesis. Similarly, HWmOct1 and HW1mOct1, each of which has nucleotide substitutions in the Oct-1 binding site from 5'-ATG CTA AT-3' to 5'-GAT CTA AT-3' and from 5'-ATA CTA AT-3' to 5'-GAT CTA AT-3', were constructed by PCR-directed mutagenesis. Both HWmC/EBP-1 and HWmC/EBP-2 that have nucleotide substitutions to destroy the binding site for C/EBP (5'-GCC AAT-3' to 5'-GCT AGC-3' and 5'-GCC AGT-3', respectively) were also constructed by PCR-directed mutagenesis. For HWmOct1/mC/EBP-1, the CAAT-box of HWmOct1 was destroyed by the same procedure.
Transfection and luciferase assay.
The human cell lines Tera-1 (ATCC HTB-105), HEK-293 (ATCC CRL-1573), MCF-7 (ATCC HTB-22), HCT116 (KCLB 10002), HepG2 (KCLB 58065) and HeLa (KCLB 30022) were obtained from the American Type Culture Collection and Korean Cell Line Bank, respectively. Cells were seeded at 2x105 cells per 60 mm diameter plate and transfected the next day with a calcium phosphate/DNA precipitate containing 3 µg each of target and effecter plasmid DNAs as previously described (Gorman et al., 1982). To control for variation in transfection efficiency, 2 µg of plasmid pCH110 (Pharmacia) containing the E. coli lacZ gene under control of the SV40 promoter was cotransfected. After 48 h, the level of expression from the target gene (luciferase activity) was analysed and values obtained were normalized to the
-galactosidase activity measured in the corresponding cell extracts. Each experiment was repeated at least three times.
Slot blot analysis.
Total cellular RNA was prepared from either Tera-1 or HeLa cells using Trizol (Gibco). Five µg of RNA was spotted onto a nitrocellulose membrane (Hybond-N; Amersham) and hybridized with an appropriate probe using a Roche Detection Starter Kit II. For preparation of probes, cDNA fragments were amplified with Taq polymerase using sense primers 5'-CTG CAC TCT TCT GGT CCG T-3' and 5'-ACC ACA GTC CAT GCC ATC AC-3' and antisense primers 5'-TGG AGG TAC CTT CAT GGT T-3' and 5'-TAC AGC AAC AGG GTG GTG GA-3' for HERV-W and glyceraldehyde-3-phosphate dehydrogenase (G3PDH), respectively, and were labelled using a Roche DIG labelling kit. Spots on the filters were quantified using BIO-PROFIL image analysis software (Vilber Lourmat, France).
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RESULTS |
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Identification of transcriptional regulatory elements in the U3 region of HERV-W LTR
According to the above results, most of the transcription stimulatory activity of the LTR resides in the U3R region. To determine whether the R region has an enhancer activity, the sequence from nt 259 to 347 corresponding to the R region was deleted to generate HW1259. Such a deletion did not change the luciferase activity at all, suggesting the absence of enhancer activity in the R region. In addition, the R region alone did not increase the basal luciferase activity of pGL2, as demonstrated with HW259347. Therefore, we concluded that the U3 region contains all of the regulatory sequences required for the transcriptional activation of HERV-W LTR and decided to analyse it in detail in order to elucidate the transcriptional activation mechanism of the LTR.
Based on computer sequence analysis, Schön et al. (2001) suggested the presence of five putative transcription factor binding sites in addition to a TATA box in the U3 region of the LTR, which are also completely conserved in our isolate (Fig. 1B
). To determine which of these regulatory sites is critical for the transcriptional regulation of the LTR, additional luciferase constructs were prepared as shown in Fig. 1(B)
. Deletion of the sequence between nt 1 and 143 hardly affected the LTR activity, as demonstrated with HW143347. In addition, the sequence between nt 1 and 143 had little effect on the basal activity of pGL2 empty vector, suggesting that the three putative TF binding sites located in the 5' half of the U3 region might be dispensable for the LTR activity. Interestingly, further deletion up to nt 160 increased the LTR activity up to 3-fold, suggesting the presence of a negative regulatory element between nt 143 and 160. This was also demonstrated with HWdOct1, which contains an internal deletion of the sequence from nt 144 to 160. Conversely, additional deletion of 30 nt between nt 161 and 190 reduced the LTR activity approximately-5 fold, indicating the presence of an enhancer sequence within this region. This was also demonstrated with HWdC/EBP, which has an internal deletion of the sequence from nt 179 to 190. HW191347, which includes a TATA box and the R region of the LTR, still exhibited a relatively high luciferase activity, approximately 8-fold higher than that of empty vector. Considering the absence of a regulatory activity in the R region, most of the luciferase activity from HW191347 might be due to the sequence from 161 to 259, which includes a TATA box.
Recently, Schön et al. (2001) suggested a negative regulatory role of the Sp1 site identified at nt 220 to 233 in all the inactive HERV-W LTR isolates, but absent from all the active HERV-W LTR isolates. As none of the HERV-W LTR isolates we obtained contain such a regulatory site, we artificially introduced it into HW1347 at the corresponding site and tested its effect on the LTR activity to determine whether the Sp1 site actually has a repressive activity for transcription. However, the luciferase activity from HWSp1 was approximately 3-fold that of HW1347 (Fig. 1B
), which is opposite to the prediction of Schön et al. (2001)
.
The importance of Oct-1 and C/EBP sites for the transcriptional regulation of HERV-W LTR
The silencer and enhancer sequences defined in this study contain transcription factor binding sites for Oct-1 and CCAAT/enhancer binding protein (C/EBP), respectively (Fig. 1B; Schön et al., 2001
). To prove that the Oct-1 binding site located between nt 161 and 190 is responsible for the silencer activity, we constructed HWmOct1, which contains GAT instead of ATG at the first 3 nt of the Oct-1 binding site (Fig. 2
). Such nucleotide substitutions severely damage this binding site (Douville et al., 1995
). As expected, the luciferase activity from HWmOct1 was approximately 3-fold higher than that of HW1347. In addition, destruction of the CAAT-box by nucleotide substitutions as in HWmC/EBP-1 and HWmC/EBP-2 significantly reduced the LTR activity, confirming that the C/EBP binding site acts as an enhancer for transcription (Fig. 2
). HWmOct1/mC/EBP-1, which contains substitutions at both TF binding sites, exhibited a luciferase activity intermediate between that of HWmOct1 and HWmC/EBP-1, probably due to the loss of both silencer and enhancer activities.
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Cell type-specific promoter activity of HERV-W LTR
To characterize the cell type-specific promoter activity of the HERV-W LTR, the luciferase construct HW1347 was transfected into several human cell lines, including teratocarcinoma (Tera-1), cervical carcinoma (HeLa), colon cancer (HCT116), hepatocarcinoma (HepG2), embryonal kidney (HEK-293) and breast cancer (MCF-7) cells. The promoter activity of the LTR was measured by luciferase assay and was normalized to the -galactosidase activity measured in the corresponding cell extracts to compensate for the differences in transfection efficiency and expression rate. The luciferase activity of HW1347 was highest in Tera-1 cells and lowest in MCF-7 cells (Fig. 3
A). The activity in HeLa was higher than in MCF-7, which is consistent with the previous report by Schön et al. (2001)
. When the amount of HERV-W RNA was measured by slot blot analysis, Tera-1 had approximately 2-fold higher HERV-W transcripts compared to HeLa cells (Fig. 3B
), which is consistent with the results of the luciferase assay shown in Fig. 3(A)
.
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DISCUSSION |
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The HERV-W LTR possesses bidirectional promoter activity, acting in either the forward or reverse orientation. A similar reverse promoter activity of the U3 region has also been reported in several cases including the HERV-K LTR (Domansky et al., 2000), the HERV-H LTR (Feuchter & Mager, 1990
), the murine IAP LTR (Christy & Huang, 1998
) and the HIV-LTR (Peeters et al., 1996
). Therefore, at least some retroviral LTRs might promote transcription of downstream as well as upstream genes. Possible functions of this reverse promoter activity in the retroviral LTRs, as suggested by Domansky et al. (2000)
, may include involvement in inhibition of flanking cellular gene transcription, production of double-stranded RNA or the regulation of synthesis of positive-strand-derived proteins by antisense RNA.
Recently, Schön et al. (2001) divided HERV-W LTRs into two groups by sequence comparison and phylogenetic analysis. They found that all LTR sequences strong in promoter activity belong to group I whereas all weak LTR sequences were allocated to group II. In addition, to explain the difference in promoter activity between two groups, they argued for a negative regulatory role of the Sp1 site identified at nt 220 to 233 in the inactive HERV-W LTR isolates, but absent in the active HERV-W LTR isolates. However, according to our results, the Sp1 site, at least in our context, did not exhibit such a silencer activity but showed an enhancer activity, stimulating transcription from the LTR. Therefore, the Sp1 site might be not responsible for the weak promoter activity observed in the members of group II. Instead, our present study suggests that the Oct-1 and C/EBP binding sites are important for determining the LTR strength. The repressive role of the Oct-1 binding site was demonstrated by the high promoter activity of HW1, which has an impaired Oct-1 site. In addition, all of the inactive HERV-W LTRs identified by Schön et al. (2001)
had an altered CAAT-box with a substituted G at the fifth nucleotide, i.e. GCCAGT, whereas all active ones did not contain such a substitution. Therefore, it is possible that the presence or absence of functional binding sites for Oct-1 and C/EBP may determine whether the HERV-W LTR is active or not. We obtained much lower promoter activity when the fifth nucleotide of the CAAT-box was substituted, A into G.
Our present study also demonstrated that the promoter activity of the HERV-W LTR varies significantly depending on the cell type. Neither the Oct-1- nor the C/EBP binding site was required for the cell type-specific activity of the HERV-W LTR, excluding the possibility that it results from differential distribution of Oct-1 or C/EBP depending on the cell type. Instead, the basic promoter, including a TATA box located at the 3' end of the U3, was enough to confer cell type specificity, suggesting that the efficiency of assembly of basic transcription machinery at the TATA box of HERV-W might be different depending on the cell type. However, the possibility that other unidentified regulatory sites present at nt 191 to 259 are responsible for the specificity cannot be excluded.
In conclusion, in this study we analysed the regulatory sequences of the HERV-W LTR in detail in order to understand the cell type-specific activity as well as the transcriptional regulatory mechanism of the LTR. Further studies might be necessary to elucidate the exact mechanism of transcription from the HERV-W LTR. First of all, it is important to clarify whether and how binding of Oct-1 to the site defined in this study represses the promoter activity of the HERV-W LTR. The other question to be solved is how the TATA alone confers cell type specificity. In addition, the repressive activity of the U5 region was partially defined in this study. It might be necessary to analyse more HERV-W isolates to divide HERV-W LTRs into two groups based on the regulatory sequences defined in this study.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 December 2002;
accepted 6 April 2003.