From the Terry Fox Laboratory, British Columbia Cancer Agency and Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, V5Z 1L3, Canada
Received for publication, July 24, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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To examine the potential regulatory involvement
of retroelements in the human genome, we screened the transcribed
sequences of GenBankTM and expressed sequence tag
data bases with long terminal repeat (LTR) elements derived from
different human endogenous retroviruses. These screenings detected
human transcripts containing LTRs belonging to the human endogenous
retrovirus-E family fused to the apolipoprotein CI (apoC-I) and
the endothelin B receptor (EBR) genes. However, both genes are known to
have non-LTR (native) promoters. Initial reverse
transcription-polymerase chain reaction experiments confirmed and
authenticated the presence of transcripts from both the native and LTR
promoters. Using a 5'-rapid amplification of cDNA ends protocol, we
showed that the alternative transcripts of apoC-I and EBR are initiated
and promoted by the LTRs. The LTR-apoC-I fusion and native apoC-I
transcripts are present in many of the tissues tested. As expected, we
found apoC-I preferentially expressed in liver, where about 15% of the
transcripts are derived from the LTR promoter. Transient transfections
suggest that the expression is not dependent on the LTR itself, but the
presence of the LTR increases activity of the apoC-I promoter from both
humans and baboons. The native EBR-driven transcripts were also
detected in many tissues, whereas the LTR-driven transcripts appear
limited to placenta. In contrast to the LTR of apoC-I, the EBR LTR
promotes a significant proportion of the total EBR transcripts, and
transient transfection results indicate that the LTR acts as a strong
promoter and enhancer in a placental cell line. This investigation
reports two examples where LTR sequences contribute to increased
transcription of human genes and illustrates the impact of mobile
elements on gene and genome evolution.
A very high proportion of mammalian DNA consists of retroelements
that have arisen via RNA reverse transcription and reintegration into
the genome (1). Retroelements are found in most, if not all, species,
where they have amplified to high copy numbers during evolution (2).
The sheer number of such mobile elements suggests that they affect the
host genome, and several observations indicate that retroelements
impact on the species in a number of ways by acting as insertional
mutagens or contributing regulatory functions to genes (3). While
transposable elements can be harmful to their host, the vast majority
of transposable elements present in humans are derived from ancient
transpositional events which are fixed in Old World primates. Potential
long term effects of the majority of these elements must be either
neutral or beneficial; otherwise they would be eliminated by selection.
Human DNA contains essentially two classes of retrosequences, (i) the
non-long terminal repeat
(non-LTR)1 retroposons
represented by LINE and Alu sequences, and (ii) the LTR retroelements
in which the endogenous retroviruses (ERVs), solitary LTRs derived from
ERVs, and other LTR-like sequences fall (4). Human ERVs (HERVs) are
classified into different families based on sequence similarity and
monophyletic clustering (5, 6). The thousands of ERVs and solitary LTRs
that are present in human DNA are the result of infections and
transposition events during primate evolution. Solitary LTRs are common
features in the human genome, and they probably arose from a
recombination event between the 5' and 3' LTR of a full-length
provirus. Despite their evolutionary age, many ERVs are still
transcriptionally active in human cells, where different ERV families
show quite different sites and levels of transcription (7). The LTR and ERV elements are especially interesting in this regard, since they
naturally possess enhancer, promoter, and polyadenylation functions
within their LTRs, which probably accounts for differences in
transcription of the various HERV families. Besides promoting transcription of retroviral genes, several studies have demonstrated that ERVs and LTRs can assume gene regulatory functions (8-10). For
example, the paratoid-specific expression of amylase in humans is
dependent and under control of an HERV-E element (11). HERV-E also
appears to be involved in the expression of human pleiotrophin in
placenta (12). It has also been demonstrated that a HERV-K LTR encodes
the last 67 amino acids of one form of the leptin receptor OBR (13).
These findings indicate that an LTR insertion adjacent to or within a
gene could have a variety of effects without destroying gene function.
Such new insertions may alter tissue specific gene expression or
enhance the general transcription levels of the gene, which could be
selectively advantageous.
We are using LTR sequences to study the involvement of retroelements in
gene regulation. Specifically, we have searched the expressed sequence
tag and transcribed subset of GenBankTM for chimeric
retroviral gene sequences. Here, we report two human genes that are
affected by ERV LTRs, the apolipoprotein C-I (apoC-I) gene and the
endothelin B receptor (EBR) gene. We show that these two genes use a
HERV-E LTR as an alternative promoter, demonstrate the presence of the
chimeric transcripts in human tissues, and test the significance of the
LTRs at the genomic loci of apoC-I and EBR.
Reverse Transcription and PCR Amplification--
Reverse
transcription was done with Superscript II (Life Technologies, Inc.)
using the same reaction conditions as described previously (14). PCR
was carried out using 0.1-0.5 volumes of each cDNA (0.1-0.5 µg
of the initial RNA) per reaction. RNA samples were either obtained from
CLONTECH or prepared from different sections of
placenta, as described previously (15).
The following primers were used to detect the different transcript
forms shown in Fig. 2 (see Table I for primer sequences): LTR-apoC-I
fusion transcript, primers APO-LTR1/APO-Ex1; native apoC-I transcript,
primers APO-N/APO-Ex1; LTR-EBR fusion transcript, primers
EBR-L1/EBR-Ex1; native EBR transcript, primers EBR-N/EBR-Ex1. Amplification was done by using 0.5 volumes of cDNA (see above) with the following cycling profile: one initial incubation of 95 °C
for 1 min followed by 35 cycles (for the apoC-I amplifications) or 30 cycles (for the EBR amplifications) of 95 °C for 30 s, 63 °C
for 30 s, and 72 °C for 30 s, and one final elongation at
72 °C for 5 min. In the semiquantitative RT-PCR of different EBR transcript forms (Fig. 5), the following primer combinations were used:
LTR-EBR fusion transcript, primers EBR-L1/EBR-Ex2; native EBR
transcript, primers EBR-N/EBR-Ex2; total EBR transcript, primers EBR-Ex3/EBR-Ex2. In these experiments, 0.15 volumes cDNA was used in the same PCR profile as described above but with lower cycling (25-28 cycles) to avoid saturation effects during the amplification. The intensity of the amplification products was measured after 25 cycles from ethidium bromide-stained gels using the 1D Image Analysis
software (Eastman Kodak Co.).
5'-RACE--
Placental and brain Marathon-ready cDNA
libraries were purchased from CLONTECH, and a
5-µl cDNA library was used in 5'-RACE analysis as described in
the protocol supplied with the library. The first PCR amplification was
performed using EBR exon-specific primer (Table I) EBR-ex4 and the AP1
primer (provided by the supplier) and with the apoC-I primer APO-ex1
and primer AP1. The nested PCR was performed by EBR primer EBR-ex5 and
the AP2 primer (provided by the supplier) and with the apoC-I primer
APO-ex2 and AP2. The following temperature profile was used for all
amplifications: one initial denaturing at 95 °C for 1 min followed
by 35 cycles at 95 °C for 30 s and annealing and extension at
68 °C for 4 min. The 5'-RACE products were cloned using the pGEM-T
vector system I (Promega). Clones were selected for sequencing after
hybridization using retrovirus-specific oligonucleotides
APO-LTR1 and EBR-L1 (Table I).
Primer Extension--
The oligonucleotide primer APO-ex3,
complementary to exon 3 of apoC-I, was radiolabeled with
Locus-specific PCR--
Locus specific PCR was performed
essentially as described previously (16). Genomic DNA prepared from
marmoset (New World Monkey), baboon (Old World Monkey), gibbon,
orangutan, gorilla, chimpanzee, and human cell lines (17) was used in
PCR. Primers APO-P1 (5'-GGTTTTTACAGTGTCATCCAGCT-3')/APO-P2 (5'-
GATTCAGGTTGGTGCTGAGTG-3') were used to detect the presence or absence
of the solitary LTR in the apoC-I locus of different primates. The LTR
upstream of the EBR locus was amplified by primer EBR-F1
(5'-AACATCCTCTGTCTCTCTCC-3'; sequence flanking the LTR integration) and
primer EBR-LTR1 (5'-GATCGACCCCTGACCTAACC-3'; sequence from the LTR).
The apoC-I and EBR primers were specified from GenBankTM
accession numbers AB012576 and AL139002, respectively.
Plasmid Constructs--
The 5'-flanking regions of apoC-I and
EBR were amplified from human genomic DNA and subcloned upstream of the
luciferase gene in the promoterless luciferase reporter plasmid pGL3B
(Promega). To facilitate directional cloning into pGL3B, primers were
designed with terminal sequences specific for restriction enzyme
recognition. The restriction enzyme adaptor is indicated after each
primer (see below), where the following suffixes are used: K,
KpnI; B, BglII; Ba, BamHI; X,
XbaI; Xh, XhoI. The numbers in parenthesis after
each primer indicate the start and end positions of the primer upstream
in the flanking DNA sequence, relative to the first nucleotide of the
initiation codon of the two genes. The primer sequences are available
upon request. Because the exact distances of the EBR LTR and the
hepatic control region (HCR) to EBR and apoC-I are uncertain, the
primer sequences used to amplify the EBR LTR and the HCR are shown
below, where primer sequence in lowercase type indicates the
restriction enzyme recognition sequence.
The following EBR constructs were made. For pEBR-NP, the 5'-flanking
region of the native EBR transcription initiation site was isolated
using primers EBR-NP1K (
The following apoC-I constructs were made. For pAPO-P, the 5'-flanking
region of the apoC-I transcription initiation site was isolated using
primers APO-P1K (
The following baboon apoC-I constructs were made. For pBAPO-P, the
baboon apoC-I locus was amplified from baboon genomic DNA with primers
APO-P1K (
The HCR was isolated from human DNA using PCR and primers HCR1Xh
(5'-ccgctcgagTTAGAGAACAGAGCTGCAGGCT-3') and HCR2Xh
(5'-ATGCCCCGACCCCGAAGCctcgagcgg-3'). The primer sequences were derived
from positions 36815-36836, and positions 37201-37218 of
GenBankTM accession number AF050154, respectively. The PCR
product was digested with XhoI, and the purified fragment
was introduced into the pGL3B SalI site of all apoC-I
constructs, which is 3' to the luciferase gene.
Cell Lines and Transient Transfections--
Plasmid DNA was
purified by using the Qiagen plasmid midi kit (Qiagen) prior to
transfections. HepG2 (human hepatoblastoma cells; ATCC HB-8065) cells
were cultured in Dulbeccos's minimal essential medium supplemented
with 10% fetal calf serum. Cells were seeded 24 h prior to
transfections in six-well plates at a density of 3 × 105 cells/well. Transient transfections of HepG2 were done
by cotransfecting 1.5 µg of plasmid DNA and 50 ng of pRL-TK vector
(Promega) using calcium phosphate (Cellphect; Amersham Pharmacia
Biotech) as described in the protocol supplied with the reagent. JEG-3
cells (human choriocarcinoma; ATCC HTB-36) were maintained in RPMI
supplemented with 5% fetal calf serum. JEG-3 cells were seeded in
six-well plates at a density of 2 × 105 cells/well
and cotransfected 24 h later with 1.0 µg of plasmid DNA and 200 ng of pRL-TK using 7 µl of LipofectAMINE (Life Technologies, Inc.),
as described in the protocol from the supplier. After 24 h, the
cells were lysed, and the luciferase activities were measured using the
Dual-Luciferase Reporter Assay System (Promega) and normalized to the
internal control. Transfections were performed in triplicates and
repeated at least twice.
DNA Sequencing--
Double-stranded plasmid DNAs were sequenced
using an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit
and an ABI 373 sequencer (PerkinElmer Life Sciences).
Identification and Characterization of Chimeric
Transcripts--
Using the strategy outlined in Fig.
1, we searched GenBankTM and
the human expressed sequence tag data bases using the LTR and the
leader region of published HERV elements (7). Transcripts with only the
U3-R part of the LTR and no other HERV sequence probably represent
mRNA polyadenylated by the LTR, whereas transcripts with R-U5 or
R-U5-leader probably represent promotion by an LTR. During data base
screenings, we encountered two transcripts where HERV-E (18) sequences
were fused to the EBR gene (accession number D90402) and the
apoC-I gene (accession number W79313). The structure of these
transcripts suggests that the EBR utilizes the splice donor (SD) in the
leader region of the HERV element, which is located downstream of the
5' LTR (Fig. 1) of an integrated provirus. The same SD is used in
subgenomic splicing of HERV-E envelope transcripts, suggesting that
this represents the original SD of HERV-E (19). The apoC-I fusion
transcript represents another possible LTR-driven transcript type,
which is derived from a solitary LTR and reads into the flanking
non-HERV region.
To authenticate the presence of fusion transcripts, we synthesized
primers corresponding to the retroviral and the gene-specific regions
of the identified transcripts. By using this primer combination in
RT-PCR, it is possible to detect the presence and the relative abundance of the fusion transcripts in human tissues. Both of the genes
were previously reported as having a different promoter region,
separated from the retroviral LTR (20-22). We will refer to these two
regions as the "native" apoC-I and EBR promoter. To detect any
biases of the LTR and native transcripts, we also used a primer unique
to transcripts of the native promoters of the two genes. Results of the
RT-PCR on a panel of RNAs derived from different human tissues are
shown in Fig. 2. The LTR-promoted EBR
transcript is restricted to placenta, where its levels appear comparable with that of the widely expressed native transcript (Fig.
2B). In the case of apoC-I, transcripts from the native promoter in liver were high as expected (20) but are also detectable by
PCR in many of the other RNAs tested (Fig. 2A). Transcripts from the solitary LTR were detected in two distinct forms (see Fig.
3A), both of which were also
detected in many tissues. The result of this experiment clearly
demonstrates the presence of fusion transcripts between LTRs of HERV-E
and the genes for EBR and apoC-I. The LTRs at the EBR and apoC-I loci
vary in their tissue specificity, with the EBR LTR being much more
restricted in activity. Sequencing of the PCR products verified the
nature of the fusion transcripts, where the two fusion transcript forms of apoC-I are the result of differential splicing in the 5' UTR (Fig.
3).
Genomic Structure and Transcript Forms--
To confirm that the
apoC-I and EBR fusion transcripts initiate within the LTRs and do not
represent transcripts from a promoter located upstream of the LTRs, we
isolated the 5'-ends of both LTR fusion gene transcripts. Using a
5'-RACE protocol, we established that both the apoC-I and EBR fusion
transcript initiate within their LTRs (see below and Fig. 3).
Sequencing of several 5'-RACE clones showed that the apoC-I and EBR
initiation site is located downstream of a previously reported TATA box
of HERV-E (18, 19). This is the TATA also used by other HERV-E
proviruses because a full-length transcribed HERV-E element
(GenBankTM accession number M74509) starts 2 bp downstream
of the initiation site of the apoC-I LTR. In the case of the EBR fusion
transcript, the sequence representing the longest 5'-UTR also began
within the LTR, but at a position 3' (90 bp) to the apoC-I initiation site.
Both the apoC-I and EBR genomic loci were partially characterized at
the time of our initial studies. The only retroviral remnant of the
original proviral insertion at the apoC-I locus is a solitary LTR,
which is located 300 bp upstream of the native apoC-I promoter. The two
initiation sites are separated by 390 bp, where the initiation sites of
the native and LTR promoters are located 180 and 575 bp upstream of the
apoC-I initiation codon, respectively (Fig. 3). The EBR LTR was not
present in the reported 2-kb sequence upstream of the EBR native
promoter (GenBankTM accession D13162), which is located
~250 bp upstream of the EBR initiation codon (21, 22). A genomic
clone containing both the HERV-E proviral element and the EBR genomic
locus was recently deposited in GenBankTM (accession number
AL139002). The sequence of this clone is in a preliminary state of
annotation and contains unordered pieces of DNA. The parts of the LTR
and leader region that were identified in EBR 5'-RACE (see above) are
identical to the proviral element of AL139002. It has been previously
reported that several other alternative transcripts (named EDNRB Evolutionary Age of the LTRs--
Using primers flanking the
integration sites of the LTRs in PCR of different primate DNAs, we
earlier assigned the time of integration of various HERV-K elements
during primate evolution (16). Using the same approach, we were able to
determine when the two HERV-E LTRs integrated in the primate lineage.
The apoC-I LTR was detected in all hominoids, whereas Old and New World
monkeys did not have this LTR integrated in the apoC-I locus,
suggesting that the integration took place after the divergence of
hominoids and the Old World monkeys, about 20-30 million years ago
(Ref. 24). Since we could detect the presence of the EBR LTR both in
baboons and hominoids, but not in New World monkey, we conclude that
the LTR at the EBR locus is older than the apoC-I LTR, because it
integrated after the split between the New and Old World monkeys, ~30-40 million years ago (24). Sequence comparison of the 5' and 3'
LTRs of the EBR HERV-E revealed that they are 12% divergent. The same
time estimate of 30-40 million years is obtained by assuming that the
two LTRs diverged an average of 6% since integrating in the primate
lineage, taking a pseudogene divergence rate of 0.15-0.21% per
million years into account (6, 25).
Estimation of the Proportion of Transcripts Contributed by the LTR
Promoters--
We could not discriminate between the LTR-driven and
native transcripts using Northern blot analysis for either the apoC-I and EBR genes. For the apoC-I transcripts, all are in sizes ranging from 400 to 600 bases, and the resolution in this area of agarose gels
is poor. For EBR, the only unique sequence of the LTR-driven transcript
is from the retroviral part (Fig. 3), and it is not feasible to use
this region as a probe because of the repetitive nature of the LTR
sequences in human DNA.
We instead performed a primer extension protocol using RNAs from
several tissues and an oligonucleotide derived from exon 3 of apoC-I.
Using this strategy, we detected transcripts of sizes corresponding to
the native and the LTR-driven transcripts. We only detected transcripts
corresponding to the shorter, double-spliced apoC-I LTR fusion
transcript. As expected, the relative level of transcription was
highest in liver (Fig. 4), which is the
major site of apoC-I transcription (20). By densiotometry, we estimated that the short transcript derived from the LTR promoter represents ~15% of the total in liver. Other sites of transcription
(e.g. testis, lung, and brain) were also detected using this
analysis. However, in these tissues the level of transcription was
lower than in liver. We also used a fragment spanning the coding region of apoC-I in Northern hybridization (not shown). The signal in liver
was at least 40-50 times stronger than for any of the other tissues,
indicating that the primer extension analysis underestimated the level
of apoC-I mRNA in liver. A possible explanation of this could be
saturation effects in the primer annealing step of the extension
protocol or poor quality of the liver RNA.
To estimate the relative level of the LTR-EBR fusion transcript, we
performed a low cycle PCR protocol. This was done by serial dilution of
the input reverse transcribed RNA to avoid saturation effects during
amplification. Because the LTR-driven EBR fusion transcript was
detected in placenta, we tested RNA prepared from different parts of
the placenta in amplifications with primers specific for the native and
the LTR-driven transcripts. We also used primers specific for the exons
only, which would allow estimation of the total level of EBR mRNA
in the different samples (Fig. 5).
Depending on the origin of the cDNA used, ~50-65% of the total amount of EBR transcripts were estimated by densitometry to be derived
from the native promoter, and 25-30% of the total was derived from
the LTR promoter. As has been reported previously, we saw no evidence
for EBR expression in amnion (26).
Significance of the HERV-E LTRs in Expression of ApoC-I and
EBR--
To investigate the significance of the LTR in expression
regulation of the apoC-I gene, we inserted the native promoter region, which naturally contains the LTR and the native promoter, upstream of a
promoterless luciferase reporter plasmid (pGL3B). We also tested the
activity of the LTR by itself and the native construct where the LTR
was removed. We then performed transient transfections to test the
relative levels of promoter activity of the different constructs. The
LTR was also inserted at a distance (see "Experimental Procedures")
in constructs with the apoC-I promoter where the LTR was removed, to
test for the possibility that the LTR acts as an enhancer of the native
promoter. The expression in liver is completely dependent on a distal
HCR (27), and we saw no promoter activity of the apoC-I constructs
without the presence of this HCR. The results of the transfections of
HepG2 (liver) cells with a variety of apoC-I constructs are shown in
Fig. 6 and suggest that the LTR by itself
is not contributing significantly to the overall expression levels of
apoC-I in liver. However, when the LTR is removed from the apoC-I
locus, the promoting activity of the region drops about 40% in HepG2,
suggesting that the presence of the LTR in the apoC-I locus contributes
to the overall activity of the native promoter region. However, we
found no evidence that the LTR alone acts as an enhancer in liver cells
when positioned at a greater distance from the native promoter.
A test of the effect the LTR had at the time of integration in the
primate lineage would be to insert the LTR into the apoC-I locus of a
species that naturally lacks the LTR. Our analysis showed that all
hominoids have the LTR integrated in the apoC-I locus but that it is
absent in the baboon. The sequence of apoC-I baboon locus has been
determined (28), and sequence alignments of the human and baboon loci
verified the absence of the LTR in the baboon (not shown). We inserted
the LTR into the baboon apoC-I locus at the orthologous site, and
compared the relative promoting activity between the constructs with
and without the LTR. The LTR insertion into the baboon locus resulted
in increased expression, similar to that seen in the human locus,
suggesting that the LTR had a similar effect when it first integrated
in the primate lineage (Fig. 6).
To investigate the effect of the LTR in EBR expression, the native EBR
promoter region or the LTR alone were inserted upstream of the
luciferase gene of pGL3B. We also inserted the LTR at a distance, in
direct and opposite orientation with respect to the native EBR promoter
region, to test for potential enhancing effect of the LTR on the EBR
native promoter region. The choriocarcinoma cell line JEG-3 and the
liver cell line HepG2 were transiently transfected with these
constructs, and the results are shown in Fig.
7. In both JEG-3 and HepG2, the activity
of the native EBR promoter segment alone is low, and it is evident that
the native EBR promoter is dependent on an enhancer element not present
in the constructs or on a factor that is absent in the cell lines. However, when the LTR was inserted in either direction at a distance with respect to the native promoter (see "Experimental
Procedures"), a significant increase in activity was observed,
indicating that the LTR can act as an enhancer of the native promoter
region extrachromosomally. When constructs containing only the LTR
upstream of the luciferase gene were transfected into JEG-3, a very
high activity was observed compared with that seen in HepG2 and in
comparison with the other constructs in JEG-3 or the SV40 promoter
control plasmid pGL3P. The high activity of the LTR in JEG-3 and absent
activity in HepG2 agrees with the RT-PCR results, where the LTR-EBR
fusion transcripts were detected only in placenta. As an independent
control of enhancing activity of the LTR, constructs with the LTR
upstream of the SV40 promoter (pGL3P) were transfected into JEG-3.
Independent of the orientation of the LTR with respect to the SV40
promoter, a 7-10-fold increase in activity was seen relative to
constructs with the SV40 promoter alone (data not shown), suggesting
that the LTR also enhances the SV40 promoter in placental
cells.
In this study, we detected and characterized alternative
transcripts of the apoC-I and EBR genes with HERV-E sequences at their
5' termini. Both fusion transcripts are expressed in a variety of human
tissues and were shown by 5'-RACE to initiate downstream of a putative
TATA box within HERV-E LTRs, demonstrating that the LTRs are
alternative promoters for these genes in humans. For apoC-I, we found
that only a minor fraction of transcripts is derived from the LTR
promoter in liver. The significance of apoC-I in other tissues is not
known, and the general transcription levels are lower than observed in
liver. However, the LTR and native promoters appear to be equally
active in many of the other tissues tested.
In the case of EBR, it should be noted that the LTR-EBR fusion
transcript was first isolated from a placental library by Arai et
al. (29) but was considered to be a gene rearrangement or artifact
due to the LTR in the 5'-UTR, which differed from the originally
described UTR region of EBR (21, 22). In our 5'-RACE of placental
cDNA, the major form corresponded to transcript sizes derived from
the native promoter, demonstrating that this is the most abundant
transcript form in placenta. However, the semiquantitative RT-PCR
analysis using cDNA derived from different parts of the placenta
indicated that 25-30% of the total was derived from the LTR promoter,
depending on the placental cDNA used. In decidua and chorion, the
total level of amplified EBR was estimated to be higher than was seen
for the LTR and native derived amplification products combined, which
could be accounted for by the minor EDNRB Although the LTRs of the apoC-I and EBR locus are 88% identical in
sequence, the expression pattern of the two fusion transcripts is
different, where activity of the LTR-EBR is restricted to placenta and
the apoC-I LTR-derived transcripts are detected in many tissues. It is
possible that restrictive expression of the LTR-EBR transcript is due
to methylation of the HERV locus in adult tissues. Methylation is a
widely used mechanism employed by mammalian cells to restrict the
expression of unwanted gene products and retroelements (30, 31). The
apoC-I LTR may be protected from methylation and thereby expressed in
adult tissue, due to its close proximity to the native apoC-I promoter
region. Another explanation for the different transcription pattern,
although less likely, is that acquired nucleotide substitutions have
specifically destroyed or created transcription factor binding sites in
the two LTRs. The nucleotide divergence of the LTRs is probably a
direct effect of substitutions acquired after their integration into
the genome. We estimated that the LTR integrated into the EBR locus
about 30-40 million years ago and that the LTR integrated into the
apoC-I locus 20-30 million years ago. It is likely that HERV-E
elements were actively transposing in the primate lineage during this
time period, because the previously characterized HERV-E element 4-14 and the HERV-E of the pleiotrophin locus are of similar age (32, 33).
As is the case for many other HERV families, no recent integrations involving this endogenous family have been observed, indicating that
the HERV-E elements are deeply fixed in the primate lineage.
Transient transfections were performed to test the significance of the
LTRs in the genomic loci of apoC-I and EBR. Although these experiments
only monitor the extrachromosomal interactions, the results using the
apoC-I constructs supported the in vivo results, where the
LTR alone was shown not to contribute significantly to the overall
expression levels of apoC-I in liver. However, when the LTR was removed
from the apoC-I locus, the promoting activity of the apoC-I locus
dropped about 40% in HepG2 cells. This result suggests that the
presence of the LTR in the apoC-I locus contributes to the overall
activity of the native promoter region, perhaps by providing
position-dependent cis-acting elements, which work in
combination with the native regulatory sequences. The genes encoding
the three human apolipoproteins E, CI, and CII are located in a 45-kb
cluster on chromosome 19 (34) and encode proteins with the ability to
associate with lipids (35). The different apolipoproteins have distinct
roles in lipid metabolism, where apoC-I is implicated to interact with
apolipoprotein E in regulating the plasma lipid levels and in
prolonging the residence time of lipoprotein particles in the
circulation (36). Our analysis shows that all hominoids have the LTR
integrated in the apoC-I locus, but it is absent in baboon. By
introducing the LTR in the baboon apoC-I locus, we observed an
increased expression relative to that seen for the natural baboon
locus. At the time of integration, it is possible that the LTR was
tolerated by either its neutral or beneficial effect on individuals. It
is obvious that the presence of the LTR would have been selected
against if it had a strong impact on apoC-I expression, resulting in
hyperlipemic individuals (35), which has been suggested as a possible
explanation for silencing of a second apoC-I (the apoC-I') gene in
humans (37). Although both the in vivo and transfection
results suggest that the LTR has a moderate positive effect on the
expression levels of apoC-I, one possibility is that the LTR replaced
an existing function, for example the silenced second apoC-I gene.
Another possibility is that the LTR had a selective advantage when it was first acquired, for example in ensuring the export of lipoprotein to peripheral tissues, thereby maintaining important cellular functions
during periods of limited food supply.
In contrast to the LTR at the apoC-I locus, a significant portion of
the EBR transcripts is derived from the LTR promoter in placenta. The
LTR also increases the activity of the native EBR promoter region in
transient transfection experiments, suggesting that this LTR has a dual
role in acting both as promoter and enhancer for the expression of EBR
in placenta. In human placenta, endothelins (ETs) are implicated in the
fetoplacental circulation via ETB and ETA receptors, and as growth
factors of placental cells (38, 39). The role for ETs and ET receptors
in placental development is supported by studies in rats, where an
increase in ET and ETB receptor density coincides with a rapid increase
in placental growth (40), whereas elevated ET concentrations are
observed in cases of placental growth retardation (41). Although the exact biological consequences of the interactions of ETs and the ET
receptors in different parts of the placenta are complex and not well
understood, our studies show that the LTR contributes significantly to
expression of EBR. While the LTR-induced increase of EBR density in
placenta might be an evolutionary event without physiological
significance, another possibility is that an increased receptor density
would serve as a clearance for the high levels of ETs that are present
in the placenta, which in turn have implications in placental
development and uteroplacental functions.
As is exemplified in this study, the capacity of LTRs and other
retroelements to promote or, in other cases, polyadenylate genes is
easily detectable because retroelement sequences are present within the
transcript. Their enhancing potential is not readily detectable,
because the element will not be part of the transcript. Effects due to
retroelement enhancement on gene expression are likely to be more
common due to less constraint on the distance and orientation of the
element with respect to genes. It is probable that such elements have
been used as evolutionary tools in the genomes of many organisms, in
that they may enable switches in the regulation of tissue specificity
and levels of gene expression. Such genomic "retroelement
experiments" resulting in sudden biochemical changes may have played
an important role in adaptation. In humans, LTRs and other
retroelements are implicated in the evolution of tissue-specific gene
functions; for example, leptin is under control of a MER11 repeat
element that acts as an enhancer for this gene in placenta (42).
However, leptin is not expressed in mouse placenta because the MER11
element is absent in mice. Other examples where gene control elements
have evolved during primate evolution involve replacement of an
existing enhancer element (in the case of amylase) or creation of a
novel regulatory UTR region (in the case of pleiotrophin) by HERV-E
insertions (11, 12). It is intriguing that HERV-E elements are
repeatedly found involved in gene regulatory functions although these
elements are not as numerous as some other HERV families in the human
genome (7). Although a selective advantage for the LTR insertions is
not apparent, it is possible that the chromosomal location of HERV
sequences or conserved LTR functions may influence gene expression.
In summary, we have identified two HERV-E elements that mediate
increased transcription of the EBR and apoC-I genes in humans by
donation of promoter and enhancer functions from their LTRs and add to
the list where LTRs have been co-opted to serve gene regulatory functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primers used for RNA analysis
-32P, and 1.2 × 106 dpm of the labeled
primer was incubated with 5 µg of total RNA in a 10-µl solution
containing 50 mM KCl, 20 mM Tris-HCl, pH 8.4, 2.5 mM MgCl2, 0.1 mg/ml bovine serum albumin at
61 °C for 20 min. The samples were then transferred to ice, and 300 units of Superscript II and 15 units of RNase inhibitor were added to
the reaction and adjusted to a volume of 20 µl with a final
concentration of 50 mM KCl, 20 mM Tris-HCl, pH
8.4, 2.5 mM MgCl2, 0.1 mg/ml bovine serum
albumin, and 0.5 mM dNTPs. The primer extension reaction was performed by incubating samples at 25 °C for 10 min, 42 °C for 50 min, and 95 °C for 10 min. The reaction products were
separated on a 6% polyacrylamide gel containing 7 M urea
and visualized by exposing to x-ray film at
70 °C. The intensity
of the extension products were measured using the ImageQuant software
after incubation on PhosphorImager plates (Molecular Dynamics, Inc.,
Sunnyvale, CA).
1259/
1239) and EBR-NP2B (
208/
187).
Digested and purified amplification products were inserted into
KpnI/BglII-digested pGL3B. For pEBR-LTR, the
complete LTR was amplified with primers EBR-LTR1K
(5'-ggggtaccTAAGGGAGGATACCACC-3')/EBR-LTR2B (5'-GCAGCTTCTCCTGCTACAagatcttc-3') and inserted into
KpnI/BglII-digested pGL3B. pEBR-NP+LTR-S and
pEBR-NP+LTR-A were made by introducing the LTR at a distance of the
native promoter region of construct pEBR-NP. The full LTR was amplified
with LTR-specific primers, EBR-LTR1Ba
(5'-cgggatccTAAGGGAGGATACCACC-3')/EBR-LTR2Ba
(5'-GCAGCTTCTCCTGCTACAggatcccg-3'). Purified and
BamHI-digested amplification products were introduced into
the BamHI site of construct pEBR-NP, which is located 2 kb from the KpnI/BglII site on pGL3B. Constructs
introduced either in sense (LTR-S) or antisense (LTR-A) with respect to
the native EBR promoter region were isolated. The location of the
primers was based on the initiation codon at position 1260 of
GenBankTM accession number D13162. The LTR primers were
derived from GenBankTM accession number AL139002.
1271/
1249)/APO-P2B (
165/
145) and inserted into
KpnI/BglII-digested pGL3B. This construct
contains both the native and LTR promoter regions. For pAPO-LTR, the
complete LTR of the apoC-I locus was amplified with primers APO-LTR1K
(
920/
901)/APO-LTR2B (
484/
465) and introduced in the
KpnI/BglII site of pGL3B. For pAPO-P-noLTR, the
LTR was removed from the apoC-I locus by amplifying the non-LTR parts
of pAPO-P with primers APO-P1K (
1271/
1249)/APO-P3X (
941/
924)
and APO-P4X (
455/
439)/APO-P2B (
165/
145). The two amplification
products were digested with XbaI, ligated together, and
introduced after KpnI/BglII digestion into pGL3B.
This construct has the same structure as the pAPO-P except that the LTR
is absent. pAPO-P-noLTR+LTR-S and pAPO-P-noLTR+LTR-A were made by
introducing the LTR at a distance of the native apoC-I promoter region
lacking the LTR (construct pAPO-P-noLTR). The full LTR was
amplified with LTR-specific primers, APO-LTR1Ba
(
920/
901)/APO-LTR2Ba (
484/
465). Purified and
BamHI-digested amplification products were introduced into
the BamHI site of construct pAPO-P-noLTR. Constructs
introduced either in sense (LTR-S) or antisense (LTR-A) with respect to
the apoC-I promoter region were isolated. The location of the primers is with respect to the apoC-I initiation codon at position 27457 of
GenBankTM accession number AB012576.
782/
760)/APO-P2B (
160/
140) and inserted into
KpnI/BglII-digested pGL3B. For pBAPO-P+LTR,
construct pBAPO-P was amplified with primers APO-P1K
(
782/
760)/APO-P5Ba (
463/
444) and APO-P6Ba
(
435/
414)/APO-P2B (
160/
140). The two amplification products
were digested with BamHI and ligated together. This
religated fragment was inserted into pGEM-T (construct pBAPO-GEM). The
LTR that was amplified with primers APO-LTR1Ba/APO-LTR2Ba (see above) was introduced into the BamHI site of pBAPO-GEM. After
selection of LTR-positive clones, the KpnI/BglII
cassette (containing the LTR in the baboon apoC-I at the same
orthologous site as in humans) was subcloned into pGL3B. Positions of
the primers are from GenBankTM accession number L13176 and
with respect to the initiation codon of the baboon apoC-I at position 1017.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
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Fig. 1.
General structure and transcriptional
regulation mediated by LTRs. Regulatory regions are located within
the U3 region, and the transcription initiation site defines the U3/R
boundary. Polyadenylation signals are located within R, where the
polyadenylation site defines the R/U5 boundary. An SD that is used for
subgenomic splicing is located in the leader region downstream of the
5' LTR of a provirus.
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Fig. 2.
Detection of fusion transcripts by RT-PCR of
apoC-I and EBR in different human tissues. A,
upper panel, LTR-apoC-I fusion transcripts were
detected by using LTR and apoC-I exon-specific primers in RT-PCR.
Lower panel, amplification products derived from
primers detecting transcripts of the native apoC-I promoter.
B, upper panel, detection of LTR-EBR
fusion transcripts by using leader (derived from the provirus) and EBR
exon primers. Lower panel, result of
amplification using primers specific for transcripts derived from the
native EBR promoter. Primer sequences are shown in Table I. Expected
amplification product sizes were obtained for the different primer
combinations. The numbers to the left are sizes
of the DNA markers.
View larger version (66K):
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Fig. 3.
Genomic structure and transcripts forms of
apoC-I and EBR. A, the structure of genomic DNA of
apoC-I, where the position of the solitary LTR (arrow) is
shown with respect to apoC-I exons (rectangles). The native
promoter (P) is indicated upstream of exon 2B. An
asterisk indicates the start of the protein-coding region in
exon 3. Below this is a schematic illustration of three
different forms of apoC-I transcripts. The two LTR-apoC-I forms were
determined by RT-PCR and 5'-RACE. The apoC-I form derived from the
native promoter was reported previously (20). Distances are not drawn
to scale. B, DNA sequence of the promoter regions upstream
of apoC-I. The solitary LTR sequence is shown in lowercase
letters and framed. Putative TATA regions are
boxed (located upstream of exon 1 and exon 2B). Nonintronic
transcribed sequence is shown on a black
background, where exon 1 initiates in the LTR, and exon 2B
is the start site derived from the native promoter. The first
nucleotide of exon 2B and the translational start in exon 3 is
underlined. The numbers to the right
refer to exons shown in Fig. 3A. C, genomic
structure of the EBR locus. The proviral element is shown as
filled rectangles and arrows, where
the 5' and 3' LTRs and the SD in the leader are shown. The proviral
element is located about 21 kb upstream of the native promoter
(P). The translational start of EBR is indicated with an
asterisk. A schematic representation of the different forms
of EBR transcripts are shown below. The LTR-EBR fusion
transcript was determined by RT-PCR and 5'-RACE. The EBR transcript of
the native promoter was reported before (21, 22). Distances are not
drawn to scale. D, genomic sequence upstream of the
initiation sites of the two EBR transcripts. The LTR sequence is
framed and shown in lowercase type. A putative
TATA present in the LTR is boxed. Nonintronic transcribed
sequence is highlighted on a black background.
The first part of exon 1 is located in the LTR. Exon 1 is joined to
exon 2B by splicing. The splice donor is located in the proviral leader
region, and the splice acceptor (SA), defining the start of
exon 2B, is located >21 kb downstream. Transcripts derived from the
native promoter define the start of exon 2A. The first nucleotide of
exon 2B and the translational start is underlined. The
numbers to the right refer to exons shown in
C.
)
are created by initiation 560 and 940 bp upstream of the ATG codon and
alternative splicing of the 5'-UTR (23). The 5' LTR leader of the
HERV-E element is located over 20 kb from the EBR gene and is joined by
splicing to the same splice acceptor in the 5'-UTR as are the spliced
EDNRB
transcripts. The genomic organization and the structures of
the different transcripts at these two loci are shown in Fig. 3. Due to
the retroviral sequence, the fusion transcripts have partially different 5'-UTRs compared with the native forms, but all maintain the
same apoC-I and EBR coding regions.
View larger version (41K):
[in a new window]
Fig. 4.
ApoC-I primer extension analysis. An
oligonucleotide complementary to exon 3 of apoC-I (see Fig. 3) was used
to analyze the relative abundance of the different transcript forms.
Extension products of about 160 and 305 bp, corresponding to expected
sizes of the native (163-bp) and type II LTR fusion (304-bp) transcript
were detected. Sizes were estimated by comigrating DNA markers. Shown
below the gel is the relative abundance of the transcript
forms in the different tissues. All values were adjusted to the total
observed in liver (as percentages). The amounts estimated from the
LTR-apoC-I fusion transcript and the native transcript form are shown
as filled and open rectangles,
respectively.
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Fig. 5.
RT-PCR of EBR transcripts in placenta.
The relative abundance of the LTR and native transcript forms was
estimated by low cycle RT-PCR. The LTR-driven form was detected by
using primers derived from exon1/exon3, the native form with primers
from exon 2A/exon3, and the total EBR was amplified using primers
derived from exon 2B/exon3 (see Fig. 3). RT-PCR was done on cDNA of
different section of placenta: villi (V), decidua
(D), chorion (C), amnion (A). Sizes of
the DNA markers are shown to the left. Expected
amplification product sizes were obtained using the different primer
combinations. The relative abundance of the different transcript forms
is shown to the right of the gel. Total EBR levels of the
different samples are indicated with a bar.
Filled and open rectangles show the
values of the native and LTR-driven forms, respectively. All values
were adjusted to the total seen in decidua.
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Fig. 6.
Effect of the LTR on apoC-I promoter activity
in human and baboon. The native human apoC-I, baboon
(B-APOCI), and LTR fragments were inserted upstream of the
luciferase (luc) vector pGL3B. Constructs where the LTR was
removed from the human or added to the baboon apoC-I promoter region
were used as a comparison with the native constructs.
Numbers 1-7 shown on the left refer
to constructs pBAPO-P+LTR, pBAPO-P, pAPO-LTR, pAPO-P-noLTR, pAPO-P,
pGL3P, and pGL3B, respectively. On the right are the results
of the luciferase activities obtained from the different constructs
after transient transfection in HepG2. All values are normalized to
-fold activity in respect to pGL3B.
View larger version (21K):
[in a new window]
Fig. 7.
Effect of the LTR on EBR promoter
activity. The native promoter region and the LTR fragments were
inserted upstream of the promoterless luciferase (luc)
reporter vector pGL3B, as shown on the left, where the
numbers 1-6 refer to constructs pEBR-LTR,
pEBR-NP+LTR-S, pEBR-NP+LTR-A, pEBR-NP, pGL3P, and pGL3B, respectively.
Luciferase activity after transient transfection in HepG2
(black rectangles) or JEG-3 (striped
rectangles) cells, normalized to -fold activity relative to
pGL3B is shown to the right.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
forms (see "Results"),
because these transcripts are also expressed in placenta (23).
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ACKNOWLEDGEMENTS |
---|
We thank Doug Freeman, Paul Kowalski, and Holly Stamm for technical assistance.
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FOOTNOTES |
---|
* This work was supported by a grant from the Medical Research Council (MRC) of Canada with core support provided by the British Columbia Cancer Agency, by the Crafoord Foundation and the Royal Physiografic Foundation, Lund, Sweden.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a fellowship from the Swedish Cancer Foundation and
Knut and Alice Wallenberg Foundation, Sweden.
§ Supported by a studentship from the MRC of Canada.
¶ To whom correspondence should be addressed: Terry Fox Laboratory, BC Cancer Agency, 601 West 10th Ave., Vancouver, British Columbia V5Z 1L3, Canada. Tel.: 604-877-6070 (ext. 3185); Fax: 604-877-0712; E-mail: dixie@interchange.ubc.ca.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M006557200
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ABBREVIATIONS |
---|
The abbreviations used are: LTR, long terminal repeat; ERV, endogenous retrovirus; HERV, human ERV; apoC-I, apolipoprotein C-I; EBR, endothelin B receptor; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; RACE, rapid amplification of cDNA ends; kb, kilobase pair(s); bp, base pair(s); HCR, hepatic control region; SD, splice donor; ET, endothelin.
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