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Long Terminal Repeats Are Used as Alternative Promoters for the
Endothelin B Receptor and Apolipoprotein C-I Genes in Humans*
Received for publication, July 24, 2000, and in revised form, September 28, 2000
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M006557200
Patrik Medstrand‡, Josette-Rene´ e Landry§, and Dixie L. Mager¶
From the Terry Fox Laboratory, British Columbia Cancer Agency and Department of Medical Genetics, University of
British Columbia, Vancouver, British Columbia, V5Z 1L3, Canada
To examine the potential regulatory involvement of
retroelements in the human genome, we screened the
transcribed sequences of GenBank
TM
and expressed se-
quence tag data bases with long terminal repeat (LTR)
elements derived from different human endogenous ret-
roviruses. 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 (na-
tive) promoters. Initial reverse transcription-polymer-
ase chain reaction experiments confirmed and authen-
ticated the presence of transcripts from both the native
and LTR promoters. Using a 5ⴕ-rapid amplification of
cDNA ends protocol, we showed that the alternative
transcripts of apoC-I and EBR are initiated and pro-
moted 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 ex-
pressed 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 ap-
pear 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 illus-
trates the impact of mobile elements on gene and ge-
nome evolution.
A very high proportion of mammalian DNA consists of ret-
roelements 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 sev-
eral 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 trans-
posable elements can be harmful to their host, the vast major-
ity 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 retrose-
quences, (i) the non-long terminal repeat (non-LTR)
1
retro-
posons 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 monophyl-
etic 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 en-
hancer, 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 demon-
strated that a HERV-K LTR encodes the last 67 amino acids of
one form of the leptin receptor OBR (13). These findings indi-
cate 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.
* 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. This
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 Labora-
tory, BC Cancer Agency, 601 West 10th Ave., Vancouver, British Co-
lumbia V5Z 1L3, Canada. Tel.: 604-877-6070 (ext. 3185); Fax: 604-877-
0712; E-mail: dixie@interchange.ubc.ca.
1
The abbreviations used are: LTR, long terminal repeat; ERV, en-
dogenous 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.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 3, Issue of January 19, pp. 1896–1903, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org1896
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
GenBank
TM
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.
EXPERIMENTAL PROCEDURES
Reverse Transcription and PCR Amplification—Reverse transcrip-
tion 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
g 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 tran-
script, 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 vol-
umes 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 meas-
ured 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-
l 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, comple-
mentary to exon 3 of apoC-I, was radiolabeled with
␥
-
32
P, and 1.2 ⫻10
6
dpm of the labeled primer was incubated with 5
g of total RNA in a
10-
l solution containing 50 mMKCl, 20 mMTris-HCl, pH 8.4, 2.5 mM
MgCl
2
, 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
l with a final concentration of 50 mMKCl, 20 mM
Tris-HCl, pH 8.4, 2.5 mMMgCl
2
, 0.1 mg/ml bovine serum albumin, and
0.5 mMdNTPs. The primer extension reaction was performed by incu-
bating 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 Murea and visualized by exposing to x-ray film at ⫺70 °C.
The intensity of the extension products were measured using the Im-
ageQuant software after incubation on PhosphorImager plates (Molec-
ular Dynamics, Inc., Sunnyvale, CA).
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. Prim-
ers 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 inte-
gration) and primer EBR-LTR1 (5⬘-GATCGACCCCTGACCTAACC-3⬘;
sequence from the LTR). The apoC-I and EBR primers were specified
from GenBank
TM
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 restric-
tion 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 (⫺1259/⫺1239) and EBR-NP2B
(⫺208/⫺187). Digested and purified amplification products were in-
serted into KpnI/BglII-digested pGL3B. For pEBR-LTR, the complete
LTR was amplified with primers EBR-LTR1K (5⬘-ggggtaccTAAGG-
GAGGATACCACC-3⬘)/EBR-LTR2B (5⬘-GCAGCTTCTCCTGCTACAa-
gatcttc-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⬘-GCAGCTTC-
TCCTGCTACAggatcccg-3⬘). Purified and BamHI-digested amplifica-
tion 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)
TABLE I
Primers used for RNA analysis
Primer Sequence Position
a
APO-ex1 5⬘-AGCCGCATCAAACAGAGTGAACTT-3⬘⫹157/180
APO-ex2 5⬘-TCCTCCTGCTACATTCTGAGTGG-3⬘⫺477/⫺455
APO-ex3 5⬘-ACGTGCCTTGGATAAGCTGAAG-3⬘⫹95/⫹114
APO-LTR1 5⬘-GTCTGAGGAATTTTGTCTGCGGCT-3⬘⫺500/⫺477
APO-N 5⬘-CCAAGCCCTCCAGCAAGGATTC-3⬘⫺182/⫺161
EBR-ex1 5⬘-AGTCTATGTGCTCTGAGTATTGAC-3⬘⫹571/⫹594
EBR-ex2 5⬘-GACTGGCCATTTGGAGCTGAGAT-3⬘⫹496/⫹518
EBR-ex3 5⬘-CTTCTGGAGCAGGTAGCAGCATG-3⬘⫺20/⫹3
EBR-ex4 5⬘-GACGCCACCCACTAAGACCTTATG-3⬘⫹138/⫹161
EBR-ex5 5⬘-GACGCCTTCTGGAGCAGGTAGCA-3⬘⫺25/⫺3
EBR-L1 5⬘-CATGGAGGATCAACACAGTGGCT-3⬘⫺21,500
b
EBR-N 5⬘-TTACTTTTGAGCGTGGATACTGGC-3⬘⫺166/⫺143
a
Positions relative to the first nucleotide of the translational initiation; positions upstream (⫺) in the genomic DNA or downstream (⫹)inthe
cDNA.
b
Approximate position (see “Experimental Procedures.”).
LTRs Act as Alternative Promoters for EBR and ApoC-I 1897
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 GenBank
TM
accession number D13162. The LTR primers were
derived from GenBank
TM
accession number AL139002.
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 (⫺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 prod-
ucts 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 (con-
struct pAPO-P-noLTR). The full LTR was amplified with LTR-specific
primers, APO-LTR1Ba (⫺920/⫺901)/APO-LTR2Ba (⫺484/⫺465). Puri-
fied 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 GenBank
TM
accession number AB012576.
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 (⫺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. Posi-
tions of the primers are from GenBank
TM
accession number L13176 and
with respect to the initiation codon of the baboon apoC-I at position
1017.
The HCR was isolated from human DNA using PCR and primers
HCR1Xh (5⬘-ccgctcgagTTAGAGAACAGAGCTGCAGGCT-3⬘) and
HCR2Xh (5⬘-ATGCCCCGACCCCGAAGCctcgagcgg-3⬘). The primer se-
quences were derived from positions 36815–36836, and positions
37201–37218 of GenBank
TM
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 cul-
tured 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 ⫻10
5
cells/well. Transient transfections of
HepG2 were done by cotransfecting 1.5
g of plasmid DNA and 50 ng of
pRL-TK vector (Promega) using calcium phosphate (Cellphect; Amer-
sham 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 ⫻10
5
cells/well and
cotransfected 24 h later with 1.0
g of plasmid DNA and 200 ng of
pRL-TK using 7
l 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 re-
peated 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).
RESULTS
Identification and Characterization of Chimeric Tran-
scripts—Using the strategy outlined in Fig. 1, we searched
GenBank
TM
and the human expressed sequence tag data bases
using the LTR and the leader region of published HERV ele-
ments (7). Transcripts with only the U3-R part of the LTR and
no other HERV sequence probably represent mRNA polyade-
nylated 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, suggest-
ing 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 syn-
thesized 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 ret-
roviral 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 re-
stricted 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).
FIG.1. General structure and transcriptional regulation me-
diated by LTRs. Regulatory regions are located within the U3 region,
and the transcription initiation site defines the U3/R boundary. Poly-
adenylation 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.
LTRs Act as Alternative Promoters for EBR and ApoC-I1898
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 up-
stream 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
(GenBank
TM
accession number M74509) starts 2 bp down-
stream of the initiation site of the apoC-I LTR. In the case of
the EBR fusion transcript, the sequence representing the long-
est 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 char-
acterized 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 pro-
moter (GenBank
TM
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 GenBank
TM
(ac-
cession number AL139002). The sequence of this clone is in a
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 am-
plification using primers specific for transcripts derived from the native
EBR promoter. Primer sequences are shown in Table I. Expected am-
plification product sizes were obtained for the different primer combi-
nations. The numbers to the left are sizes of the DNA markers.
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 (rectan-
gles). 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 tran-
scripts. The two LTR-apoC-I forms were determined by RT-PCR and
5⬘-RACE. The apoC-I form derived from the native promoter was re-
ported previously (20). Distances are not drawn to scale. B, DNA se-
quence 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 transla-
tional start of EBR is indicated with an asterisk. A schematic represen-
tation 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 back-
ground. 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 pro-
moter 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.
LTRs Act as Alternative Promoters for EBR and ApoC-I 1899
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⌬)
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 tran-
scripts at these two loci are shown in Fig. 3. Due to the retro-
viral 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.
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 ap-
proach, we were able to determine when the two HERV-E LTRs
integrated in the primate lineage. The apoC-I LTR was de-
tected in all hominoids, whereas Old and New World monkeys
did not have this LTR integrated in the apoC-I locus, suggest-
ing 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 diver-
gence 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 analy-
sis for either the apoC-I and EBR genes. For the apoC-I tran-
scripts, 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 ret-
roviral 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 tran-
scripts. We only detected transcripts corresponding to the
shorter, double-spliced apoC-I LTR fusion transcript. As ex-
pected, 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, indicat-
ing 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 tran-
script, we performed a low cycle PCR protocol. This was done by
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 prod-
ucts of about 160 and 305 bp, corresponding to expected sizes of the
native (163-bp) and type II LTR fusion (304-bp) transcript were de-
tected. 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.
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.
LTRs Act as Alternative Promoters for EBR and ApoC-I1900
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 expres-
sion 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 re-
porter 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 re-
gion. 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 extrachromo-
somally. 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 de-
tected 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. Inde-
pendent 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.
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 tran-
sient transfection in HepG2. All values are normalized to -fold activity
in respect to pGL3B.
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, respec-
tively. Luciferase activity after transient transfection in HepG2 (black
rectangles) or JEG-3 (striped rectangles) cells, normalized to -fold activ-
ity relative to pGL3B is shown to the right.
LTRs Act as Alternative Promoters for EBR and ApoC-I 1901
DISCUSSION
In this study, we detected and characterized alternative
transcripts of the apoC-I and EBR genes with HERV-E se-
quences at their 5⬘termini. Both fusion transcripts are ex-
pressed 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, demonstrat-
ing 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, de-
pending 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⌬forms (see “Results”), because these transcripts are
also expressed in placenta (23).
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 re-
stricted to placenta and the apoC-I LTR-derived transcripts are
detected in many tissues. It is possible that restrictive expres-
sion 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 ex-
pression 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 char-
acterized 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 signifi-
cance of the LTRs in the genomic loci of apoC-I and EBR.
Although these experiments only monitor the extrachromo-
somal 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 pres-
ence 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 combi-
nation with the native regulatory sequences. The genes encod-
ing 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 regu-
lating the plasma lipid levels and in prolonging the residence
time of lipoprotein particles in the circulation (36). Our analy-
sis 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 expres-
sion 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 obvi-
ous that the presence of the LTR would have been selected
against if it had a strong impact on apoC-I expression, result-
ing 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 pos-
itive 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 dur-
ing 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 pro-
moter in placenta. The LTR also increases the activity of the
native EBR promoter region in transient transfection experi-
ments, suggesting that this LTR has a dual role in acting both
as promoter and enhancer for the expression of EBR in pla-
centa. 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 bio-
logical 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, polyadeny-
late 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,
LTRs Act as Alternative Promoters for EBR and ApoC-I1902
in that they may enable switches in the regulation of tissue
specificity and levels of gene expression. Such genomic “retro-
element 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 al-
though 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.
Acknowledgments—We thank Doug Freeman, Paul Kowalski, and
Holly Stamm for technical assistance.
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LTRs Act as Alternative Promoters for EBR and ApoC-I 1903
