(Received for publication, August 8, 1995)
From the
We have identified a second hepatic control region (HCR-2) in the human apolipoprotein (apo) E gene locus that confers liver expression of the human apoE gene in transgenic mice. This HCR-2 sequence is located 27 kilobases downstream of the apoE gene and 10 kilobases downstream of the previously described liver-specific enhancer (HCR-1). Nucleotide sequence analysis of the HCR-2 region revealed a sequence that shares 85% identity to the functional 319-base pair domain of HCR-1. To test its activity, transgenic mice were prepared with a fusion construct containing a human apoE gene fragment, which is not normally expressed in the liver, ligated to a 632-base pair region containing the HCR-2 sequence. This construct resulted in high levels of liver-specific apoE transgene expression, indicating that HCR-2 can function as a hepatic enhancer and has an activity similar to that of HCR-1. Hence, these findings suggest that there are at least two hepatic control regions, HCR-1 and HCR-2, capable of controlling the liver expression of this human apolipoprotein gene locus.
The genes encoding human apolipoprotein (apo) ()E,
apoC-I, and apoC-II are located within a 45-kb cluster on chromosome
19(1, 2) . We recently identified and characterized
another human gene in this cluster, the apoC-IV gene, that shares
structural characteristics with the other apolipoprotein genes of this
locus(3) . The human locus also contains an apoC-I`
pseudogene(4) , located between the apoC-I and apoC-II genes,
which appears to have arisen from a duplication event early in the
primate lineage, approximately 39 million years
ago(5, 6) .
The apoE, apoC-I, and apoC-II genes code for apolipoproteins that are components of plasma lipoproteins. These apolipoproteins have evolved distinct functions in lipid metabolism. Apolipoprotein E mediates lipoprotein clearance from the plasma by acting as a ligand for the low density lipoprotein receptor (7, 8) and the low density lipoprotein receptor-related protein(9) . A role for apoE in neuron growth and homeostasis, as well as the pathology of Alzheimer's disease, has been suggested by recent studies (for review, see (10) ). Apolipoprotein C-II is an essential cofactor for lipoprotein lipase and therefore has an important role in the hydrolysis of lipoprotein triglycerides(11) . The precise function of apoC-I is uncertain, but it may inhibit the apoE-mediated cellular uptake of lipoproteins(12, 13) . This would suggest a role in modulating lipoprotein catabolism. It may also function as one of several cofactors for the enzyme lecithin:cholesterol acyltransferase(14) .
The primary site of synthesis for
apolipoproteins E, C-I, and C-II is the
liver(4, 15, 16) . Previous studies indicated
that the regulatory elements required for hepatic expression of the
human apoE and apoC-I genes are contained in a specific region, known
as the hepatic control region, denoted HCR(17) . The expression
of various genomic constructs containing the human apoE and/or apoC-I
genes in transgenic mice demonstrated that a 774-bp region, located 15
and 5 kb downstream of the apoE and apoC-I genes, respectively, directs
high level liver expression of both genes(17) . More recent
studies in our laboratory have determined that full activity of the HCR
is provided by a 319-bp sequence, which contains at least three domains
that are cooperatively involved in directing high level and
liver-specific expression of the human apoE transgene. ()A
154-bp subfragment of this region has been reported to have liver
enhancer capability(18) .
We now report the identification
of a second HCR sequence (denoted HCR-2) at this locus, located
5.5 kb downstream of the apoC-I` pseudogene. HCR-2 shares 85%
homology to the functional 319-bp HCR (henceforth referred to as HCR-1)
sequence. In addition, we show that HCR-2, which appears to have arisen
from the duplication event that formed the apoC-I` pseudogene, has
retained the necessary sequences for directing high level and
liver-specific expression of human apoE mRNA in transgenic mice.
Figure 1: Alignment of nucleotide sequences. The genomic sequences of HCR-1 and a related sequence, denoted HCR-2, were aligned using the DNA alignment program in GeneWorks 2.4 (IntelliGenetics, Inc.); identical nucleotide positions are boxed. The boundaries of the Alu family elements (in reverse orientation) are indicated by an arrow below the corresponding sequences. The 5` sequence of the pC4.1 cDNA (see text and Fig. 4) is shown above HCR-2, with the homologous 173-bp sequence in boldface, and the second exon of the apoC-IV gene is shown in lower case. The consensus splice site donor sequence located in HCR-2 is shown by the bar. The relative genomic locations of the two related HCR sequences are shown in Fig. 2.
Figure 4: The HCR-related sequence of a liver-derived cDNA clone, pC4.1. The relative genomic location of the 173-bp HCR-related sequence of the partial pC4.1 cDNA is indicated by the filled box, with the exons corresponding to the human apoC-IV gene shown by the open boxes. The identified transcripts of the exons are shown by arrows.
Figure 2:
Hepatic control regions in the human
apoE/C-I/C-IV/C-II gene locus. A, the apolipoprotein gene
locus maps indicate the proposed duplication of an 10-kb fragment
that formed the present day human locus, which resulted in two copies
of both the ancestral apoC-I gene and HCR sequences. The relative
locations of the human apoE, C-I, C-II, and C-IV genes, and the apoC-I`
pseudogene are indicated by the closed boxes. All genes lie in
the same transcriptional orientation, 5`
3`. The positions of
HCR-1 and HCR-2 are shown by the closed ovals. B, the
genomic location of the 10.5-kb BamHI (B) fragment
that was examined is shown.
These results suggest
that HCR-2, which is located approximately 10 kb downstream of HCR-1
(see Fig. 2), arose from the duplication event that formed the
apoC-I` pseudogene. Comparison of the 3`-flanking nucleotide sequence
of both HCR-1 and HCR-2 showed that the sequence homology ended at two
reverse Alu sequences (indicated in Fig. 1); nonhomologous
sequences were identified immediately downstream of the Alu sequences
(data not shown). It is possible that these Alu repeats represent the
3` boundary of the duplication event, in which a 10-kb genomic
fragment containing the ancestral human apoC-I gene and HCR duplicated
to form the present day arrangement observed at this locus. The 5`
boundary of this ancestral fragment appears to be located just upstream
of the apoC-I gene or the apoC-I` pseudogene, as the nucleotide
sequence homology extends only 279 bp upstream of both genes (4) .
To determine if the HCR-2 sequence was capable of directing expression in the liver, like HCR-1(17) , a 632-bp genomic sequence containing HCR-2 was ligated to the 5` end of the apoE gene HEG1 fragment (Fig. 3). The resulting construct, HCR-2.HEG1, was used to generate transgenic mice. We examined two independent founder lines of transgenic mice for expression of the HCR-2.HEG1 apoE transgene in six different tissues, including the liver. These transgenic lines incorporated 5 and 13 copies of the HCR-2.HEG1 construct, respectively. By RNase protection analysis, each transgenic line showed the same pattern of human apoE transgene expression (Fig. 3), with both the liver and kidney expressing high levels of human apoE mRNA. The levels of transgene expression in both tissues were comparable with the expression levels that were previously reported using HEG1 constructs containing the 774-bp HCR-1 sequence(17) . Therefore, like the HCR-1 sequence, the 632-bp HCR-2 sequence can direct liver-specific and high level expression of the human apoE gene.
Figure 3: Expression of human apoE mRNA in HCR-2.HEG1 transgenic mice. Transgenic animals were generated with the construct HCR-2.HEG1, shown above. Total RNA was isolated from each tissue sample (obtained from a founder with five copies of the HCR-2.HEG1 construct); 5 µg of total cellular RNA were analyzed, and protected fragments were resolved by electrophoresis in 6% polyacrylamide gels containing 7 M urea. Autoradiograms of the dried gel following RNase protection analysis of human apoE mRNA or mouse actin mRNA in mouse liver, small intestine (jejunum), kidney, spleen, brain, and lung, and cultured HepG2 cells (tested for apoE mRNA only) are shown. The bands shown here correspond to the expected protected human apoE mRNA or mouse actin mRNA fragments (291 and 250 bp, respectively). HEG1 without HCR-2 is expressed in only the kidney, as described previously(19) . The human apoE hybridization probe does not cross-react with mouse apoE mRNA in this assay(17, 19) .
The specific roles of two HCR domains in the apoE gene locus remain to be determined. It is possible that each gene in the locus is controlled primarily by an individual HCR. Previous findings demonstrated that HCR-1 is sufficient to direct high levels of apoE and apoC-I expression in the liver(17) . Our results here indicate that HCR-2 also can function to control the expression of the apoE gene. However, the downstream location of HCR-2 suggests that this latter domain may have a more important function in directing apoC-IV and apoC-II gene expression. Alternatively, the activities of HCR-1 and HCR-2 might be combined to yield increased levels of liver expression for one or more genes.
It is noteworthy that the duplication event that gave rise to two copies of the apoC-I gene (which would include the HCR domain) was estimated to have occurred early in the primate lineage(5, 6) . The corresponding mouse (25) and rat (26) apoE gene loci lack the equivalent of the human apoC-I- pseudogene. Therefore, unlike the human locus, the mouse and rat gene loci may contain only one HCR-like domain located between the apoC-I and apoC-IV genes(27) . Thus, the liver-specific expression of the apoE gene locus may be controlled by a single HCR domain in nonprimate species.
The presence of this 173-bp sequence at the 5` end of a human liver-derived cDNA clone (pC4.1) suggests that a genomic region positioned upstream of this sequence may initiate transcription in the liver. However, the adjacent 5` sequence of HCR-2 lacks typical promoter motifs, such as TATA boxes or Sp1-binding sites (see Fig. 1), suggesting that a conventional gene promoter is not present in this region. While it is possible that this 173-bp sequence represents a distant exon of the apoC-IV gene, previous studies of both the human and mouse apoC-IV genes provide compelling evidence that this gene consists of only three exons in both species(3, 27) . RNase protection analysis of human liver total RNA using an antisense probe to the 173-bp sequence did not detect any liver RNA transcript (data not shown), indicating that this sequence is not highly expressed in the liver and that the partial cDNA insert of pC4.1 probably results from a rare transcript. Thus, the pC4.1 sequence may represent an alternative splicing product of this rare primary transcript (see Fig. 4). Similar transcripts have been reported for the cynomolgus monkey and Balb/c mouse apoC-IV and apoC-II genes, in which alternative splicing resulted in extended apoC-II transcripts that also contained exons of the apoC-IV gene(3, 27) . Since they amount to only minor amounts of the transcribed products of both genes, the functional significance of these hybrid transcripts is not clear(3, 28) .
The similarity of this 173-bp sequence to an exon is consistent with the presence of AA at its 3` end and GTAAGT at the 5` end of the adjacent intron-like region (see Fig. 1). This potential exon-intron boundary sequence (AA/GTAAGT) is similar to the consensus ((A/C)G/GT(A/G)AGT) for a splice site donor sequence(29) . Interestingly, a consensus sequence for a splice acceptor site is not found adjacent to the 5` end of the 173-bp exon, which indicates the absence of an upstream exon. It is noteworthy that the consensus splice site donor sequence is not found in the corresponding HCR-1 sequence (Fig. 1), which suggests that HCR-1 is unlikely to produce comparable transcripts (i.e. containing both HCR-1 and apoC-I` sequences).
Thus,
it appears that part of HCR-2 can direct rare transcription of a
sequence that resembles an exon. It is interesting that the
corresponding segment of HCR-1 (i.e. the region upstream of
the sequence homologous to the exon-like 173-bp region of HCR-2) is the
domain that is required for directing high level and liver-specific
expression of the human apoE gene. In addition, it has been
shown that a 154-bp segment of this 5` portion of HCR-1 can confer
transgene expression in the liver of transgenic mice(18) .
Thus, the key regulatory region of HCR-1, and possibly HCR-2, may be
functionally distinct from the 3` portion that was detected as a
transcribed sequence.
In summary, we have identified a new
regulatory sequence, HCR-2, in the human apoE/C-I/C-IV/C-II gene locus
that is closely related to HCR-1. HCR-2, like HCR-1, can function to
direct high level and liver-specific expression of the human apoE gene
in transgenic mice. Previous studies reported that no liver enhancers
were located within 30 kb of the 5`-flanking region of the apoE
gene(17) , and our preliminary analysis of apoC-II gene
constructs in transgenic mice have indicated that no liver enhancer
lies within 3 kb of its 3`-flanking sequence. Thus, there
are two HCR domains in an
80 kb genomic segment that contains four
genes that are expressed in the liver. The potential roles of the two
liver-specific control regions in this gene locus remain to be
determined. Our present findings suggest that the tissue-specific
expression of the human apoE gene and other genes at this locus may be
more complicated than previously believed.