(Received for publication, July 5, 1995)
From the
The specificity of expression in the liver of the human apolipoprotein (apo) E/C-I gene locus is determined by a hepatic control region (HCR) that is located 15 kilobases downstream of the apoE gene. DNase I footprint studies of this sequence using nuclear extracts identified a region of the HCR that is enriched in nuclear protein-binding sites. Nuclease analysis of chromatin revealed liver-specific DNase I-hypersensitive sites that were associated with this region, and additional liver-specific nuclease-sensitive sites associated with the apoE gene were identified. The HCR domain has a limited binding affinity for the nuclear scaffold. The specific domain required for liver expression was tested by ligating subfragments of the HCR to the apoE gene and examining their activity in transgenic mice. A segment of 319 nucleotides that contained several potential regulatory sequences was required for full activity of liver-specific transcription with shorter segments yielding much lower levels of expression in the liver. All constructs that contained a fully active HCR were expressed in approximately a copy-dependent manner, suggesting that transgene expression was independent of integration position. Taken together, the properties of the HCR are consistent with its function as a locus control region for the liver-specific expression of the apoE gene.
Human apolipoprotein (apo) E, a 299-amino-acid
glycoprotein of M
= 35,000(1) , is
a major component of various plasma lipoprotein classes, including
chylomicron remnants, very low density lipoproteins, and high density
lipoproteins(2, 3) . It facilitates the redistribution
of cholesterol from peripheral tissues to the liver and is required for
the receptor-mediated uptake of chylomicron
remnants(2, 4) . Although apoE is produced by many
different tissues, more than 90% of the circulating apoE in human
plasma comes from liver hepatocytes(2) . Receptor binding
defective variants of apoE having the E2 phenotype are associated with
type III hyperlipoproteinemia and premature
atherosclerosis(2) . A commonly occurring apoE variant, the E4
allele, has been linked to an enhanced occurrence of the neurofibulary
tangles and plaque deposits characteristic of Alzheimer's
disease(5, 6, 7) .
The human apoE gene is 3.6 kb in length (8, 9) and is located at the 5` end of a 45-kb cluster of apolipoprotein genes on chromosome 19, all of which have the same transcriptional orientation(10) . The apoC-I gene (4.7 kb) is located 5.3 kb downstream of the apoE gene and a 4.4-kb apoC-I` pseudogene is located 7.5 kb still further downstream. The apoC-II gene (3.3 kb) is found about 16 kb downstream of the apoC-I` pseudogene. Each of these genes contains four exons, with the introns located in similar intragenic positions, suggesting that this gene family evolved from a common ancestor(1, 9) .
Expression of the apoE gene in the liver was shown initially by Simonet et al.(11, 12) to require the presence of a distal downstream tissue-specific enhancer. Subsequent studies by Simonet et al.(12) demonstrated that this hepatic control region (HCR) was located 19 kb downstream of the transcription start site of apoE gene and 9 kb downstream of the beginning of apoC-I gene. The HCR contained the sequences necessary to direct expression of both the apoE and apoC-I genes to hepatocytes with no liver specificity contributed by the promoters of either gene(13) . Any construct that lacked the HCR was not expressed, even at low levels, in the transgenic mouse liver. Full liver-specific activity of the HCR was provided by a 0.77-kb fragment (construct LE6 in (13) ). However, the presence of a previously characterized enhancer element, that lacks tissue specificity, in the promoter of the apoE gene (14, 15) was required for transcriptional activation. These results suggested that interactions of a unique hepatocyte-specific combination of distal elements in the HCR with an activator sequence in the promoter directed the expression of the apoE/C-I/C-II locus in the liver.
The current study extends our earlier findings (13) and characterizes more precisely (16) the features of the HCR that define its activity. We find that a genomic fragment of about 300 nucleotides is required for full levels of HCR activity, that at least three nuclear protein binding sequences are involved, and that the HCR is characterized by prominent liver-specific DNase I hypersensitivity. These results further define the unique qualities of the HCR that determine liver-specific activity.
The nuclei were diluted to 10 A/ml with Buffer A (without EDTA) containing 5%
glycerol and 5 mM MgCl
. Then, 0.5-ml aliquots were
incubated on ice for 10 min with different amounts of DNase I. The
reactions were quenched by addition of 25 µl of 0.5 M EDTA, 25 µl of 5 M NaCl, 5 µl of 10% SDS, 12.5
µl of 1 M Tris, and 10 µl of 10 mg/ml proteinase K and
incubation at 50 °C for 1 h. Aliquots were extracted with phenol,
and DNA was precipitated with ethanol and then resuspended in 0.1
TE buffer. DNA was digested with appropriate restriction
enzymes, resolved by electrophoresis in a 0.8% agarose gel, and
transferred to nylon membranes. The membranes were hybridized to
radioactively labeled DNA probes at 42 °C overnight, washed to a
final stringency of 0.1
SSC (0.15 mM NaCl, 15 mM sodium citrate) and 0.1% SDS at 55 °C for 30 min, and then
examined by autoradiography.
Total cellular RNA was isolated from various tissues of transgenic
mice and examined by RNase protection analysis as described (29) followed by quantitation via the Fuji Bas1000 Bio-imaging
analyzer. The quantity of the total cellular RNA in each reaction was
monitored by comparison to the amount of mouse -actin measured by
the same assay using a mouse
-actin antisense RNA probe (Ambion,
TX). For each construct, at least two, and up to five, independent
transgenic lines were analyzed.
Figure 1: Sequence analysis of LCR region. Shaded boxes represent footprint regions. Arrows with letters indicate the TGTTTGC motif. Triangles indicate DNase I-hypersensitive sites revealed by in vitro footprinting study. The line above nucleotides 462 to 774 indicates Alu family sequence homology.
Figure 2: DNase I footprinting of HCR. Single end-labeled DNA fragments were incubated with various amounts of nuclear extracts from mouse liver or HeLa cells and digested with different amounts of DNase I. The digested DNA was extracted and resolved on sequencing gels. Four end-labeled fragments were used. Panel A, PstI-AvaII fragment, labeled on 3` end, top strand; panel B, full-length HCR region, labeled on 5` end, bottom strand; panel C, AvaII-XbaI fragment, labeled on 5` end, bottom strand; panel D, full-length HCR region, labeled on 3` end, top strand. Liver = mouse liver nuclear extract; HeLa = HeLa cell nuclear extract; G + A and G = Maxam-Gilbert reaction. Solid bars indicate footprints. Dots represent DNase I-hypersensitive sites, and their sequence locations are indicated.
DNase I-hypersensitive sites were detected at footprint regions as a consequence of nuclear protein binding ( Fig. 1and Fig. 2). These sites were abundant in the region around footprint 1. It is noteworthy that this region has four tandem copies of the TGTTTGC motif and two upstream closely related sequences, indicated by the arrow bars in Fig. 1. In all but one of these sequences, a DNase I-hypersensitive site was present in the TGTTTGT motif between the two G nucleotides at a T adjacent to a G. The most prominent hypersensitive site detected in the 3` strand (nucleotide 114) was located within one of these motifs although an unambiguous footprint was not detected here. The hypersensitive site at nucleotide 156 in footprint 1 was prominent in both DNA strands. Several DNase I hypersensitive sites were detected elsewhere in the HCR fragment where clear footprints were not observed, possibly reflecting weak or unstable protein binding. Some DNase I-hypersensitive sites were detected in the Alu family sequence region. Both footprint 5, adjacent to the Alu sequence, and footprint 6, within the Alu sequence, had nearby hypersensitive sites. Since subsequent studies (described below) showed that this region was not required for HCR activity, the significance of DNase I sensitive sites in the Alu sequence domain remains unclear.
Figure 3: Gel shift assays. Mouse liver nuclear extract or in vitro translated nuclear factors were incubated with end-labeled oligonucleotides that were synthesized according to footprint sequences and DNA binding consensus sequence of nuclear factors TF-LF2 (21) and HNF-4(22) , respectively. Unlabeled oligonucleotides were used as specific competitors. The samples were analyzed by PAGE, and autoradiograms of dried gels are shown. ML = mouse liver.
The radioactively labeled footprint 1
oligonucleotide had prominent gel-shift bands, and low levels of these
bands were still detectable in the presence of a 100-fold excess of
nonlabeled competitor. The footprint 1 sequence did not bind C/EBP,
HNF1, nor HNF4 (data not shown), but it did bind HNF3, yielding a
gel-shift band that corresponded to the major band produced by liver
extracts (Fig. 3, panel 1).
Footprint 2 contains an
identity of eight nucleotides with a sequence in the TF-LF2 element
located at nucleotide -480 in the promoter of the rat transferrin
gene(21) . In gel-shift assays, an oligonucleotide
corresponding to the transferrin regulatory sequence competed
effectively for nuclear protein binding by the HCR footprint 2 probe (Fig. 3). This result suggests that the TF-LF2 factor (or factor
family) recognizes the HCR footprint 2 sequence. The footprint 2
sequence did not bind C/EBP, HNF1, HNF3
, or HNF4 (data not
shown).
The 5` portion of footprint 3 has a limited homology to the
consensus binding sequence for HNF4(22) ; however, the HNF4
produced by an in vitro transcription/translation system bound
very weakly to the footprint 3 oligonucleotide (data not shown).
Nevertheless, the oligonucleotide of the footprint 3 sequence was an
effective competitor for HNF4 binding to its own consensus high
affinity binding sequence (22) . These results suggest that a
member of the HNF4 factor family may contribute to generating footprint
3. Although footprint 3 extended over a length of 41 nucleotides, only
one major gel-shift band was observed. The footprint 3 sequence did not
bind C/EBP, HNF1, or HNF3
(data not shown).
Gel-shift
assays confirmed the binding of a nuclear protein to the footprint 4
sequence (Fig. 3, panel 4). However, the factors HNF1,
HNF3, HNF4, and C/EBP did not bind to this element (data not
shown).
Footprint 5 of the HCR contains a GATA motif. This motif is
a core sequence in regulatory elements that bind a family of erythroid
transcription factors most closely identified with globin gene
promoters and the globin locus control
region(32, 33, 34, 35) . However,
GATA-binding factors also have been identified in nonerythroid tissue (24) . An oligonucleotide corresponding to a high affinity
GATA-1-binding site in the -globin promoter (24) was used
as a competitor in HCR gel-shift assays. The major gel-shift band
produced by the footprint 5 oligonucleotide with liver extract was
competed by the erythroid GATA-1 oligonucleotide (Fig. 3),
indicating a potential role for a GATA binding factor in HCR function.
The sequence of footprint 6 has a limited homology to the consensus
binding sequence of C/EBP(23) . Protein produced by a
C/EBP expression vector bound to the footprint 6 oligonucleotide,
yielding a band in the gel-shift assay that corresponded to one of the
liver extract-generated bands (Fig. 3). Thus, it is likely that
a member of this transcription family interacts with this sequence.
Figure 4: DNase I sensitivity of human apoE gene with upstream flanking sequence and the LE1 fragment(11) . Nuclei isolated from liver, kidney, or brain of transgenic mice bearing the HEG.LE1 constructs (13) were digested with increasing concentrations of DNase I for 10 min on ice: lanes 1-6 correspond to 0, 48, 100, 200, 800, and 1,600 units/ml DNase I. Genomic DNA from the treated nuclei then was purified and digested with restriction enzymes SacI (panels A, C, and D) and EcoRI (panel B). Samples were separated on 0.8% agarose gel then blotted onto nylon membranes. DNase I hypersensitive sites (DHS) were revealed by hybridization with a radioactively labeled probe (indicated by bars) from the ends of each of the fragments that had been derived from restriction digestions with different restriction enzymes. p indicates the parent bands. p1 and p2 indicate the parent bands due to tail-to-tail and head-to-tail integration of the transgene (present in about 70 copies). Arrows indicate DNase I-hypersensitive sites. Asterisks indicate liver specific DHS. S = SalI, Sa = SacI, E = EcoRI, H = HindIII, Sp = SphI, Ps = PstI, X = XbaI, and B = BamHI.
Figure 5: Summary of hypersensitive sites in HEG.LE1. Arrows indicate DNase I-hypersensitive sites. Asterisks indicate liver specific DHS. S = SalI, Sa = SacI, E = EcoRI, H = HindIII, Sp = SphI, Ps = PstI, X = XbaI, and B = BamHI.
To determine if tissue-specific nuclease cleavage sites were associated with the HEG.LE1 transgene, isolated intact nuclei from the liver, brain, and kidney of transgenic mice were incubated briefly with DNase I. Then, the DNA was extracted and hybridized to probes from different regions of the construct. As shown in Fig. 4, panels A-D, and summarized in Fig. 5, 20 nuclease-sensitive sites were identified in chromatin that were distributed throughout the transgene. Most sites were detected in all tissues examined. However, five sites were liver-specific: one in the proximal promoter, two in the second intron, one in the HCR domain, and one just 3` of the LE6 HCR segment. The most prominent liver-specific DNase I-sensitive sites were DHS 11 in the second intron and DHS 17 in the 5` region of the HCR. DHS 17 is located in the region of footprint 1, a sequence shown to be enriched in nuclease-sensitive sites by the footprinting analysis ( Fig. 1and Fig. 2).
Figure 6: Subfragments of the HCR. Filled ovals with numbers indicate DNA footprint locations, arrows indicate DNase I-hypersensitive sites detected by the DNA footprinting experiments. The subfragments are identified by the LES nomenclature, their locations in the LE6 HCR domain are given, and their liver-specific enhancers activities are indicated by shaded boxes.
Figure 7:
Expression pattern of HCR subfragments.
RNase protection assays with RNAs from various tissues of transgenic
mice are shown. The quality and content of the RNAs were checked by
RNase protection assays with a mouse -actin probe, with a typical
result shown in the right bottom panel. The bands shown here
correspond to the expected protected bands for human apoE mRNA and
mouse actin mRNA fragments.
As shown in Table 1, subfragments LES2, LES3, and LES7 directed similar levels of expression in the liver. This activity is equivalent to that of the reference LE1 transgenic line that includes the complete HCR domain. These results suggest that the region containing footprints 4, 5, and 6 may not be required for the full transcriptional activity of the HCR and that the Alu sequence domain may not contribute to HCR activity. Further reduction of HCR length to delete the sequences containing footprint 3 (LES12) and footprint 2 (LES11) resulted in a 4-fold to more than 10-fold reduction in HCR activity. A construct having only the 5`-terminal 118 nucleotides (LES14) showed no expression in the liver (data not shown). The LES13 fragment that deleted 72 nucleotides of the 5` portion of the HCR but retained footprints 1, 2, and 3 sequences together with nearly all of the 5` DNase I-hypersensitive sites had low activity in the liver, equivalent to that of the LES12 fragment. The LES6 construct had no detectable liver expression, and a comparison of its activity to that of the LES3 construct, in which deletion of the 5`-terminal 122 nucleotides is the only difference, suggests that this segment is essential to the activity of the HCR. Taken together, these results indicated that the minimum region required for HCR activity includes the footprint 1 sequence and the adjacent DNase I-hypersensitive regions, as contained in the LES11 fragment.
Figure 8:
Affinity of the HCR for the nuclear
scaffold. Plasmids carrying HEG.LE6 and subfragments of LE6 were
digested with HindIII and XbaI, then end-labeled with
[P]dCTP. The nuclear scaffolds were isolated
from ICR mouse liver nuclei, then digested with HindIII, BamHI, and XhoI. Probes and digested scaffolds were
incubated at 37 °C for 1 h, then pellets and supernatants were
separated by centrifugation. DNA from each fraction was extracted, then
resolved by electrophoresis in 1.0% agarose gels. The gels were dried
and autoradiographed. Samples were labeled as T = total
probe, S = 1/2 supernatant fraction, and P = pellet fraction.
Previous studies in our laboratory mapped the liver-specific HCR of the human apoE/C-I locus to a 774-bp fragment (LE6) located about 19 kb downstream of apoE promoter and about 9 kb downstream of apoC-I promoter(13) . The expression of apoE and apoC-I genes in the liver of the transgenic mice were totally dependent on this distal 3` regulatory region. In the absence of the HCR, there was no expression of the apoE gene in the liver. No sequences in the promoter, even extending 30 kb upstream, were found that conferred expression of the apoE gene (or the apoC-I gene) in the liver. The finding that all of the liver expression capability of the apoE gene is contained entirely within the downstream HCR makes this tissue specificity unique among genes that are expressed in the liver. The typical mechanism for the hepatic expression of other genes positive promoter elements that provide low levels of liver expression, with higher levels of transcription in the liver dependent upon distal enhancer sequences. The results presented here have described several distinct properties of the apoE gene HCR that contribute to its unique activity.
Nuclear protein DNase I footprint analysis and gel-shift assays of the LE6 HCR revealed several different protein families that bind to this fragment. Most of these families consist of multiple members, often present together in the same cell type, binding to DNA sequences that are characteristic of a particular family. Thus, members of the HNF3 and HNF4 transcription factor families may influence HCR activity, whereas the potential roles of the GATA-binding and the C/EBP families are unclear. Further study beyond the scope of this preliminary investigation would be required to identify the particular member in each factor family that binds to the HCR domain. The HNF3, HNF4, and C/EBP transcription factor families contribute to the liver-specific expression of many genes (i.e. most of the genes listed in Table 2). In data not shown here, we determined that the frequently used liver transcription factor HNF-1 (generated by in vitro translation of a cDNA expression vector) does not bind to any footprint sequence in the HCR. Furthermore, none of the transcription factors investigated in this study bind to footprint sequences that were previously identified (1, 15) in the apoE gene promoter (data not shown), consistent with its inability to direct gene expression to the liver(13, 14, 15) .
The unique sequence domain at the 5` end of the LE6 fragment (nucleotides 1-461) appears to constitute the functional HCR. The minimum length having full activity is a 319-nucleotide region located at the 5` terminus. This segment contains three major footprints, two of which are liver-specific, and prominent DNase I-hypersensitive character, which is also liver-specific. Analysis of subfragments of the HCR in apoE transgene constructs demonstrates that shorter fragments have a 4-fold or greater reduction in transcriptional activity. Thus, while the footprint 1 element together with adjacent nuclease-sensitive regions can direct liver-specific expression, full activity requires a sequence of about 300 nucleotides.
Three copies of the TGTTTGC motif are found in the footprint 1 sequence, and three more copies are located nearby. Five of these sequences are associated with DNase I-hypersensitive sites, and together they probably account for the prominent liver-specific nuclease hypersensitivity that was detected in whole nuclear chromatin at this location (Fig. 4, panel D, site 17). The highly conserved TGTTTGC motif is found in the promoters and/or enhancers of many genes that are expressed in the liver at moderate to high levels (Table 2). In the human transferrin gene promoter, this motif binds a transcription factor that is specific for single-stranded DNA(37) . It is of interest that the TTTG motif is conserved in the core DNA binding sequence of high mobility group (HMG) proteins(38) . The HMG proteins act by introducing pronounced bending in DNA at the binding site(39) , an action expected to result in enhanced DNase I hypersensitivity. Perhaps, the binding of nuclear proteins to the TGTTTGC motif in the footprint 1 sequence of the HCR may induce a substantial conformational change in the DNA duplex.
Nuclease-sensitive sites are a characteristic feature of transcriptionally active chromatin in eukaryotes (reviewed in (40) ), and they are associated with a wide variety of regulatory elements. We mapped 20 DNase I-hypersensitive sites in whole nuclei that were distributed throughout the HEG1.LE1 transgene, with five of them being specific to the liver. Since HEG1 alone, without an HCR domain, is not expressed in the liver(11) , it was notable to find one liver-specific site in the proximal promoter and two liver-specific DNase I-hypersensitive sites in the second intron. These regions may participate in the hepatic expression of the apoE gene, even though transgene expression data shows that the far downstream HCR is sufficient for directing liver tissue-specific transcription.
The
shortest HCR construct analyzed that resulted in full liver-specific
activity was the 319-nucleotide LES2 subfragment. A comparison to the
3.8-kb LE1 parent HCR fragment (13) indicates that transgenic
mice generated with either construct yield similar apoE mRNA levels per
gene copy number (Table 1). The range of 2-70 transgene
copies in the six independent lines examined for these two constructs
suggests that HCR activity is relatively independent of the position of
construct integration into the genome. This property is similar to the
action of the locus control region of the human -globin
gene(41, 42) .
Position-independent expression of exogenous genes in stably transfected cultured cells or transgenic mice has been shown to be a consequence of the presence of nuclear scaffold binding regions(41, 42) . The finding of limited nuclear scaffold binding character in the unique sequence portion of the HCR, but not in other regions of the HEG1 apoE gene fragments, is consistent with the observed HCR action. Perhaps sequences adjacent to this portion of the HCR are required for conferring more stringent position independence.
Our results differ from the recently reported findings of others(16) . They employed nuclear extracts from cultured hepatoma cells to identify two DNase I footprints in a fragment that corresponded to our nucleotides 191-223, but they did not detect footprint 1. They also concluded that a 154-bp fragment (corresponding to our nucleotides 78-231) constituted the liver-specific enhancer. Their use of cultured tumor cells, potential differences in nuclear extract protocols, as well as a transgene test vector with a comparatively short 5`-flanking sequence, may have led to their different conclusions regarding the nature of the HCR.
The results of our current study, taken together with previously published data(11, 12, 13) , argue that a domain of nucleotides 6-325 is required for full HCR activity. The footprint 1 sequence region is essential for HCR function, and it is associated with prominent liver-specific DNase I hypersensitivity. It also seems likely that DNase I nuclease-sensitive sites adjacent to the HCR, within the second intron, and within the proximal promoter region may facilitate HCR activity. In addition, a potential nuclear scaffold attachment capability may contribute to HCR action in directing position independent gene expression in the liver. While these additional elements may contribute to the unique character of the HCR, the required information that determines the liver-specific expression of the apoE gene is contained within the unique combination of elements that constitute the far downstream HCR.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U32510[GenBank].