From the Genome Sciences Department, Lawrence Berkeley National Laboratory, Berkeley, California 94720
Received for publication, January 22, 2001, and in revised form, March 12, 2001
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ABSTRACT |
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The ABCA1 transporter is one of the
limiting steps in cellular cholesterol efflux. To study the expression
and activity of the human ABCA1 gene in vivo we
have examined mice containing two human BAC transgenes with different
5' ends. Mice containing a 255-kilobase (kb) BAC transgene, including
70 kb upstream of the previously defined exon 1, demonstrated a pattern
of tissue-specific expression mimicking that of the endogenous mouse
gene. Compared with macrophages from control mice, macrophages from
these transgenics had increases in apoA-I cholesterol efflux heightened
in response to increases in cell cholesterol content. The observed
increase in macrophage apoA-I-mediated cholesterol efflux was not
accompanied by alterations in plasma high density lipoprotein in the
transgenics. Although mice containing a smaller 171-kb human BAC
transgene, lacking the previously described exon 1 and
ABCA1 promoter, did not express human ABCA1 in macrophages,
they did express the human transgene in liver at levels comparable with
those of the orthologous mouse gene. Analysis by 5' rapid amplification
of cDNA ends of liver mRNA from these animals revealed a new
ABCA1 exon 1 (exon 1A) and a previously unrecognized promoter. Analysis
of human tissue revealed that exon 1A containing transcripts
accounted for a high proportion of the ABCA1 mRNAs present in
human liver. This analysis of ABCA1 transgenics showed that the
expression of human ABCA1 transgenes can result in increased
cholesterol efflux from macrophages, unaccompanied by changes in plasma
high density lipoprotein, and identified a new ABCA1
promoter in humans.
A major mechanism postulated to contribute to the
athero-protective effects of high plasma
HDL1 is reverse cholesterol
transport. This is a process whereby HDL participates in the transfer
of excess cholesterol from peripheral tissues to the liver for
catalysis (1, 2). Specifically HDL is believed to protect against the
accumulation of lipids in the vasculature by removing cholesterol and
phospholipids through an active transport pathway (3, 4). This pathway
is impaired in Tangier disease (5-7), a rare recessive disorder
characterized by the accumulation of lipids in peripheral tissues,
dramatic reductions in plasma HDL, and an increased risk for
cardiovascular disease (8, 9). Recent studies have demonstrated that
ABCA1, an ATP binding cassette transporter, plays a key role in
apolipoprotein-mediated cholesterol transport (10) and that mutations
in the ABCA1 gene are the underlying genetic defect in
Tangier disease (11-15). These results imply that the ABCA1
transporter is a rate-limiting factor in reverse cholesterol transport
and plays a key role in the protection against atherosclerosis.
Due to the importance of ABCA1 in HDL formation and
cholesterol efflux, great efforts have been made to define the genetic components and the cellular mediators determining ABCA1 expression levels. Various in vitro (10, 16-18) and in vivo
(19) studies of ABCA1 expression have shown that this gene is tightly
regulated by the cholesterol status of the cell. Studies have defined
the human ABCA1 promoter as spanning nucleotides Although both mice and humans with reduced ABCA1 have been
characterized and shown to have reduced HDL levels and decreased cellular cholesterol efflux (8, 11, 24-27), the in vivo
effect of increased expression of this gene and its regulation have not been characterized. Accordingly, we have examined transgenic mice with
two different human ABCA1-containing BAC transgenes. The mice with the
human ABCA1 transgene with more human 5' DNA demonstrated largely
appropriated regulation of the human ABCA1 transgene. Surprisingly, the
increased cholesterol efflux in macrophages was not accompanied by
changes in plasma HDL levels. Analysis of the mice with a 5' truncated
ABCA1 transgene led to the discovery of a new ABCA1 promoter
and alternatively spliced transcript that is present at high levels in
the livers of humans.
Mice--
The ABC1 transgenics examined in this study were
created and maintained in a pure FVB background as described previously
(28). The structure of the two BAC transgenes analyzed in this study is
depicted in Fig. 1. Both BAC1 and BAC2 encode the complete ABCA1
protein including the ATG methionine initiation site present in exon 2. The 255-kb BAC1 comprises the entire structural gene plus 70 kb of DNA
upstream of the 5' initiation start site and at least 35 kb of DNA
downstream of the polyadenylation signal. The 171-kb BAC2 lacks exon 1 and the 5' initiation site and contains 13 kb of DNA upstream of exon 2 and ~35 kb downstream of the polyadenylation signal. Studies of gene
expression, cholesterol efflux, and plasma HDL were performed on at
least two lines of BAC1 and BAC2 transgenic mice.
Diets and Plasma HDL Measurements--
Mice were fed either
Purina Mouse Chow (no. 5001) or a Western-type diet with 1.5 g/kg of
cholesterol (Harlan Teklad, TD 88137) for 2 and 4 weeks. Blood samples
were collected after an overnight fast, and HDL cholesterol was
measured by standard methods described previously (29).
Expression Analysis of Human and Mouse ABCA1--
Total murine
RNAs from lung, liver, spleen, proximal, medial, and distal small
intestine, and testis were extracted using the TRIZOL method (Life
Technologies, Inc., catalog no. 15596). Total RNA from human tissues
including liver, heart, lung, kidney, adrenal, intestine, brain, and
testis was purchased from CLONTECH. Reverse
transcriptions were performed using 5 µg of total RNA, random
hexamers (New England Biolabs), and Superscript II reverse transcriptase (Life Technologies, Inc.) at 42 °C for 60 min. The amount of cDNA generated was measured using the TaqMan Syber-green quantitative PCR assay with a 7700 ABI PRISM sequence detector system
(PerkinElmer Life Sciences). The primer pairs used to detect the mouse
endogenous Abca1 gene (mouse exons 2F and 4R), the human ABCA1 transgene (exons 3F and 4R), the human exon 1A transcript (exons
1AF and 2R), and the human exon 1 transcript (exons 1F and 2R) are
listed in Table I. The relative amount of ABCA1 transcript was
normalized to 18 S RNA amplification values retrieved from the same
sample. The control 18 S RNA used to correct for the total RNA amounts
in each sample was purchased from Ambion, and a ratio of 0.8:1.2 for
primers/competimers was used for all the samples. PCR was
performed with an initial hot start of 3 min at 94 °C followed by 40 cycles of 94 °C for 10 s, 57 °C for 15 s, and 72 °C
for 30 s.
Isolation of Peritoneal Macrophages--
Control and transgenic
mice were injected intraperitoneally with 5 ml of thioglycolate
solution. On the fifth day after injection, the elicited cells were
isolated by peritoneal lavage with 5 ml of sterile phosphate-buffered
saline. The cells were then pelleted by centrifugation for 10 min at
1,000 rpm and recovered in RPMI 1640 medium with 10% FBS. One million
cells/well were plated on 24-well plates. The cells were allowed to
attach to the plates for 4 h and were then washed with RPMI 1640 medium with 1% FBS.
Cholesterol Efflux--
Peritoneal macrophages were labeled with
1 µCi/ml/well of 3H-cholesterol (48 Ci/mmol) for
24 h. At the same time, cells were incubated with 0.3 mM 8-(4-chlorophenylthio)-adenosine-3':5'-cyclic monophosphate (cAMP), 50 µg of protein/ml of acetylated LDL, or 1%
FBS. Prior to cholesterol efflux measurements, cells were equilibrated (30 min at 37 °C) with 0.2% bovine serum albumin in RPMI 1640 medium followed by brief rinses with serum-free RPMI. Cells were exposed to 25 µg/ml lipid-free human apoA-I for 2, 4, 8, 14, and 22 h. Medium recovered from cells was centrifuged (2,000 × g, 10 min) to remove cellular debris, and the supernatant
was used to measure 3H-cholesterol in the medium. Cell
lipids were extracted using isopropanol, and efflux results were
expressed as a percentage of the initial cellular 3H
appearing in the medium at each time point.
5' Rapid Amplification of cDNA Ends (RACE)--
5' RACE was
performed to determine the 5' end of the human ABCA1 transgene
expressed in mouse liver. Total RNAs from the liver tissue of the BAC2
transgenic mouse were extracted using the TRIZOL method (Life
Technologies, Inc.). First strand cDNA was generated from 1 µg of
total RNA using Superscript reverse transcriptase (Life Technologies,
Inc.) at 42 °C for 60 min. An aliquot of the first strand cDNA
was then amplified using a human ABCA1-specific primer, exon 4R (Table
I), and a universal primer
(CLONTECH). PCR was performed with 40 cycles of
94 °C for 10 s, 57 °C for 15 s, and 72 °C for
30 s. The amplified products were analyzed by agarose gel
eletrophoresis and cloned into the TA cloning vector (Invitrogen) using
conditions recommended by the manufacturer. Twenty-four single colonies
were picked and sequenced from both ends using the ABI Prism BigDye
terminator cycle sequencing kit (Applied Biosystems Division, Foster
City, CA) with a 377 automated DNA-sequencing instrument (Applied
Biosystems Division).
Expression of Human ABCA1 in Mouse Tissues--
The tissue
expression patterns of two human ABCA1 transgenes have been reported by
Qiu et al. (28). We have examined several additional
tissues (Fig. 2A), and the results support the theory that BAC1 contains the DNA necessary for appropriate
tissue-specific expression of ABCA1. BAC2 contains the entire coding
sequence of human ABCA1 and 13 kb upstream of exon 2 (Fig.
1). Although this transgene lacks the
previously described promoter (20), it is expressed in liver and testis
and at very low levels in all other tissues examined (Fig.
2B).
To determine how the transgene is regulated in macrophages, we measured
the transgene expression changes in cholesterol-loaded and cAMP-treated
mouse peritoneal macrophages. Both cholesterol and cAMP analogs induce
the apolipoprotein-mediated cholesterol efflux and ABCA1 expression
(10, 16-18, 30). The BAC1 transgene was expressed in peritoneal
macrophages and induced by acetylated LDL in the same manner as the
endogenous mouse gene (Fig. 3). The
response to cAMP treatment was different for the human transgene and
the endogenous mouse gene. The human transgene was down-regulated by
the cAMP treatment, whereas the endogenous mouse gene was highly induced (Fig. 3). This may reflect differences in the kinetics of the
response between the human and mouse ABCA1 genes in
peritoneal macrophages. The response of ABCA1 to cAMP has previously
been reported to differ between mouse and human cells, with mouse cells showing a high and immediate cAMP induction and human cells showing no
induction or requiring a longer cAMP incubation time (17).
Cholesterol Efflux in Peritoneal Macrophages from BAC1
Transgenics--
To address the question of whether the expression of
the human transgene confers functional activity, we measured the efflux of cholesterol to apoA-I using peritoneal macrophages from BAC1 and
control mice. Apolipoprotein-mediated efflux was ~2-fold greater in
macrophages expressing the human ABCA1 transgene compared with controls
(Fig. 4A). Macrophages from
the transgenic and control mice responded similarly to acetylated LDL
exposures with nearly 4-fold increases in efflux compared with the
nonexposure cholesterol efflux levels (Fig. 4B). These
findings are consistent with an up-regulation of ABCA1 gene
expression with cholesterol loading of macrophages and demonstrate the
human transgene mimicking the response of the endogenous mouse
transporter.
Plasma HDL Cholesterol--
The background for investigating the
effect of the increased ABCA1 expression on plasma HDL in the
transgenics were studies demonstrating that decreased activity
of ABCA1 results in dramatic reductions in plasma HDL in mice (25-27)
and humans (8, 11, 24). The effect of the increased cholesterol efflux
noted in isolated macrophages from the BAC1 transgenic mice on plasma
HDL in these animals was assessed. Plasma HDL cholesterol in transgenic and control mice on Mouse Chow and Western diet was measured, and no
significant differences were noted on either diet (Fig. 5).
5' RACE--
To map the transcription start site of the liver
transcript, we performed 5' RACE on the total liver RNA extracted from
the BAC2 transgenics. The sequence of the 5' RACE product revealed a
transcript with an alternative exon 1 and correctly spliced exons 2, 3, and 4 (Fig. 6A). This newly
discovered exon, named exon 1A, is located 2,210 base pairs upstream of
exon 2. The exon 1A is 136 base pairs in length (Fig.
6B).
Analysis of the Promoter Sequence--
A comparison of the
sequence of exon 1A with the genomic sequence identified a conserved 5'
splice site located adjacent to this exon. Moreover, the sequence
contains the classical TATA box and a CAAT site upstream of the
initiation site, suggesting that it may be part of the promoter
responsible for the transcriptional control of exon 1A (Fig.
6B). Transfac searches (31) and visual analysis of the
promoter region have detected the presence of possible binding sites
for several sterol-sensing nuclear receptors including LXRs,
steroid and xenobiotic receptor, liver receptor homologue-1 (LRH-1),
and hepatic nuclear factor-3 (Fig. 6B). These receptors have
been shown to mediate transcriptional regulation of many genes
implicated in cholestererol homeostasis including the ABCA1
gene (32). Although these DNA motifs have not been proven to
interact with the transcription factors, they are likely targets for
studying the regulatory function of ABCA1 in liver and testis.
Expression of the Exon 1A Transcripts in Human Tissues--
In the
BAC1 transgenics, both exon 1 and exon 1A transcripts are present in
all tissues with the highest exon 1A expression level in the liver
(data not shown). The observation that BAC1 transgenic mice express
both exon 1 and exon 1A transcripts suggested that exon 1A may not be a
cryptic start site. To determine whether the exon 1A transcripts are
present in human tissues and to examine their potential physiological
relevance, we investigated the expression of exons 1 and 1A in several
human tissues (Fig. 7). Exon 1A was found
to be very abundant in human liver, whereas the exon 1 transcripts were
found to be more abundant in other peripheral tissues such as lung or
adrenals. These results indicate that exon 1A occurs naturally in
human tissues and is the predominant ABCA1 transcript in liver.
The expression pattern of the BAC1 transgene, including 70 kb of
DNA upstream of exon 1, suggests that this human transgene likely
contains many of the necessary sequences required for the proper
regulation of ABCA1 in vivo. Although the observation that expression of this transgene was associated with increased
apoA-I-mediated cholesterol efflux in peritoneal macrophages was
predicted, the finding that this was not associated with changes in
plasma HDL on two different diets was surprising. An increase in plasma
HDL was expected given the fact that both human and mice with decreased ABCA1 expression have decreased HDL values (8, 11, 24-27) and that
up-regulation of the gene by rexinoids results in increased HDL plasma
values (19).
One of the many possible explanations for the lack of changes in plasma
HDL in the ABCA1 transgenics is that the increased efflux in these
animals may be too low to impact plasma HDL. Induction of the
ABCA1 gene by rexinoids, which results in enormous increases in ABCA1 expression in macrophages, is associated with only moderate increases in plasma HDL cholesterol levels (19). Another explanation for the absence of measurable increases in plasma HDL in BAC1 transgenics relates to the fact that plasma HDL only measures the
steady-state level of this lipoprotein in plasma. The ABCA1 transgene
may also affect HDL catabolism downstream of the efflux process,
possibly in the liver, where it might compensate for the increased
efflux occurring at peripheral tissue caused by increased ABCA1
expression. Finally, it can be hypothesized that there are other
limiting parameters necessary for HDL formation such as apoA-I, which
is not concomitantly up-regulated upon ABCA1 over-expression. Through
the analysis of higher expressing ABCA1 transgenics and the crossing of
ABCA1 and apoA-I transgenics coupled with more in-depth
metabolic studies should help elucidate the relationship between
increased expression of ABCA1 and plasma HDL levels.
A surprising result of this study was the expression of the smaller
human BAC2 transgene in liver and testis despite the absence of the
previously defined ABCA1 promoter (20). The 5' RACE analysis of liver RNA from animals expressing this transgene identified a new
ABCA1 exon (exon 1A) preceded by a sequence containing the hallmarks of
a classical promoter. This new transcriptional start giving rise to a
properly spliced alternative variant was noted to be the predominant
ABCA1 form in human liver, supporting the physiological relevance of
this transcript. Other alternatively spliced variants have been
detected previously in HepG2 cells (21), but they do not correspond to
the exon 1A variant identified in the present study; and although they
change the amino acid sequence of the protein, their function has not
been further characterized. The ABCA1 liver variant described in the
present study has a different 5' untranslated region but no alterations
in its protein sequence. This variant ABCA1 may confer a new level of
regulation to the gene either at the transcriptional level, through the
use of an alternate promoter, or the translational level, with the new
5' untranslated region possibly affecting the intracellular site and
efficiency of ABCA1 translation.
Several features of the newly identified exon 1A transcription variant
suggest that it may contribute to tissue-specific control of ABCA1
expression. This includes the detection of possible LXR/RXR and
LRH-1/steroidogenic factor-1 binding sites at the 5'-flanking region of
exon 1A. LXRs are ligand-activated transcription factors that mediate
the regulation of several genes implicated in cholesterol homeostasis
(32, 33). LRH-1 is a monomeric nuclear receptor that functions as a
tissue-specific transcription factor and acts as a competence factor
mediating the liver-specific regulation of the cholesterol 7 Modification of HDL metabolism has long been considered a target for
therapeutic interventions in the prevention of atherogenesis (for a
recent review, see Ref. 38). With the discovery of ABCA1 and its impact
on HDL metabolism effecting cholesterol efflux and intestinal
absorption of dietary cholesterol (17, 19, 27), ABCA1 has become a
major target for intervention. Because of the expression of ABCA1 in
many tissues and its multiple apparent functions (25, 39-41), only
through in vivo studies can we begin to assess the relevant
physiological effects of increasing its expression on lipid homeostasis
and atherogenesis. The results of the present analysis of the in
vivo effects of the human ABCA1 transgene illustrate the
complexity of ABCA1 regulation and its impact on HDL formation and
metabolism. Analysis of the atherogenesis susceptibility of these BAC
transgenics with increases in cholesterol efflux unaccompanied by
measurable changes in plasma HDL cholesterol should provide important
insights into the utility of altering the expression of ABCA1
expression on atherogenesis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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200 to
80 upstream of exon 1 (20). This promoter contains a DR-4 element
that binds the LXR and RXR heterodimers and transactivates gene
expression when bound to oxysterols and retinoic acid (21-23). This
mechanism explains in part the tight regulation of the ABCA1
gene by sterols and supports the idea that the response of ABCA1 to
increased cholesterol leads to an accelerated clearance of cholesterol
by macrophages maintaining intracellular cholesterol homeostasis.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primers used in the TaqMan system and 5' RACE PCRs
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Map of the human ABCA1 BACs. The
schematic representation of the exon/intron structure in the human
ABCA1 gene is shown at the top. The newly
identified exon (exon 1A or E1A) is located 2,210 base pairs upstream
of exon 2. The ATG initiation codon and the sequence encoding for the
first 21 amino acids of the ABCA1 protein is located in exon 2. The
expressed sequence tags represent the open reading frames of two
identified expressed sequence tags with unassigned functions. BAC1 is
255 kb long, and it contains 70 kb upstream of exon 1 and ~35 kb
downstream of the 3' untranslated region. The 171-kb BAC2 lacks exon 1 and its promoter but contains the new exon 1A and its promoter together
with the complete coding sequence of ABCA1.
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Fig. 2.
The expression of human ABCA1
genes in transgenic mice. Total RNA was isolated from
different tissues of a BAC1 (A) and a BAC2 (B)
transgenic mouse. RNA levels of the ABCA1 genes were
measured using a TaqMan quantitative assay using mouse Abca1 primers
located in exons 2 and 4 and human ABCA1 primers in exons 3 and 4 (Table I). The relative transcript amounts were obtained by normalizing
the mouse and human ABCA1 signals to the 18 S signals retrieved from
the same tissues. The error bars indicate
the S.D. of four TaqMan measurements. Similar results were obtained
from at least two independent lines of BAC1 and BAC2 transgenic mice.
SIP, SIM, and SID are abbreviations
for the proximal, medial, and distal parts of the small intestine,
respectively.
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Fig. 3.
The expression of mouse and human ABCA1 in
peritoneal macrophages from BAC1 transgenics. Peritoneal
macrophages isolated from five BAC1 transgenic mice were isolated as
indicated under "Experimental Procedures." One million cells/well
were plated in 24-well plates, allowed to attach to the plates for
4 h, and then washed with RPMI 1640 medium with 1% FBS. Two wells
were incubated for 24 h with 1% FBS, 50 µg of protein/ml of
acetylated LDL (acLDL), or 0.3 mM
8-(4-chlorophenylthio)-adenosine-3':5'-cyclic monophosphate
(cAMP). RNA levels of the ABCA1 genes were
measured using a TaqMan quantitative assay using mouse Abca1 primers
(A) located in exons 2 and 4 and human ABCA1 primers
(B) located in exons 3 and 4. The relative transcript
amounts were calculated by normalizing the mouse and human ABCA1
signals to the 18 S signal retrieved from the same RNA. Each
column represents an average measurement of duplicate TaqMan
experiments.
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Fig. 4.
3H-Cholesterol efflux in
peritoneal macrophages from BAC1 transgenics. A,
ApoA-I-mediated cholesterol efflux. Five BAC1 ( ) and five control
mice (
) were used to isolate the peritoneal macrophages as described
under "Experimental Procedures." The percentage of cholesterol was
measured at the time intervals given after the addition of
lipid-free human apoA-I. Each number represents an average
measurement of two independent wells. B,
3H-cholesterol efflux of cells incubated with or without
acetylated LDL (AcLDL). Peritoneal macrophages were isolated
from five BAC1 (gray columns) and five control mice
(white columns). The efflux of cholesterol to apoA-I
after 24 h was measured in the presence or absence of acetylated
LDL. Each column represents an average measurement of two
independent wells for each treatment.
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Fig. 5.
Plasma HDL cholesterol in BAC1
transgenics. HDL cholesterol was measured in five 4-month-old BAC1
transgenic mice ( , three male and two female) and five littermate
control mice (
, three male and two female) on Mouse Chow diet
or Western diet for 2 and 4 weeks. Error bars indicate the
S.D. of the five samples in each group.
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Fig. 6.
Alternative splice variant in BAC2
transgenics and the sequence of exon 1A and its putative promoter.
A, the structure of the alternative splice variant found in
the liver mRNA of BAC2 transgenics. The 5' RACE product contains
exon 1A and correctly spliced exons 2, 3, and 4. B, the
sequence of exon 1A and the upstream promoter region. The exon 1A
sequence is underlined and bold. The
transcription initiation site mapped by 5' RACE PCR is indicated with
+1. The putative CAAT site, TATA box, and several possible
transcription factor binding sites are given in bold
capitals. The DNA motifs resembling DR-4- and DR-3-like elements
are possible binding sites for LXR/RXR and steroid and
xenobiotic receptor/RXR, respectively. The DNA motifs similar to the
binding sites for the liver receptor homologue-1 (LRH-1; NR5A2) and for
the steroidogenic factor-1 are indicated as LRH-RE/SF-1. The
DNA sequences resembling the binding sites for hepatic nuclear factor-3
and sterol regulatory element-binding protein are indicated as
HNF3 and SREBP, respectively.
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Fig. 7.
The expression of exons 1 and 1A in human
tissues. The relative expression of exon 1 (A) and exon
1A (B) transcripts was measured from the total RNA of
different human tissues (CLONTECH) in a TaqMan PCR
quantitative assay described under "Experimental Procedures." The
signals for each tissue were corrected for the total amount of RNA
given by the 18 S signal.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase gene by LXR
(34-36). Steroidogenic factor-1 has DNA
binding requirements similar to LRH-1 and controls the
oxysterol-mediated transactivation of genes involved in steroidogenesis and gonad development (37). The presence of these DNA motifs in the
5'-flanking region of exon 1A can potentially account for the
expression of BAC2 in liver and testis. The putative exon 1A promoter
might allow for an alternate regulation of ABCA1 by liver-specific
by-products of cholesterol catabolism and in that way contribute to the
coordinate regulation of ABCA1 in the different steps of reverse
cholesterol transport.
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ACKNOWLEDGEMENT |
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We thank Dr. Trudy Forte for providing purified apoA-I for this study.
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FOOTNOTES |
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* This work was supported by NHLBI, National Institutes of Health, Programs for Genomic Applications Grant HL66728-01, the National Institutes of Health Grant HL63897-01, the Swedish Medical Research Council and the Knut and Alice Wallenberg Stiftelse for postdoctoral fellowships (to L. B. C.), and National Institutes of Health Grant HL59483-01A2 (to J. K. B.). Research was conducted at the E. O. Lawrence Berkeley National Laboratory and performed under Department of Energy Contract DE-AC0376SF00098, University of California.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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Genome Sciences Dept., Lawrence Berkeley National Laboratory, One Cyclotron Rd., MS-84-171 Berkeley, CA 94720. Tel.: 510-486-5072; Fax: 510-486-4229; E-mail: Emrubin@lbl.gov or JFCheng{at}lbl.gov.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M100565200
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ABBREVIATIONS |
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The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP binding cassette transporter 1; kb, kilobase(s); LXR, liver X receptor; RXR, retinoid X receptor; BAC, bacterial artificial chromosomes; PCR, polymerase chain reaction; Abca1, mouse ATP binding cassette transporter 1; FBS, fetal bovine serum; LDL, low density lipoprotein; apoA-I, apolipoprotein A-I; RACE, rapid amplification of cDNA ends; LRH-1, liver receptor homologue-1.
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