Vol. 176, May
No.
15,
3, 1991
AND
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
APOLIPOPROTEIN
B UPSTREAM SUPPRESSOR SITE CAN DECREASE APOLIPOPROTEIN
WHICH
Robert
S. ROSS,* Andrew
Molecular
Disease
*Department Received
March
National
Heart, Lung Bethesda,
of Medicine,
13.
: IDENTIFICATION OF AN ELEMENT B TRANSCRIPTION
C. Li, Jelfrey M. Hoeg, Uwe K. Schumacher, and H. Bryan Brewer, Jr.
Branch,
University
1116-1122
and Blood Maryland
Institute,
of California
Stephen
National
San Diego,
J. Demosky,
Institutes
La Jolla,
Jr.,
of Health,
California
I991
Elevated plasma levels of apolipoprotein B (apoB) may predispose to development of premature coronary atherosclerosis. We have identilied the first well localized domain of the apoB gene which can effect negative regulation of its transcription. This region binds trans-activating factors present only in apoB producing cell lines. Mutagenesis of this region causes up-regulation of its transcriptional activity. We have termed this element apoB upstream suppressor site (aBUSS) and its trans.activators the apoB repressor proteins (ARP). 0 1991 Academic aBUSS and ARP may play important roles in the transcriptional modulation of apoB. Press, Inc.
Summary
Atherosclerotic
coronary
States. Multiple
epidemiologic
apolipoprotcin
(apo)
Specifically,
artery studies
moictics
increased
to bc highly
concentrations (HDL)
development
of premature
atherosclerotic
modulation
ApoB kb mRNA. unique
investigated premature domains
transcript
lipoproteins
(1,2,5,6).
metabolism,
lipoproteins
incrcascd
risk
(LDL),
concentrations,
disease
can produce (s-10).
(11-13).
Reduction
of apoB
For
with
have
but therapy
for
apoB, been
Therapeutic
of a 43 kb gene (7). Transcription
mechanism
CAD.
apoA1
and HDL
editing
important
corrclatcd
levels of certain
causes of mortality
in the United
(Ip) and their
development and Lp(a)
implicated
associated
of CAD
(l-6).
as well as decreased as risk
modalities
presently
has not been
specilically
stratifies
for
exist that enable directed
toward
of the apolipoproteins.
is the product
This single
RNA
of LDL
regulation
and
is one of the leading
plasma
of low density
lipoproteins
transcriptional
(CAD)
have shown
high-density
appropriate
disease
this reason,
for negative
two isoproteins
Regulation
of apoB
biosynthesis
we have studied regulation
occurs
could
designated
impact
and intestine
apoB-100
at the transcriptional
the 5’ flanking
of apoB
in liver
and apoB-48
a 14.1
by utilizing
a
level has been only recently
on the predisposition
sequence
to produce
for development
of the apoB gene in order
of
to locate
transcription,
METHODS Plasmid Constructions: Standard techniques were used for all cloning procedures and plasmid isolations (14). The parent plasmid (pNLBCAT), from which all 5’ to 3’ deletions of the apoB regulatory unit were derived, has been previously described (15). Deletions were constructed by using convenient restriction enzyme (RE) sites in the pNLBCAT clone or by RE digestion followed by exonucleasc III/mung bean nuclease digestions utilizing protocols as recommended by the manufacturer. The 5’site of each deletional mutant was confirmed by dideoxy sequencing (16). The promoterless chloramphenicol acetyltransferase (CAT) plasmid pSVOCAT and pRSVCAT containing the Rous sarcoma virus (RSV) LTR were utilized as negative and positive controls respectively for
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1116
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
all CAT transfection (tfxn) studies (17,18). pRSVL in which the RSV LTR controls the luciferase gene was utilized to control for tfxn efticiency (19). Deletional and Substitution Mutagenesis: Deletional and substitution mutagenesis of the wild-type apoB promoter from -899 to + 121 was performed using two approaches. The parent plasmid was digested with RE that cut at unique sites surrounding the area of mutagenesis and either, 1) synthetic oligonucleotides (oligos) were synthesized, annealed and ligated into the digested apoB-CAT plasmid, or 2)oligo primers corresponding IO the desired mutation were utilized in the polymcrase chain reaction (PCR) (21,22). PCR products were then isolated and ligated into the digcstcd plasmid. Mutations wcrc confirmed via standard didcoxy scqucncing methods (16). Heterolocous Promoter Constructs: Plasmid pBLCAT2 (ATCC# 37527) containing the Herpes Simplex Virus thymidine kinase (tk) linker scanning mutant LS -115/-105 controlling the CAT gene was used (20). Doublestranded apoB oligos were ligated into the unique BamHI site in pBLCAT2. When longer regions of the apoB 5’ flanking region were to be utilized as regulatory elements, they were amplified using the PCR and cloned into pBLCAT2. All insert sequences were conlirmed with chain termination sequencing. pBLCAT3 (ATCC# 3752S), a promoterless CAT plasmid which is identical to pBLCAT2 except for the hsv/tk sequence, was used as a negative control plasmid in all pBLCAT2 studies. TtYinSfeCtiOn studies. CAT and Luciferase assays: Transient tfxn studies were performed via the calcium phosphate co-precipitation technique as previously described (15). Protein concentrations were measured on all cellular extracts by the method of Bradford (25). CAT and luciferase assays were performed as previously reported (23-24). Studies were performed in cell lines known to produce apoB (Hep G2, a human hepatoma line, and Caco-2 a colonic carcinoma line) as well as control, non-apoB producing lines KB, an epithelial carcinoma cell line, NIH-3T3 tibroblasts, and chincse hamster ovary (CHO) cells. Kadiolabeline of DNA fragments: DNA fragments to be used as probes were end-labeled with [‘“PI dATP by standard procedures (14). The labeled oligo was then used as one primer in the PCR to generate the appropriate DNA fragment and was then purilicd by preparative polyacrylamide gel electrophorcsis. All fragments were further evaluated as to scqucncc specificity using chemical cleavage DNA sequencing methods (26). Nuclear extract urenaration and eel retardation assays: Nuclear extracts from Hep G2, Caco-2, and KB cells were prepared by the method of Dignam (27). Gel mobility shift assays were performed using the method of Strauss and Varshavsky (28) utilizing 3-5 ug of poly [(dA-dT)-(dAdT)] as non-specific competitor DNA. Southwestern Rlotting: Southwestern blotting was performed by the method of Schreiber et.al. (30) utilizing endlabeled double-stranded apoB oligos as probes. DNA Sectuence analysis: DNA sequence analysis was carried out using the Fasta program on the Bionet National Computer Resource.
RESULTS Regulatory transcriptional
domains
activity
in Hcp G2 and Caco-2
in lhc apoB
AND
gene
of 5’ dclctional
mutants
lines, howcvcr
no activity
DISCUSSION
from
-899 to +121
in transient
transfection
was dctectcd
studies
in the non-apoB
identified (Fig.
by evaluation 1). CAT
producing
activity lines CHO,
of relative was detected KB or 3T3
Cat Gene
ApoB Promoter f121
-833
___
-6RR
+121
cr’
/---------I +121
-512 /
were
H
_._
+191
w
-273
+121 4
-243
f121 4 -85
+121 H -3
+121
FIGURE
1. Schematic 5’ deletional constructs and their relative transcriptional activity in Hep G2 cells. representations of the S-3 deletional constructs are depicted in the panel on the left. The location in base pairs relative to the apoB transcriptional start site is indicated by the numbers under each construct. The right panel contains an autoradiograph of a representative CAT assay from Hep G2 cells.
ApoB
1117
Vol.
176,
No.
BIOCHEMICAL
3, 1991
2 Y :: :! >” 5 id cc
2.2
-
AND
BIOPHYSICAL
COMMUNICATIONS
n
n KB 0 CACO 0 HEP G2
1.8-
RESEARCH
r
1.4l-
-899
-639
-512
SITE RELATIVE
-376
-278
-24:
TO ApoB TRANSCRIPTIONAL
START
FIGURE 2. The relative CAT activity of 5’ apoB deletional mutants transfected into Hep G2, Caco-2 and KB cells. Percent :rcetylation was determined and compared to the positive RSVCAT control. Each CAT activity was normalized for transfection efticiency and the quantity of cellular protein. Each bar represents at least 3 separate experiments with transfections of each delctional mutant within each experiment.
(data
not shown).
2). Progressive CAT
deletion
activity
apoB.
Dclctional
from
Further
allowed
moderate cxprcssion.
nuclear
protein
No.
15,
3, 1991
AND
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
APOLIPOPROTEIN
B UPSTREAM SUPPRESSOR SITE CAN DECREASE APOLIPOPROTEIN
WHICH
Robert
S. ROSS,* Andrew
Molecular
Disease
*Department Received
March
National
Heart, Lung Bethesda,
of Medicine,
13.
: IDENTIFICATION OF AN ELEMENT B TRANSCRIPTION
C. Li, Jelfrey M. Hoeg, Uwe K. Schumacher, and H. Bryan Brewer, Jr.
Branch,
University
1116-1122
and Blood Maryland
Institute,
of California
Stephen
National
San Diego,
J. Demosky,
Institutes
La Jolla,
Jr.,
of Health,
California
I991
Elevated plasma levels of apolipoprotein B (apoB) may predispose to development of premature coronary atherosclerosis. We have identilied the first well localized domain of the apoB gene which can effect negative regulation of its transcription. This region binds trans-activating factors present only in apoB producing cell lines. Mutagenesis of this region causes up-regulation of its transcriptional activity. We have termed this element apoB upstream suppressor site (aBUSS) and its trans.activators the apoB repressor proteins (ARP). 0 1991 Academic aBUSS and ARP may play important roles in the transcriptional modulation of apoB. Press, Inc.
Summary
Atherosclerotic
coronary
States. Multiple
epidemiologic
apolipoprotcin
(apo)
Specifically,
artery studies
moictics
increased
to bc highly
concentrations (HDL)
development
of premature
atherosclerotic
modulation
ApoB kb mRNA. unique
investigated premature domains
transcript
lipoproteins
(1,2,5,6).
metabolism,
lipoproteins
incrcascd
risk
(LDL),
concentrations,
disease
can produce (s-10).
(11-13).
Reduction
of apoB
For
with
have
but therapy
for
apoB, been
Therapeutic
of a 43 kb gene (7). Transcription
mechanism
CAD.
apoA1
and HDL
editing
important
corrclatcd
levels of certain
causes of mortality
in the United
(Ip) and their
development and Lp(a)
implicated
associated
of CAD
(l-6).
as well as decreased as risk
modalities
presently
has not been
specilically
stratifies
for
exist that enable directed
toward
of the apolipoproteins.
is the product
This single
RNA
of LDL
regulation
and
is one of the leading
plasma
of low density
lipoproteins
transcriptional
(CAD)
have shown
high-density
appropriate
disease
this reason,
for negative
two isoproteins
Regulation
of apoB
biosynthesis
we have studied regulation
occurs
could
designated
impact
and intestine
apoB-100
at the transcriptional
the 5’ flanking
of apoB
in liver
and apoB-48
a 14.1
by utilizing
a
level has been only recently
on the predisposition
sequence
to produce
for development
of the apoB gene in order
of
to locate
transcription,
METHODS Plasmid Constructions: Standard techniques were used for all cloning procedures and plasmid isolations (14). The parent plasmid (pNLBCAT), from which all 5’ to 3’ deletions of the apoB regulatory unit were derived, has been previously described (15). Deletions were constructed by using convenient restriction enzyme (RE) sites in the pNLBCAT clone or by RE digestion followed by exonucleasc III/mung bean nuclease digestions utilizing protocols as recommended by the manufacturer. The 5’site of each deletional mutant was confirmed by dideoxy sequencing (16). The promoterless chloramphenicol acetyltransferase (CAT) plasmid pSVOCAT and pRSVCAT containing the Rous sarcoma virus (RSV) LTR were utilized as negative and positive controls respectively for
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1116
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
all CAT transfection (tfxn) studies (17,18). pRSVL in which the RSV LTR controls the luciferase gene was utilized to control for tfxn efticiency (19). Deletional and Substitution Mutagenesis: Deletional and substitution mutagenesis of the wild-type apoB promoter from -899 to + 121 was performed using two approaches. The parent plasmid was digested with RE that cut at unique sites surrounding the area of mutagenesis and either, 1) synthetic oligonucleotides (oligos) were synthesized, annealed and ligated into the digested apoB-CAT plasmid, or 2)oligo primers corresponding IO the desired mutation were utilized in the polymcrase chain reaction (PCR) (21,22). PCR products were then isolated and ligated into the digcstcd plasmid. Mutations wcrc confirmed via standard didcoxy scqucncing methods (16). Heterolocous Promoter Constructs: Plasmid pBLCAT2 (ATCC# 37527) containing the Herpes Simplex Virus thymidine kinase (tk) linker scanning mutant LS -115/-105 controlling the CAT gene was used (20). Doublestranded apoB oligos were ligated into the unique BamHI site in pBLCAT2. When longer regions of the apoB 5’ flanking region were to be utilized as regulatory elements, they were amplified using the PCR and cloned into pBLCAT2. All insert sequences were conlirmed with chain termination sequencing. pBLCAT3 (ATCC# 3752S), a promoterless CAT plasmid which is identical to pBLCAT2 except for the hsv/tk sequence, was used as a negative control plasmid in all pBLCAT2 studies. TtYinSfeCtiOn studies. CAT and Luciferase assays: Transient tfxn studies were performed via the calcium phosphate co-precipitation technique as previously described (15). Protein concentrations were measured on all cellular extracts by the method of Bradford (25). CAT and luciferase assays were performed as previously reported (23-24). Studies were performed in cell lines known to produce apoB (Hep G2, a human hepatoma line, and Caco-2 a colonic carcinoma line) as well as control, non-apoB producing lines KB, an epithelial carcinoma cell line, NIH-3T3 tibroblasts, and chincse hamster ovary (CHO) cells. Kadiolabeline of DNA fragments: DNA fragments to be used as probes were end-labeled with [‘“PI dATP by standard procedures (14). The labeled oligo was then used as one primer in the PCR to generate the appropriate DNA fragment and was then purilicd by preparative polyacrylamide gel electrophorcsis. All fragments were further evaluated as to scqucncc specificity using chemical cleavage DNA sequencing methods (26). Nuclear extract urenaration and eel retardation assays: Nuclear extracts from Hep G2, Caco-2, and KB cells were prepared by the method of Dignam (27). Gel mobility shift assays were performed using the method of Strauss and Varshavsky (28) utilizing 3-5 ug of poly [(dA-dT)-(dAdT)] as non-specific competitor DNA. Southwestern Rlotting: Southwestern blotting was performed by the method of Schreiber et.al. (30) utilizing endlabeled double-stranded apoB oligos as probes. DNA Sectuence analysis: DNA sequence analysis was carried out using the Fasta program on the Bionet National Computer Resource.
RESULTS Regulatory transcriptional
domains
activity
in Hcp G2 and Caco-2
in lhc apoB
AND
gene
of 5’ dclctional
mutants
lines, howcvcr
no activity
DISCUSSION
from
-899 to +121
in transient
transfection
was dctectcd
studies
in the non-apoB
identified (Fig.
by evaluation 1). CAT
producing
activity lines CHO,
of relative was detected KB or 3T3
Cat Gene
ApoB Promoter f121
-833
___
-6RR
+121
cr’
/---------I +121
-512 /
were
H
_._
+191
w
-273
+121 4
-243
f121 4 -85
+121 H -3
+121
FIGURE
1. Schematic 5’ deletional constructs and their relative transcriptional activity in Hep G2 cells. representations of the S-3 deletional constructs are depicted in the panel on the left. The location in base pairs relative to the apoB transcriptional start site is indicated by the numbers under each construct. The right panel contains an autoradiograph of a representative CAT assay from Hep G2 cells.
ApoB
1117
Vol.
176,
No.
BIOCHEMICAL
3, 1991
2 Y :: :! >” 5 id cc
2.2
-
AND
BIOPHYSICAL
COMMUNICATIONS
n
n KB 0 CACO 0 HEP G2
1.8-
RESEARCH
r
1.4l-
-899
-639
-512
SITE RELATIVE
-376
-278
-24:
TO ApoB TRANSCRIPTIONAL
START
FIGURE 2. The relative CAT activity of 5’ apoB deletional mutants transfected into Hep G2, Caco-2 and KB cells. Percent :rcetylation was determined and compared to the positive RSVCAT control. Each CAT activity was normalized for transfection efticiency and the quantity of cellular protein. Each bar represents at least 3 separate experiments with transfections of each delctional mutant within each experiment.
(data
not shown).
2). Progressive CAT
deletion
activity
apoB.
Dclctional
from
Further
allowed
moderate cxprcssion.
nuclear
protein
