Negative and positive promoter elements contribute to tissue specificity of apolipoprotein B expression

Gene, 77(1989)113-121

113

Elsevier GEN 02939

Negative and positive promoter elements contribute to tissue specificity of apolipoprotein B expression (Recombinant DNA; chloramphenicol acetyltransferase assay; enhancer; silencer; low-density lipoproteins; promoter; transcriptional regulation; liver- and intestine-specific gene expression; HeLa cells; phage I vectors)

Peter Carlsson and Gunnar Bjursell Department of Medical Biochemistry, Universityof Giiteborg, S-400 33 Gijteborg (Sweden) Received

by D.T. Denhardt:

Revised:

19 December

Accepted:

4 October

1988

1988

20 December

1988

SUMMARY

Apolipoprotein B (ApoB) is a major constituent of the plasma lipoproteins. In adult mammals it is synthesized in two different tissues, liver and intestine. We have examined the promoter elements involved in determining the cell specificity of up& expression, using a c~oramphenicol acetyltr~sferase assay and the cell lines HepG2, CaCo-2 and IIeLa. The human apoB promoter contains: (i) a strong, cell-specific, positive element which can act on a heterologous promoter. This element is located between pos -111 and -33 and is built up by three subdomains, two positive and one negative; (ii) a large, negative element between pos -639 and -129, which reduces promoter activity in apoB expressing cells (HepG2 and CaCo-2) and block activation of the promoter by the SV40 enhancer in non-expressing cells (HeLa); (iii) a positive element in the noncoding part of exon 1 which retains its activity if placed upstre~ from the other regulatory elements and stimulates transcription from the simian virus 40 promoter in all three cell lines. The same sequence elements appear to be important for expression in cells of hepatic (HepG2) and intestinal (CaCo-2) origin.

In humans, two different proteins, with partly different functions, are made from the single apoB gene. ApoB-100 is a very large protein of 512 kDa which is synthesized by the liver. It is essential for the formation of triglyceride-rich VLDL and is the sole

protein component of their cholesterol-rich remnants, LDL. The intestinal species, ApoB-48, is colinear with the N-terminal half of ApoB-100. Recently, a novel RNA-editing mechanism was discovered which introduces a stop codon in the intestinal upoB transcript and results in the formation of ApoB-48 by tissue-specific truncation of translation

Correspondence

counts/mm;

INTRODUCTION

Biochemistry, Goteborg

to:

P.

Carlsson,

University

(Sweden)

Department

of Goteborg,

Tel. (31)853459;

of

Box 33031,

Medical S-400

33

Fax (31)853746.

ethidium

national

unit(s);

iipoproteins; Abbreviations:

ApoB,

apolipoprotein

ApoB; BGal, P-galactosidase; transferase;

Cm, chloramphenicol;

0378-I 119/89/$03.50

B; a@,

gene coding

for

bp, base pair(s); CAT, Cm acetyl-

0 1989 Elsevier

CoA,

coenzyme

Science Publishers

A; cpm,

B.V. (Biomedical

DMEM,

EtdBr,

kb, kilobase

nt, nucleotide(s);

pos, nt position simian

‘Dulbecco’s

bromide;

modified

FCS,

medium; IU, inter-

or 1000 bp; LDL, low-density oiigo, oligodeoxyrjbonucleotide;

relative to the start point of transcription;

virus 40; UMS,

see Wood

et al. (1986); VLDL, very-low-density

Division)

Eagle’s

fetal calf serum;

SV40,

et al. (1984) and McGeady lipoprotein(s).

114

(Powell

et al., 1987; Chen

et al., 1987). ApoB-48

on chylornicrons and lacks properties of ApoB-100.

and polyadenylation signals, was excised from pSVEcat (Herbomel et al., 1983) by HindIIIcleavage followed by filling-in of ends and partial

As one of the key proteins in mammalian lipid metabolism, ApoB is believed to play an important role in the development of atherosclerosis and coronary

EcoRI digestion. This fragment was inserted between the EcoRI and blunted BamHI sites in the polylinker of the apoB-pTZ18R recombinant. This

artery disease (Editorial, Lance& 1988). We have examined the functional elements

places 898 bp of 5’-flanking DNA and 122 bp of exon 1 in front of the CAT-coding sequence, leaving a uniqueXba1 site from the polylinker in between. To

appears in the circulation the LDL receptor-binding

of the

apoB gene promoter. To understand what governs the dual tissue specificity of apoB expression the__ behavior

of a set of promoter

different

cell lines

was

deletion

analyzed.

mutants

HepG2

in

(Aden

et al., 1979; Knowles et al., 1980) is a human hepatoma cell line which synthesizes ApoB-100 in a manner analogous to that ofthe liver; CaCo-2 (Hughes et al., 1987) is derived from a human colon carcinoma and was used as a model of intestinal apoB expression. Finally, HeLa cells were chosen as an example of a cell type that does not express apoB. In addition to deletion mutants, a number of chimeric promoter constructs, made up of parts of the SV40 early and apoB promoters, were assayed for their transcriptional activity. Promoter strength was measured as CAT activity produced transiently after transfection, when the promoter construct had been inserted upstream from the CAT-coding sequence.

MATERIALS AND METHODS

(a) Cloning the human apoB promoter region A human, genomic 1 EMBL3 library (provided by Dr. B. Servenius) was screened with a cDNA probe from the 5’ end of the human apoB mRNA (Protter et al., 1986). The insert of one positive clone was mapped with restriction enzymes, confirmed by comparison with Southern blots of genomic DNA, and the fragments surrounding the start of transcription (Protter et al., 1986) were subcloned and sequenced.

abolish background activity, scripts initiated in plasmid transcription

terminator

due to spurious transequences, the UMS

(Wood

et al., 1984; Mc-

Geady et al., 1986) was inserted above the apoB promoter. A 360-bp SstI-NcoI fragment was bluntend ligated into the filled-in Hind111 site of the polylinker; this restores the upstream Hind111 site and leaves unique SphI and PstI sites between UMS and the apoB promoter. To add upstream sequences up to pos -1800, the fragment between the Hind111 site of the polylinker and the unique ApaI site at pos -153 in pBP-898 was replaced by a 1.65-kb genomic HindIII-ApaI fragment, resulting in pBP-1800. The reference plasmid pSVep was created by exchange of the HindIII-XbaI fragment of pBP-898 for a 327-bp HindIII-StuI fragment from pSVEcat, which contains the SV40 early promoter. As a negative control plasmid the pUMScat was made by deleting the apoB promoter (SphI-XbaI) from pBP-898, so that UMS was placed immediately upstream from the CAT-coding sequence. All other constructs were either deletions or combinations of parts of these plasmids. (c) Nucleotide

sequence determinations

(b) Plasmid constructs

The 1020-bp PvuII fragment that spans pos -898 to + 122 was digested with restriction enzymes, subcloned in M13mp18 and M13mp19, and sequenced with dideoxy chain terminators (Sanger et al., 1977; Biggin et al., 1983). The G + C-rich region between pos -22 and + 120 was resolved by substitution of 7-deaza-dGTP for dGTP in the sequencing reactions. To verify plasmid constructs and define

To create the paternal apoB-CAT plasmid (pBP898), a 1020-bp PvuII fragment, spanning pos -898 to + 122, was cloned into the Hz%11 site of plasmid vector pTZ18R (Pharmacia). A 1.63-kb cassette, containing the CAT-coding region and SV40 splice

deletion breakpoints, single-stranded plasmid DNA was prepared after superinfection of plasmid-harboring Escherichia coli TG 1 (supE, thi, hsdD 5, A(lacproAB), [F’, traD36, proAB+, ZacPZAMlS]) with helper-phage M13K07 (Szczesna-Skorupa et al., 1988), and sequenced with relevant oligo primers.

115

MEM to 0.45 ml of the cell suspension,

(d) Cell culture

placed in sterile l-ml disposable The cell culture medium consisted

of DMEM

with

Electroporation

cuvettes

mixed and (Kartell).

was done with a BTX Transfector-

and 100 pg/ml of

300 with 3.5-mm electrode gap and the settings were:

streptomycin (Gibco). HepG2 and CaCo-2 were grown on plastic coated with collagen (type I, from rat tail, Sigma) and HeLa cells directly on the plastic.

1800pF, 190 V for CaCo-2, 1400 pF, 200 V for HepG2 and 1600 pF, 180 V for HeLa. The cells were

10% FCS, 100 IU/ml

of penicillin

(e) Transfections

by

Plasmids to’ be used for transfections Sephacryl S-500 (Pharmacia)

(16 x 510 mm column)

in 10 mM

were purified gel filtration Tris * HCl

pH

8.0/l mM EDTA/0.3 M NaCl. This efficiently removes low M; impurities and was found to be superior to CsCl/EtdBr banding in preparing plasmid DNA that gives high and reproducible transfection efficiencies. To each 60-mm culture dish, 10 pg CAT (pBP898) plasmid was cotransfected with 2.4 pg pRSV-/?gal (Edlund et al., 1985) using one of the following methods:

kept in the cuvette for 90 s after discharge and then transferred to a 60-mm dish containing 6 ml medium. The cells were washed 16-20 h later and supplied with fresh medium. The choice of transfection the

following

considerations

(ii) Calcium phosphate coprecipitation Precipitates were made as described (Graham and Van der Eb, 1973) with the modifications of Weber et al. (1984) and added to the medium of subconfluent cultures in 60-mm dishes. About 6 h after addition of the precipitates, the cells were shocked for 90 s with :15% glycerol in Hepes-buffered saline (Gorman, 1985) and then supplied with fresh medium. (iii, Electroporation Growing cells were trypsinized and resuspended in Opti-MEM at a concentration of 6-10 x lo6 cells/ml. Plasmid DNA was added in 50 ~1 Opti-

:

was based on

lipofection

and

Ca * phosphate coprecipitation are simple to perform on large number of samples; lipofection is more efficient and reproducible, but limited by lipofectin not being commercially available. Electroporation is, once optimized, also efficient and reproducible, but is more labor-intensive and uses more cells. As long as sufficient efficiency was obtained, the results were independent of transfection method. (f) CAT and /?-galactosidase

(I, Lipofection Plasmid DNA was diluted in 1.5 ml Opti-MEM (Gibco) and mixed with 1.5 ml Opti-MEM containing 20 pg (HeLa) or 30 pg (HepG2 and CaCo-2) lipofectin (Felgner et al., 1987; provided by Syntex Research Inc. and Life Technology Inc.). Slightly subconfluent monolayers of cells in 60-mm dishes were washed twice with serum-free medium before adding the 3-ml DNA-lipofectin complex in OptiMEM. After 3 (HeLa) to 20 (HepG2 and CaCo-2) h incubation, 3 ml of DMEM, with 10% FCS, was added and incubation continued.

method

assays

Cells were harvested 48-72 h after transfection and enzyme extracts were made by three freeze/thaw cycles (Gorman, 1985). One aliquot (40 ~1 out of 150-200 ~1 total extract volume) was taken out for the BGal assay (Miller, 1972) and the rest was treated for 15 min at 65’ C. Extract (5-50 ~1) was used in the diffusion CAT assay described by Neumann et al. (1987). [ “C]Butyryl-CoA (NEN, 100 nCi/assay), rather than acetyl-CoA, was used, the reactions were allowed to proceed at room temperature and the samples counted in a liquid scintillation counter at constant time intervals (lo-20 min) during approx. 3 h. Cpm vs. time was plotted for each sample and CAT activity calculated from the slope of the curve within its linear range. The values (cpm/h/pmol plasmid) were normalized against j?Gal activities to compensate for variations in transfection efliciency. In each experiment the activity of pSVep was defined as 100% and estimates of the other constructs’ relative activities were calculated as the mean of two to six transfections. Average experiment-to-experiment variation of the normalized values was + 20.4% (standard deviation) of the calculated means.

116

% 2

B

HeLa

n-n-ll-p 8. CaCo-2

J HepG2

10

c

HeLa

HepG2

r IlaCo--2

!1 .I

3*c; HepG2

Fig. 1. Relative CAT activities. (Panel A) Activity produced by 5’ deletions. upoB promoter fragments with their 5’ end at the specified positions and the 3’ ends at pos + 122 were inserted in front of the CAT-coding sequence. All constructs,

except -1800,

are flanked

by the UMS transcription terminator at their ii’-end. None ofthese constructs produced any detectable CAT activity (i.e.,
117

RESULTS

negative element was deleted (not shown). Since this

AND DISCUSSION

activity was eliminated

upon inclusion

(a) The apoll promoter directs CAT expression in a

assume that it reflects artefactual

cell-specific

mid sequences.

manner

When the apoB-CAT construct containing apoBsequences from pos -1800 to + 122 was transfected to HepG2

and CaCo-2

cells, high levels of CAT

Extension

detected in HeLa cells. This shows that enough information to ‘direct cell-specific expression from the is contained

within

this

reduces

region.

promoter

type. Our interpretation of these data is that a minor positive element is located around pos -96, downstream from the distal negative element, beginning below pos -111. The major positive element is situated downstream from pos -84 and the proximal negative element is jammed between these two positive elements. To test this model we made a number of 3’ deletions which were inserted upstream from the SV40 early promoter, stripped of the 72-bp enhancer. The SV40 promoter provides the TATA box and a basal activity (approx. 0.3% of pSVep in all cell types) from the 21-bp repeats. The ability of apoB sequences to stimulate the activity of this promoter in different cell lines is shown in Fig. 1B. Sequences upstream from pos -33 increase expression 15 to 20

In the initial experiments, a similar series of 5’deletions that was used did not contain the UMS transcription terminator upstream from the apoB promoter fragments. These constructs produced a low but distinct activity in HeLa cells when the distal

sequence.

the 21-bp repeats, An identical

(Panel C) Activity produced terminator

the TATA box and cap site from the early transcription

construct

without

by internal

and the CAT-coding

deletions.

sequence.

activity (i.e., < 0.1%) when transfected 72-bp enhancer

any inserted

(i.e., a 145-bp PvuII-SphI

fragment

which contains

in the apoB promoter

5’ deletions

extension

of the 5’ deletions

and the asterisked

amounts

apoB promoter

Thin lines refer to deleted regions.

from the indicated

boxes symbolize

of pSVep in each cell line.

unit). These chimeras

produced

into HeLa cells. (Panel D) Activation

upstream

by equimolar

apoB sequences

The indicated

from pos -129 to -111

tion to pos -84 increased the promoter activity twoand-a-half to three times, followed by a stepwise 30-fold reduction when sequences down to pos -69 were deleted. The remaining activity produced by the -69 and -45 constructs was only 0.7% in HepG2 and 0.2% in CaCo-2 and represents the residual expression from the TATA box (at pos -29) and sequences down to pos + 122. When the rest of the apoB promoter was deleted, UMS being located directly in front of the CAT-coding sequence, no activity (< 0.1%) could be detected in either cell

Deletion of sequences from the 5’ end down to pos -639 had little effect on expression, but further deletions resulted in a gradual increase in CAT activity. The main negative domain seems to be located between pos -261 and -129, and the -129 construct produces an activity five to six times higher than the -11800 in both HepG2 and CaCo-2 (Fig. 1A). Inspection of the nucleotide sequence in this region (Fig. 2A) reveals a dyad symmetry between pos -226 and -124. Twelve bp separate the two inverted repeats which are not identical; they differ in 9 out, of 45 bp and the upper one has a 1-nt insertion at pos -204.

contains

of 5’ deletions

had no effect, but deletion to pos -96 resulted in a dramatic drop in CAT expression: to l/lo in HepG2 and to l/zo in CaCo-2. Further extension of the dele-

Although it is difficult to make a fair comparison of a promoter’s strength in two different cell lines, the lower CAT activity observed in CaCo-2 compared to HepG2 is consistent with the relative levels of apoB expression in these two cell types. (h) A distal negative element activity in expressing cells

of plas-

(c) Two tissue-specific positive elements, one superior and one subordinate, flank a proximal negative element

expression (18% and 6.8% of pSVep, respectively) were obtained, while no activity (< 0.1%) could be

apoB promoter

of UMS we

influence

approx.

fragments

0.3 y0 relative

CAT activity

in all cell types.

were inserted between the UMS transcription

None of these constructs of the apoB promoter

one intact

were linked to the CAT-coding

72-bp repeat

produced

any detectable

by the SV40 enhancer.

and 51 bp of the second)

or (far right) directly

in front of the CAT sequence.

the SV40 enhancer.

CAT activity is expressed

CAT

The SV40

was inserted

Numbers

refer to

as y0 of that produced

118

A -890 -870 -850 -830 CTGGGCAGAGGGAGCGAGCGCTGECTCAAGTTTCCGGTGGG~TGGGCAG~~~~TAG~GAG~GG~~GATG~ATGA

-810 "-790 -770 -750 GAAGGTTCCAGATGTCTATGAGGAACATGACGTGTCCTGTCCACTACTCTGCTTT~C~~CC~~CGCCTCCCCACCACTGG -730 -710 -690 -670 AGGAAACCTAGAAGCTGGTGCAGGNYlTCCTCCTCTCAACAACCCAAGAACACTTTCCACAAGAGGGGTGGGCCCTCGGA -650 -630 -610 -590 GGTTGCTCTTCCCCAGAGGCCTCTCCTCGCTGGCGTTTCTTGAAGACAGATACTTGGACTCCTGCTGGGACCAGGCAGGC -570 -550 -530 -51u CACCCATCCTCAGGGGCAGTGACTGGTCACTCACCAGACCT~~~TG~ATCCCCCTT~~CTCT~CTGCC~~AGCACGGG~T

-250 -230 -210 -190 GGCCCCACTGAGACACAGGAAEEGCC~CGCC~~~~~A~TG~GACGCTTGGGG~GGG~~~~ACC~GGGA~CCAGC~CC -170 -150 -130 -110 TGGTGGCTGCGGCTGCATCCCAGCTGGGCCCCCTCCCCGAGGCTCTTC~G~CTC~A~A~AAGCCAGTGTAG~G~A )----- ll_~--l.."l~l--------- -- --_--_ ____

-10 ?lO TGCGGGCGCCGGCCGCGCATTCCCBLCGGGACCTG

30 50 GGGGCTGAGTGCCCTTCTCC~AGhGCCCGCC

B

i-1

-327

-261 -

-2QTATA_23 -

/I

W-P

I

mRNh

+

D +129 \

ATG --96

Fig. 2. Structure of the upoB promoter, (A) Nucleotide sequence of the upoB promoter region. Numbers refer to the transcriptional start point (= $ 1). The transcribed region is underlined and the sequence ends with the start codon ATG. Putative Spl-binding sites are boxed and the TATA motif double boxed. Dashed arrows show the positions of the inverted repeats with dashes underlining the regions of complem~nt~it~. jBf Schematic presentation of the upoB promoter. The approximate location of control elements acting positively ( t- ) or negatively ( - ) on transcription. Divergent arrows synbohze the inverted repeats in the distal negative element.

in HepG2 and CacO-2 wkhout affecting the basal level activity in HeLa cells. Even higher activity (30 to 40 times) is obtained when the greater part of the distal negative element is removed through 5’ deletion to pos -150. The expected decrease (twoand-a-half times) is observed when the minor positive and proximal negative elements are removed by extension ofthe 5’ deletion to pos -84. As soon as the 3 ’ deletions extend above pos -66, activity drops to the 0.3 y0 basal level This suggests that the minor positive element is active only when the major positive element is also present. Similarly, the two negative elements fail to influence expression if the major positive has been deleted; no variation in the basal level is observed when progressive 3’ deletions from ti~~tx

pos -85 to -241 are compared. These results support a hierarchical model in which assistant transcription factors indirectly modulate transcriptional activity, through a coordinating activator rather than on RNA polymerase II itself. If such a coordination factor binds to the major positive element, the other regulatory elements would display their natural properties, in context of the SV40 promoter, only when the major positive element is present. Combination of the results of 5’ and 3’ deletions can be used to define the borders of the major positive element. Extension of 5’ deletions from pas -84 to -78 reduce expression by one third, which sets the 5’ border at or just below -84. For expression in HepG2, 3’ deletions up to pos -66 have no signill-

119

cant effect, whereas

the further deletion

completely

the enhancement

activity.

destroys

to pos -85 of promoter

That the 19 bp from pos -84 to -66

are

enough to mediate cell-specific, transcriptional activation is proven directly by the 16-fold increase in expression observed when this fragment is inserted upstream from the SV40 early promoter (Fig. 1B). The same is true also for CaCo-2, although in this cell type 3’ deletions to pos -33 consistently activities than corresponding constructs

give higher deleted to

pos -66. (d) A general, positive element in the noncoding part of exon 1 When sequences from pos -4 to + 122 are deleted from pBP-898, an almost complete loss of CAT expression is observed in both HepG2 and CaCo-2. This deletion removes the natural cap site and the 122 bp of apoB exon 1 that would otherwise be a part of the CAT mRNA. The importance of this region for promoter function is hard to estimate, because several factors that will influence CAT expression, such as initiation efficiency, mRNA stability and translation, can be affected by mutations in it. When the corresponding region (pos. -12 to + 122) was inserted upstream from the other regulatory elements, at pas, -639, the downstream deletion was partly cured; this is shown by a six- to eight-fold increase in expression (Fig. 1C). The stimulatory effect on expression, which is independent of position, exerted by this part of exon 1, suggests that at least part of the 25-fold decrease observed when it is deleted is due to removal of a positive promoter element. To further characterize this element, the -8 to + 122 fragment was inserted between the SV40 early promoter and the CAT coding sequence, in the same orientation as in the apoB promoter. As ratios, the CAT activities produced by this construct, as against that of pSVep, were 1.31 & 0.15 for HepG2, 0.98 f 0.06 for CaCo-2 and 1.69 ? 0.27 for HeLa (mean f standard deviation). Thus the presence of apoB exon 1 sequences in the CAT transcript, when driven by a strong promoter, has little influence on CAT expression. When the 72-bp enhancer was deleted from both these plasmids, expression decreased dramatically, as expected. The ratios, however, increased to 4.2 ? 0.87 for HepG2, 3.3 + 0.53 for

TABLE

I

Comparison

ofthe putative

the consensus

Spl-binding

sites in apoB exon 1 with

sequence

Sequence a + 19 GGGGC

GGGCGG

T a Identified ware package

+ 55

(reverse)

t C + 106

(reverse)

cT

+ 115 GGGGCGGc G

+28

t GAGT

+ 64 TGGGCGGG

GGC Spl consensus

sequence b

AAT by the FitConsensus (Devereux

b From Kadonaga

program

of the UWGCG

soft-

et al., 1984).

et al. (1986).

CaCo-2 and 3.5 f 0.60 for HeLa. Since the SV40 cap site and the mRNA sequence are not changed when the enhancer is removed, attribution to a positive element of a stimulatory effect which is overshadowed if the enhancer is present seems to be the most plausible explanation. In context of the SV40 promoter, this element also enhances expression in HeLa cells. Three decanucleotide sequences that resemble the consensus of Spl binding sites are found in the non‘coding part of exon 1 (Table I). Recognition sequences for transcription factor Spl, or GC boxes, contribute to the activity of many cellular and viral promoters and are typically located close to and upstream from the transcriptional start point (Kadonaga et al., 1986). That the three GC boxes in apoB exon 1 actually bind Spl or influence transcription cannot be inferred from mere sequence comparisons, but they are likely candidates account for the positive activity of this region.

to

(e) The distal negative element contributes to tissue specificity To investigate whether the apoB promoter, when combined with a general positive element, has the potential to function in non expressing cells, we added the SV40 72-bp enhancer upstream from a number of apoB 5’ deletions. When the SV40 enhancer is positioned at pos -150, -96 or -84 the activities produced will be around 200% of that of pSVep in all cell types (Fig. 1D). In contrast the enhancer immediately in front of the CAT-coding

120

sequence produces 7 -10%) showing that the apoB promoter, if activated by a general transcriptional enhancer, is as efficient in HeLa cells as in the apoB expressing cell types. A slight decline in activity is observed

in HepG2 and CaCo-2 when the enhancer

is moved up to pos -898, but the activity is still seven to ten times greater than that produced

by the cor-

of apoB expression is a consequence of the promoter utilizing a subset of transcription factors, present in both hepatocytes and enterocytes. Alternatively, related but cell-specific factors may occupy the same promoter regions. On the supposition that some of the inscription gene activities

responding construct without enhancer. In HeLa the situation is quite different: when the enhancer is

important

moved from pos -150 to -261 (just upstream

different

from

the inverted repeat in the distal negative element) activity falls to r/6; when it is moved to pos -898 the fall is to less than I/40. Thus the distal negative element appears to block enhancer activity, or its access to the promoter, in a cell-specific manner. To maintain tissue specificity, this may be a necessary compliment to tissue-spec~c, positive elements. The promoter could otherwise be activated in the wrong cell types by enhancers in other loci, located . 9 in ~1s. Negative elements, or silencers, were originally discovered in yeast (Brand et al,, 1985) and have been found in a number of eukaryotic promoters and enhancers. When inducible or tissue-specific and closely associated with general, positive elements, the net result will be an enhancer that is inducible (Goodbourn et al., 1986) or tissue specific (Imler et al., 1987; Nir et al., 1986). Silencers can also counteract enhancers at a distance, a kind of activity that is shown by members of certain classes of middle repetitive sequences (Laimins et al., 1986; Baniahmad et al., 1987), which have been suggested as a mechanism to fence transcriptional activity within defined domains (Laimins et al., 1986). The apoB distal negative element is identified by two distinct abilities: (i) to decrease activity from the associated promoter in upobl-expressing cells (Fig. 1A) and (ii) to block enhancer activity in nonexpressing cells (Fig. 1D). Our results do not allow us to determine whether these abilities are different manifestations of the same activity, or reflect cohabitation of two distinct elements.

factors are involved in re~lation of with different tissue distribution, the

‘shared-subset’

model

mechanism positive

predicts

cooperation

in maintaining

elements

as an

specificity. The

of the upoB promoter

seem to cooperate, since promoter activity ceases if any one of them is deleted. That negative elements may be necessary to enforce this mutual dependence is illustrated by the 1: 60 difference (Fig. 1C) in expression between the -150/-84 internal deletion (removes the minor positive and proximal negative elements, 0.46% in HepG2) and the -84 5’ deletion (also removes the distal negative element, 28.8%). (g) Conclusions (1) A fragment of the human apoB gene that spans pos -1800 to + 122, relative to the transcription start point, has promoter activity in upoB-expressing cells of both hepatic and intestinal origin, but no activity in a non-expressing cell type. (2) A large negative element is located upstream from the promoter elements, from pos -129 and about 500 bp upwards. It reduces promoter activity in apoB-expressing cells and blocks the activity of a heterologous enhancer specifically in non-expressing cells. (3) The proximal region between pos -111 and -33 is a strong, positive element which can act on a heterologous promoter. It is active only in apoBexpressing cells and contains one negative and two positive subdomains. (4) The noncoding part of exon 1 contains a general positive element. Three putative Sp 1 binding sites in this region may be responsible for the activity. (5) With respect to promoter-element requirements for up&&expression, cells of intestinal and hepatic origin are very similar.

(f) The same promoter regions are important for hepatic and intestinal up& expression ACKNOWLEDGEMENTS

The similarities between HepG2 and CaCo-2 with respect to promoter sequence requirements are striking. This suggests that the dual tissue specificity

We would like to thank M. Hermansson and E. Bjdrck for excellent technical assistance, Drs. A.A.

121

Protter, M. Yaniv, G. F. Vande Woude, G. Akusj&vi and T. Edlund for sharing plasmids and Dr. B. Servenius for the human genomic library. Syntex Research Inc. and Life Technology Inc. kindly provided lipofectin. We also thank S. Enerb&ck and S.O. Olofsson for valuable discussions. This work was supported by grants from the Swedish Medical Research Council (M.F.R.), the National Swedish Board for Technical Development (S.T.U.) and King Gustaf V’s foundation.

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P., De Crombrug~e,

efficiencies

of eukaryotic

tiated cells, as assayed phenicol Prolif Hughes,

T.E., Sasak, W.V., Ordovas,

Fava, S. and Schaefer, study of intestinal

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can

C. and Renkawitz,

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be compensated

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of the chicken

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lyso-

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heavy

T.J. and Hong, G.F.: Buffer gradient

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Biochem.

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