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EBP-related proteins
Gene, 118 (1992) 231-238 0 1992 Elsevier Science
GENE
Publishers
B.V. All rights reserved.
231
0378-l 119/92/SO5.00
06604
The carbamyl phosphate synthetase promoter for C/EBP-related proteins (Recombinant
DNA;
Monique Laga&“*, Gordon C. Shorea ‘I Department
contains multiple binding sites
rat liver; footprinting)
Ing Swie Goping”,
Christopher
R. Muellerb,
Maribeth
Lazzaro”
and
of Biochemistry. McGill University, Montreal, Quebec, Canada; and ’ Cancer Research Laboratory, Queen’s Uniwr.sity, Kingston. Ontario.
Canada. Tel. 1613)545-6751 Received
by R. Rachubinski:
24 January
1992; Revised/Accepted:
14 February/21
February
1992; Received
at publishers:
18 May 1992
SUMMARY
The promoter of the gene (CPS) encoding rat carbamyl phosphate synthetase I has been mapped 5’ to a segment of about 525 nucleotides upstream from the transcription start point and, when analyzed in liver nuclear extracts, contained six well-defined protein-recognition elements, designated CPS sites Z-VI. All six elements were recognized, with varying affinities, by CAAT and enhancer-binding protein (C/EBPa) produced in bacteria. Oligodeoxyribonucleotides corresponding to CPS site II or to the C/EBPa-recognition element of the ALB promoter, site D, competed with the six CPS-promoter elements in footprinting assays. However, mutagenesis of the C/EBPr-recognition element, 5’-GTTGCAAC, at the core of site II was sufficient to abolish transactivation of the CPS promoter by C/EBPc( in co-transfected HepG2 cells. These findings indicate that the CPS promoter contains multiple recognition elements for factors with DNA-binding specificities similar to C/EBP proteins. Activation by C/EBPa, however, requires promoter site II.
INTRODUCTION
The mammalian urea-cycle enzyme, carbamyl phosphate synthetase (CPS), is an essential detoxifying enzyme that is located in the mitochondrial matrix compartment, and is
Correspondence to: Dr. G.C. Shore, Department
of Biochemistry,
McGill
University, 3655 Drummond St., McIntyre Medical Science Bldg., Montreal, Quebec H3G lY6, Canada. Tel. (514)398-7282; Fax (514)398-7384. * Present address:
I’Institut du Cancer de Montreal,
Montreal,
H2L 4M1, Canada.
Quebec
Abbreviations: C/EBP,
ALE, albumin-encoding
CAAT and enhancer-binding
synthetase
(E.C.
6.3.4.16);
strand(ed);
kb, kilobase
Hopital Notre-Dame,
Tel. (514)876-7078. gene (promoter); protein;
CPS, gene encoding or 1000 bp; LUC,
bp, base pair(s);
CPS, carbamyl rat CPS;
luciferase;
phosphate ds, double
LUC, gene en-
coding LUC; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; polymerase chain reaction; tsp, transcription start point(s).
PCR,
produced at detectable levels in only two cell types: hepatocytes and epithelial cells of the small intestinal mucosa (Ryall et al., 1985; and references therein). In the rat, expression of the CPS gene begins, in both tissues, at about day 16 of fetal development and reaches maximal (adult) levels shortly after birth (Ryall et al., 1985; Adcock et al., 1984). The enzyme is one of a cohort of proteins that contributes to the phenotype of the terminally differentiated hepatocyte; manipulations leading to hepatic dedifferentiation are usually accompanied by loss of CPS expression (Rozen et al., 1983). Here, we have identified protein-recognition elements in the CPS promoter and characterized their ability to interact with protein(s) exhibiting properties similar to those of C/EBP, a transactivator of gene transcription in liver (Friedman et al., 1989) and in a limited number of other tissues (adipose and intestine, and at lower levels in lung and skin; Birkenmeier et al., 1989). C/EBP (now called
232
+ strand
- strand
12345678
12345678 -1q3
‘7 proximal probe
Fig. 1. DNase combined
I protection
analysis
with phosphorylation
erate three probes
covering
of the CPS promoter
with T4 polynucleotide
CPS promoter
regions
in liver nuclear
extracts.
kinase and [;>-3’P]ATP
from nt -163 to t 7, -547
Restriction followed
digests of the plasmids,
by polyacrylamide-gel
to - 140. and
-547 to -235,
pGEM161CPS
clectrophorcsis,
or pGEMCPS412,
were employed
to gen-
labelled on either the ( t ) or ( - ) strand.
Adult rat liver nuclear extracts were prepared according to Gorski ct al. (1986) with the modifications described in Maire et al. (1989); various amounts of nuclear extract, indicated above each lane (pg protein), were used in DNase I protection assays which were carried out according to Lichtsteiner et al. (1987), with minor modifications; 1 fmol of probe (5000 cpm) and 500 ng ds poly(dI)-poly(dC)/2 mM Na vanadate/l mM KF were included per reaction. A pool of the reagents was combined with nuclear reactions containing less than 10 pg of extract
extracts and the binding reactions conducted for 15 min at 4°C before DNase I was added. For protein, bovine serum albumin was added to augment the quantity of protein to
233 + strand
- strand
0 0.5 2 5 1020400
Extra<
nt
2 Q.e) nt -473
-244
-412
-336 -371
-259 -441
-484
-516
12345678
12345678
-547
-140 distal probe
10 pg per reaction Sequencing reactions were performed using the same labelled probes and, together with the footprint reactions, were analyzed by electrophoresis in an 8% polyacrylamide gel containing 7 M urea to determine the positions of the protected regions, as described by Howell et al. (1989). Shown
are autoradiograms
(B) the distal probe (shown
of analyses
-547 to -235, see Fig. 2) was employed refer to positions hypersensitive
using (A) the proximal
probe (shown
as a line below the autoradiogram, to gain better resolution
relative to the tsp. The brackets
and sequence
as a line below the autoradiogram,
nt -140 to -547,
in the 5’ region of the promoter numbers
nt - 163 to + 7, see Figs. 2 and 3) and
see Fig. 2) labelled on either the + or (not shown). Numbers
on the right denote the footprint
regions.
strand.
The third probe (nt
on the left of each autoradiogram The arrowheads
indicate
DNase
I
sites.
C/EBPcc; Cao et al., 1991) is a member of a relatively complex family of proteins that exhibit diverse DNA recognition specificities (Lamb and McKnight, 1991). The immediate members of the family are designated C/EBPa, p, y, and 6 (Cao et al., 1991; Williams et al., 1991). A related factor, DBP, can substitute for C/EBPa in activating transcription of the ALB gene by binding to ALB promoter site D (Mueller et al., 1990).
RESULTS
AND DISCUSSION
(a) DNase I footprinting of the CPS promoter region Three probes were designed to cover the CPS promoter region from nt + 4 to -547 and were analyzed by DNase I protection analysis on the coding ( + ) and noncoding ( - ) strands. A total of six well-defined footprints were observed using liver nuclear extracts and were designated
234 sites I to VI (Fig. 1). The sequences derived from the reproducible results from a number of footprint analyses are given in Fig. 2. Site I (nt -79 to -97) corresponds to a CAAT motif that we had previously identified by sequence homology (Lagace et al., 1987). Site II (nt -103 to -122) was previously identified as a recognition site for a nuclear factor with properties similar to those of C/EBPa (Howell et al., 1989); methylation interference localized the contact site to the
-5.50 (+)5’-GTTTTGATTA (-)3’-CAAAACTAAT
GTGTTACCAA CACAATGGTT
TAGCTTTAAA ATCGAAATTT
ATCTGGAATG TAGACCTTAC
TAGAGTAATG ATCTCATTAC
V
VI -500
TTGTAATAAA AACATTATTT
GCTGTAGTAT CGACATCATA
ACTGCTTCAT TGACGAAGTA
TAGTAATCCT ATCATTAGGA
CTTCCCAAAT GAAGGGTTTA
-450
AATTCAGATG TTAAG’KTAC
TTT’PCTGTTT AA?.AGACXiA
ATTTGTGAAG TAAACACTTC
GAAAGTTCCA CTTTCAAGGT
GTTTCTTTAC CAAAGAAATG
-400
TGGATGTGGT ACCTACACCA
TTCCATCATC AAGGTAGTAG
TTTTCCCAAG AAAAGGGTTC
CAGAACGTGT GTCTTGCACA
CCAGGTTTGG GGTCC?LA?.CC
-350
GTTTTCTTGA CAAAAGAACT
AATCXUATA TTAGGTTTAT
TCTTCTGAAG AGAAGACTTC
TAAACGAAGC ATTTGCTTCG
ATCATTTCCA TAGTAAAGGT
A -300
ACWAGTAAA TGAGTtATTT
GCGTGTGACC CGCACACTGG
TTTTGTATTA AAAACATAAT
TTAGAGAGGT AATCTCTCCA
ACTGTCCTTC TGACAGGAAG
-250
AAATAAGCAG TTTATTCGTC
TACACCTAAG ATGTGGATTC
GCTGTTTACC CGACAAATGG
ACGCCWGGT TGCGGACCCA
CATGTTATTC GTACAATAAG
-200
AGCTACACAA TCGATGTGTT
GCAACTCTTT CGTTGAGAAA
TGATGGCTAG ACTACCGATC
CT’ECTTXT GAACGAACGA
GCAGCAGTAA CG’KGTCATT
-150
CATGATT’EC GTACTAAAGG
ACAGGAGAAG TGTCCTCTTC
GTGCCATTTG CACGGTAAAC
TTATGTTGCA AATACAACGT
ACTTGTATGA TGAACATACT
-100
CATGTCCATT GTACAGGTAA
‘XMCiWCW TGGACATCAG CCTTGTAGAG ACCTGTAGTC
CTTGGGAGGA GAACCCTCCT
GGGGCTGAGG CCCCGACTCC
-50
AGGGGAGGAG TCCCCTCCW
CTGTAGACGG GACATCTGCC
GCTATTTaAT CGATAAATTA
GGCAGAATGA CCGTCTTACT
ATGGGGAGT; TACCCCTCAG
GTGCAGTCAG
CCTTCAGCCC
CAACTGCACT
GTCTCCACAC
III
II
I
-1
+1
+51
AGCTTTCCTT
CCCACTGGTT
ACAAGCAAAT
TGGACAACM
AATCTCATGA
+101
GATATTTGTG
ATTTAATTTT
AGTCACAAAA
CATCTTCAAA
ATG
Fig. 2. Sequences
of the six footprints
on the U’S promoter
conferred
by liver nuclear extracts
region (as based on data in Fig. 1.). The heavy lines
above and below the CPS promoter line the protected were reproducibly
sequence
(LagacC et al., 1987) out-
regions on the ( + ) or ( - ) strands, respectively, that obtained by footprint analyses in liver nuclear extracts.
The 5’-3’(
+ ) and 3’-5’(-)
strands
nt numbers
are as indicated
in Fig. 1. The nt labeled
are marked
region of DNA representing the 0.S promoter quence and only the 5’-3’( + ) strand of nt +
and the corresponding
I
+ 1 is the
tsp.The
is shown as the ds seto + 143, which rcpre-
sents a portion of the transcribed sequence, is shown. Sequences in bold type represent a CAAT motif within site I (Laga& et al., 1987). The C/EBPz-recognition element within site II is underlined with dashes. Italicized
sequences
comprised
of more than one protein-binding
(b) Binding of recombinant C/EBPa CPS promoter region
site.
to multiple sites in the
The extent of binding of C/EBPa to the CPS promoter was examined using the protein produced in bacteria. The recombinant C/EBPa was in the form of a protein extract that had been partially purified by ion-exchange chromatography (Landschulz et al., 1988). It was found to protect all sites, i.e., sites I, II, III, V and VI, and half of site IV (nt -330 to -348) in footprint analyses (Fig. 3).
IV
V
kAGATCGCT
inverted repeat sequence, 5’-GTTGCAAC, located at nt -109 to -116 (Howell et al., 1989). Sites II, III, V and VI all exhibit (Fig. 2) a certain degree of similarity to a region of site D (5’-TGATTTTGTAAT) in the ALB promoter, a site that can be recognized by C/EBPz, as well as by other members of the C/EBP family (Friedman et al., 1989; Lamb and McKnight, 1991). Site IV contains an internal hypersensitive site (Fig. lB), which may mean that this region is
within sites II,III,V and VI
denote sequences related
to ALB site D (SW sections a, c and Fig. 4). A hypersensitive IV is indicated by arrowheads at nt -337 on the (+ ) strand on the ( - ) strand.
site in site and -338
(c) Competition with oligos corresponding to CPS site II and ALB site D Fig. 4 demonstrates that oligos corresponding either to site D or to site II can effectively compete with all six sites of the CPS promoter, albeit site VI was competed weakly by the oligo corresponding to site D. No such competition was seen using a nonspecific oligo of similar length (data not shown). Interestingly, both oligos (coding for ALB site D and CPS site II) can compete the entire footprint at site IV (Fig. 4), a site which is recognized only on one half by recombinant C/EBPa (Fig. 3). (d) Transactivation of the CPS promoter by C/EBPa in vivo An expression plasmid encoding C/EBPx (Friedman et al., 1989) was transiently co-transfected into HepG2 cells (which contain relatively low amounts of endogenous C/EBPa; Friedman et al., 1989) with a plasmid containing the LUC reporter gene linked to regions of the CPS promoter extending to nt -35 and -597 (containing just the TATA element or the TATA element plus footprints I-VI, respectively). Negligible promoter activity was associated with the region extending to nt -35 (Fig. 5), indicating that initiation of transcription from the promoter was absolutely dependent on the upstream activating elements. Cotransfection with optimal concentrations of the C/EBPa expression plasmid resulted in a two- to threefold stimulation of CPS promoter activity but, again, no activity was observed with the control (i.e., the -35 region). The C/EBPa recognition element at the core of site II, 5’-GTTGCAAC (Howell et al., 1989), in the CPS promoter, was mutated to ATCTAGAA. A synthetic oligo
235 - 5 10 20 40 - - - - Extract (ug) _ _ _ - - - 20200C/BBP(ng)
-
5 102040 - - - -
- - - - 20200 -
III -516-
123456789
123456789
-163 ‘7 proximal probe Fig. 3. Binding (Landschulz
of the bacterial
expression
et al., 1988) was incubated
product
distal probe
of recombinant
with the proximal
-140
547
C/EBPG( to the CPS promoter
region.
Partially
below the autoradiograms) of the CPS promoter labelled on the coding strand (see Figs. 1 and 2) at the concentrations dicated, and a standard DNase I footprint analysis (see Fig. 1) was carried out. For comparison, liver nuclear extracts (pg protein)
containing
indicated.
Other symbols
this mutation
and methods
purified
C/EBPG( from bacteria
(nt - 163 to + 7, left panel) or the distal (nt -547 to - 140, right panel) probes (shown by lines (ng of protein per reaction) inwere also used at the amounts
are as in Fig. 1.
did not bind any detectable
pro-
tein in a gel retardation assay (Goping et al., 1992). The mutation was found to have a slight inhibitory effect on the activity of the promoter in transfection assays (Fig. 5). However, the mutation at site II abolished C/EBPadependent stimulation of the promoter (Fig. 5), suggesting that site II may be essential for mediating the C/EBPc( response in transfected cells. (e) Conclusions (I) The CPS promoter contains (at least) six proteinbinding elements, all of which can be recognized by factors
with DNA-recognition properties similar to those of C/EBPa. (2) Transactivation of the CPS promoter by C/EBPa in co-transfected HepG2 cells (as well as in rat Reuber H35 cells; data not shown) was abolished by a mutation of the C/EBPcc-recognition element, 5’-GTTGCAAC, at the core of site II. Although the extent of transactivation by C/EBPcr was relatively low compared to corresponding stimulation of the ALB promoter in HepG2 cells (e.g., see Friedman et al., 1989), it was comparable to the levels seen for the promoters of the genes encoding phosphoenolpyruvate carboxykinase (Park et al., 1990) and alcohol dehy-
A
_
-541
-484 -
_ - 10 50 250
20
and methods
elements
J
-140
1 VI
are as in Fig. 1.
out using the coding ( + ) strand
tq an oligo corresponding
distal nrohe
1234567
Ic
-
5 10
““_
_
-
were carried
of the C‘PS promoter
I
Site D (molar e
Extract(w)
of liver nuclear extracts
Other svmbols
in the presence
comoetitor.
analyses
competition
proximal probe
- 20 100500
20
B
510
-163 ‘;/
- 10100
20
proximal probe
123456
_ _ -
-
-516-
(pg)
-547
Site cn iolar e
Extract
-
20
I
distal probe
123456
c
5 10
-140
either to site D of the rat ALB promoter or to site II of the C‘PS promoter. (A) Standard DNase I proteclion 163 to + 7. left panel) and distal (nt -547 to -140, right panel) probes of the of both the proximal (nt 1989) of the rat ALB promoter (27.mer; nt -93 to -115 plus a HirzdIlI site overhang) was added CPS promoter (see Figs. 1 and 2). except that unlabelled ds oligo coding for site D (Friedman et al., to the reaction as competitor, at a molar excess indicated above the autoradiogram. (B) Same as A but using a 30-mer ds oligo coding for site II of the CPS promoter (nt -98 LO - 127. see Fig. 2) as
-163 ‘7
1234567
_
Fig. 4. Footprint
_
-510
237 drogenase
(Stewart
et al.,
1991).
Transactivation
C/EBPa, therefore, appears to require site II. (3) Although the remaining sites share similar
2.5
recognition properties it is also known that sequences, including McKnight, 1991). It sites can be occupied cell.
by protein
with site II and can bind C/EBPa, C/EBPa can recognize diverse DNA those for other factors (Lamb and is likely, therefore, that these binding as well by other factors in the intact
ACKNOWLEDGEMENTS
We are very grateful to S.L. McKnight and colleagues (Baltimore) for recombinant C/EBPa and the C/EBPcr expression plasmid. We thank M. Therien, J. Drouin and B.W. Howell for helpful suggestions throughout this work. This study was supported by operating grants from the National Cancer Institute of Canada and Medical Research Council, and by studentships from these agencies to I.S.G. and M.L., respectively.
Fold induction by C/EBP,
TATA
-35M
1
2.7
Fig. 5. Effects of C/EBPz moter
activity
promoter
on wild-type
in co-transfected
regions extending
(mm) CPS pro-
(wt) and mutant
HepG2
cells. Plasmids
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(Friedman
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were prepared
lysates
containing
48 h after the addition
of
(deWet et al., 1987) as follows. Cell
final concentrations
of 0.5”;
(W/V) NP-40/10
mM
Tris,HCl pH 7.8, were prepared and LUC activity was measured in an automated luminometer (LKB, Uppsala, Sweden). The reaction was initiated by injection
of 1.6 vol of a mixture to give final concentrations
0.66 mM luciferinl.6 were recorded. emission
mM ATP/3.3
1.0 relative unit of LUC activity corresponds
units/100
pg protein.
tained from two independent extending to nt -35 or -597
of
mM MgCI,. The peak light emissions
Shown
are an average
to 184 light
of the results ob-
experiments. A diagram of the constructs (Fig. 2) is given below the histogram. The
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in wt
(designated
mut
(shaded
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GENE
Publishers
B.V. All rights reserved.
231
0378-l 119/92/SO5.00
06604
The carbamyl phosphate synthetase promoter for C/EBP-related proteins (Recombinant
DNA;
Monique Laga&“*, Gordon C. Shorea ‘I Department
contains multiple binding sites
rat liver; footprinting)
Ing Swie Goping”,
Christopher
R. Muellerb,
Maribeth
Lazzaro”
and
of Biochemistry. McGill University, Montreal, Quebec, Canada; and ’ Cancer Research Laboratory, Queen’s Uniwr.sity, Kingston. Ontario.
Canada. Tel. 1613)545-6751 Received
by R. Rachubinski:
24 January
1992; Revised/Accepted:
14 February/21
February
1992; Received
at publishers:
18 May 1992
SUMMARY
The promoter of the gene (CPS) encoding rat carbamyl phosphate synthetase I has been mapped 5’ to a segment of about 525 nucleotides upstream from the transcription start point and, when analyzed in liver nuclear extracts, contained six well-defined protein-recognition elements, designated CPS sites Z-VI. All six elements were recognized, with varying affinities, by CAAT and enhancer-binding protein (C/EBPa) produced in bacteria. Oligodeoxyribonucleotides corresponding to CPS site II or to the C/EBPa-recognition element of the ALB promoter, site D, competed with the six CPS-promoter elements in footprinting assays. However, mutagenesis of the C/EBPr-recognition element, 5’-GTTGCAAC, at the core of site II was sufficient to abolish transactivation of the CPS promoter by C/EBPc( in co-transfected HepG2 cells. These findings indicate that the CPS promoter contains multiple recognition elements for factors with DNA-binding specificities similar to C/EBP proteins. Activation by C/EBPa, however, requires promoter site II.
INTRODUCTION
The mammalian urea-cycle enzyme, carbamyl phosphate synthetase (CPS), is an essential detoxifying enzyme that is located in the mitochondrial matrix compartment, and is
Correspondence to: Dr. G.C. Shore, Department
of Biochemistry,
McGill
University, 3655 Drummond St., McIntyre Medical Science Bldg., Montreal, Quebec H3G lY6, Canada. Tel. (514)398-7282; Fax (514)398-7384. * Present address:
I’Institut du Cancer de Montreal,
Montreal,
H2L 4M1, Canada.
Quebec
Abbreviations: C/EBP,
ALE, albumin-encoding
CAAT and enhancer-binding
synthetase
(E.C.
6.3.4.16);
strand(ed);
kb, kilobase
Hopital Notre-Dame,
Tel. (514)876-7078. gene (promoter); protein;
CPS, gene encoding or 1000 bp; LUC,
bp, base pair(s);
CPS, carbamyl rat CPS;
luciferase;
phosphate ds, double
LUC, gene en-
coding LUC; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; polymerase chain reaction; tsp, transcription start point(s).
PCR,
produced at detectable levels in only two cell types: hepatocytes and epithelial cells of the small intestinal mucosa (Ryall et al., 1985; and references therein). In the rat, expression of the CPS gene begins, in both tissues, at about day 16 of fetal development and reaches maximal (adult) levels shortly after birth (Ryall et al., 1985; Adcock et al., 1984). The enzyme is one of a cohort of proteins that contributes to the phenotype of the terminally differentiated hepatocyte; manipulations leading to hepatic dedifferentiation are usually accompanied by loss of CPS expression (Rozen et al., 1983). Here, we have identified protein-recognition elements in the CPS promoter and characterized their ability to interact with protein(s) exhibiting properties similar to those of C/EBP, a transactivator of gene transcription in liver (Friedman et al., 1989) and in a limited number of other tissues (adipose and intestine, and at lower levels in lung and skin; Birkenmeier et al., 1989). C/EBP (now called
232
+ strand
- strand
12345678
12345678 -1q3
‘7 proximal probe
Fig. 1. DNase combined
I protection
analysis
with phosphorylation
erate three probes
covering
of the CPS promoter
with T4 polynucleotide
CPS promoter
regions
in liver nuclear
extracts.
kinase and [;>-3’P]ATP
from nt -163 to t 7, -547
Restriction followed
digests of the plasmids,
by polyacrylamide-gel
to - 140. and
-547 to -235,
pGEM161CPS
clectrophorcsis,
or pGEMCPS412,
were employed
to gen-
labelled on either the ( t ) or ( - ) strand.
Adult rat liver nuclear extracts were prepared according to Gorski ct al. (1986) with the modifications described in Maire et al. (1989); various amounts of nuclear extract, indicated above each lane (pg protein), were used in DNase I protection assays which were carried out according to Lichtsteiner et al. (1987), with minor modifications; 1 fmol of probe (5000 cpm) and 500 ng ds poly(dI)-poly(dC)/2 mM Na vanadate/l mM KF were included per reaction. A pool of the reagents was combined with nuclear reactions containing less than 10 pg of extract
extracts and the binding reactions conducted for 15 min at 4°C before DNase I was added. For protein, bovine serum albumin was added to augment the quantity of protein to
233 + strand
- strand
0 0.5 2 5 1020400
Extra<
nt
2 Q.e) nt -473
-244
-412
-336 -371
-259 -441
-484
-516
12345678
12345678
-547
-140 distal probe
10 pg per reaction Sequencing reactions were performed using the same labelled probes and, together with the footprint reactions, were analyzed by electrophoresis in an 8% polyacrylamide gel containing 7 M urea to determine the positions of the protected regions, as described by Howell et al. (1989). Shown
are autoradiograms
(B) the distal probe (shown
of analyses
-547 to -235, see Fig. 2) was employed refer to positions hypersensitive
using (A) the proximal
probe (shown
as a line below the autoradiogram, to gain better resolution
relative to the tsp. The brackets
and sequence
as a line below the autoradiogram,
nt -140 to -547,
in the 5’ region of the promoter numbers
nt - 163 to + 7, see Figs. 2 and 3) and
see Fig. 2) labelled on either the + or (not shown). Numbers
on the right denote the footprint
regions.
strand.
The third probe (nt
on the left of each autoradiogram The arrowheads
indicate
DNase
I
sites.
C/EBPcc; Cao et al., 1991) is a member of a relatively complex family of proteins that exhibit diverse DNA recognition specificities (Lamb and McKnight, 1991). The immediate members of the family are designated C/EBPa, p, y, and 6 (Cao et al., 1991; Williams et al., 1991). A related factor, DBP, can substitute for C/EBPa in activating transcription of the ALB gene by binding to ALB promoter site D (Mueller et al., 1990).
RESULTS
AND DISCUSSION
(a) DNase I footprinting of the CPS promoter region Three probes were designed to cover the CPS promoter region from nt + 4 to -547 and were analyzed by DNase I protection analysis on the coding ( + ) and noncoding ( - ) strands. A total of six well-defined footprints were observed using liver nuclear extracts and were designated
234 sites I to VI (Fig. 1). The sequences derived from the reproducible results from a number of footprint analyses are given in Fig. 2. Site I (nt -79 to -97) corresponds to a CAAT motif that we had previously identified by sequence homology (Lagace et al., 1987). Site II (nt -103 to -122) was previously identified as a recognition site for a nuclear factor with properties similar to those of C/EBPa (Howell et al., 1989); methylation interference localized the contact site to the
-5.50 (+)5’-GTTTTGATTA (-)3’-CAAAACTAAT
GTGTTACCAA CACAATGGTT
TAGCTTTAAA ATCGAAATTT
ATCTGGAATG TAGACCTTAC
TAGAGTAATG ATCTCATTAC
V
VI -500
TTGTAATAAA AACATTATTT
GCTGTAGTAT CGACATCATA
ACTGCTTCAT TGACGAAGTA
TAGTAATCCT ATCATTAGGA
CTTCCCAAAT GAAGGGTTTA
-450
AATTCAGATG TTAAG’KTAC
TTT’PCTGTTT AA?.AGACXiA
ATTTGTGAAG TAAACACTTC
GAAAGTTCCA CTTTCAAGGT
GTTTCTTTAC CAAAGAAATG
-400
TGGATGTGGT ACCTACACCA
TTCCATCATC AAGGTAGTAG
TTTTCCCAAG AAAAGGGTTC
CAGAACGTGT GTCTTGCACA
CCAGGTTTGG GGTCC?LA?.CC
-350
GTTTTCTTGA CAAAAGAACT
AATCXUATA TTAGGTTTAT
TCTTCTGAAG AGAAGACTTC
TAAACGAAGC ATTTGCTTCG
ATCATTTCCA TAGTAAAGGT
A -300
ACWAGTAAA TGAGTtATTT
GCGTGTGACC CGCACACTGG
TTTTGTATTA AAAACATAAT
TTAGAGAGGT AATCTCTCCA
ACTGTCCTTC TGACAGGAAG
-250
AAATAAGCAG TTTATTCGTC
TACACCTAAG ATGTGGATTC
GCTGTTTACC CGACAAATGG
ACGCCWGGT TGCGGACCCA
CATGTTATTC GTACAATAAG
-200
AGCTACACAA TCGATGTGTT
GCAACTCTTT CGTTGAGAAA
TGATGGCTAG ACTACCGATC
CT’ECTTXT GAACGAACGA
GCAGCAGTAA CG’KGTCATT
-150
CATGATT’EC GTACTAAAGG
ACAGGAGAAG TGTCCTCTTC
GTGCCATTTG CACGGTAAAC
TTATGTTGCA AATACAACGT
ACTTGTATGA TGAACATACT
-100
CATGTCCATT GTACAGGTAA
‘XMCiWCW TGGACATCAG CCTTGTAGAG ACCTGTAGTC
CTTGGGAGGA GAACCCTCCT
GGGGCTGAGG CCCCGACTCC
-50
AGGGGAGGAG TCCCCTCCW
CTGTAGACGG GACATCTGCC
GCTATTTaAT CGATAAATTA
GGCAGAATGA CCGTCTTACT
ATGGGGAGT; TACCCCTCAG
GTGCAGTCAG
CCTTCAGCCC
CAACTGCACT
GTCTCCACAC
III
II
I
-1
+1
+51
AGCTTTCCTT
CCCACTGGTT
ACAAGCAAAT
TGGACAACM
AATCTCATGA
+101
GATATTTGTG
ATTTAATTTT
AGTCACAAAA
CATCTTCAAA
ATG
Fig. 2. Sequences
of the six footprints
on the U’S promoter
conferred
by liver nuclear extracts
region (as based on data in Fig. 1.). The heavy lines
above and below the CPS promoter line the protected were reproducibly
sequence
(LagacC et al., 1987) out-
regions on the ( + ) or ( - ) strands, respectively, that obtained by footprint analyses in liver nuclear extracts.
The 5’-3’(
+ ) and 3’-5’(-)
strands
nt numbers
are as indicated
in Fig. 1. The nt labeled
are marked
region of DNA representing the 0.S promoter quence and only the 5’-3’( + ) strand of nt +
and the corresponding
I
+ 1 is the
tsp.The
is shown as the ds seto + 143, which rcpre-
sents a portion of the transcribed sequence, is shown. Sequences in bold type represent a CAAT motif within site I (Laga& et al., 1987). The C/EBPz-recognition element within site II is underlined with dashes. Italicized
sequences
comprised
of more than one protein-binding
(b) Binding of recombinant C/EBPa CPS promoter region
site.
to multiple sites in the
The extent of binding of C/EBPa to the CPS promoter was examined using the protein produced in bacteria. The recombinant C/EBPa was in the form of a protein extract that had been partially purified by ion-exchange chromatography (Landschulz et al., 1988). It was found to protect all sites, i.e., sites I, II, III, V and VI, and half of site IV (nt -330 to -348) in footprint analyses (Fig. 3).
IV
V
kAGATCGCT
inverted repeat sequence, 5’-GTTGCAAC, located at nt -109 to -116 (Howell et al., 1989). Sites II, III, V and VI all exhibit (Fig. 2) a certain degree of similarity to a region of site D (5’-TGATTTTGTAAT) in the ALB promoter, a site that can be recognized by C/EBPz, as well as by other members of the C/EBP family (Friedman et al., 1989; Lamb and McKnight, 1991). Site IV contains an internal hypersensitive site (Fig. lB), which may mean that this region is
within sites II,III,V and VI
denote sequences related
to ALB site D (SW sections a, c and Fig. 4). A hypersensitive IV is indicated by arrowheads at nt -337 on the (+ ) strand on the ( - ) strand.
site in site and -338
(c) Competition with oligos corresponding to CPS site II and ALB site D Fig. 4 demonstrates that oligos corresponding either to site D or to site II can effectively compete with all six sites of the CPS promoter, albeit site VI was competed weakly by the oligo corresponding to site D. No such competition was seen using a nonspecific oligo of similar length (data not shown). Interestingly, both oligos (coding for ALB site D and CPS site II) can compete the entire footprint at site IV (Fig. 4), a site which is recognized only on one half by recombinant C/EBPa (Fig. 3). (d) Transactivation of the CPS promoter by C/EBPa in vivo An expression plasmid encoding C/EBPx (Friedman et al., 1989) was transiently co-transfected into HepG2 cells (which contain relatively low amounts of endogenous C/EBPa; Friedman et al., 1989) with a plasmid containing the LUC reporter gene linked to regions of the CPS promoter extending to nt -35 and -597 (containing just the TATA element or the TATA element plus footprints I-VI, respectively). Negligible promoter activity was associated with the region extending to nt -35 (Fig. 5), indicating that initiation of transcription from the promoter was absolutely dependent on the upstream activating elements. Cotransfection with optimal concentrations of the C/EBPa expression plasmid resulted in a two- to threefold stimulation of CPS promoter activity but, again, no activity was observed with the control (i.e., the -35 region). The C/EBPa recognition element at the core of site II, 5’-GTTGCAAC (Howell et al., 1989), in the CPS promoter, was mutated to ATCTAGAA. A synthetic oligo
235 - 5 10 20 40 - - - - Extract (ug) _ _ _ - - - 20200C/BBP(ng)
-
5 102040 - - - -
- - - - 20200 -
III -516-
123456789
123456789
-163 ‘7 proximal probe Fig. 3. Binding (Landschulz
of the bacterial
expression
et al., 1988) was incubated
product
distal probe
of recombinant
with the proximal
-140
547
C/EBPG( to the CPS promoter
region.
Partially
below the autoradiograms) of the CPS promoter labelled on the coding strand (see Figs. 1 and 2) at the concentrations dicated, and a standard DNase I footprint analysis (see Fig. 1) was carried out. For comparison, liver nuclear extracts (pg protein)
containing
indicated.
Other symbols
this mutation
and methods
purified
C/EBPG( from bacteria
(nt - 163 to + 7, left panel) or the distal (nt -547 to - 140, right panel) probes (shown by lines (ng of protein per reaction) inwere also used at the amounts
are as in Fig. 1.
did not bind any detectable
pro-
tein in a gel retardation assay (Goping et al., 1992). The mutation was found to have a slight inhibitory effect on the activity of the promoter in transfection assays (Fig. 5). However, the mutation at site II abolished C/EBPadependent stimulation of the promoter (Fig. 5), suggesting that site II may be essential for mediating the C/EBPc( response in transfected cells. (e) Conclusions (I) The CPS promoter contains (at least) six proteinbinding elements, all of which can be recognized by factors
with DNA-recognition properties similar to those of C/EBPa. (2) Transactivation of the CPS promoter by C/EBPa in co-transfected HepG2 cells (as well as in rat Reuber H35 cells; data not shown) was abolished by a mutation of the C/EBPcc-recognition element, 5’-GTTGCAAC, at the core of site II. Although the extent of transactivation by C/EBPcr was relatively low compared to corresponding stimulation of the ALB promoter in HepG2 cells (e.g., see Friedman et al., 1989), it was comparable to the levels seen for the promoters of the genes encoding phosphoenolpyruvate carboxykinase (Park et al., 1990) and alcohol dehy-
A
_
-541
-484 -
_ - 10 50 250
20
and methods
elements
J
-140
1 VI
are as in Fig. 1.
out using the coding ( + ) strand
tq an oligo corresponding
distal nrohe
1234567
Ic
-
5 10
““_
_
-
were carried
of the C‘PS promoter
I
Site D (molar e
Extract(w)
of liver nuclear extracts
Other svmbols
in the presence
comoetitor.
analyses
competition
proximal probe
- 20 100500
20
B
510
-163 ‘;/
- 10100
20
proximal probe
123456
_ _ -
-
-516-
(pg)
-547
Site cn iolar e
Extract
-
20
I
distal probe
123456
c
5 10
-140
either to site D of the rat ALB promoter or to site II of the C‘PS promoter. (A) Standard DNase I proteclion 163 to + 7. left panel) and distal (nt -547 to -140, right panel) probes of the of both the proximal (nt 1989) of the rat ALB promoter (27.mer; nt -93 to -115 plus a HirzdIlI site overhang) was added CPS promoter (see Figs. 1 and 2). except that unlabelled ds oligo coding for site D (Friedman et al., to the reaction as competitor, at a molar excess indicated above the autoradiogram. (B) Same as A but using a 30-mer ds oligo coding for site II of the CPS promoter (nt -98 LO - 127. see Fig. 2) as
-163 ‘7
1234567
_
Fig. 4. Footprint
_
-510
237 drogenase
(Stewart
et al.,
1991).
Transactivation
C/EBPa, therefore, appears to require site II. (3) Although the remaining sites share similar
2.5
recognition properties it is also known that sequences, including McKnight, 1991). It sites can be occupied cell.
by protein
with site II and can bind C/EBPa, C/EBPa can recognize diverse DNA those for other factors (Lamb and is likely, therefore, that these binding as well by other factors in the intact
ACKNOWLEDGEMENTS
We are very grateful to S.L. McKnight and colleagues (Baltimore) for recombinant C/EBPa and the C/EBPcr expression plasmid. We thank M. Therien, J. Drouin and B.W. Howell for helpful suggestions throughout this work. This study was supported by operating grants from the National Cancer Institute of Canada and Medical Research Council, and by studentships from these agencies to I.S.G. and M.L., respectively.
Fold induction by C/EBP,
TATA
-35M
1
2.7
Fig. 5. Effects of C/EBPz moter
activity
promoter
on wild-type
in co-transfected
regions extending
(mm) CPS pro-
(wt) and mutant
HepG2
cells. Plasmids
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mM ATP/3.3
1.0 relative unit of LUC activity corresponds
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pg protein.
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