(Received for publication, October 12, 1995; and in revised form, November 14, 1995)
From the
Blood coagulation Factor X and its activated form Factor Xa play an essential role in the midphase of the clotting cascade. To delineate the mechanisms governing the liver-specific expression of Factor X, we have previously characterized the complete 2.8 kilobase pairs of the 5`-flanking region of Factor X and demonstrated that the first 209 base pairs is sufficient to confer maximal promoter activity in HepG2 cells, a hepatoma cell line that expresses Factor X. We have also shown that mutations at ACTTTG and CCAAT elements located at -56 to -51 and -120 to -116, respectively, significantly reduce the promoter activity. In this report, we demonstrate that Factor X mRNA is primarily but not exclusively expressed in the liver. Using DNase I footprinting analysis, we determine four protein binding sites within the 209-base pair fragment, designated site 1 (-73 to -44), site 2 (-128 to -94), site 3 (-165 to -132), and site 4 (-195 to -169). Using gel mobility shift assays in combination with competition and supershift experiments, we demonstrate that hepatocyte nuclear factor 4 and Sp1 bind at site 1, the site which contains the ACTTTG element. Methylation interference assays reveal that HNF-4 and Sp1 contact adjacent sites with minor overlap. HNF-4 and Sp1 appear to bind site 1 in a mutually exclusive fashion. We also demonstrate that HNF-4 can transactivate the Factor X promoter in HeLa cells; mutation at the adjacent Sp1 site further increases the transactivation. Heteromeric transcription factor NF-Y was identified as the protein that binds the CCAAT box at site 2. We conclude that HNF-4 and NF-Y play crucial roles in modulating the activity of the proximal promoter of Factor X.
The vitamin K-dependent proteins F.VII, ()F.IX, F.X
and prothrombin are the precursors of the major enzymes of the
coagulation cascade. In order to have activity in coagulation, these
proteins must undergo
-carboxylation of glutamic acid residues at
the N terminus, a reaction catalyzed by the enzyme
-glutamyl
carboxylase, which requires vitamin K as a cofactor(1) . The
vitamin K-dependent clotting factors are synthesized predominantly or
exclusively in the liver, but the mechanisms controlling liver-specific
expression are not understood in detail. We have chosen F.X as a
paradigm for study, because of its central role in coagulation (when
activated, it converts prothrombin to thrombin) and because, in
contrast to the case of F.IX, a well characterized cell line exists,
HepG2 cells, which expresses F.X(2) . In previous
work(3) , we had defined the start sites of transcription of
the F.X gene and had also carried out a functional characterization of
the F.X promoter, which demonstrated that the proximal 209 bp of the
promoter were adequate to confer maximal activity in HepG2 cells. Using
site-directed mutagenesis and reporter gene assays, we defined two
areas within the F.X promoter that bound proteins from HepG2 nuclear
extracts and that were required for promoter activity. In this report,
we have used DNase footprinting to define 4 protein-binding sites in
the proximal promoter. Using gel shift assays with nuclear extracts or
purified proteins, and supershift assays with well characterized
antibodies, we have determined the identity of the transcription
factors that bind at the two proximal sites. In the case of the most
proximal site, site 1, we show that two transcription factors, one
ubiquitous (Sp1), and the other found in only a few tissues (HNF-4)
bind at the site in a competitive fashion; we present evidence that
HNF-4 binds at a similar site in the promoters of F.VII and F.IX. For
site 2, which contains a CCAAT box, we have shown that the ubiquitous
transcription factor NF-Y binds at this site; this is distinct from the
transcription factor that binds at the CCAAT box in the F.IX promoter,
which occupies a similar position with respect to the translation start
site.
In
competition experiments, unlabeled competitors were added prior to the
addition of probes. In supershift experiments, antibodies were added 10
min after the addition of probes. Antiserum against amino acids
445-455 of rat HNF-4 was a gift from Dr. Frances
Sladek(9) . The epitope for mouse monoclonal antibody
NF-YA1a was mapped to the first glutamine-rich domain of NF-YA.
NF-YB is a polyclonal antiserum raised against recombinant NF-YB.
Both
NF-YA1a and
NF-YB were gifts from Dr. Diane
Mathis(10) . Affinity-purified polyclonal antibody against
amino acids 436-454 of Sp1 was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Pf7 (gift from Drs. Weimin Zhong and
James Darnell) (9) containing HNF-4 cDNA was transcribed by T3
RNA polymerase and translated in a rabbit reticulocyte lysate system
according to the manufacturer's specifications (Promega).
The methylation interference assay was carried out as described by Hendrickson et al.(11) . Oligonucleotides used in methylation interference were labeled as in electrophoretic mobility shift assay except that only one strand was labeled. Recombinant Sp1 used in the methylation interference studies was purchased from Promega (Madison, WI).
Human growth hormone reporter gene
constructs FXpGH-279, FXpGH-mutACTTTG, FXpGH-mutACTTAG, and
FXpGH-mut
GGGGCG have been described previously(3) .
FVIIpGH-309 was generated by ligation of a polymerase chain
reaction-generated 309-bp fragment of F.VII 5`-flanking region
(-309 to -1, with reference to the translation start site
as +1) into the XbaI site in p0GH. FIXpGH-420 was
generated by the same strategy with 420 bp of the F.IX promoter
fragment. HNF-4 expression plasmid pLEN4S contains a full-length rat
HNF-4
1 cDNA under the control of the human metallothionein
promoter and the SV40 enhancer (gift from Dr. James
Darnell)(9) . pLEN(-) was generated by removing the HNF-4
cDNA insert from pLEN4S.
Figure 1:
Northern blot analysis of the Factor
X mRNA in adult human tissues. A, multiple tissue Northern
blots were hybridized with a human Factor X cDNA probe and
autoradiographed (upper panel). Each lane contains
approximately 2 µg of poly(A) RNA. Blots were
stripped and rehybridized with a
-actin cDNA probe to normalize
for the differences in loading and transfer among lanes (lower
panel). The positions of the marker are indicated on the left. Note that lanes 1-8 and lanes
9-16 are on two separate blots and were electrophoresed for
different lengths of time. The size of the F.X mRNA is 2 kb; the
-actin transcripts are present as 2-kb (ubiquitous) and 1.8-kb
(skeletal muscle-specific) messages. B, densitometric scanning
of the F.X mRNA levels in various tissues are normalized against
-actin mRNA levels and plotted as percentage of the level in
liver.
Figure 2: DNase I footprint analysis of the Factor X promoter. A, a probe spanning -279 to +1 of the Factor X gene plus 215 bp of the p0GH sequence was end-labeled at +1 and incubated with 50 µg of nuclear extract (NE) from either HepG2 (lane 4) or HeLa (lane 5) cells and digested with 2.5 units of DNase I. Lanes 1 and 2 contain Maxam and Gilbert sequencing ladders. Lane 3 contains DNA probe digested with 0.1 unit of DNase I in the absence of nuclear extracts. Regions protected by nuclear extracts are bracketed and designated as sites 1, 2, 3, and 4. Note that the boundaries of site 1 as defined by HepG2 nuclear extract are different from those defined by HeLa nuclear extract. Site P is only protected by HeLa nuclear extracts but not by HepG2 nuclear extracts and is within the p0GH vector sequence. The panel on the right contains the same reactions electrophoresed longer to better define the boundaries of sites 2, 3, and 4. B, a detailed map of the protein-protected sites in the Factor X promoter. The sites protected by HepG2 nuclear extracts are denoted by thin lines and those protected by HeLa nuclear extracts by thick lines.
Figure 3:
Site 1 binds two distinct proteins and
forms two DNA-protein complexes. A, sequences of the
oligonucleotides used in Fig. 3are shown. Only one strand is
shown for each oligonucleotide. The numbering system uses translation
start site as +1. B, an oligonucleotide spanning
-68 to -39 of the Factor X gene (FXSite1) was labeled and
incubated with 10 µg of nuclear extract (N.E.) from cell
lines and rat tissues as indicated. Two DNA-protein complexes are
designated complex A and complex B. The position of the free probe is
marked as F. C, an oligonucleotide spanning site 1
was incubated with 12 µg of nuclear extract (N.E.) from
human liver (lanes 1-12), 2 µl of in vitro translated HNF-4 in rabbit reticulocyte lysate (lane 13),
or 2 µl of unprogrammed rabbit reticulocyte lysate (lane
14). Lanes 2-5 contain unlabeled site 1
oligonucleotide as cold competitors in 20 , 100
, 500
, and 1000
molar excess. Lanes 7-10 contain unlabeled APF-1 (a strong HNF-4 binding site derived from
-87 to -66 of apolipoprotein C III gene) as cold
competitors in 20
, 100
, 500
, and 1000
molar excess. Lane 12 contains 1 µl of HNF-4 antiserum.
The positions of the antibody-HNF-4-DNA complexes are indicated by SS. D, probes containing the ACTTTG common motif from
Factors VII, IX, X, and APF-1 were incubated with 12 µg of human
liver nuclear extracts (N.E.) (lanes 1-8). One
µl of HNF-4 antiserum was added in lanes 5-8.
Positions of the immune complexes (supershift) are marked as SS. Two DNA-protein complexes specific to the APF-1 probe are
marked by asterisks. E, the Factor X site 1 probe was
incubated with 15 µg of nuclear extract (N.E.) from either
HepG2 (lanes 1-3) or HeLa cells (lanes
4-6). In lanes 2 and 5, 200
molar
excess of unlabeled Sp1 consensus oligonucleotide was added. In lanes 3 and 6, 1 µl of Sp1 antibody was added.
The position of a minor complex is denoted by an asterisk.
Figure 5: Increasing amounts of Sp1 diminish the binding of HNF-4 to site 1. Labeled Factor X site 1 oligonucleotide was incubated with 12 µg of human liver nuclear extracts (N.E.) in the presence of 0, 0.2, 1, and 2 footprint units (lanes 1-4) of recombinant Sp1.
Figure 4:
Methylation interference analysis of the
contact points of HNF-4 and Sp1 to site 1. A, On the left, Factor X site 1 was labeled on one strand (sense strand
or antisense strand) and partially methylated. The methylated probe was
incubated with 100 µg of human liver nuclear extract and analyzed
in regular electrophoretic mobility shift assay. The free probe
fraction (F) and the complex B fraction (B, which
contains HNF-4) were excised from the gel, cleaved with piperidine to
reveal the G/A ladder, and resolved on a denaturing gel. On the right, the site 1 oligonucleotide was incubated with 10
footprint units of recombinant Sp1 and analyzed as described above. The
free fraction (F) and bound fraction (B) were
analyzed. The contact points revealed are marked as .
indicates methylated positions that partially interfere with the
binding. * indicates that methylation at these nucleotides enhances the
protein binding. B, summary of results of methylation
interference analysis at site 1.
Figure 6: HNF-4 transactivates the Factor X promoter through the ACTTTG motif. Ten µg of FXpGH wild type and mutant constructs were cotransfected with 10 µg of either pLEN(-) (shaded bars) or pLEN4S (black bars) into HeLa cells. The expression level of FXpGH-279 in the presence of pLEN(-) was taken as 1. The expression level of FXpGH + pLEN4S was compared with that of FXpGH + pLEN(-), and the average -fold increase is shown at the top of each column. The results represent an average of three independent transfection experiments.
Figure 7: Site 2 contains a CCAAT box and binds to a ubiquitous protein. An oligonucleotide spanning the region from -133 to -104 of Factor X (FXCCAATwt) was incubated with 10 µg of nuclear extract (N.E.) from cell lines and rat tissues as indicated. The position of the major DNA-protein complex is indicated by an arrow.
Figure 8:
The protein which binds the CCAAT box in
Factor X site 2 is NF-Y. A, sequences of the oligonucleotides
used are shown. B, FXCCAATwt oligonucleotide was incubated
with 15 µg of nuclear extract (N.E.) from HepG2 cells (lanes 1-21). Unlabeled competitors added were FXCCAATwt (lanes 2-6), FXCCAATmut (lanes 7-11),
FIXCCAATwt (lanes 12-16), and MHC class II E Y Box (lanes 17-21) at 10
, 50
, 100
,
500
, and 1000
molar excess. C, labeled
oligonucleotides as indicated were incubated with 15 µg of nuclear
extract (N.E.) from HepG2 (lanes 1-4) or HeLa
cells (lanes 5-8). D, labeled FXCCAATwt
oligonucleotide was incubated with 15 µg of nuclear extract (N.E.) from HepG2 cells. Lanes 2-4 contain 0.5,
1, and 2 µg of monoclonal antibody
NF-YA1a, respectively. Lanes 5-7 contain 0.5, 1, and 2 µl of polyclonal
antiserum
NF-YB, respectively. Positions of the immune complexes
(supershift) are denoted SS1 and SS2.
Second, incubation of
radiolabeled probes from either the F.X CCAAT box, or the Y box of the
MHC class II gene E, with nuclear extracts from HepG2 or HeLa
cells, results in complexes of the same mobility (lanes 1, 4,
5, and 8, Fig. 8C). Note that this
complex is not of the same mobility as that formed when the F.IX CCAAT
sequence (lanes 3 and 7), which occurs at
approximately the same position in its promoter, is used as probe,
suggesting that different proteins bind at the CCAAT boxes in F.IX and
F.X. It had been demonstrated previously that this sequence in the F.IX
promoter recognizes the unrelated transcription factor
NF-1(19) .
Third, a monoclonal antibody against the first
glutamine-rich domain of NF-YA (NF-YA1a) and a polyclonal
antiserum against NF-YB (
NF-YB) (10) result in supershift
of the complex formed by FXCCAATwt (Fig. 8D). A faster
migrating complex only present in the HepG2 nuclear extracts (Fig. 7) was not supershifted by
NF-YA1a, but was
supershifted by
NF-YB. This complex likely contains the
heterodimer of an alternatively spliced form of NF-YA (which lacks the
first Q-rich domain(20) ) with NF-YB. Taken together, these
data provide strong evidence that the protein binding at site 2 is
NF-Y.
A longstanding but poorly documented assumption regarding
procoagulant proteins has been that expression of these proteins is
confined to the liver. Our data indicate that, although liver is the
major site of F.X mRNA synthesis, F.X mRNA is also found in lung,
heart, ovary, and small intestine. Similar findings have been reported
for other vitamin K-dependent coagulation proteins: Jamison and Degen (21) have reported that prothrombin mRNA is found in uterus,
placenta, kidney, spleen, and small intestine in addition to liver; and
more recently, Stitt et al.(22) have reported the
presence of protein S mRNA in uterus, heart, placenta, lung, smooth
muscle, kidney, spleen, and ovary. Like thrombin and protein S, which
have recently been demonstrated to activate intracellular signaling
cascades through binding to specific cell surface receptors (a G
protein-coupled receptor in the case of thrombin(23) , the Tyro
3/Axl family of receptor tyrosine kinases in the case of protein
S(22) ), F.Xa has also been shown to bind to a specific cell
surface receptor, EPR-1, found on monocytes and endothelial
cells(24) . Occupation of the EPR-1 receptor by F.Xa triggers a
mitogenic response through a signal transduction process that is not
yet well understood. Thus, in contrast to F.VII ()and
F.IX(25) , vitamin K-dependent proteins for which expression is
clearly confined to the liver, our data suggest that F.X belongs to
another group of vitamin K-dependent proteins with a wider tissue
distribution and more protean biological effects.
In previous work we had determined that the proximal 209 bp of the F.X promoter were adequate to confer maximal promoter activity in a reporter gene assay. In this work, we have used DNase I footprint analysis with nuclear extracts from HepG2 and HeLa cells to identify four protein-binding sites within this promoter element. A previously reported footprint analysis of this region carried out with HepG2 extracts identified only one of these sites, site 1(26) . The failure to detect other sites within the probe used by these investigators may reflect a difference in the quality and concentration of transcription factors in the nuclear extracts used, or it may be a function of the incubation conditions used, since failure to saturate a protein-binding site may render it difficult to detect on a footprint. For the same reasons, of course, we cannot state with certainty that the four sites we have mapped are the only protein-binding sites within this fragment.
We chose the two proximal protein-binding sites for further study. In previous studies, we had established the functional significance of both of these sites(3) . Additionally, site 1 was of special interest, because it is the only one of the four for which the footprint differs between HepG2 and HeLa cells. We have now identified the cognate binding proteins for each site. Site 1 forms two DNA-protein complexes, designated A and B. Complex B is present only in nuclear extracts from liver, kidney, and HepG2 cells, whereas complex A is present (in varying amounts) with all extracts tested. Competition experiments, gel shift assays using in vitro translated HNF-4, and supershift experiments document that the protein giving rise to complex B is HNF-4. HNF-4 belongs to the nuclear receptor superfamily of transcription factors; these are characterized by two highly conserved regions, one in the N terminus which binds DNA, and another in the C terminus that is required for ligand binding, dimerization, and activation. Because of the high degree of conservation in the DNA-binding domain, other members of the superfamily can demonstrate similar binding specificity. For example, COUP-TF and ARP-1 have been demonstrated to compete with HNF-4 for binding sites in the promoters of apolipoprotein CIII(27) , ornithine transcarbamylase(28) , and erythropoietin(29) . In the supershift experiment presented in Fig. 3D, the two residual bands seen with the APF-1 probe (marked by asterisks next to lane 8) likely represent complexes containing these (or other) related proteins. The absence of any distinct residual bands in the supershift experiments with F.VII, F.IX and F.X (lanes 5-7) suggests that these promoters are probably not recognized by other members of the nuclear receptor superfamily.
Recently published data (30) demonstrate that HNF-4 forms stable homodimers and fails to heterodimerize with a number of nuclear receptors that were tested. In the supershift experiment presented here (Fig. 3C, lane 12), the presence of 2 supershifted complexes raises the question of heterodimerization, but the data presented by Jiang et al.(30) make it more likely that the two complexes arise from the binding of either one (faster moving complex) or two (slower moving complex) antibody molecules to the homodimer in the supershift complex.
The promoters of F.VII, F.IX, and F.X all contain the ACTTTG motif and all bind HNF-4 (Fig. 3D). The molar amounts of labeled DNA were identical in these experiments. Thus HNF-4 has greater affinity for the F.X promoter than for the promoters of F.VII and F.IX. Comparison of the nucleotide sequences to a 13-bp consensus HNF-4 binding site proposed by Sladek (31) showed that the F.X promoter sequence gave the best match (12/13 nucleotides). The F.VII and F.IX promoters did not exhibit as close a match (11/13 and 10/13, respectively).
In addition to the binding experiments, we showed that HNF-4 can transactivate all three promoters in HeLa cells. The transactivation effect in our system ranged from 2-fold in the case of F.X to 4.5-fold in the case of F.VII. These results are different from those previously published by Reijnen et al.(32) , who reported a >300-fold transactivation of the F.IX promoter by HNF-4 in cotransfection experiments using the same HNF-4 expression vector. Technical considerations likely account for the differences in results. To assess transactivation, we compared our results to a base-line control cotransfected with the expression plasmid without the HNF-4 insert. Use of mock-transfected cells as base line, as reported by Reijnen et al.(32) , results in a much higher fold transactivation effect.
Based on competition with an Sp1 consensus oligonucleotide and on supershift by an Sp1 antibody, the protein in complex A that binds to site 1 is identified as Sp1. Sp1 is a ubiquitous transcription factor, but as is clearly evident in Fig. 3B, its concentration in different tissues varies considerably. These differences are supported by a study of the developmental expression of Sp1 in the mouse(33) , which documents low levels of Sp1 in the liver, and 70-fold higher levels in the thymus, the highest expressing tissue. One possibility is that relative levels of HNF-4 and Sp1 may influence sites of expression of F.X, with the highest levels of expression being seen in the liver where HNF-4 levels are high and Sp1 levels are low.
Methylation interference analysis of the contact points of HNF-4 and Sp1 on site 1 indicates that the two transcription factors bind at adjacent sites with minor overlap. The contact region of HNF-4 includes at least 11 nucleotides centered around the ACTTTG element, whereas the contact region of Sp1 surrounds the GGGGCG element. The overlap of the HNF-4 and Sp1 binding sites suggests that they may bind to site 1 in a mutually exclusive manner. This notion is further supported by the fact that addition of increasing amounts of Sp1 reduces the binding of HNF-4 in a gel mobility shift assay (Fig. 5). Based on this hypothesis, one would predict that Sp1 could repress expression of Factor X by excluding the binding of HNF-4 at the neighboring site and that loss of the Sp1 site might result in higher level expression. Experimental confirmation of the latter prediction can be seen in Fig. 6, where HNF-4 transactivation of the F.X promoter in HeLa cells improves from 2- to 3.5-fold when the Sp1 binding site is destroyed. In previous work (3) , we had shown that mutation of the Sp1 site does not result in an increase in expression in HepG2 cells. This is most likely due to the fact that the concentration of Sp1 in HepG2 cells is low (Fig. 3B), so that loss of the Sp1 binding site has little effect. In contrast, in HeLa cells, where Sp1 is abundant, loss of the Sp1 binding site has a noticeable effect on transactivation. Whether repression by Sp1 plays any physiologic role in cells that express HNF-4 but express only low levels of F.X (e.g. kidney) is not yet clear.
Two lines of
evidence support our contention that the ubiquitous transcription
factor NF-Y binds at site 2. First, the specific complex formed between
site 2 and HepG2 nuclear extracts can be competed away by a strong NF-Y
binding site (the Y box from MHC class II E gene), and second, the
DNA-protein complexes can be supershifted by antibodies against NF-YA
and NF-YB. It is of interest that the CCAAT box from the F.IX gene,
which resides at a similar location in the F.IX promoter, cannot
compete away the complex formed between site 2 and HepG2 nuclear
extracts. Crossley and Brownlee (19) have demonstrated that the
F.IX CCAAT box binds to NF-1, a transcription factor that is not
structurally related to NF-Y(34) . Thus in contrast to the
situation with HNF-4, which binds to the conserved ACTTTG element in
all three promoters (F.VII, F.IX, and F.X), the conserved CCAAT boxes
of F.IX and F.X demonstrate binding to distinct and unrelated cognate
proteins.
Our findings are summarized in Fig. 9. The proximal 209 bp of the F.X promoter, which are sufficient to confer maximal activity in HepG2 cells, contains four protein-protected sites as identified by DNase I footprinting. Only one of these, site 1, shows a unique footprint pattern when results with HepG2 nuclear extracts are compared with extracts from non-hepatic cell lines. We have demonstrated that site 1 binds two distinct transcription factors, HNF-4, a liver-specific factor, and Sp1, a ubiquitous factor. Methylation interference assays and transactivation experiments suggest that these two factors bind at overlapping sites in a competitive manner. In reporter gene assays in HepG2 cells, a mutation that abolishes HNF-4 binding (but does not affect Sp1 binding) reduces promoter activity to 17.2% of wild type, but mutation of the Sp1 site has virtually no effect (90.2%)(3) . We have also demonstrated that the ubiquitous transcription factor NF-Y binds at site 2; a mutation at this site also markedly reduces promoter activity to 11.8% of wild type(3) . These data confirm that sites 1 and 2 are critical for activity of the F.X promoter. Ongoing experiments are directed at determining the identity of the proteins that bind at sites 3 and 4 and their role in the regulation of F.X transcription.
Figure 9: Summary of the cis and trans elements of the Factor X promoter. The translation start site is designated as +1. The boundaries of the protein-protected sites defined by the DNase I footprint assays are shown. The identity of the transcription factors binding at sites 1 and 2 is noted. The promoter activities in HepG2 cells of constructs containing mutations at these sites (expressed as a percent of wild type) are shown below the sites.