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INTRODUCTION |
The important role of endothelial cells in many physiological and
pathophysiological processes has made the regulation of gene expression
in these cells a subject of intensive investigations (1). A number of
endothelial cell-specific promoters have been identified (2). Several
trans-acting factors including GATA2 (3, 4), GATA3, GATA6 (5), Ets (6,
7), SCL/Tal-1 (8), SP1 (9), vezf (10), Oct1 (11, 12), and
NF1 (13) in various combinations have been shown to participate in the regulation of these endothelial specific promoters. Although some of
these transcription factors demonstrate endothelial preferential or
specific expression pattern, a single endothelial specific transcription factor that could regulate the activities of all these
promoters is not characterized.
Emerging concepts in the mechanism of gene regulation suggest that in
addition to DNA-binding transcription factors, cofactors that can
either confer cell type specificity and/or participate in modification
of chromatin structures also play an important role in the mechanism of
regulation of gene expression (14). Cell type-specific coactivators can
confer cell type-specific function to commonly expressed trans-acting
factors. Oct1-dependent activation of
TIE-2 gene in endothelial cells was reported to be
dependent on interaction of Oct1 with an endothelial restricted cofactor (12). FOG1 and FOG2 are examples of cofactors that regulate
the function of GATA transcription factor family members GATA1 and
GATA2 (15, 16).
States of chromatin structures that contain a gene also play a central
role in regulation of gene expression. Functions of many DNA-binding
trans-acting factors are regulated by cofactors with
chromatin-modifying properties including histone
acetylation/deacetylation, phosphorylation, and methylation (17).
Specifically, many coactivators with histone acetyltransferase
(HAT)1 and deacetylase (HDAC)
functions are recruited to the promoters by complex formation with
trans-acting factors (14). Recruitments of coactivators with chromatin
modifying functions to specific promoters participate in determining
the role of a trans-acting factor as an activator or a repressor.
Coactivators such as CREB-binding protein, P300, and PCAF with
HAT activities can acetylate histones, thus conferring an active
chromatin structure to the promoter, leading to activation (18, 19).
These coactivators may also acetylate specific trans-acting factors,
such as p53 and GATA1, and therefore increase their transcriptional
activity (20, 21). Coactivators with HDAC function in contrast remove
acetyl groups from histones and confer an inactive chromatin state to
the promoters, thus leading to repression (14). A DNA-binding
trans-acting factor may function as an activator or a repressor
depending on its interaction with cofactors that have HAT or HDAC
function. GATA2, while being an activator of gene expression in
hematopoietic stem cells, can also function as a repressor when
complexed with HDAC3 (22). Trans-acting factors YY1 and NF-
B can
also function as either an activator or a repressor depending on their
interaction with HATs or HDACs (23, 24). The binding sequence of YY1
also appears to participate in whether this factor functions as a
repressor or an activator (25).
Analyses of the endothelial specific regulation of the von Willebrand
factor (VWF) promoter have resulted in characterization of several
trans-acting factors that participate in regulation of the activity of
this promoter. An Ets and a member of the GATA family of trans-acting
factors were shown to function as activators while Oct1 and NF1 were
shown to function as repressors of the VWF promoter (7, 13, 11,
26).
Recently, we have demonstrated (27) that the NFY transcription factor
functions as both an activator and a repressor of the VWF promoter
activity. This dual repressor/activator function of NFY appears to be
modulated through its differential binding sequences. Two distinct DNA
sequences with no homologies are used as binding sites for NFY in the
VWF promoter. NFY functions as an activator when interacting with the
sequence CCAAT (previously reported NFY consensus binding site) at
position
18 in the VWF promoter, whereas it functions as a repressor
when interacting with a novel binding sequence (sequences +226 to +234)
located in the first exon (27).
Based on these results, we have hypothesized that the function of NFY
may be dependent on the cofactors that interact with this ubiquitous
trans-acting factor and thus modulate its trans-acting function as
either an activator or a repressor. In this report, we demonstrated
that the function of NFY as a repressor is mediated through cell
type-specific recruitment of HDACs to the VWF promoter, resulting in
the promoter inactivation in non-endothelial cells. We also identified
GATA6 as the member of the GATA trans-acting factor family that
interacts with the VWF promoter and demonstrates a cell type-specific
association of GATA6 and NFY. We hypothesized that these cell-specific
associations of DNA-binding trans-acting factors and cofactors provide
a molecular basis for determining the endothelial specific activation
pattern of the VWF gene expression.
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MATERIALS AND METHODS |
Cell Culture, Transfection, and Plasmid Generation--
HEK293
cells and human umbilical vein endothelial cells (HUVEC) were grown and
maintained as described previously (27, 28). Generation of plasmids
HGH-1, HGH-1K, and HGH-1KY and transfection of HEK293 cells were
carried out as described previously (27). For trichostatin A (TSA)
treatment experiments, stably transfected HEK293 cells were grown in
100-mm culture dishes for 24 h prior to addition of TSA (100 ng/ml) (Sigma) to the media. Growth hormone assay was carried out as
described previously (26). Growth hormone assays were performed on
transfected cells prior to addition of TSA and 48 h after TSA treatment.
Gel Mobility Supershift Assay--
Nuclear extract from HUVEC
was prepared by the method of Schreiber et al. (29).
Double-stranded oligonucleotides corresponding to the sequences +206 to
+236 of the VWF promoter were radioactively labeled using
[32P]ATP and polynucleotide kinase as described
previously (26). Nuclear extract (12 µg) was incubated with 1 µg of
appropriate antibodies (or no antibody) in a 19-µl reaction mixture
containing 50 mM KCl, 10 mM HEPES (pH 7.9), 5 mM MgCl2, 1 mM EDTA, 5% glycerol, and 1 µg of poly(dI-dC). Following 15 min of incubation on ice, 1 µl of the probe (8000 cpm) was added to each reaction mixture and
incubated at room temperature for 30 min. Complexes were resolved on a
5% non-denaturing polyacrylamide gel in 1× Tris borate EDTA buffer
(pH 8.3) as described previously (27).
Immunoprecipitation and Western Blot Analysis
(IP/Western)--
IP/Western blots were performed as described
previously (30). Nuclear extracts from HUVEC and HEK293 cells (50 µg)
were pre-cleared and immunoprecipitated with appropriate antibodies (5 µg), and immunoprecipitated samples were subjected to Western blot
analysis as described previously (30).
Chromatin Immunoprecipitation--
The ChIP experiments were
performed according to Boyd et al. (31) and the
chromatin immunoprecipitation protocol provided by Upstate
Biotechnology, Inc. (Lake Placid, NY) with modifications. The cells
were exposed to 1% formaldehyde (Fisher) for 10 min at 22 °C to
obtain cross-linked chromatin. Reactions were stopped by addition of
glycine to a final concentration of 0.125 M. To harvest the
cells, the plates were rinsed with cold phosphate-buffered saline,
incubated with 0.2× trypsin/EDTA (Invitrogen) in phosphate-buffered saline, and then scraped. The cells were collected by centrifugation and washed twice in cold phosphate-buffered saline containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 100 ng/ml
aprotinin, and 100 ng/ml leupeptin. The cell numbers were determined at
the last wash. The cell pellets were resuspended in buffer A (5 mM HEPES (pH 7.9), 85 mM KCl, 0.5% Nonidet
P-40, 0.5 mM PMSF, 100 ng/ml leupeptin and aprotinin) and
incubated on ice for 20 min, followed by the addition of Nonidet P-40
(final concentration of 0.5%) and brief vortexing. Nuclear pellets
obtained by brief centrifugation were resuspended in buffer S (1% SDS,
10 mM EDTA, 50 mM Tris-HCl (pH 8.1), 0.5 mM PMSF, and 100 ng/ml leupeptin and aprotinin) and
incubated on ice for 15 min. The cross-linked chromatin were subjected
to sonication using VibraCellTM (Sonics and Material Inc.,
Danbury, CT) to obtain DNA fragments of ~200 bp. Fragmented chromatin
was pelleted by brief centrifugation, and samples corresponding to
~106 cells were diluted 10 times in buffer C (0.01% SDS,
1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris
(pH 8.0), 167 mM NaCl, 1 mM PMSF, and 100 ng/ml
leupeptin and aprotinin). Prior to immunoprecipitation the samples were
pre-cleared with the addition of 80 µl of A/G protein for 2 h at
4 °C. An aliquot of the pre-cleared chromatin (10 µl) was saved
for analysis of total input, and the remaining samples were
immunoprecipitated with 5 µg of appropriate antibodies or no
antibody. The immunoprecipitated chromatin was washed sequentially with
wash buffers 1, 2, 3, and 4 (wash buffer 1: 150 mM NaCl, 50 mM Tris (pH 8.0), 0.1% SDS, 0.5% deoxycholic acid, 1%
Nonidet P-40; wash buffer 2: 0.1% SDS, 1% Nonidet P-40, 50 mM Tris (pH 8.0), 0.5 M NaCl; wash buffer 3:
0.25 M LiCl, 1% Nonidet P-40, 0.5% deoxycholate, 50 mM Tris (pH 8.1), 1 mM EDTA; and wash buffer 4:
10 mM Tris (pH 8.1), 1 mM EDTA). Then the
chromatin was eluted by a 15-min incubation in 150 µl of fresh buffer
E (1% SDS, 50 mM NaHCO3). To reverse
cross-linking, the samples were incubated at 65 °C for 4 h in a
buffer containing 200 mM NaCl and 1 µg of RNase A
followed by treatment with proteinase K and ethanol precipitation. The
pellets were collected by microcentrifugation, resuspended in 20 µl
of H2O, and subjected to PCR analysis. The PCRs were performed with appropriate primer pairs. The human VWF promoter region
form
30 to +155 was amplified with the following primers: primer 1, 5'-ATTAAAAGGAGGCCAATCCCCTGTTGTGGC-3', and primer 2, 5'-CTGCTGCAAAGGCTCAATCAGGTCTGCTA-3'. The VWF promoter region form +155
to +247 was amplified with the following primers: 2T,
5'-GCTGAGAGCATGGCCTAGGGTGGTGGGCGGCAC-3'; primer 3, 5'-CCCCTGCAAATGAGGGCTGCGGCTATCTCCAAG-3'. The VWF-HGH plasmids in stably
transfected cells were amplified with HH1T, 5'-TAGCAGACCTGATTGAGCCTTTGCAGCAGC-3' (according to the VWF sequence +127 to +156), and HGH-2, 5'-AGT GGT TCG GGG AGT TGG GCC TTG GGA TCC-3'
(according to the HGH sequence +1 to +30). Human growth hormone
gene starting from the position +1 was cloned in the multiple cloning
site in plasmid p
HGH (provided by Nichols Institute, San Juan
Capistrano, CA). The VWF promoter in all VWF-HGH plasmid was cloned
immediately upstream of the +1 position of the human growth hormone gene.
The VCAM-specific primers are VCAM-3T,
5'-CCCCACCCCCTTAACCCACATTGGATTCAG-3', and VCAM-4B,
5'-TCCTCTCTCTGTCCTGGCAAAAGAAGACAC-3'.
-Actin promoter-specific
primers used are primer-actin1, 5'-TGCCTAGGTCACCCACTAATG-3', and
primer-actin 2, 5'-GTGGCCCGTGATGAAGGCTA-3'. PCRs contained 1 µl of
immunoprecipitated chromatin or diluted total input chromatin, 50 ng of
each primer, 25 µl of TaqPCR Master Mixture (Qiagen, Germany) in a total reaction volume of 50 µl. After 25-35 cycles of
amplification, PCR products were run on a 1.5% agarose gel and
analyzed by ethidium bromide staining.
Antibodies--
The antibodies used are as follows: anti-GATA2,
anti-GATA4, anti-GATA-6, anti-HDAC2, and mouse IgG from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA); anti-HDAC1, anti-acetylated
histone H3, and anti-acetylated histone H4 from Upstate Biotechnology,
Inc. (Lake Placid, NY); and anti-NFY-A from Pharmingen.
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RESULTS |
The VWF Promoter Activity Is Associated with Acetylated Histone
H4--
We have demonstrated recently (27) that NFY transcription
factor functions both as an activator and a repressor of the VWF promoter activity through two distinct binding sequences. Because NFY
is shown to associate with cofactors that function as HATs (30, 32,
33), we hypothesized that the dual function of NFY may be modulated
through recruitment of HATs and HDACs depending on its binding
sequence. To test this hypothesis, we carried out ChIP experiments to
first determine whether NFY interacts with the VWF promoter in the
context of chromatin in vivo and whether the VWF promoter
activity is associated with the status of histone acetylation.
For these analyses, we carried out ChIP experiments of the endogenous
VWF promoter in human umbilical vein endothelial cells (HUVEC) and
HEK293 cells. These two cell types were chosen as models of human VWF
expressing (HUVEC) and non-expressing (HEK293) cell types.
To determine directly whether NFY interacts with chromatin containing
the VWF promoter, we used antibodies that specifically recognize the
NFY-A subunit of NFY complex to immunoprecipitate native chromatin from
these two cell types. We then determined the presence of the VWF
promoter fragments in the immunoprecipitated fractions using PCR
analysis with the VWF-specific primers that amplify the +155 to +247
sequences of the VWF promoter (Fig.
1A). These sequences contain
the novel NFY-binding site that functions as a repressor, although
precipitated chromatin fragments may also include the VWF promoter
sequences that contain the upstream CCAAT-NFY-binding site. Primers
that specifically recognize the actin promoter sequences were used as
control.

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Fig. 1.
NFY and acetylated histone H4 are associated
with the VWF promoter. A, schematic representation of
the VWF promoter with trans-acting factor-binding sites is shown.
Arrows show the positions of the PCR primers used in ChIP
analyses. B, ChIP analyses using anti-NFY-A antibody were
carried out. The immunoprecipitated chromatin from HUVEC and HEK293
cells was subjected to PCR analyses using primers that amplify the
sequences +155 to +247 for detecting the VWF promoter and specific
primers (described under "Materials and Methods") to detect the
actin promoter (as control) sequences. The input panel
represents the non-immunoprecipitated chromatin fraction used as
template for PCR. The amplified VWF and actin fragments were 92 and 160 bp, respectively. C, chromatin from HUVEC and HEK293 cells
was immunoprecipitated with no antibody (Ab), anti-NFY-A,
anti-acetylated histone H3 (AH3), and anti-acetylated
histone H4 (AH4) antibodies. The immunoprecipitated
chromatin was subjected to PCR using primers that amplify the sequences
30 to +155 of the VWF promoter. The amplified PCR fragments were
analyzed on 1.5% agarose gels. The input panel represents
the non-immunoprecipitated chromatin fraction used as template for
PCR.
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The results demonstrated that NFY-A-immunoprecipitated chromatin in
both HUVEC and HEK293 cells was specifically amplified with
the VWF-specific primers but not with the actin-specific primers (Fig.
1B, upper panel). Both the actin and the
VWF-specific primers amplified the appropriate gene fragments from
non-immunoprecipitated chromatin input, demonstrating PCR efficiency
(Fig. 1B, lower panel). The fragment sizes
amplified by the VWF and the actin primers were 92 and 160 bp,
respectively. These data demonstrated that NFY interacts with
endogenous VWF promoter but not the actin promoter in the context of
chromatin in both HUVEC and HEK293 cells (Fig. 1B).
To demonstrate directly whether the active VWF promoter is associated
with acetylated histones, we used antibodies that specifically recognize acetylated histone H3 and acetylated histone H4 to
immunoprecipitate native chromatin from these two cell types. We then
determined the presence of the VWF promoter fragment in the
immunoprecipitated fractions using PCR analysis with the VWF-specific
primers that amplify a 185-bp fragment corresponding to the sequences
30 to +155 region of the VWF promoter (Fig. 1A). We also
included anti-NFY-A antibody in these analyses to confirm the
interaction of NFY with the VWF promoter sequences with these set of
primers. Immunoprecipitated samples in absence of antibodies were used
as control for PCR analysis.
The results of these analyses confirmed the interaction of NFY
transcription factor with the VWF promoter in both cell types (Fig.
1C, lanes 3 and 4) and demonstrated
that the VWF promoter is similarly associated with acetylated histone
H3 in both HUVEC and HEK293 cells (Fig. 1C, lanes
7 and 8). However, acetylated histone H4 was shown to
be specifically associated with the VWF promoter in HUVEC (Fig.
1C, lane 5), thus demonstrating that the state of
acetylation of histone H4 is correlated to the VWF promoter activity.
Inhibition of the VWF Promoter Activity through NFY Is Correlated
to Histone H4 Hypoacetylation of the Promoter--
It was demonstrated
previously that a fragment of the VWF promoter spanning the sequences
90 to +155 functions as a core promoter and is activated in both
endothelial and non-endothelial cells, while a fragment that spans the
sequences
90 to +247 functions as an endothelial specific promoter,
and its activity is repressed in non-endothelial cells (26, 27).
Mutation and transfection analysis in HEK293 cells demonstrated that
the inhibition of this promoter function in non-endothelial cells is
mediated through NFY binding to the novel binding site located at the
sequences +226 to +234 (27). To determine whether repression/activation of this promoter fragment in transfected cells is correlated to changes
in acetylation status of associated histones, we performed ChIP
analysis of the stably transfected HEK293 cells.
The cells were stably transfected with plasmids HGH-1, HGH-1K, and
HGH-1KY that contain human growth hormone gene fused to various VWF
promoter fragments. Plasmid HGH-1 contains the core VWF promoter
fragment (the sequences
90 to +155) that is active in all cell types
including HEK293 cells. Plasmid HGH-1K contains wild type sequences
90 to +247 of the VWF promoter that has significantly reduced
activity (compared with HGH-1) in HEK293 cells. Plasmid HGH-1KY
contains sequences
90 to +247 with 3-base substitution mutations at
the repressor-NFY-binding site. We have reported previously that this
mutation inhibits NFY interaction with the VWF DNA sequences and
results in promoter activation in HEK293 cells to a similar level as
that of the core promoter (HGH-1). The levels of growth hormone
expression (that is an indicator of these VWF promoters activities)
from stably transfected HEK293 cells were reported previously (27) and
confirmed by repeating the growth hormone assay from the transfected
cells as shown in Fig. 2A.

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Fig. 2.
Interaction of NFY with the repressor element
in the VWF promoter decreases association of acetylated histone H4 with
the VWF promoter sequences in HEK293 cells. A,
schematic representations of the VWF-HGH plasmids containing the wild
type and mutant VWF promoter fragments are shown on the left.
Solid triangle, labeled NFY represents the mutation in the
repressor NFY-binding site. HEK293 cells were stably transfected with
VWF-HGH plasmids, and analyses of growth hormone expression were
performed as described previously (27). Bar graph represents
percent activity of each promoter fragment as compared with that of
HGH-1. B, chromatin from HEK293 cells stably transfected
with HGH-1, HGH-1K, and HGH-1KY (described in A) were
immunoprecipitated with anti-acetylated histone H3 (AH3) and
anti-acetylated histone H4 (AH4) antibodies.
Immunoprecipitated chromatins were subjected to PCR analyses using the
actin-specific primers described in Fig. 1B and VWF-specific
primers that amplify VWF gene sequences 30 to +155 (described under
"Materials and Methods"). Amplified DNA fragments were analyzed on
1.5% agarose gel. C, HEK293 cells stably transfected with
HGH-1, HGH-1K, and HGH-1KY (described in A) were exposed to
100 ng/ml TSA, and the levels of growth hormone prior to and 48 h
after exposure to TSA were determined. The level of growth hormone
expression from each plasmid prior to TSA treatment was considered as
100% (shown by hatched bar), and the level of expression
post-TSA treatment (shown by solid bar) was determined as
percentage of that obtained prior to TSA treatment. Results are
averages of six independent experimental points for each plasmid.
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To determine correlation of these VWF promoter activities to the states
of histone acetylation, ChIP analyses were performed using
anti-acetylated histone H3 and H4 antibodies and the VWF-specific primers (amplifying a 185-bp fragment corresponding to sequences
30
to +155) for PCR detection as described above for Fig. 1C. Primers that were used in PCR analysis detect endogenous VWF as well as
transfected VWF-HGH sequences. However, we do not expect any changes in
the levels of acetylated histones that are associated with endogenous
VWF in these cells. Thus any differences observed reflect differences
in the levels of acetylated histones that are associated with
transfected VWF promoter fragments. As control we also used
actin-specific primers (as described for Fig. 1B) to
demonstrate equal association of actin promoter fragment with acetylated H3 and H4 histones in all transfected cells.
The results demonstrated that there were no significant differences in
the levels of acetylated histone H3 that were associated with the VWF
promoter sequences among all three transfected cell lines (Fig.
2B, lanes 1-3). However, acetylated histone H4
levels that were associated with the VWF promoters significantly
differed in these cell lines (Fig. 2B, lanes
4-6). The promoters that were active, HGH-1 and HGH-1KY,
had significantly higher levels of acetylated histone H4 compared to
HGH-1K (Fig. 2B, compare lanes 4 and 6 to lane 5), which had reduced activity as determined by growth hormone analysis (as shown in Fig. 2A).
These data demonstrated that the VWF promoter fragments with deletion
or mutation of the novel NFY-binding sites (HGH-1 and HGH-1KY) were
active in HEK293 cells and had increased association with acetylated
histone H4 compared with the VWF promoter fragment that had an intact
NFY-binding site and reduced activity (HGH-1K).
Because specific mutation of NFY-binding site in the HGH-1KY plasmid
was correlated to an increased association of acetylated histone H4
with the VWF promoter fragment, we hypothesized that NFY may recruit
histone deacetylases to the VWF promoter through interaction with the
repressor element (the novel NFY-binding site located at the sequences
+226 to +234), thus reducing the level of acetylation of histone H4
associated with the VWF promoter and leading to promoter inactivation.
Thus, mutation of the NFY-binding site that inhibits NFY interaction
with the VWF promoter would be expected to inhibit recruitment of
histone deacetylases to the promoter. This results in increased level
of histone H4 acetylation, consequently leading to promoter activation.
To test this hypothesis, we first performed experiments in which the
effects of histone deacetylase inhibitor trichostatin A on the level of
the activities of these promoter fragments were determined. HEK293
cells stably transfected with HGH-1, HGH-1K, and HGH-1KY were untreated
or treated with TSA (100 ng/ml), and the growth hormone expressed was
determined prior to and 48 h post-treatment. The level of growth
hormone in cells expressing each plasmid was considered as 100%
prior to treatment.
The results demonstrated that there was an ~50% increase in the
activity of the VWF promoter fragment that contained the intact novel
NFY-binding site (Fig. 2C, HGH-1K), whereas TSA
treatment had no effect on the activity of the VWF promoter fragments
with deletion or mutation of novel NFY-binding sequences (Fig.
2C, HGH-1 and HGH-1KY).
The results are consistent with the hypothesis that NFY may recruit
HDAC(s) to the VWF promoter, and thus only the activity of the VWF
promoter fragment that contains the intact NFY-binding site (HGH-1K) is
affected by treatment with HDACs inhibitor.
HDACs Specifically Interact with NFY and the VWF Promoter in
Non-endothelial Cells--
To determine whether histone deacetylases
were associated with the VWF promoter sequences, we performed chromatin
immunoprecipitation using antibodies that specifically recognize HDAC1
and HDAC2. Chromatin-HDAC complexes were isolated from HUVEC and HEK293
cells and subjected to PCR analysis using the VWF-specific primers that amplify the 92-bp fragment of the VWF corresponding to sequences +155
to +247 as described for Fig. 1B. The results demonstrated that both HDAC1 and HDAC2 were specifically associated with endogenous VWF promoter sequences in VWF-non-expressing HEK293 cells (Fig. 3A, lanes 5 and
6) but not in VWF-expressing HUVEC (Fig.
3A, lanes 2 and 3). The VWF-specific
primers amplified the appropriate VWF fragment from all
non-immunoprecipitated input chromatin used as control (Fig.
3A, lower panel).

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Fig. 3.
HDACs are specifically associated with NFY
and the VWF promoter in HEK293 cells. A, chromatin from
HUVEC and HEK293 cells was immunoprecipitated with no antibody
(Ab), anti-HDAC1, and anti-HDAC2 antibodies. The input
panel serves as positive control and represents the total
chromatin used as template that was not subjected to
immunoprecipitation. The ChIP and input chromatin were used as
templates for PCR to detect the VWF promoter sequences. Primers
amplified the sequences +155 to +247 of the VWF gene (shown in Fig.
1A). The lane labeled represents
sham-immunoprecipitated chromatin (no antibody). The amplified PCR
fragments were analyzed on 1.5% agarose gel. B, nuclear
extracts (30 µg) prepared from HUVEC and HEK293 cells were subjected
to Western blot analysis and hybridized to anti-HDAC1 and
anti-HDAC2-specific antibodies. C, nuclear extracts (50 µg) prepared from HUVEC and HEK293 cells were immunoprecipitated with
IgG, anti-NFY-A, and anti-HDAC1 antibodies (represented as
IP-Ab). The immunoprecipitated complexes were analyzed by
Western blot analysis and hybridized to anti-NFY-A antibody
(represented as W-Ab). D, chromatin from HEK293
cells stably transfected with HGH-1K and HGH-1KY were
immunoprecipitated with no antibody, anti-HDAC1, and anti-HDAC2
antibodies. The input panel serves as positive control and
represents the total chromatin used as template that was not subjected
to immunoprecipitation. The ChIP and input chromatin were used as
templates for PCR to detect VWF-HGH sequences specifically in the
transgene. The primers corresponded to the +153 to +183 region of the
VWF gene and the +1 to +30 region of the human growth hormone
gene.
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To determine whether both HEK293 and HUVEC cells express HDAC1 and
HDAC2 proteins, Western blot analysis was performed using anti-HDAC1
and -2 antibodies. The results demonstrated that both HDACs are present
at similar levels in HUVEC and HEK293 cells (Fig. 3B). These
results demonstrate that the lack of association of HDACs with VWF
promoter in HUVEC is not because of the absence of HDAC proteins in
these cells.
To investigate the role of NFY in recruitment of histone deacetylases,
we performed immunoprecipitation/Western blot analysis to determine
interaction of NFY and histone deacetylases. For these analyses we used
anti-HDAC1, anti-NFY-A (as positive control), and anti-IgG (as negative
control) antibodies to immunoprecipitate the corresponding proteins
from HEK293 and HUVEC cells and subjected the immunoprecipitated
protein complexes to Western blot analysis using anti-NFY antibody. The
results demonstrated that NFY and HDAC1 are associated in both cell
types; however, the level of association is significantly higher in
HEK293 cells than in HUVEC (Fig. 3C, lanes 3 and
6). There were no associations of immunoprecipitated IgGs
with NFY in either cell type (Fig. 3C, lanes 1 and 4), whereas immunoprecipitated NFY was detected with
anti-NFY-A antibody in both cell types (Fig. 3C, lanes
2 and 5), thus demonstrating the specificity of the
NFY-A antibody in Western blot analysis.
This result demonstrates that NFY interacts with histone deacetylases,
and this association is specifically reduced in endothelial cells.
To demonstrate directly that HDACs are recruited to the VWF promoter
through interaction with NFY, we performed ChIP analysis in HEK293
cells stably transfected with plasmids HGH-1K and HGH-1KY. For these
analyses chromatin from stably transfected cells were immunoprecipitated with anti-HDAC1 and anti-HDAC2 antibodies as described for Fig. 3A; however, the primers that were used
for PCR analysis corresponded to the VWF promoter sequences +153 to +183 and the human growth hormone sequences +1 to +30. By using these
sets of primers, a 150-bp fragment corresponding to the sequences
spanning the chimeric VWF and human growth hormone gene sequences in
the HGH-1K and HGH-1KY transgenes (and not the endogenous VWF
promoter sequences) were amplified, thus avoiding ambiguity in
interpretation of the data as to whether the results represent the
association of HDAC with endogenous VWF or the transgene sequences. The
results demonstrated that HDAC1 and HDAC2 are associated with the VWF
promoter sequences in the HGH-1K plasmid that contains the wild type
NFY-binding site (Fig. 3D, lanes 2 and
3) but not with VWF sequences in the HGH-1KY plasmid that
contains the base substitution mutations in the NFY-binding site (Fig.
3D, lanes 5 and 6). The VWF and
HGH-specific primers amplified the appropriate 150-bp fragments from
all non-immunoprecipitated input chromatins used as control (Fig.
3D, lower panel). These data demonstrate that
HDACs are associated with the VWF promoter and that mutation of the
NFY-binding site inhibits HDAC association with the VWF promoter, thus
demonstrating that NFY binding to the novel repressor element is
necessary for the recruitment of the HDACs to the VWF promoter.
Based on these results, we hypothesize that NFY can function as a
repressor by recruiting histone deacetylases and that there may be a
cell type-specific mechanism of regulating NFY-HDAC1 association.
Transcription Factor GATA6 Interacts with the VWF Promoter and
Associates with NFY in a Cell Type-specific Manner--
Our previous
analysis of the VWF promoter demonstrated that a GATA transcription
factor interacts with the VWF promoter sequences, and this interaction
was necessary for the promoter activation in endothelial cells (26).
Because the binding site for the GATA factor is adjacent to the novel
NFY-binding site, we hypothesized that the GATA factor is involved in
regulation of the VWF promoter through a cell type-specific mechanism
that may involve interaction of GATA and NFY.
To test this hypothesis, first we needed to determine which member of
the family of GATA transcription factors interacts with the VWF
promoter sequences. For these analyses we performed supershift gel
mobility assays using the VWF sequences +209 to +239 (which were
previously shown to bind the GATA factor (26)) as probe and nuclear
extracts prepared from HUVEC. Nuclear extracts were preincubated with
specific antibodies recognizing human GATA2, GATA4, and GATA6 prior to
addition of the probe. We excluded GATA1 and GATA5 antibodies from
these studies because previous reports (34, 35) have demonstrated that
these factors are not expressed in endothelial cells. GATA3
transcription factor was also reported to mediate T-cell-specific gene
expression, thus suggesting that it is an unlikely candidate for the
regulation of the VWF gene expression (36). The result of supershift
gel mobility assay demonstrated that the only antibody that generated a
supershift was anti-GATA6 antibody. There were no differences in the
pattern of complex formation among nuclear extracts that were
preincubated in the absence of antibodies and those that were
preincubated with the antibodies of IgG, GATA2, or GATA4 (Fig.
4A).

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Fig. 4.
GATA6 interacts with the VWF promoter in
HUVEC and HEK293 cells and specifically associates with NFY in HEK293
cells. A, supershift gel mobility experiments were
carried out using nuclear extract prepared from HUVEC and a
radioactively labeled oligonucleotide probe corresponding to the DNA
sequences +206 to +236 of the VWF gene (containing the GATA-binding
site). Nuclear extracts (12 µg) were incubated with no antibody ( ),
anti-GATA2, anti-GATA4, anti-GATA6, and IgG prior to the addition of
the probe (8000 cpm), and complexes were analyzed on a non-denaturing
5% polyacrylamide gel as described under "Materials and Methods."
Arrows show the position of the specific major complexes
C1 and C2, and SS represents the
supershifted complex. B, nuclear extracts (30 µg) from
HUVEC were subjected to Western blot analysis and hybridized to
anti-GATA6 and anti-GATA2 antibodies as described under "Materials
and Methods." C, chromatin from HUVEC was
immunoprecipitated with no antibody ( ), anti-GATA2, or
anti-GATA6 antibody. The immunoprecipitated chromatin fractions were
used in PCRs with specific primers to amplify the 92-bp VWF promoter
sequences corresponding to the +155 to +247 region (VWF
panel) and VCAM-specific primers to amplify the 160-bp VCAM
promoter sequences containing the two GATA-binding sites (VCAM
panel). The non-immunoprecipitated chromatin fractions were used
as control with the VWF-specific primers that amplify the VWF sequences
shown in upper VWF panel. D, chromatin from HUVEC
and HEK293 cells was immunoprecipitated with no antibody or anti-GATA6
antibody. The immunoprecipitated chromatin was used as template for PCR
to detect the VWF promoter sequences. The primers amplified the VWF
sequences +155 to +247 (shown in Fig. 1A). The amplified PCR
fragments were analyzed on 1.5% agarose gel. E, nuclear
extracts (50 µg) prepared from HUVEC and HEK293 cells were
immunoprecipitated with IgG, anti-NFY-A, and anti-GATA6 antibodies
(represented as IP-Ab). The immunoprecipitated complexes
were analyzed by Western blot analysis and hybridized to anti NFY-A
antibody (represented as W-Ab).
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GATA2 transcription factor is expressed in endothelial cells and
participates in the regulation of a number of genes including VCAM and
PECAM that are preferentially expressed in endothelial cells (3, 5).
However, supershift gel mobility assay demonstrated that GATA2 does not
interact with VWF promoter sequences, whereas GATA6 appears to bind to
the GATA site. To determine the presence of GATA2 and GATA6 factors in
HUVEC, we performed Western blot analysis of the nuclear proteins. The
results demonstrated that while both GATA2 and GATA6 are expressed in
HUVEC, the level of GATA6 transcription factor appears to be
significantly higher in these cells (Fig. 4B). However, this
may be due to the efficiencies of the antibodies used to detect GATA2
and GATA6. To demonstrate directly whether GATA6 and/or GATA2 interact
with VWF promoter sequences in vivo, we performed ChIP assay
using anti-GATA6 and anti-GATA2 antibodies and chromatin prepared from
HUVEC. Immunoprecipitated chromatin was subjected to PCR analysis using
the VWF-specific primers (amplifying the 92-bp fragment corresponding
to VWF sequences +155 to +247) as described above for Fig.
1C. As control we used VCAM-specific primers that amplify a
160-bp region of the VCAM promoter (corresponding to the
sequences
320 to
161) containing two GATA-binding sites (37), which
were reported previously (38) to bind GATA2. VCAM promoter was also
shown to bind GATA6 in response to tumor necrosis factor-
stimulation (5). The results of ChIP analysis demonstrated that GATA6
but not GATA2 interacts with endogenous VWF promoter sequences in HUVEC
(Fig. 4C, panel VWF, lanes 2 and
3), whereas both GATA2 and GATA6 interact with the VCAM
promoter sequences (Fig. 4C, panel VCAM,
lanes 2 and 3).
These data confirm the results of gel mobility experiments and
demonstrate that GATA6 specifically interacts with the VWF promoter
sequences in HUVEC.
To demonstrate whether GATA6 interaction with the VWF promoter
sequences is specific to endothelial cells, we performed ChIP assay
using anti-GATA6 antibody and chromatin prepared from HUVEC and HEK293
cells. Immunoprecipitated chromatin was subjected to PCR analysis using
the VWF-specific primers (amplifying the 92-bp fragment corresponding
to the VWF sequences +155 to +247) as described above. The results
demonstrated that GATA6 interacts with the endogenous VWF promoter
sequences both in HUVEC and HEK293 cells (Fig. 4D). These
analyses demonstrated that there was no preferential binding of GATA6
to the VWF promoter in endothelial cells compared with non-endothelial cells.
These results suggest that although GATA6 binding is necessary for VWF
promoter activation, it is not sufficient to determine differential
regulation of VWF promoter activity in endothelial and non-endothelial
cells. However, this does not exclude the possibility that there may be
differential interaction of NFY-HDAC and the adjacent GATA6 factor in
endothelial and non-endothelial cells that may contribute to the
mechanism of differential regulation of the VWF promoter.
To test this hypothesis, we performed immunoprecipitation/Western blot
analysis to determine the pattern of interaction of NFY and GATA6 in
these two cell types. Mouse IgG, anti-NFY-A, and anti-GATA6 antibodies
were used to immunoprecipitate the corresponding proteins, and the
complexes were subjected to Western blot analysis using anti-NFY-A
antibody. The results demonstrated that GATA6 is specifically
associated with NFY in HEK293 cells but not in HUVEC (Fig.
4E).
Although these results do not determine whether GATA6 and NFY directly
interact with each other, they demonstrate an association of these
factors that is cell type-specific. Based on these results, we
hypothesize that the activating function of GATA6 may be modulated through its interaction with other factors including NFY and
potentially HDACs that are also associated with NFY. Such interactions
may modify or inhibit activating potential of GATA6 in non-endothelial cells without interfering with its DNA binding properties. In endothelial cells, GATA6 may be specifically modified so that it does
not interact with NFY and/or it may interact with different cohorts of
activators/coactivators to maintain and enhance its transcriptional
activating properties.
 |
DISCUSSION |
Regulation of the VWF promoter fragments that function in an
endothelial specific manner involves a number of cis- and trans-acting factors that positively and negatively affect transcription in cell
culture (7, 11, 13, 26). A major component of this cell type-specific
regulatory mechanism is inhibition of transcription in non-endothelial
cells that is achieved through the function of repressors interacting
with negative regulatory elements. The 734-bp fragment of the VWF
promoter that exhibits endothelial specific activation pattern was
shown previously to contain NF1- and Oct1-binding sites that function
as repressors (11, 13). Deletion of 5'-flanking sequences of the VWF
promoter that contain these repressor elements did not result in
promoter activation in non-endothelial cells. However, deletion of an
additional 100-bp fragment spanning the sequences +155 to +247 at the
3' end of the VWF promoter resulted in promoter activation in
non-endothelial cells as well as in endothelial cells (26). We have
demonstrated recently (27) that these VWF sequences (+155 to +247)
contain a novel binding site for transcription factor NFY that
functions as a repressor of the VWF promoter activity. In the absence
of NF1 and Oct binding, specific base substitution mutations that inhibited NFY interaction with this novel binding sequence was sufficient for the promoter activation in non-endothelial cells (27).
We have also shown that NFY interacts with a consensus CCAAT sequence
in the VWF promoter, and through this interaction, it functions as an
activator of the VWF promoter (27, 30). This dual repressor/activator
function of NFY appears to be modulated through its differential
binding sequences (27). Based on these results, we have hypothesized
that NFY function may be dependent on cofactors that interact with this
ubiquitous trans-acting factor and thus modulate its trans-acting
function as either an activator or a repressor.
Previous reports (39) on NFY function have demonstrated that this
trans-acting factor mediates nucleosomal assembly and participates in
regulation of the activities of the promoters through enhancement of
the function of neighboring cis-acting elements.
Coactivators PCAF and p300 with histone acetylating activities were
shown to be associated with NFY (32, 33). Thus, recruitment of these
histone-modifying coactivators to the promoters may be a mechanism by
which NFY functions as an activator. The role of NFY in mediating the
state of histone acetylation of its target promoter sequences is
demonstrated by the effect of histone deacetylase inhibitors on the
activity of these promoters. Activation of human MDR1,
Xenopus HSP70, and transforming growth factor-
type II receptor in response to HDAC inhibitors TSA, sodium butyrate, and MS-275 is mediated by NFY (42-44). HDAC inhibitors were shown to
initiate interaction of NFY with HATs leading to activation of
transforming growth factor-
type II receptor promoter in human breast cancer cell lines, whereas basal association of NFY and HATs
were reported in other systems (44).
We have also demonstrated that the interaction between NFY and the
CCAAT sequence is required for the VWF promoter up-regulation in
response to irradiation and that NFY and PCAF association in endothelial cells is increased in response to irradiation (30).
Because coactivators with histone acetylating function may contribute
to trans-activating function of NFY, we hypothesized that cofactors
with histone deacetylating function may contribute to NFY function as
repressor. Based on this hypothesis, VWF promoter activity is expected
to correlate to the level of acetylation of the histones that are
associated with the VWF promoter sequences. In addition, inhibition of
NFY binding to the repressor element is expected to result in changes
in acetylation pattern of histones associated with the VWF promoter.
Our analyses demonstrated that the endogenous VWF promoter sequences in
endothelial cells are specifically associated with acetylated histone
H4. Such association was not observed in HEK293 cells that do not
express the VWF gene. Furthermore, transfection studies in HEK293 cells
demonstrated that the core VWF promoter fragment (sequences
90 to
+155, which does not contain repressor elements and is active in HEK293
cells) is also associated with acetylated histone H4, whereas
endothelial specific promoter fragment (sequences
90 to +247, that
contains the repressor NFY-binding site and is not active in HEK293
cells) has significantly decreased association with acetylated histone
H4. Direct evidence that NFY binding to the repressor element mediates
the decreased association of acetylated H4 with VWF promoter was
obtained when the endothelial specific promoter fragment with mutation
in the repressor NFY-binding site was shown to have similar levels of
association with acetylated H4 and similar levels of activity as that
of the VWF core promoter fragment. In addition, histone deacetylase
inhibitor TSA was shown to increase the activity of the transfected VWF
promoter fragment that contains an intact repressor NFY-binding site
but not that of mutant or core promoter fragment. These results are
consistent with the hypothesis that NFY factor recruits histone
deacetylases to the VWF promoter when functioning as repressor. Further
evidence to support this hypothesis was obtained by IP/Western blot
analysis that demonstrated the association of NFY and HDAC1
specifically in HEK293 cells, and also ChIP analysis demonstrating that
HDACs are specifically recruited to the endogenous VWF promoter
sequences in HEK293 cells but not in HUVEC. Furthermore, HDACs were
shown to be associated with VWF-HGH transgene (HGH-1K)
containing the wild type but not that containing the mutated
NFY-binding site (HGH-1KY) in transfected HEK293 cells.
These data not only support the hypothesis that the function of NFY as
a repressor is mediated through recruitment of HDACs, they also
demonstrate that there is cell type specificity to the pattern of NFY
association with HDACs.
Based on these results, we also hypothesize that there are two pools of
NFY-containing complexes in cells, one that is associated with HATs and
one that is associated with HDACs. This hypothesis is consistent with
previous observations that HDAC inhibitors increase
NFY-dependent promoter activation as discussed above. Based
on this hypothesis, HDAC inhibitors may release NFY from HDAC complexes
and thus increase the level of NFY that can associate with HATs.
There are observations that in some cell types there are basal
NFY-HAT complexes (42) while in others this complex is observed only in
response to inducers or HDAC inhibitors (44), and there is cell type
specificity to the pattern of NFY-HDAC and potentially NFY-HAT complex
formations. Recently, the dual function of NFY as both activator and
repressor of transcription of SHP-1 gene in MCF7
cells was reported (40). Although in this report two distinct binding
sites for NFY were also involved, both binding sequences consisted of
inverted CCAAT sequence. The binding of NFY to the distal CCAAT element
was shown to enhance transcription, whereas binding to the proximal
site was reported to repress the promoter activity. Furthermore, the
mutation of the proximal site eliminated the enhancing effect of TSA on
the promoter activity (40). This further supports our hypothesis that
NFY can interact with complexes that contain HDACs and function as repressor.
Other ubiquitously expressed transcription factors such as YY1 and
NF-
B are also shown to function as activators and repressors based
on their interaction with HATs and HDACs (23, 24). Specifically, the
binding sequence for YY1 is implicated in the role of this factor as
activator or repressor (25). Thus, NFY provides another example of a
ubiquitously expressed trans-acting factor whose function can be
modulated based on its interaction with specific type of coactivators.
The cell type-specific pattern of association of NFY with HDACs also
provides a mechanism for this ubiquitously expressed factor to
participate in cell type-specific regulation of gene expression.
Interaction of NFY with other DNA binding transcription factors is also
reported to participate in the regulatory function of NFY in mediating
promoter activation (41). We have reported previously (26) the presence
of a GATA-binding sequence in the VWF promoter that is necessary for
promoter activation in endothelial cells. This GATA element is situated
directly adjacent to the repressor NFY-binding sequence, thus raising
the possibility that the interaction of NFY and the GATA factor that
binds to the GATA site may be involved in cell type-specific regulation
of the VWF promoter activity (27). To test this hypothesis we first
identified GATA6 as the member of the GATA transcription factor family
that interacts with the GATA site in the VWF promoter, and we then determined the pattern of GATA6 and NFY interaction in endothelial and
non-endothelial cells. Our results demonstrated that GATA6 is
specifically associated with NFY in HEK293 cells but not in HUVEC.
Because these results were obtained by IP/Western blot analysis, they
do not demonstrate whether this association is direct or indirect;
however, the cell type specificity of the association suggests a
potential mechanism for participation of these factors in regulating
the endothelial cell-specific activation of the VWF promoter. Based on
these data we propose a hypothesis (Fig.
5). The complexes including NFY-HDAC and
GATA6 are formed in non-endothelial cells, which may result in
deacetylation of GATA6 as well as recruiting HDACs to the VWF promoter.
This complex may inhibit the transcriptional activity of GATA6 as well
as deacetylation of the histones on the VWF promoter, thus rendering
the promoter inactive in non-endothelial cells. The lack of this
complex formation in endothelial cells may result in the presence of a
pool of GATA6 transcription factors that are not deacetylated and,
thus, can function as an activator of the VWF promoter. This hypothesis does not exclude the possibility that endothelial cell-specific transcription factors or coactivators may interact with GATA6 to
prevent association of GATA6 with NFY-HDAC complexes or recruit HATs
specifically to GATA6 and/or disrupt NFY/HDAC associations specifically
in endothelial cells. Regardless of the potential mechanism, the
presence of NFY in a complex that contains GATA6 and HDAC specifically
in non-endothelial cells provides a potential mechanism for cell
type-specific regulation of the VWF promoter by these transcription
factors that does not depend on their cellular expression pattern.
However, potentially cell type-specific post-translational modifications of these factors may lead to these different associations and complex formations, thus leading to their differential regulation of the VWF promoter in endothelial and non-endothelial cells.

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Fig. 5.
Model describing the activation and
repression function of NFY-binding site in the VWF promoter. The
model represents the VWF region corresponding to sequences +155 to
+247. The solid cylinders represent potential nucleosomes,
and circles with Ac represent acetylation. In endothelial
cells a complex composed of NFY, GATA6, and potentially an endothelial
specific HAT (unknown thus represented as HATs?) may be
formed. The putative HAT may acetylate the GATA6 and NFY as well as
histone H4 in the nucleosomes, and these modifications of trans-acting
factors and nucleosomes could facilitate promoter activation. In
non-endothelial cells, the absence of endothelial specific HATs could
result in the increased pool of NFY associated with HDACs, and a
complex consisting of NFY-HDAC-GATA6 may be recruited to the VWF
promoters which deacetylate the histone H4 in the nucleosomes and
potentially maintain a deacetylated form of GATA6 and NFY. These may
contribute to inhibition of the promoter activity by deacetylation of
histone H4 and potentially inhibiting the activating function of GATA6
and NFY, thus turning the entire complex to a repressor.
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