Transcriptional Repression of the Cystic Fibrosis Transmembrane
Conductance Regulator Gene, Mediated by CCAAT Displacement
Protein/cut Homolog, Is Associated with Histone
Deacetylation*
SiDe
Li
,
Libia
Moy
,
Nanci
Pittman
,
Gongliang
Shue
,
Barbara
Aufiero§¶,
Ellis J.
Neufeld
,
Neal S.
LeLeiko
, and
Martin J.
Walsh
**
From the
Department of Pediatrics, Division of
Pediatric Gastroenterology and Liver Diseases, the ** Program in
Molecular, Cellular, Biochemical, and Developmental Sciences, and the
§ Department of Medicine, Division of Molecular Medicine,
Mount Sinai School of Medicine, New York, New York 10029 and the
Medical Division of Hematology-Oncology, Children's Hospital,
Harvard Medical School, Boston, Massachusetts 02115
 |
ABSTRACT |
Human cystic fibrosis transmembrane
conductance regulator gene (CFTR) transcription is tightly
regulated by nucleotide sequences upstream of the initiator sequences.
Our studies of human CFTR transcription focus on
identifying transcription factors bound to an inverted CCAAT consensus
or "Y-box element." The human homeodomain CCAAT displacement
protein/cut homolog (CDP/cut) can bind to the Y-box element through a cut repeat and homeobox. Analysis
of stably transfected cell lines with wild-type and mutant human
CFTR-directed reporter genes demonstrates that human
histone acetyltransferase GCN5 and transcription factor ATF-1 can
potentiate CFTR transcription through the Y-box element. We
have found 1) that human CDP/cut acts as a repressor of
CFTR transcription through the Y-box element by competing
for the sites of transactivators hGCN5 and ATF-1; 2) that the ability
of CDP/cut to repress activities of hGCN5 and ATF-1
activity is contingent on the amount of CDP/cut expression; 3) that histone acetylation may have a role in the regulation of gene
transcription by altering the accessibility of the CFTR Y-box for sequence-specific transcription factors; 4) that trichostatin A, an inhibitor of histone deacetylase activity, activates
transcription of CFTR through the Y-box element; 5) that
the inhibition of histone deacetylase activity leads to an alteration
of local chromatin structure requiring an intact Y-box sequence in
CFTR; 6) that immunocomplexes of CDP/cut
possess an associated histone deacetylase activity; 7) that the
carboxyl region of CDP/cut, responsible for the
transcriptional repressor function, interacts with the histone
deacetylase, HDAC1. We propose that CFTR transcription may
be regulated through interactions with factors directing the modification of chromatin and requires the conservation of the inverted
CCAAT (Y-box) element of the CFTR promoter.
 |
INTRODUCTION |
The gene responsible for cystic fibrosis encodes the
cystic fibrosis transmembrane
conductance regulator
(CFTR)1 protein (1, 2). The
expression of CFTR is confined primarily to
specific epithelial cell types and is ordinarily expressed in low
levels. The low levels and cell type-specific expression of
CFTR appear to be dictated primarily by sequences upstream of the transcriptional start sites of CFTR which correspond
to functional promoter sequences (3, 4). The elements required for
active, cell type-specific CFTR transcription lie in a
narrow band of nucleotide sequences, proximal to the multiple
transcript initiation (5). The levels of CFTR transcripts in
individual cell types appear to correspond to the ability of specific
epithelia to modulate expression of CFTR variably in
response to 1) levels of cAMP (6), 2) the stimulation of protein kinase
A and C activities (7-9), and 3) phorbol esters (10) likely through
signaling pathways or mechanisms that converge ultimately on gene
transcription (for review, see Ref. 11).
The tight transcriptional control of CFTR requires a
conserved inverted CCAAT (Y-box) element (12). Despite the absence of a
TATA signal, the human CFTR gene can initiate RNA
transcription through multiple, discrete start sites requiring
conserved residues within the Y-box. Thus, an intact CCAAT consensus on
the opposing strand of the human CFTR appears to be a
requirement for accurate transcript initiation (12). Both basal and
cAMP-mediated regulation of CFTR transcription have
implicated the Y-box (12) in tandem with a weak CRE nucleotide
consensus downstream (13). CFTR transcription appears to be
influenced by additional cis-acting elements, including a
site located in the first intron of the human CFTR (14). The activation of CFTR transcription by cAMP correlates with
increasing sensitivity to DNase I overlapping the Y-box element (12).
This may reflect the role of trans-acting factors in
modifying local chromatin structure. Although it has been shown
previously that individual members of bZIP families of transcription
factors have the capacity to bind and activate CFTR
transcription (12, 13), little is known about the parameters and
proteins involved in directing transcription of the CFTR in
vivo.
Histone modification plays an important role in regulating gene
transcription (15, 16). The level of acetylation of conserved residues
on the core histones H3 and H4 appears to plays a critical role in the
regulation of gene transcription (17), and the presence of histone
acetyltransferase activity correlates with the activation of other
genes (18, 19). Conversely, it is believed that gene repression is
associated with the increased activity of histone deacetylases (17,
20). Although it is accepted that histone acetyltransferases and
deacetylases are tightly associated with transcriptional machinery
through several well characterized transcriptional cofactors or adaptor
proteins (21-23), little is known about how such activities are
directed to a specific gene. Several genes encoding histone
acetyltransferase and deacetylase activities have been identified in
human. Interestingly, many of these genes encode both transcriptional
coactivators and corepressors, such as hGCN5, P/CAF, p300/CBP, TAF250,
and HDAC1/2 (for review see Ref. 18). Recent observations have
suggested that transcriptional cofactors such as p300/CBP and HDAC1/2
encode proteins for the enzymatic histone acetyltransferase and
deacetylase activities, respectively (22, 24). These proteins can form
complexes with sequence-specific nuclear hormone receptors and
transcription factors (25-28). Although these cofactors do not possess
sequence-specific DNA binding activities, a transcriptional cofactor
can target a specific gene when tethered to a gene promoter through a
heterologous DNA binding domain as shown previously (29). Therefore,
transcriptional cofactors may regulate the level of histone acetylation
of a specific gene through interactions with sequence-specific
transcription factors.
Our present objective was to identify proteins bound to the human
CFTR promoter through the Y-box nucleotide consensus of the
CFTR. We have now identified two such proteins:
CDP/cut, a homeodomain repressor, and the transcription
factor complex CBF·NF-Y. CBF·NF-Y is a highly conserved complex of
heterotrimeric subunits shown to interact with CCAAT sequence elements
common to many eukaryotic promoters (30, 31). Conserved residues within
subunits of the CBF·NF-Y complex, which include a region homologous
to the histone-fold motif found in histones H2A and H2B, are necessary for subunit interaction to form the heterotrimeric complex (32). Domains of CBF·NF-Y, located in two of the subunits (CBF-c and CBF-b)
are necessary for the transactivation by the CBF·NF-Y complex (33).
The CBF·NF-Y-bound complex can form tight interactions with known
histone acetyltransferases hGCN5 and P/CAF to transactivate transcription (27). Mammalian CCAAT displacement
protein/cut (CDP/cut) repressor is a
180-190-kDa polypeptide (34-36) closely related to the cut
protein of Drosophila (37), which determines cell fate in
Drosophila (38, 39). CDP was first described as a
CCAAT-box-binding protein in the sea urchin sperm histone promoter
(40). It was defined subsequently as a gene-specific transcriptional
repressor associated with the differential regulation of the
gp91phox gene in myeloid cell differentiation
(41). Human CDP/cut also binds to promoters of the
c-myc, N-cam, thymidine kinase, c-mos, and histone genes in a variety of cell types (42-46) and is implicated in the regulation of gene transcription through sequence elements associated with nuclear matrix attachment (47). The competing activities between CBF·NF-Y and CDP/cut have been
postulated as a mechanism to regulate several genes through the CCAAT
motif (44, 48, 49).
The objective of this study was to determine further whether
transcription of CFTR is regulated by nuclear factors
associated with histone acetylation. Because the regulation
CFTR transcription is potentiated through cAMP and the
CREB/ATF activators (12, 13), we postulate that mechanisms directing
the recruitment of histone acetyltransferase coactivators could,
conversely, direct a histone deacetylase complex through a separate set
of CCAAT-binding factors within the context of the human
CFTR promoter Y-box element. In this report, we present
evidence that the Y-box element of the CFTR promoter is a
site that intersects histone acetylation and deacetylation processes
with mechanisms to modulate transcription of CFTR.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
Pancreatic carcinoma (PANC1) and histiocytic
lymphoma (U937) cells were obtained from the American Type Culture
Collection (Rockville, MD). PANC1 cells stably transfected with human
CFTR promoter-directed human growth hormone transgenes
pCF-197wt/hGH and pCF-197
CCAAT/hGH and the pØGH promoterless vector
have been described (12). All PANC1 cell cultures were maintained in
Dulbecco's modified Eagle's medium in glucose (4.5 g/liter) (Life
Technologies, Inc.). U937 cell were maintained in RPMI. Medium was
supplemented with 10% fetal calf serum (Hyclone, Logan, UT) and 1 × gentamycin (Life Technologies, Inc.). Cell cultures were maintained
in 5% CO2 incubator at 37 °C.
Expression Plasmids--
Plasmids for hGCN5 (50) (Dr. Yoshihiro
Nakatani, NIH, Bethesda, MD) and ATF-1 (51) (Dr. Michael Green,
University of Massachusetts, Amherst) cDNAs were generously
provided as gifts. Fragments containing cDNAs were amplified by
polymerase chain reaction (PCR) using primers carrying BamHI
adaptors and subcloned into a mammalian expression plasmid
pAsc.CMV-SVneoVA.2
The expression of ATF-1 and hGCN5 cDNAs was directed by a
cytomegalovirus immediate-early promoter in plasmid
pAsc.CMV-SVneoVa. The expression vector for full-length
human CDP/cut, pMT2-CDP, was described previously (34) as
well as the bacterial expression plasmids for pGEXcut3-HD,
pGEXcut3-term, and pGEXcut3+HD-term (35).
Expression and purification of CDP/cut fragments by
subcloning into the pGEX2TK vector (Amersham Pharmacia Biotech) and
expression in Escherichia coli bacterial strain BL-21 as
glutathione S-transferase (GST) fusion proteins were as
described previously (12).
Cell Transfections and Reporter Gene Analysis--
Cells were
plated at a density of 5 × 105 cells/35-mm plate
(approximately 75% confluent) 18 h prior to transient
transfections. Transfections were carried out as described previously
(12). Briefly, plasmid expression reporter and 100 ng of internal
control pRSV/CAT were diluted to a final volume of 50 µl in 20 mM Hepes buffer, pH 7.6. The DNA mixture was transferred to
a tube containing 30 µl of LipofectAMINETM (Life Technologies, Inc.)
diluted in 1 ml of Opti-MEM (Life Technologies, Inc.). The mixture was
incubated for 15 min at room temperature. This medium mixture was used
to replace the existing medium in the cell cultures and rocked gently, briefly, for even distribution of the mixture. Transfected cells were
incubated for 6 h at 37 °C in a 5% CO2 atmosphere.
At this time, medium was replaced with fresh culture medium and
cultured for an additional 48 h before assaying the cultures and
culture medium for chloramphenicol acetyltransferase activity and human growth hormone (hGH) (12). The levels of hGH secreted into the medium
from transfected cells were assayed by hGH enzyme-linked immunosorbent
assay (Boehringer Mannheim) according to the manufacturer's instructions. hGH expression was normalized to the level of
chloramphenicol acetyltransferase expression directed by the Rous
sarcoma virus long terminal repeat sequences in each experiment.
Preparation of Nuclear Extracts, Electrophoretic Mobility Shift
Assays, and DNase I Protection Assay--
Nuclear extracts from human
PANC1 cells were prepared essentially as described (52) with
modifications to the preparation of nuclear extracts described
previously (12). The extracts were frozen under liquid N2
and stored at
80 °C until further use. The concentrations of the
nuclear extracts were determined by colorimetric quantitation assays
according to the manufacturer's directions (Bio-Rad). The
double-stranded oligonucleotide corresponding to sequences of the
CFTR inverted CCAAT element
5'-tggggggaattggaagccaaatgacatcac-3' was synthesized and labeled at the
5'-end by incubating with [
-32P]ATP and T4
polynucleotide kinase. The binding reactions were carried out at
4 °C for 30 min with 0.1 ng of the 32P-labeled
oligonucleotide, 0.5 µg of nonspecific competitor DNA (poly(dI·dC)), and either 2 or 10 µg of PANC1 nuclear extract in
the following buffer conditions: 20 mM Hepes, pH 7.9, 60 mM KCl, 0.1 mM EDTA, 5% (v/v) glycerol. For
competition experiments, unlabeled competitor oligonucleotides
corresponding to nucleotide sequences of the CFTR gene
promoter bearing a mutant inverted CCAAT element
(5'-tggggggaagaagccaaatgacatcac-3') and mutant
(5'-tggggggaattggaagccaaataacatgac-3') were
added in 100 × molar excess to the binding reactions prior to the
introduction of labeled oligonucleotides. All DNA-protein complexes
were resolved by electrophoresis on 5% polyacrylamide:bisacrylamide (80:1) in 0.25 Tris acetate and EDTA at 10 volts/cm at 4 °C.
Antibody supershift assays were performed similarly, with the exception that polyclonal antiserum (1 µg) to CBF-c (33), CDP/cut,
or preimmune serum was added along with bovine serum albumin (5 µg) directly to the DNA-nuclear extract mixture and allowed to incubate at
4 °C for an additional 30 min. DNA-protein and antibody complexes were fractionated on 5% polyacrylamide:bisacrylamide (80:1) in 0.25 Tris acetate and EDTA at 10 volts/cm at 4 °C.
Deoxyribonuclease I (DNase I) protection assay was performed using
nuclear extracts prepared from untreated and 8-Br-cAMP-treated cells on
the 127-bp fragment from the region of the CFTR promoter between
198 and
71 upstream of the translation start site. A DNA
fragment was generated with the primers 5'-gggcagtgaaggcgggggaaagagc-3' and 5'-ctgggtgcctgccgctcaaccctt-3'. The 5'-oligonucleotide
5'-gggcagtgaaggcgggggaaaga-3' was labeled by using
[
-32P]ATP and T4 polynucleotide kinase. Addition of
the [
-32P]ATP-labeled oligonucleotide (~ 5 pmol) to
the PCR was used to generate a 32P-labeled DNA fragment.
The DNA fragment was excised and purified from a 5% polyacrylamide
gel. DNase protection experiments were carried out as described
previously (12). To confirm the location of protection from DNase
hydrolysis, corresponding Maxam-Gilbert [G +A] reactions were
conducted on the 32P-labeled DNA fragment.
Endonuclease Sensitivity Assays--
Nuclei from trichostatin A
(TSA)-treated, 8-Br-cAMP/TSA-treated, and untreated PANC1 cells were
digested with BspMI (500 units, New England Biolabs,
Beverly, MA) at 37 °C for 2 h in digestion buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 60 mM KCl, 0.1 mM EDTA, 5 mM
MgCl2, 5% (v/v) glycerol, and 0.5 mM
dithiothreitol). After digestion with BspMI in
vivo, DNA was extracted with a phenol/chloroform/isoamyl alcohol
(24:24:1) mixture and recovered by ethanol precipitation. Extracted DNA
was digested further with the restriction enzymes HindIII
and Eae1, fractionated on 1.8% agarose gels by
electrophoresis, and transferred onto nylon membrane filters. Filters
were hybridized to a 32P-labeled DNA probe as indicated.
Filters were washed three times using high stringency washing
conditions with a final wash (0.1 × SSC at 68 °C) for 15 min.
Filters were exposed to x-ray film or evaluated with a Storm 860 PhosphoImager using ImageQuantTM software (Molecular Dynamics,
Sunnyvale, CA).
Genomic DNase I Hypersensitivity Assays--
Cells were treated
with 100 ng/ml TSA (Sigma) for 30 min at 37 °C prior to assays.
Nuclei (20 µg of DNA equivalent) from stably transfected PANC1 cells
(12) were treated with the addition of the RNase-free DNase I (Promega,
Madison, WI) with increasing concentrations from 5 to 50 units in a
buffer. Nuclease reactions were terminated by the extraction of genomic
DNA with phenol/chloroform, and DNA was then precipitated with ethanol
at
20 °C from 2 h. DNase-treated treated DNA was digested
with SacII (420 units) and SacI (340 units, New
England Biolabs) at 37 °C for 18 h in a 1 °C digestion
buffer. Digested genomic DNA was fractionated by agarose gel
electrophoresis, blotted onto membranes, and hybridized to a human
genomic CFTR 32P-labeled
SacII-SacI probe, indicated in the experiment
shown, washed, and autoradiographed.
Immunoprecipitations and GST Pull-down Assay--
U937 cells
were metabolically labeled with [35S]methionine/cysteine
(NEN Life Science Products). Nuclear lysates of 35S-labeled
U937 cells were precleared with rabbit preimmune serum and
immunoprecipitated with protein A-Sepharose (Amersham Pharmacia Biotech). The remaining supernatant was used for immunoprecipitation experiments. Immunoprecipitation of HDAC1 was performed using nuclear
lysates from metabolically 35S-labeled U937 cells
pretreated with GST to eliminate background and immunoprecipitated with
HDAC1 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) as described
(53). The GST and the GST-CDP fusion proteins were expressed as
described above and conjugated to glutathione-Sepharose beads (Amersham
Pharmacia Biotech) after low stringency washing procedure (54). The GST
and GST-CDP/cut fusion proteins, bound to conjugated
glutathione-Sepharose beads, were equilibrated in a lysis washing (LW)
buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5 mM EDTA, 10 mM Na3
(PO4), 10 mM NaF, 2.5 mM leupeptin,
10 mM phenylmethylsulfonyl fluoride, 100 µg of
aprotinin), then incubated with nuclear extracts of
35S-labeled U937 cells (2 × 105 dpm) in a
final volume of 250 µl for 3 h at 4 °C. Then the beads were
centrifuged and washed three times in LW buffer and treated with HDAC1
antiserum. The antiserum was passed over a GST column to preclear
anti-GST antibodies. After elution of GST fusion protein from the beads
with glutathione, immunoprecipitations with HDAC1 antiserum were
performed as described below. Mixtures were washed twice with LW buffer
containing 0.5% deoxycholate and immunoprecipitated with protein
A-Sepharose beads. The immunoprecipitates were washed three times with
LW buffer containing 0.5% deoxycholate, resuspended in a 2 × Laemmli gel loading buffer, and loaded onto a 12% SDS-polyacrylamide gel. Rabbit polyclonal antibodies for CDP/cut were generated
against the GST fusion containing the human CDP/cut protein
encoding the homeodomain through the carboxyl (COOH)-terminal region
using standard protocols (54).
In Vitro Protein-Protein Binding Assays--
The GST and the
GST-CDP/cut fusion proteins were expressed as described
above and immobilized on glutathione-conjugated beads, washed, and
equilibrated in HEKG binding buffer (40 mM Hepes, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 2.5 mM
leupeptin, 10 mM phenylmethylsulfonyl fluoride, 100 µg of
aprotinin, 1.0 mM dithiothreitol, and 0.5 mg of bovine
serum albumin). Transcription template for HDAC1 was created by the
reverse transcription PCR method with 0.5 µg of human HeLa cell
mRNA using a primer pair set corresponding to the translational
open reading frame (5'-cgagcaagatggcgcagacgcag-3') and terminus
(5'-cgtgagggactcagcaggaagcc-3') of human HDAC1. The correct PCR product
was obtained, verified by gel electrophoresis and nucleotide sequence
analysis, and then cloned into the vector pCR®-Blunt
according to the manufacturer's instructions (Invitrogen) to generate
the in vitro transcription template pHDAC1-T7. The 35S-labeled HDAC1 protein was synthesized in
vitro from the linearized plasmid template by the use of the
coupled transcription-translation system (TNT® system,
Promega). The 35S-labeled HDAC1 protein was incubated with
a 50% slurry of the corresponding GST-CDP/cut fusion
protein in 400 µl of HEKG binding buffer for 1 h at 4 °C with
gentle rocking. The Sepharose beads were then washed four times with 1 ml each time of HEKG binding buffer (lacking bovine serum albumin,
leupeptin, aprotinin, and phenylmethylsulfonyl fluoride). Bound
proteins were eluted in 25 µl of 50 mM Tris-HCl, pH 6.9, 0.1 mM EDTA, containing 100 mM glutathione
(Sigma). Eluted proteins were resuspended in a 2 × Laemmli gel
loading buffer and loaded onto a 12% SDS-polyacrylamide gel and
resolved by electrophoresis.
In Vitro Histone Deacetylase Assays--
Histone deacetylase
activity was assayed essentially as described (23) with 50 µl of
crude cell extract, then immunoprecipitated with rabbit polyclonal
antiserum against recombinant human CDP/cut (34) for
2.5 h at 37 °C. Immunoprecipitates of human CDP/cut were washed three times at 4 °C in a low stringent wash containing 0.1% Nonidet P-40 in phosphate-buffered saline. Pretreatment of immunoprecipitates with 100 ng/ml TSA occurred for 30 min at 4 °C
before addition of the peptide substrate. The synthetic peptide substrate was purchased commercially (Accurate Chemical, Westbury, NY)
and corresponds to the first 24 residues of the amino-terminal domain
of bovine histone H4 (23). Immunoprecipitates were then incubated with
2.5 mg (60,000 dpm) of acid-soluble peptide for 30 min at 37 °C. The
reaction was terminated with acetic acid and HCl. Released
[3H]acetic acid was obtained by extraction with ethyl
acetate and quantitated by liquid scintillation counting. Samples were
were assayed triplicate (in four trials), and the nonenzymatic release of label was subtracted to obtain the final value.
 |
RESULTS |
CBF·NF-Y Is Associated with the CFTR Gene Proximal Y-box
Element--
Because basal and cAMP-mediated CFTR
transcription requires the conservation of the inverted CCAAT motif in
the promoter of CFTR (12, 13) and is a potential consensus
to bind CBF·NF-Y (55), we reasoned that CBF·NF-Y or similar
CCAAT-binding factors could have a role in directing CFTR
transcription. To identify the nuclear proteins associated with basal
and inducible activation of CFTR transcription, we tested
whether the transcription factor CBF·NF-Y complex could bind to the
proximal Y-box consensus site of the human CFTR promoter.
Fig. 1A demonstrates the
binding of nuclear protein prepared from confluent monolayers of PANC1
cells in competitive EMSA in the presence of unlabeled competing
oligonucleotides corresponding to wild-type human CFTR
sequences or mutant CFTR oligonucleotide sequences. The
unlabeled homologous CFTR Y-box element sequence depletes
the visible complex in a competition assay. Addition of an
oligonucleotide corresponding to the same sequence with the exception
of a three-nucleotide deletion directed at the ATTGG to A
G failed to
compete for binding. Moreover, a mutation introduced into a CRE
consensus 11 bp downstream of the ATTGG sequence competed equally as
well for the binding of nuclear protein from PANC1 cells (Fig. 1).
Shown in panel B are supershift assays (EMSA) performed with
rabbit antibodies directed against the (c) subunit of CBF·NF-Y; they
indicate a supershifted band of the CBF·NF-Y complex bound to the
Y-box motif (first lane). The CBF·NF-Y supershift was
compared with nuclear extracts treated with preimmune serum and shows
no altered mobility of the DNA-protein complex in the last
lane (Fig. 1B). This result confirms that CBF·NF-Y
can bind the Y-box sequence of human CFTR and correlates with the relative consensus necessary for binding CBF·NF-Y (55). It
is important to note that previous studies by this laboratory have also
documented the ability of this site to bind C/EBP (12). Antibodies
directed against the C/EBP
and C/EBP
proteins alter complex
formation in these experiments (data not shown) as well as in other
experiments (12) but failed to immunoprecipitate any histone
acetyltransferase activity.3
However, the question that still remains is whether cell type specificity plays a role in the ability of either CBF·NF-Y or C/EBP
to bind the CFTR Y-box element to activate transcription in vivo. Here we addressed the role of CBF·NF-Y to bind
the CFTR promoter because of the purported role of
CBF·NF-Y in histone acetylation (27, 28). Therefore, we wanted to
expand our studies to include other relevant DNA-binding proteins to
act through the inverted CCAAT element.

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Fig. 1.
EMSA/supershift demonstrates that CBF·NF-Y
binds to the proximal Y-box of the human CFTR promoter. Panel
A, competitive EMSA was performed with nuclear extracts prepared
from PANC1 cells after confluent growth. Using 2 µg of nuclear
extract and 1 × 105 cpm of a 32P-labeled
oligonucleotide of the wild-type CFTR inverted CCAAT
element, binding was competed with unlabeled oligonucleotides
containing sequences of CFTR bearing mutant CCAAT and CRE
elements. Panel B, supershifted complexes containing
CBF·NF-Y bound to the radiolabeled wild-type CFTR
oligonucleotide are recognized by the antiserum against the CBF-c
subunit of CBF·NF-Y as shown in the EMSA. Panel C,
nucleotide sequence of the human CFTR promoter between 147
and 92, relative to the first open reading frame. The sequence
corresponding to the inverted CCAAT element of CFTR is
outlined in the open box. Underlined
sequences corresponding to the nucleotide consensus of the CRE are
shown and are indicated as the hatched box above the
sequence.
|
|
Activation of CFTR Transcription by the Transcription Factor ATF-1
and the Histone Acetyltransferase hGCN5--
The correlation between
the DNA-binding proteins of the Y-box element with the local structural
changes in chromatin (12) has prompted speculation that the
modification of core histones, which overlap the Y-box, may reflect a
change to chromatin structure favorable for the activation of
transcription (27, 28). As a way of determining the role of
transcription factors and chromatin accessory factors associated with
the proximal Y-box (inverted CCAAT) element to influence the
transcription of CFTR, transactivation assays were performed
on PANC1 cells carrying stably transfected CFTR
promoter-directed transgenes. Clonal populations of stably transfected
cells bearing wild-type (pCF-197wt/hGH-PANC1) and mutated Y-box
(pCF-197
CCAAT/hGH-PANC1) transgenes were used to assess the ability
of cotransfected effector genes to alter expression of the integrated
reporter gene. Six individual stably transfected PANC1 clonal
populations were isolated and tested for each of the two transgene
constructs (pCF-197wt/hGH-PANC1 and pCF-197
CCAAT/hGH-PANC1) to
confirm that repression is not the result of integration artifacts of
the two stably transfected transgenes (Fig.
2). The background expression of promoter
function was determined by including stably transfected PANC1 cells
bearing the parental hGH reporter gene construct lacking a promoter
(Fig. 2). The cDNAs tested include the cyclic AMP-responsive
transcription factor ATF-1 and the human histone acetyltransferase
hGCN5 and activate transcription of CFTR in pancreatic
carcinoma cells (Fig. 2). Analysis of reporter transgene expression
(Fig. 2) indicates that both ATF-1 and hGCN5 direct the activation of a
CFTR-directed reporter gene. The observed 4-5-fold
activation was dependent on an intact Y-box element consensus site,
suggesting that both ATF-1 and hGCN5 functionally converge on the Y-box
element to stimulate CFTR transcription. Although the
histone acetyltransferase P/CAF (56), a close homolog of hGCN5, was not
tested here, similar results have been obtained from other studies
(28), suggesting the involvement of histone acetyltransferase in the
activation of CFTR. A mutated CFTR Y-box element
failed to confer activation by either ATF-1 or hGCN5, suggesting that
the activity by each of the effectors tested requires the intact Y-box
sequence. These data support the role of histone acetyltransferase in
the activation of CFTR and the requirement for an intact
CCAAT consensus in response to the transient expression of histone
acetyltransferase activity in the PANC1 cell type.

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Fig. 2.
Transactivation of CFTR gene transcription by
human histone acetyltransferase hGCN5 and transcription factor
ATF-1. The two cell lines bearing wild-type or mutant
CFTR promoters were cotransfected with the effector plasmid
expressing the transcription factors ATF-1 or human hGCN5 with an
internal control pCMV ( -galactosidase expression). Each
transfection was normalized to levels of -galactosidase expressed to
correct for transfection efficiency. Human GH production was expressed
as pg of hGH produced in 1 ml of culture medium. Values represent the
mean ± S.E. (n 5).
|
|
The Human Transcriptional Repressor CDP/cut Binds to the Y-box
Region of the CFTR Promoter--
We had previously suggested a strong
correlation between the Y-box (inverted CCAAT) element of the
CFTR promoter and repression of CFTR
transcription (12). Previous experiments had demonstrated the absence
of protein binding with the induction of CFTR transcription by cAMP (12). Although repression of CFTR transcription
could not be confirmed by site-directed mutagenesis because of the
intrinsic nature of the inverted CCAAT in basal transcription (12),
other reports suggest that the Y-box element of CFTR could
be a target for CDP/cut (44, 48). To determine the
functional role of CDP/cut in CFTR transcription,
PANC1 cell clones (pCF-197wt/hGH-PANC1 and pCF-197
CCAAT/hGH-PANC1)
were cotransfected with an expression vector encoding the full-length
CDP/cut cDNA, pMT2-CDP (Fig.
3). Because of the lack of
CDP/cut protein in PANC1 cells compared with U937 cells by
Western blot analysis (data not shown), PANC1 cells provided the
opportunity to introduce the ectopic expression of CDP/cut
in a relatively naïve model. Results (Fig. 3) demonstrate that
the function of CDP/cut was dependent on the Y-box to
repress activation, mediated by either ATF-1 or the human histone
acetyltransferase hGCN5. Although repression of a constitutively
expressed CFTR-directed transgene was relative, a striking
reduction (20-fold) in hGH expression was apparent when either ATF-1 or
hGCN5 was used as an activator in the cotransfection experiments (Fig.
3). It is apparent that overexpression of CDP/cut can
directly antagonize either ATF-1 or hGCN5, and repression of the
constitutive expression of CFTR was consistent with the
antagonism of ATF-1 and hGCN5 transactivation, as similarly reported
with heterologous promoters (57). To determine if the behavior of
CDP/cut to repress the activity by either ATF-1 or hGCN5 is
stoichiometric, we titered the amount of the plasmid expression vector
for CDP/cut, pMT2/CDP/cut used in each
cotransfection experiment (Fig. 4). In
these experiments the cell line pCF-197wt/hGH-PANC1, bearing the
wild-type Y-box motif of CFTR, was used to determine the
ability of CDP/cut to compete with the transactivation
potential ATF-1 and hGCN5. Results shown in Fig. 4 demonstrate that the
transfected CDP/cut expression vector competes in a
dose-dependent manner with hGCN5 and to a lesser degree
ATF-1.

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Fig. 3.
CDP/cut represses gene
expression mediated through the Y-box element of a CFTR-directed
transgene. Cotransfection experiments were performed using
LipofectAMINE reagent (Life Technologies, Inc.) of stably transfected
cell lines each containing one of the three independent human
CFTR-directed transgenes under the selection of 400 µg/ml
G418 (12). Secretion of the reporter gene product hGH was monitored
from medium of stably transfected cells lines after cotransfections
performed with the parental control vector (pMT2), the expression
vectors for CDP/cut (pMT2/CDP/cut), ATF-1
(pASC/ATF1), or the histone acetyltransferase hGCN5(pASC1/hGCN5). Each
transfection was normalized with an internal control plasmid
vector (pCMV ) expressing -galactosidase to correct for
transfection efficiency. Values (n 8) represent the
mean ± the S.D. expressed as pg of hGH secreted in 1 ml of
medium.
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Fig. 4.
Dose-dependent expression of
human CDP/cut represses ATF-1 and human hGCN5
transactivation of CFTR transcription. Cotransfection with
pMT2/CDP/cut and pASC/ATF1 or pASC1/hGCN5 (bottom
panel) into the stably transfected cell lines pCF-197wt/hGH-PANC1
and pCF-197 CCAAT/hGH-PANC1 was performed and assayed for hGH
expression. Varible but linear amounts of pMT2/CDP/cut
plasmid were cotransfected with 10 and 25 µg of either pASC/ATF1 or
pASC1/GCN5. Each cotransfection was normalized with an internal control
plasmid (pCMV ) to correct for the efficiency of each transfection
with the expression of -galactosidase activity. Human GH was
expressed as pg/ml medium. Bars above and below
boxes represent the S.E. (n 5).
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To confirm whether CDP/cut does bind the CFTR
promoter, studies were conducted using nuclear extracts from U937 cells
and recombinant CDP/cut to determine the binding pattern of
CDP/cut to the Y-box consensus site of CFTR.
Results in Fig. 5A show an EMSA antibody supershift experiment performed using an oligonucleotide corresponding to a CFTR inverted CCAAT sequence (Y-box) and
antibodies generated against the CDP/cut protein and a
recombinant GST fusion protein of CDP/cut expressed in
E. coli (see "Experimental Procedures"). Results shown
in Fig. 5A indicate that CDP/cut is a component of the complex bound to the Y-box inverted CCAAT sequence of the CFTR. A subsequent DNase I footprinting experiment was
performed to establish the location of recombinant CDP/cut
binding within the human CFTR 5'-flanking region. Protection
from hydrolysis by DNase I in the presence of recombinant
CDP/cut reveals a pattern of protection overlapping the
inverted CCAAT (Y-box) motif as shown in Fig. 5B. The figure
indicates the position relative to the open reading frame of CFTR. A
GST-CDP/cut fusion protein carrying the third cut
repeat through the termination of human CDP/cut, which
includes the homeodomain, was expressed in E. coli as a GST
fusion protein and used in DNase I footprint analysis (Fig. 5B). The GST-CDP/cut fusion protein was able to
confer protection of the same region overlapping the Y-box motif of the
CFTR promoter (Fig. 5B).

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Fig. 5.
Human CDP/cut binds the
human CFTR gene. Panel A, supershift analysis by EMSA of
CDP/cut bound to the CFTR Y-box element. Using
extracts from U937 cells, gel shift analysis of the Y-box sequence
representative of 92 to 151 nucleotides of the human
CFTR promoter was radiolabeled with [32P]ATP.
EMSA, using two different polyclonal antisera directed at
CDP/cut, was performed to identify DNA-protein mobility,
shown as complex C. Panel B, protection from hydrolysis by
DNase I between the nucleotides located 92 and 151 relative to the
5'-upstream region of the first AUG codon overlapping the Y-box
nucleotide consensus of the human CFTR gene promoter is
shown. Extracts from COS-7 cells transfected with the
CDP/cut expression vector pMT2/CDP/cut and the
GST fusion protein of CDP/cut were used in a DNase I
protection experiment and compared with control nuclear extracts from
the identical cells. Corresponding Maxam-Gilbert reactions carried out
to identify the boundaries of protection rendered by the extracts are
outlined by the hatched bar adjacent to the
photograph.
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Inhibition of Histone Deacetylase Activity by TSA Corresponds with
Activation of CFTR Transcription Mediated through the Proximal Y-box
Element--
The recent findings that coactivators and transcription
factors, such as p300/CBP, C/EBP, and CBF·NF-Y, encode or are
tethered to enzymatic activity associated with histone acetylation
provide rationale for investigating whether local chromatin,
overlapping the Y-box, may be a direct target for histone acetylation.
To test this hypothesis, we demonstrate a relationship between histone acetylation and transcription of CFTR. TSA is a specific
inhibitor of histone deacetylase activity and has been associated with
potentiating gene transcription in cell cultures (58, 59). We
determined the relationship of TSA treatment in multiple populations of
stably transfected cells to the stimulation of reporter gene expression when directed by the human wild-type CFTR promoter. Results
of our preliminary studies indicate that administration of TSA
potentiates the expression of the hGH reporter gene and that
potentiation of a CFTR-directed transgene specifically
requires the Y-box consensus (Fig. 6). To
investigate whether transcription, potentiated by TSA, is mediated
through the Y-box motif, we then tested a stably transfected transgene
in which the Y-box was mutated and no longer bound nuclear protein
(12). In the transgene constructs shown in Fig. 6, the Y-box was either
left intact in the wild-type CFTR promoter or mutated by the
deletion of four nucleotides in the Y-box element. The control reporter
transgene with the intact Y-box responded positively to both cAMP and
TSA induction. As anticipated the reporter with the mutated Y-box
sequence was not activated by either cAMP or TSA. These results
indicate that TSA enhances transcription through the Y-box. Therefore,
the inhibition of histone deacetylase activity is directed through
interactions with the Y-box sequence of CFTR.

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Fig. 6.
Requirement of the CFTR gene Y-box element
for the induction of gene transcription by TSA. Experiments were
performed using multiple clonal populations of stably transfected cell
lines carrying wild-type and mutant CFTR-directed hGH
transgenes. Each of the two transgene constructs bearing either the
wild-type or mutant CFTR-directed reporter gene received
either TSA, cAMP, or TSA+cAMP. The production of hGH was monitored
48 h after each transfection. Values reflect the percent of hGH
production relative to the untreated PANC1 cells stably transfected
with the wild-type transgene ± the S.D. (n = 6).
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Inhibition of Histone Deacetylase Activity Is Associated with
Chromatin Accessibility of CFTR in Vivo--
To establish a
correlation between the inhibition of histone deacetylation with
increased accessibility in the CFTR promoter in
vivo, we examined whether TSA treatment alters the sensitivity of
nuclei to hydrolysis with a restriction endonuclease. The experimental strategy was based on the ability to detect the sensitivity of the
CFTR promoter in nuclei to endonuclease digestion after
treatment with the histone deacetylase inhibitor, TSA. BspMI
was chosen (Fig. 7B) because
it recognizes a nucleotide sequence immediately adjacent to the Y-box
of the CFTR promoter. PANC1 cells, treated with TSA, were
used to prepare nuclei and digested with BspMI in
vivo, DNA was purified, restricted with HindIII and
Eae1, and then digestion products were detected by Southern
blot hybridization using a 32P-labeled probe corresponding
to the HindIII/Eae1 fragment of the
CFTR promoter. Fig. 7A shows that both
TSA-treated and cAMP+TSA-treated cells display a specific pattern that
corresponds with the digestion by BspMI endonuclease. This
pattern demonstrates the presence of the 673-bp and 180-bp fragments
shown (Fig. 7A). Decreasing the amount of TSA, added to
PANC1 cell cultures, reflects the loss of specific BspMI
cleavage. The treatment of PANC1 cells with both 8-Br-cAMP and TSA (100 ng/ml) complemented the sensitivity to BspMI cleavage. Thus,
TSA by itself and in combination with 8-Br-cAMP directs
BspMI cleavage of nucleotide sequences adjacent to the Y-box
of CFTR, suggesting that histone acetylation contributes to
an alteration of chromatin structure proximal to the Y-box element of
the CFTR promoter.

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Fig. 7.
Endonuclease sensitivity of the CFTR promoter
by TSA. Panel A, PANC1 cells were treated with increasing
amounts of TSA (10, 100, and 250 ng/ml) or TSA (100 ng/ml) with 500 µM 8-Br-cAMP. Nuclei were digested with BspMI,
genomic DNA was then extracted followed by digestion with
HindIII and Eae1. Digested DNA was fractionated
by electrophoresis, transferred to filters, and hybridized to the
indicated probe. The upper most band and two lower
bands represent undigested or BspMI-digested bands,
respectively. In the last lane, TSA-treated nuclei were
digested with BspMI, and genomic DNA was extracted and
loaded on to the gel in the absence of HindIII and
Eae1 digestion. Plasmid DNA, corresponding to the identical
genomic region, was digested in vitro (naked DNA) and used
as a control for comparison (not shown). Molecular weight DNA markers
were used to confirm the length of the fragments. The lengths of
undigested and digested fragments are shown to the left.
kb, kilobases. Panel B, diagram of
CFTR and the corresponding endonuclease digestion. The
inverted CCAAT (Y-box) element is indicated as the hatched
box. Double arrows indicate the anticipated
fragments after digestion with BspMI. The shaded
bar indicates the length of the 32P-labeled probe used
corresponding to the restriction fragment of CFTR.
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DNase I Hypersensitivity in the CFTR Promoter Requires an Intact
Proximal Y-box Element--
Because the status of histone acetylation
may determine the structure of chromatin, we examined whether increased
sensitivity to DNase I hydrolysis gives a pattern consistent with the
stimulation of CFTR transcription. The experiment shown in
Fig. 8 examined whether mutation to the
intact Y-box element alters sensitivity to nuclease hydrolysis
surrounding the CFTR promoter/hGH gene junction from a
stably transfected transgene. Nuclei from multiple clonal populations
of stably transfected pancreatic carcinoma PANC1 cell lines were pooled
and used to determine whether a mutation directed at the Y-box sequence
alters the pattern of nuclease sensitivity consistent with the
treatment with TSA. Shown is an autoradiograph of a Southern blot
hybridization (Fig. 8) depicting the relationship of an intact Y-box to
DNase I hydrolysis of nuclei after treatment with TSA and cAMP. The
top panels demonstrate a stably transfected PANC1 cell line,
pCF-197wt/hGH-PANC1, bearing the intact wild-type Y-box sequence of the
human CFTR-directed transgene (Fig. 8). In the comparative
experiment, mutation directed at the Y-box nucleotide consensus in cell
line pCF-197
CCAAT/hGH-PANC1 failed to confer the same sensitivity to
DNase I hydrolysis after treatments with either cAMP or TSA.
Experiments were also performed in the absence of both TSA and cAMP in
each of the transgenic constructs and demonstrate a pattern
corresponding to the lack of DNase I hypersensitivity within the
CFTR promoter (not shown). Although this is consistent with
the activation of CFTR transcription, the comparison that is
made in this experiment was planned only to reflect differences between
wild-type and mutant sequences of the inverted CCAAT element.
Therefore, results shown in Fig. 8 suggest that increased sensitivity
to DNase I by cAMP or TSA is dependent on the intact Y-box sequence.
Our results suggest that the intact inverted CCAAT sequence regulates
the accessibility of chromatin after treatment with TSA and cAMP. This
observation is consistent with the induction of the activation of gene
transcription (shown in Fig. 6) and accessibilty of chromatin to
BspMI cleavage (shown in Fig. 7). We support the notion that
the acetylation of histones, mediated by the CCAAT sequence element,
contributes to the activation of CFTR-directed
transcription. However, we do not know the precise mechanism for this
result, nor do we suggest that CBF·NF-Y (or other CCAAT-binding
factor) is responsible for the targeted recruitment of histone
acetyltransferase activity to CFTR. We observed that DNase I
hypersensitivity may correlate closely with the accessibility of the
sequences in chromatin by potential trans-acting factors.
Access to the CCAAT element in chromatin may be stimulated further by
histone acetylation, and this, in turn, potentiates further remodeling
of chromatin through the association of histone acetyltransferases
consistent with other findings (27, 28).

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Fig. 8.
DNase I hypersensitivity of the Y-box in the
human CFTR gene promoter by trichostatin A. Panel A,
schematic diagram depicting the transgenic constructs used in the
stably transfected cell lines. The diagram indicates the structural
relationship of the human CFTR gene promoter to sequences
fused with the hGH gene. The sequences beneath the diagram
are representative of the wild-type and mutant CFTR
promoters used to construct the minigenes in stably transfected cell
lines. The probe used for the Southern hybridization analysis is
indicated as the SacII-SacI fragment of the
transgene. Panel B, stably transfected cells were treated
with TSA at 100 ng/ml for 24 h. Nuclei were prepared and digested
with increasing amounts of DNase I. Genomic DNA was extracted by
organic phase extraction, digested with restriction enzymes
SacII and SacI, fractionated by gel
electrophoresis, transferred on to nitrocellulose filters, followed by
hybridization with a 32P-labeled probe as indicated.
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HDAC1 Is Associated with the COOH-terminal Region of
CDP/cut--
Recent observations have shown that repressors of
transcription alter chromatin structure through the association of
histone deacetylase activities (60, 61). Studies also have shown the association of a novel mammalian transcriptional corepressor, mSin3A,
with the histone deacetylases HDAC1 and HDAC2 (23, 60). Interactions
are mediated through a paired amphipathic helix domain conserved within
several proteins and associated with protein-protein interactions
(60-62). We tested the hypothesis to determine if human
CDP/cut, which binds to the Y-box of the CFTR,
may recruit the interactions of a histone deacetylase through a domain
similar to the paired amphipathic helix motif of mSin3A (57, 60). To
determine if histone deacetylase activity was associated with CDP/cut in vivo histone deacetylase activity was tested on
immunoprecipitates of CDP/cut as shown in Fig.
9A. CDP/cut
immunoprecipitates possess histone deacetylase activity when using a
synthetic peptide substrate corresponding to the first 24 amino-terminal residues of histone H4 (23). However, only background
levels of histone deacetylase activity were detected in the
immunoprecipitates when the antiserum was blocked with the specific
immunogen, recombinant CDP/cut. To determine if the
deacetylase activity is present, as described in Figs. 6 and 7, we
tested whether TSA would inhibit the activity associated with the
immunoprecipitates of CDP/cut. Results show that treatment
of the immunoprecipitates of CDP/cut with TSA reduces the
acetylation by approximately 60-70% (Fig. 9A). This
suggests that the associated histone deacetylase activity in the
CDP/cut immunoprecipitates is sensitive to TSA treatment.
Using a truncated human CDP/cut corresponding to the
carboxyl-terminal region of human CDP/cut, we generated
three GST fusion proteins representative of the carboxyl terminus of
CDP/cut (shown in Fig. 9B). Previous studies have
shown that the repressor function of CDP/cut was confined to
the COOH-terminal region of the CDP/cut gene product (57).
In the experiment shown in Fig. 9C, GST fusion proteins were
expressed and purified. Proteins shown in Fig. 9C correspond to the regions of human CDP/cut which encode fusion proteins
overlapping the homeodomain through the end of CDP/cut, the
cut3 domain to the COOH-terminal end of CDP/cut,
and the cut3 through the homeodomain shown as
GST-CDP/cut (HD-term), GST-CDP/cut
(cut3-term), and GST-CDP/cut (cut3-HD), respectively. Each of these
GST-CDP/cut fusion proteins was used as a target to attract
interactions to the COOH-terminal third of CDP/cut of
extracts from U937 cells metabolically labeled with
[35S]methionine/cysteine, followed by the GST pull-down
assay with glutathione-agarose beads. The 35S-labeled
protein complexes, bound to the GST-CDP/cut fusion proteins, were precipitated with glutathione-conjugated agarose and then characterized by immunoprecipitation with a rabbit polyclonal antibody
directed against HDAC1 fusion protein (Fig. 9B). The polyclonal antibody and 35S-labeled protein extracts were
preabsorbed with recombinant GST protein prior to the
immunoprecipitation. Results of the immunoprecipitation experiment
(Fig. 9D) show the association of the recombinant
CDP/cut fusion protein with HDAC1 (55 kDa) (60, 61) as the
candidate protein indicated in Fig. 9D. Immunoprecipitation
of 35S-labeled extracts is shown in the first
lane and indicates the immunoprecipitation of HDAC1 from protein
extracts of U937 cells as well as two consistent bands corresponding to
molecular masses of 150 and 50 kDa.

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Fig. 9.
Association of CDP/cut with
HDAC1 activity. Panel A, in vitro histone
deacetylase activity of anti-CDP/cut immunoprecipitates are
shown from human U937 cell extracts. Cell extracts (approximately 10 mg) were immunoprecipitated using anti-human CDP/cut
polyclonal antibodies. +block indicates that the
anti-CDP/cut antibodies were preabsorbed with recombinant
CDP/cut fusion proteins. +TSA indicates that the
anti-CDP/cut immunoprecipitates were pretreated with 100 ng/ml TSA for 30 min at 4 °C before being assayed for histone
deacetylase activity. Panel B, truncated COOH-terminal
constructs of CDP/cut in-frame with the GST protein were
used to purify 35S-labeled proteins bound to the
carboxyl-terminal domain of CDP/cut. Several fusion proteins
encoding a fusion of GST in-frame with the following corresponding
COOH-terminal truncations of the human CDP/cut protein are:
GST-CDP/cut (HD-term), GST-CDP/cut
(cut3-HD), and GST-CDP/cut
(cut3-term). Proteins were characterized by
SDS-polyacrylamide gel electrophoresis and shown with a corresponding
molecular mass marker. The proteins were expressed in E. coli strain BL21 after induction with 0.1 mM isopropyl
1-thio- -D-galactopyranoside for 1 h at 37 °C.
Bacteria lysates expressing GST fusion proteins were prepared and
purified with agarose beads conjugated with glutathione, eluted, and
electrophoresed through a 9.5% SDS polyacrylamide gel before staining with Commassie Blue dye.
Panel C, immunoblot analysis of HDAC1 from nuclear extracts
isolated from PANC1 and U937 cells. Panel D, immunoblot
analysis and immunoprecipitation of HDAC1 from a GST-CDP pull-down
assay. From left, first lane,
unlabeled U937 cell extract immunoprecipitated with HDAC1 antiserum was
separated from the remainder of the gel as an individual lane,
transferred onto a filter, and immunoblotted with HDAC1 antiserum.
Second lane, immunoprecipitation of HDAC1 from
35S-labeled nuclear extracts of U937 cells.
Third through eighth lanes, immunoprecipitation
of HDAC1 from 35S-labeled nuclear extracts of U937 cells
after GST pull-down assay with either the GST-CDP/cut
(HD-term), GST-CDP/cut (cut3-term),
GST-CDP/cut (cut3-HD), and GST fusion protein.
The washing of the 35S-labeled proteins bound to the
GST-CDP/cut fusion protein and affixed to glutathione beads
was performed three times with LW buffer. The GST proteins were eluted
with glutathione, immunoprecipitated with HDAC1 antiserum, and washed
twice with LW buffer containing 0.5% deoxycholate followed by
precipitation with protein A-Sepharose. Autoradiographs of
SDS-polyacrylamide gels are shown with the corresponding molecular mass
determination.
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Interaction of GST-CDP with HDAC1 in Vitro--
Studies described
above suggest that 35S-labeled HDAC1, from metabolically
radiolabeled U937 cells, is somehow tethered to CDP (Fig. 9). To
confirm whether the interaction between GST-CDP and HDAC1 is a result
of a direct interaction or mediated by other factors (i.e.
mSin3A or mSin3B) we examined the nature of the interaction in
vitro. We tested the ability of 35S-labeled HDAC1,
synthesized and radiolabeled in vitro using a coupled
transcription-translation system, to bind to different domains of
CDP/cut, expressed as GST fusion proteins and immobilized on
glutathione-agarose beads. The structure of full-length
CDP/cut, depicted in Fig.
10A, is comprised of a
single coiled-coil domain, three cut repeats, and one
homeodomain in addition to the transcriptional repressor domain at the
most COOH-terminal region (57). We suggest that the repressor domain of
CDP/cut recruits the interaction with HDAC1, and we sought
to localize the region of CDP/cut to interact with HDAC1.
Consistent with our hypothesis (Fig. 10B) the COOH-terminal
end of CDP/cut, expressed as GST-CDP (cut3-term), is competent to interact with HDAC1 by this in vitro binding
assay, whereas HDAC1 exhibited no binding to the GST protein control. Furthermore, truncation of the COOH-terminal region, shown as the
GST-CDP (cut3-HD) protein (Fig. 10B), impaired
the ability of the fusion protein to interact with HDAC1. The GST-CDP
(HD-term) construct containing the homeodomain and
COOH-terminal domain maintained the interaction with HDAC1 (Fig.
10B).

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Fig. 10.
Localization of HDAC1 binding to CDP/cut.
Panel A, schematic representation of the human
CDP/cut protein with conserved domains of the protein
highlighted as open, shaded, or
hatched boxes (CC, coiled coil;
cut1, cut repeat 1; cut2,
cut repeat 2; cut3, cut repeat 3;
HD, homeodomain). Panel B, the full-length HDAC1
protein was synthesized and radiolabeled by in vitro
transcription-translation system. 35S-Labeled HDAC1 was
incubated with the various COOH-terminal constructs of
CDP/cut, expressed as GST fusion proteins, and immobilized
on glutathione-Sepharose beads. The domains of CDP/cut
included in the GST fusion protein and used in the protein
affinity-binding assay are indicated above the lane. The
nonrecombinant form of GST was employed as a negative control. The
35S-labeled HDAC1 protein, bound by the
GST-CDP/cut constructs, was eluted by soluble glutathione,
resolved by SDS-PAGE, and visualized by PhosphoImager analysis
(Molecular Dynamics, Inc). Sampled primary (1° Wash) and
secondary (2° Wash) washes of 35S-labeled
HDAC1 were taken before the elution of the bound GST-CDP (cut3-term)
fusion protein and are shown in the corresponding the third
and fourth lanes. 10% of total input of
35S-labeled HDAC1 is shown in the first lane.
Molecular weight markers (Bio-Rad) were used to confirm the size of the
bands.
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DISCUSSION |
The architecture of chromatin plays a fundamental role in the
regulation of genes. The underlying behavior of gene transcription in vivo may be determined more by the overall structure of
chromatin as a result of the cumulative function transcription factors
have in regulating local chromatin structure. The relationship between the remodeling of chromatin by the modification of histones and gene-specific transcription factors recently has become more apparent (15, 20). Since the observation of transcriptional regulatory proteins
encoding intrinsic activity for the acetylation or deacetylation of
histones, this mechanism for altering chromatin structure has gained
prominence in the study of gene regulation (19). Although it is
believed that acetylation of histones may neutralize charged lysine
residues to allow for the movement or displacement of nucleosomes along
a DNA template, making local chromatin more accessible and active for
gene transcription, a precise mechanism for this model has yet to be elucidated.
In this report, we provide evidence that the CFTR gene Y-box
element is a potential target of the CBF·NF-Y activation complex. This was demonstrated by competitive EMSA and supershift analysis (Fig.
1). CBF·NF-Y has been implicated previously in the assembly and
remodeling of chromatin structure surrounding its cognate sequence
(63). The trimeric subunit complex of CBF·NF-Y has been shown
previously to possess histone acetyltransferase activity through the
tethering of hGCN5 and/or P/CAF to CBF·NF-Y (27, 28). Previously, we
have also demonstrated the binding of the CFTR inverted
CCAAT (Y-box) sequence by the transcription factor C/EBP (12). Although
our study here has not included the role of C/EBP on CFTR
transcription, it is important to note that C/EBP can interact with
CBP/p300 in vivo (64) and is the focus of other studies. We
believe that C/EBP may play a particularly intriguing role in
CFTR transcription. The absence of C/EBP in cell lines derived from transgenic "knockout" mice, when compared with
wild-type littermates, show the lack of p300-containing complexes
associated with the CFTR Y-box. However, this observation is in
contrast to results showing CBP associated with the same Y-box element in both knockout and wild-type cell
lines.4 Interestingly, the
rate of CFTR transcription in vivo in these same
"knockout" cell lines is precisely half of that compared with the
wild-type cell lines. It is plausible to consider that the composition
of individual coactivator complexes could reflect a preferential
stoichiometric arrangement or dosage between CBP and p300 (65) and may
demonstrate some distinction and/or differential regulation between the
two coactivators.
We have demonstrated that transcription of CFTR is mediated
by the transcription factor ATF-1 and the human histone
acetyltransferase hGCN5 (Fig. 2). Transfection studies have
demonstrated the activating potential of the transcription factor ATF-1
and the human histone acetyltransferase hGCN5 to stimulate
CFTR transcription. Evidence that hGCN5 potentiates
transcription of CFTR indicates a role for histone
acetylation in the regulation of CFTR transcription. We also
show that CDP/cut has a functional role in repressing transcription of CFTR. This result suggested to us that
CDP/cut effectively competes with transcriptional activators
hGCN5 and ATF-1 to antagonize directly the activation of
CFTR transcription (Figs. 3 and 4). The binding of
CDP/cut (shown in Fig. 5, A and B)
overlaps the Y-box of CFTR; therefore, we speculate that
transcription factors bound to the Y-box may, differentially, alter the
pattern of histone acetylation in chromatin proximal to transcript
initiation to regulate transcription. In our studies, we have used the
CDP/cut protein, from nuclear extracts, transfected COS-7
cells, and from protein expressed in bacteria to show binding to the
CFTR Y-box (Fig. 5B). How does the Y-box element
function to regulate CFTR transcription in vivo?
Our studies demonstrate that inhibition of histone deacetylase activity
with TSA corresponds to the stimulation of CFTR
transcription mediated through an inverted and intact CCAAT sequence.
Evidence in this report implies that activities competing for the
association with the Y-box element regulate the level of histone
acetylation in chromatin surrounding the transcript start sites,
leading to either activation or repression of gene transcription. We
have shown that inhibition of histone deacetylase activity by TSA
potentiates the transcription of a CFTR-directed transgene
through the Y-box element. Thus, the activation of CFTR
transcription could be linked to histone acetyltransferase activity
through the inverted CCAAT nucleotide consensus (Y-box) sequence.
Examination of the CFTR promoter in vivo using a
BspMI endonuclease sensitivity assay suggests that TSA will
promote cleavage of nucleotide sequences proximal to the
CFTR Y-box element and CRE (Fig. 7). Furthermore, DNase I
hypersensitivity assays shown in Fig. 8 demonstrate that an intact
5'-ATTGG-3' sequence of human CFTR is required for DNase I
hypersensitivity of nuclei. DNase I hypersensitivity of two
CFTR-directed transgenic constructs reveals that, in fact,
the ATTGG consensus is an essential nucleotide requirement for the
nuclease hypersensitivity of the CFTR promoter in chromatin
as a result of the inhibition of histone deacetylase activity in
vivo. This has been demonstrated in several randomly selected
stably transfected clones (data not shown). Despite the inhibition of
histone deacetylase activity by TSA to stimulate CFTR
transcription through the putative alteration of chromatin structure,
overlapping the Y-box element, there is no direct evidence to show the
modification of histones within the context of the CFTR
promoter to undergo changes in acetylation patterns as a result of TSA
treatment. However, our observations suggest that the specialized
sensitivity of local chromatin to nuclease hydrolysis is linked to the
inhibition of histone deacetylase activity. We have yet to establish
whether trans-acting factors bound to the CFTR
Y-box element (such as CDP/cut, C/EBP, or CBF·NF-Y) can
target the enzymatic modification of local histones in vivo
(66). Although the correlation between histone acetylation and gene
activation is established, further studies to determine the extent of
local chromatin to undergo histone modification will have to be performed.
Our observations suggest the association of CDP/cut with
histone deacetylase activity. We propose that transcriptional
repression by CDP/cut may recruit histone deacetylases to
CFTR and that certain factors tethered to the DNA-binding
proteins (such as CDP/cut) can target the deacetylation (or
acetylation) of specific lysine residues of core histones such as those
established through similar models (67, 68). Because CFTR
transcription is potentiated through TSA we tested whether
CDP/cut may function to interact with any known histone
deacetylase. Immunoprecipitation of CDP/cut demonstrates
that along with CDP/cut, histone deacetylase activity is
coimmunoprecipitated and is inhibited by TSA. Furthermore, studies
using fusion proteins of CDP/cut indicate that
CDP/cut may recruit interactions with histone deacetylase,
HDAC1, through the COOH-terminal domain known to contain the repression
domain of CDP/cut (57). It is only implied here that
CDP/cut may repress gene transcription of CFTR
through a mechanism incorporating the histone deacetylation of
chromatin in proximity to the CFTR promoter. Although the
molecular basis for the repression mediated by CDP/cut in
vivo is not known, it could conceptually involve competition between enzymes involved in histone acetylation associated with sequence-specific transcription factors. Although this simple model
could explain the role CDP/cut may have in transcriptional repression of CFTR, still little is known about the precise
mechanism directing specific alterations in local chromatin domains
in vivo.
The acetylation state of local chromatin may represent a stoichiometric
relationship between competing acetylation and deacetylation processes.
Thus, the requirement for the Y-box in hGCN5-mediated transactivation
of CFTR suggests that the Y-box element likely binds
proteins necessary for the conformational changes in chromatin structure. There is evidence to suggest that certain transcription factors have higher affinity for their binding sites when the nucleotide sequences are embedded in chromatin assembled from hyperacetylated histones (69). It is plausible that competing activities directed by CDP/cut or CBF·NF-Y, as an example,
recruit activities favorable for either the repression or activation of CFTR transcription, respectively. Although we suggest that
CDP/cut recruits histone deacetylase activity, it is unclear
precisely how such a ternary complex of CDP/cut is assembled
in the context of the CFTR promoter Y-box element and
whether such events do take place in vivo. Future studies
will have to take into account the enzymatic modification of histones
associated with the site of transcriptional repression (and activation)
and will likely provide a more detailed model for the regulation of
CFTR transcription. Investigations to determine the extent
of acetylation directed at specific lysine residues within core
histones H4 and H3 of local chromatin will provide important
information about how such activity is targeted to the CFTR
promoter. However, our observations are consistent with the role of
both CBF·NF-Y and CDP/cut to regulate genes through the
recruitment of accessory enzymatic function to enhance the effect of
these factors either to activate or repress specific genes,
respectively (27, 28, 57). The recent demonstration that HDAC1 and the
mSin3A transcriptional corepressor are present as a complex in
vivo and physically interact (60, 61), along with results
presented here, suggests that histone deacetylase activity is targeted
to the CFTR inverted CCAAT sequence by the association with
the transcriptional repressor CDP/cut. A consistent pattern
in the GST pull-down assay with GST-CDP/cut fusion protein (Fig. 9C) indicates bands of ~150 and 50 kDa,
respectively; these bands closely correlate with the molecular mass of
mSin3A or N-CoR and HDAC2, respectively (70). In a model
proposed for the repression of Pit-1-mediated transcription it was
shown that the transcriptional repressors RPX and Msx-1,
both homeodomain proteins, are tethered to mSin3A and HDAC1 through the
mSin3A corepressor homolog N-CoR to repress transcription
(71). It is likely that CDP/cut, like CBF·NF-Y or
CREB/ATF, recruits transcriptional adaptor proteins that are comprised
of either coactivator or corepressor ternary complexes dependent on the
factor bound to the Y-box element. For transcription factors bound to a
chromatin template in both activated and repressive states of
transcription this would determine the coactivator or corepressor
composition. For example, the activation of CFTR
transcription by ATF-1 and hGCN5 may account for the presence of a
transcriptional coactivator p300/CBP-bound complex to displace further
nucleosome-mediated repression of CFTR through histone acetyltransferase activity (20). Conversely, CDP/cut bound
to the Y-box of CFTR may displace the p300/CBP coactivator
ternary complex in the activated state of transcription through the
association with histone deacetylase activity. Therefore, the amount of
CDP/cut expressed in the cell (Fig. 4) may reflect a
conversion of the exposed Y-box to a less accessible element in
chromatin as a result of increased recruitment of histone deacetylase
activity. Thus, the absence (or displacement) of CDP/cut
correlates with the ability of activating mechanisms of transcription
associated with histone acetyltransferase activities to promote
transcription. Although we do not preclude the role of other
cis-acting regulatory elements of CFTR to promote
the induction of transcription, here we indicate a correlation between
the accessibility of chromatin in CFTR transcription with
the intact ATTGG sequence of CFTR. Even though several
mechanisms control transcription in vivo, the regulation of
chromatin architecture by opposing enzymatic activities is implied here
as central to this process. Therefore, the regulation of enzymatic
function by histone acetylation factors could conceivably benefit those afflicted with cystic fibrosis through the enhancement of
CFTR expression in cells that bear mutations conferring a
mild cystic fibrosis phenotype.
 |
ACKNOWLEDGEMENTS |
We thank the laboratories of Drs. Michael
Green, Yoshihiro Nakatani, Yiannis Ioannou, and Sankar Maity for
plasmid reagents of ATF-1, hGCN5, the expression vector
pASC-8neoVA, and antiserum to CBF·NF-Y, respectively. We
are also grateful for advice received from Drs. Shelley Berger,
Yoshihiro Nakatani, Checco Ramirez, and from David Zappulla.
 |
FOOTNOTES |
*
This work was supported by awards (to M. J. W.) from the
Cystic Fibrosis Association of Greater New York, the Cystic Fibrosis Foundation, and in part from a Public Health Service Award from the
National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Present address: WRAIR, Dept. of Immunology, 14th and Dahlia
St. NW, Washington, D. C. 20307.

To whom correspondence should be addressed: Dept of Pediatrics,
Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY
10029. E-mail: walsh{at}msvax.mssm.edu.
2
Y. Ioannou, unpublished data.
3
M. J. Walsh, unpublished data.
4
M. J. Walsh, S.-D. Li, and G. Shue,
manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
CFTR, cystic
fibrosis transmembrane conductance regulator;
CRE, cyclic
AMP-responsive element;
P/CAF, p300/CBP-associated factor;
CREB, CRE-binding protein;
CBP/p300, CREB-binding protein/p300;
wt, wild-type;
HDAC1/2, histone deacetylase 1/2;
bp, base pair(s);
CBF·NF-Y, CCAAT-binding factor/nuclear factor Y;
CDP/cut, CCAAT displacement protein/cut homolog;
ATF-1, activating
transcription factor 1;
hGH, human growth hormone;
CMV, cytomegalovirus;
TK, thymidine kinase;
GST, glutathione
S-transferase;
PCR, polymerase chain reaction;
Br-cAMP, bromo cyclic AMP;
TSA, trichostatin;
LW, lysis washing;
EMSA, electrophoretic mobility shift assay;
C/EBP, CAAT
enhancer-binding protein.
 |
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