Repression of Dioxin Signal Transduction in Fibroblasts
IDENTIFICATION OF A PUTATIVE REPRESSOR ASSOCIATED WITH Arnt*
Katarina
Gradin
§,
Rune
Toftgård¶,
Lorenz
Poellinger
, and
Anna
Berghard
From the
Department of Cell and Molecular Biology,
Karolinska Institute, S-171 77 Stockholm, the ¶ Center for
Nutrition and Toxicology, Karolinska Institute, Novum, S-141 57 Huddinge, and the
Department of Cell and Molecular Biology,
Umeå University, S-901 87 Umeå, Sweden
 |
ABSTRACT |
Heterodimeric complexes of basic
helix-loop-helix/PAS transcription factors are involved in regulation
of diverse physiological phenomena such as circadian rhythms, reaction
to low oxygen tension, and detoxification. In fibroblasts, the basic
helix-loop-helix/PAS heterodimer consisting of the ligand-inducible
dioxin receptor and Arnt shows DNA-binding activity, and the receptor
and Arnt are able to activate transcription when fused to a
heterologous DNA-binding domain. However, fibroblasts are nonresponsive
to dioxin with regard to induction mediated by the DNA response element recognized by the receptor and Arnt. Here we demonstrate that Arnt is
associated with a fibroblast-specific factor, forming a complex that is
capable of binding the dioxin response element. This factor may
function as a repressor since negative regulation of target gene
induction appears to be abolished by inhibition of histone deacetylase
activity by trichostatin A. Finally, the negative regulatory function
of this factor appears to be restricted for dioxin signaling since Arnt
was able to mediate, together with hypoxia-inducible factor-1
,
transcriptional activation in hypoxic cells. Taken together, these data
suggest that fibroblast-specific inhibition of dioxin responsiveness
involves recruitment by Arnt of a cell type- and signaling
pathway-specific corepressor associated with a histone deacetylase.
 |
INTRODUCTION |
There is ample evidence for tissue-specific differences in
response to environmental contaminants such as halogenated polycyclic aromatic hydrocarbons. 2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD1; dioxin) is a
prototype compound for this class of compounds (for review, see Ref.
1). Induction of the dioxin response gene CYP1A1 shows
cell-type dependence as demonstrated both in cell culture and in
transgenic mice (2). Inducibility of CYP1A1 depends on
assembly of two transcription factors, the dioxin receptor and Arnt.
Upon ligand (i.e. dioxin) binding to the dioxin receptor, it
heterodimerizes with Arnt, and the dimer binds specifically to
xenobiotic response elements (XREs) located in promoter regions of
target genes such as CYP1A1 and CYP1B1 (3, 4). In
addition, it has been shown that changes in chromatin structure
contribute to ligand-dependent activation of
CYP1A1 transcription (5). Inactive ligand-free dioxin
receptor forms a stable heteromeric complex with the 90-kDa heat-shock
protein (hsp90) (6) and is activated by ligand to its DNA-binding state
by sequential release of hsp90 and heteromerization with Arnt.
Both the dioxin receptor and Arnt belong to the basic helix-loop-helix
(bHLH)/PAS family of transcription factors (7-9). A structural
similarity was found between the dioxin receptor, Arnt, and the
Drosophila proteins Sim and Per (7-9). This region of
homology was subsequently termed PAS
(Per-Arnt-Sim), and both HLH and
PAS motifs are required for dimerization of, for example, the dioxin
receptor and Arnt proteins (reviewed in Refs. 10 and 11). Moreover, the
ligand-binding domain of the dioxin receptor is contained within the
PAS region (reviewed in Refs. 10 and 11). Several additional members of
the bHLH/PAS family have recently been identified, including mouse Sim1
and Sim2 (12), and Drosophila trachealess (13, 14). The
locus of the mammalian homologue (Sim2) coincides with the Down's
syndrome chromosomal region (15). Moreover, the hypoxia-inducible
factor-1
(HIF-1
) (16) is also a bHLH/PAS protein that regulates
inducible expression of genes such as erythropoietin, vascular
endothelial growth factor, and a number of glycolytic enzymes in
hypoxic cells (reviewed in Ref. 17). Under conditions of low oxygen
tension, recruitment of Arnt is essential to enable HIF-1
to bind
its target DNA sequences (hypoxia response elements). The mechanism of
activation of this physiologically important class of transcription
factors is not yet understood.
It has become increasingly apparent that histone acetyltransferases and
histone deacetylases (HDACs) play important roles in transcriptional
regulation. Recent studies suggest that acetylation and deacetylation
of histones are involved in the process of chromatin assembly (reviewed
in Refs. 18 and 19). Moreover, histone acetyltransferases and HDACs
have been found to be components of some transcriptional coactivator
and corepressor complexes (for recent reviews, see Refs. 20 and 21),
respectively, which suggests that they modulate transcriptional
activity at specific promoters by locally perturbing chromatin
structure. Several of these histone acetyltransferase-containing
proteins complexes have been identified. For example, CBP/p300, P/CAF,
and SRC-1 interact with a variety of DNA-binding transcription factors
and have been shown to function as transcriptional coactivators
(reviewed in Ref. 21). Moreover, repression by nuclear receptors has
been correlated to binding of corepressors mouse Sin3, NCoR/SMRT, and the HDAC Rpd3/HDAC1 (22). Furthermore, the Mad/Max heterodimer recruits
an HDAC via mouse Sin3 and NCoR/SMRT, which seems to repress genes that
normally are activated by the Myc/Max heterodimer (23, 24).
Most studies of mechanisms underlying induction of the
CYP1A1 gene encoding cytochrome P4501A1 as well as
cytochrome P4501A1 enzymatic activity have been performed using hepatic
cells since liver is the tissue that has the highest concentration of
P4501A1 (reviewed in Ref. 25). Studies of, for example, transgenic mice carrying the CYP1A1 promoter in front of the lacZ
reporter gene show that inducibility varies in different adult tissues
(2), but the mechanism behind this phenomenon is not known. Moreover, consistent with reports on P4501A1 levels in various tissues after in vivo exposure, we have found normal fibroblasts in
culture to be nonresponsive to dioxin with regard to CYP1A1
inducibility (26, 27).
Analyses of nuclear extracts from fibroblasts show that the dioxin
receptor can be activated to its DNA-binding form by ligand (27).
Moreover, two constitutively expressed XRE-binding protein species that
are not found in responsive cells, i.e. keratinocytes and
HepG2 hepatoma cells, are present in fibroblast extracts. We report
that this constitutive fibroblast-specific XRE-binding factor(s) is a
heteromeric complex, in which one of the factors is Arnt, whereas the
other protein(s) that directly contacts the XRE remains to be
identified. More important, we also demonstrate that the HDAC inhibitor
trichostatin A (TSA) (28) in combination with dioxin can overcome
transcriptional repression in fibroblasts. These results suggest that a
cell type-restricted partner factor possibly harboring HDAC activity
modulates Arnt function in fibroblasts, resulting in efficient
interference with the dioxin receptor signaling pathway.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Treatments--
The human hepatoma cell line
HepG2 was grown in RPMI 1460 medium (Life Technologies, Inc.)
supplemented with 10% (v/v) fetal calf serum (Life Technologies,
Inc.), 100 µg/ml streptomycin, 100 IU/ml penicillin, and 0.25 µg/ml
Fungizone. Fibroblasts were prepared from neonatal foreskin by placing
the protease-treated dermis in a cell culture dish in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented as
described above until the fibroblasts had adhered to the plastic and
started to divide. Human keratinocytes were isolated from adult donors
and cultivated as described previously (29). Cells at the third to
fifth passages were routinely used. Cultures were treated with
2,3,7,8-tetrachlorodibenzofuran (TCDF; Cambridge Isotope Laboratories),
TCDD (Cambridge Isotope Laboratories) dissolved in Me2SO,
cycloheximide (Sigma), trichostatin A (Sigma), or sodium butyrate
(Sigma), whereas control cultures received solvent only, not exceeding
a final concentration of 0.1%.
Plasmid Constructions and Plasmids--
pCMV/
DBD,
pCMV/
DBD/DR83-805, and pCMV/
DBD/Arnt128-774 have been described
previously (30). The p(GRE)2T109Luc reporter gene was
created by subcloning an oligonucleotide containing a dimerized
glucocorticoid response element-binding site (5'-CACTAGAACA TCCTGTACAG TCGACCGCGG AGATCTAGAA CATCCTGTACA GTGAGCT-3') from the
tyrosine aminotransferase gene (31) into the SacI site of pT109Luc containing the herpes simplex thymidine kinase promoter with
luciferase as a reporter gene (32). The reporter gene constructs pT81
(32) and pTX.Dir (29) have been described previously.
Transfection and Transient Expression Assays--
For the
experiments shown in Fig. 3, HepG2 cells were transfected with
Lipofectin (Life Technologies, Inc.). The cells were incubated over
night in RPMI 1460 medium lacking serum with the indicated plasmid DNA
and 1.2 µg of Lipofectin/cm2. The medium was then changed
to RPMI 1460 medium supplemented with 10% serum, and the cells were
subsequently treated with either 50 nM TCDF or
Me2SO alone for 36-48 h prior to harvest. Fibroblasts were
transfected by electroporation. Briefly, the cells were trypsinized, washed, and resuspended in phosphate-buffered saline, and plasmid DNA
was added to a final volume of 800 µl. The DNA/cell mixture was
incubated for 5 min at room temperature prior to electroporation (13 ohms, 600 microfarads, and 400 V) with an Electro Cell Manipulator (Techtum). After electroporation, cells were put on ice for 10 min
before seeding out 100 × 106 cells in
25-cm2 cell culture flasks. Fibroblasts were grown for
48 h prior to treatment with 50 nM TCDF or
Me2SO alone for an additional 36-48 h. For the experiments
shown in Fig. 7A, both fibroblasts and HepG2 cells were
transfected with Fugene 6 transfection reagent (Roche Molecular
Biochemicals). Three microliters of Fugene 6 was combined with the
indicated plasmid, and the cells (10 cm2) were incubated
with the Fugene 6/DNA mixture for 6 h prior to treatment with 10 nM TCDD and/or TSA for 36-40 h. Luciferin was purchased
from BioThema.
RNA Isolation and RNA Blot Analysis--
Total RNAs were
isolated using acid-phenol extraction as described (33). Polyadenylated
RNA was prepared by using streptavidin-conjugated paramagnetic
oligo(dT) particles (Promega) according to the manufacturer's protocol. RNA was fractionated through formaldehyde-agarose gels, blotted onto nylon membranes, and UV-cross-linked. The filters were
subsequently prehybridized, hybridized, and washed according to
standard procedures (34) prior to autoradiography.
32P-Labeled probes for the cDNAs of cytochrome
P4501A1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (27),
cytochrome P4501B1 (4), murine dioxin receptor (7), human Arnt (9),
human HIF-1
, erythropoietin, aldolase A (35), and
-actin
(CLONTECH) were generated by a random priming
procedure. A complementary synthetic oligonucleotide specific for the
extra exon in the alternatively spliced N terminus of Arnt
(5'-AGTCTCTCTT TATCCGCAGA GCTCTGCTCA TC-3') (9) was synthesized and
32P-end-labeled with T4 polynucleotide kinase as described
(34).
Immunoblot Analysis--
Cellular protein was prepared by lysing
the cells in 20 mM sodium phosphate (pH 7.2), 50 mM
-glycerophosphate, 10% (w/v) glycerol, 1 mM EDTA, 150 mM NaCl, 0.1 mM
Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 5 µM pepstatin (Roche Molecular Biochemicals),
10 µg/ml leupeptin (Sigma), 10 µg/ml aprotinin (Bayer), and 0.1%
Nonidet P-40, and the lysates were cleared by centrifugation at
15,000 × g for 45 min at 4 °C. Proteins were
separated by 9% SDS-polyacrylamide gel electrophoresis and
electrophoretically transferred to nitrocellulose membranes, and the
membranes were subsequently stained by ECL Western blotting detection
(Amersham Pharmacia Biotech) using anti-dioxin receptor or anti-Arnt
antiserum at a 1:200 dilution. Preimmune serum did not, under these
conditions, give rise to any background reactivity (27). The antisera
were raised as described (36, 37) against the N-terminal parts of the
mouse dioxin receptor and the human Arnt protein, respectively.
Preparation of the 5-Bromo-2'-deoxyuridine-substituted DNA and
DNA Cross-linking--
A synthetic oligonucleotide spanning the XRE1
element of the rat cytochrome P4501A1 upstream promoter region (3)
(5'-gatcCCTCCA GGCTCTTCTC
ACGCAACTCC GGGGCACg-3', P4501A1 sequence
shown in uppercase letters) was annealed to a complementary
11-nucleotide primer (5'-GTGCCCCGGAG-3'). The primed template was
filled in with the Klenow fragment of Escherichia coli DNA
polymerase I in the presence of 100 µM dCTP, dATP,
5-bromo-2'-deoxyuridine triphosphate, and 3.3 µM
[
-32P]dGTP as described (34). The DNA contains three
bromodeoxyuridine residues within the recognition sequence for the
receptor as well as a fourth residue near one end of the
oligonucleotide (underlined bases). EMSA DNA-binding reactions
(contained in an open 1.5-ml Eppendorf tube on ice) were irradiated for
5 min with UV (Stratagene; emission wavelength = 254 nm;
intensity = 4000 microwatts/cm2) at a distance of 4-5
cm from the source. The proteins were separated by SDS-polyacrylamide
gel electrophoresis (9%), dried, and subjected to autoradiography. For
SDS-polyacrylamide gel electrophoresis analysis, prestained and
14C-labeled molecular mass marker proteins were purchased
from Bio-Rad and Amersham Pharmacia Biotech, respectively.
Nuclear Extract Preparation and EMSA--
Cells were treated
with 50 nM TCDF for 1 h or 10 µg/ml cycloheximide
for 4 h before harvest. Nuclei and protein extracted were prepared
as described previously (38). DNA-binding reactions were assembled in a
total volume of 20 µl with 10 µg of nuclear proteins at a final
concentration of 25 mM HEPES (pH 7.9), 0.2 mM
EDTA, 75 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 µM pepstatin, 5% glycerol, 4% Ficoll, 100 µg/ml
poly(dI-dC) (Amersham Pharmacia Biotech), and 12.5 µg/ml poly(dA-dT)
(Amersham Pharmacia Biotech) using a double-stranded,
32P-labeled oligonucleotide as a probe, the sequence of
which corresponded to residues
968 to
997 of the human
CYP1A1 gene (5'-CTCCGGTCCT TCTCACGCAA CGCCTGGGCA-3', sense
orientation). In indicated competition experiments, a 100-fold molar
excess of unlabeled oligonucleotides was used. A double-stranded
oligonucleotide (5'-TCTAGTGTTG GAGAACGAAT CAGCATCTGA GTAC-3') was used
as nonspecific competitor DNA, whereas unlabeled probe DNA served as
specific competitor. When using anti-dioxin receptor or anti-Arnt
antiserum, 10 µg of nuclear protein was preincubated with the diluted
antiserum (1:10) for 20 min at room temperature prior to the
DNA-binding reaction. Protein-DNA complexes were separated under
nondenaturing conditions on a 3% polyacrylamide gel (29:1) run in 50 mM Tris, 380 mM glycine, and 2.7 mM
EDTA at 4 °C.
 |
RESULTS |
Lack of Induction of Dioxin Target Genes in Human
Fibroblasts--
We have previously demonstrated cell type-specific
differences in P4501A1 inducibility and shown that normal human
fibroblasts are nonresponsive to dioxin with regard to induction of
P4501A1 mRNA levels (27). To investigate if the nonresponsive
phenotype of fibroblasts is a more generalized phenomenon, we analyzed
regulation in fibroblasts of another dioxin response gene, cytochrome
P4501B1 (4). Very low levels of constitutive P4501B1 mRNA
expression were observed by RNA blot analysis in untreated fibroblasts,
and more important, this message was not induced upon exposure of cells
to TCDF (Fig. 1, compare lanes
1 and 2). In control experiments analyzing primary
human keratinocytes, P4501B1 mRNA levels were barely detectable by
RNA blot analysis, being very similar to the low levels of constitutive
P4501B1 mRNA expression observed in primary human fibroblasts.
Treatment of keratinocytes with TCDF, however, resulted in significant
induction of P4501B1 mRNA expression (Fig. 1, compare lanes
4 and 5). Cycloheximide treatment is known to induce
P4501A1 expression in hepatic cells, keratinocytes, and fibroblasts in
the absence of ligand (27). Interestingly, also P4501B1 mRNA
expression was induced following cycloheximide treatment of fibroblasts
(Fig. 1, lane 3). Thus, although TCDF induction response is
inhibited, both CYP1A1 and CYP1B1 expression appear to be regulated by a similar, if not identical,
cycloheximide-sensitive mechanism, demonstrating that both promoters
are not irreversibly repressed in fibroblasts.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 1.
Fibroblasts are nonresponsive to dioxin.
Shown are the results from RNA blot analyses of P4501B1 and GAPDH
mRNA steady-state levels in keratinocytes and fibroblasts. Cells
were treated with 50 nM TCDF or 10 µg/ml cycloheximide
(CHX) for 24 h. Analysis of GAPDH mRNA served as a
control. Eight micrograms of total RNA was loaded in each lane. Note
that TCDF treatment of fibroblasts did not lead to induction of
P4501B1.
|
|
To examine any differences in expression levels of the dioxin receptor
and Arnt between nonresponsive and responsive cells, polyadenylated RNA
from fibroblasts and HepG2 cells was analyzed. RNA blot analysis
demonstrated that the ~6-kilobase dioxin receptor mRNA species
was expressed at similar levels in both cell types (Fig.
2A). Two Arnt mRNA
transcripts of 2.6 and 4.2 kb were present at comparable levels in both
fibroblasts and HepG2 cells, respectively (Fig. 2A). In
addition, an alternatively spliced Arnt mRNA has been described (9)
that contains an extra exon of 45 nucleotides encoding 15 amino acids
of unknown relevance. No cell type-specific differences in the presence
of this alternatively spliced Arnt mRNA were observed (Fig.
2A). Moreover, immunoblot analysis with antiserum directed
against the dioxin receptor or Arnt showed no differences between HepG2
cells and fibroblasts in expression levels of these proteins (Fig.
2B). Thus, these results suggest that nonresponsiveness of
fibroblasts to dioxin is not attributable to altered expression levels
of the dioxin receptor or its partner factor Arnt.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 2.
Dioxin receptor and Arnt levels in
fibroblasts and HepG2 cells. A, shown are blots of
poly(A)+ RNA from fibroblasts and HepG2 cells analyzed for
expression levels of dioxin receptor (DR), Arnt, and GAPDH
mRNAs. In addition, the presence of an alternatively spliced Arnt
(Alt. Arnt) mRNA species containing an extra exon was
examined. One microgram of poly(A)+ RNA was loaded in each
lane. B, 200 µg of protein from either HepG2 cytosol
(lanes 1 and 3) or fibroblast cytosol
(lanes 2 and 4) was loaded in each lane,
separated by SDS-polyacrylamide gel electrophoresis, blotted, and
analyzed for the dioxin receptor or Arnt protein using specific
antisera. The mobility of the dioxin receptor (DR) is
indicated.
|
|
Dioxin Receptor and Arnt Fusion Proteins That Are Functionally
Uncoupled from One Another Are Active in Fibroblasts--
To
investigate if the dioxin receptor and Arnt by themselves could
function as independent activators in fibroblasts, we used expression
plasmids coding for fusion proteins. These constructs contain the
N-terminal
DBD fragment (amino acids 1-500) of the human
glucocorticoid receptor that spans the major transactivating (
) and
DNA-binding (DBD) domains, but lack the large C-terminal hormone-binding domain (Fig.
3A). This DNA-binding
glucocorticoid receptor derivative replaced the dioxin receptor
DNA-binding N-terminal bHLH motif (construct p
DBD/DR), generating a
dioxin receptor with affinity for glucocorticoid response elements. In
addition, Arnt devoid of its N-terminal bHLH domain was fused to the
DBD motif (p
DBD/Arnt) (Fig. 3A). Fibroblasts and HepG2
cells were transiently transfected with the chimeric constructs, and
activity was assayed as ability to induce glucocorticoid response
element-dependent expression of a cotransfected reporter
gene (pT109(GRE)2Luc). Upon expression of
DBD/DR in
HepG2 cells, a 5-fold induction of reporter gene activity by TCDF was
obtained, whereas the activities of
DBD and
DBD/Arnt constructs
were not altered by this treatment (Fig. 3B). Interestingly,
expression of
DBD/DR produced a 3-fold induction response in
fibroblasts (Fig. 3B), and moreover,
DBD/Arnt showed high
constitutive activity in the same cells. Taken together, these results
indicate that the dioxin receptor and Arnt are functionally active in
fibroblasts when analyzed in a transcription-activating context not
involving their usual response element. This suggests that repression
in fibroblasts is mediated by the XRE sequence element, but not
directly through negative regulation of the transactivating domains of
the dioxin receptor/Arnt heterodimer.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Activity of dioxin receptor and Arnt when
fused to a heterologous DNA-binding domain. A, a
schematic representation of the human glucocorticoid receptor
(hGR), mouse dioxin receptor (mDR), human
Arnt protein (hArnt), and chimeric constructions is shown.
The transactivating ( 1) and DBDs of the glucocorticoid
receptor are indicated. In the case of the dioxin receptor and Arnt,
the sequence motifs are indicated: bHLH, PAS (containing repeats A and
B), and Q-RICH (glutamine-rich sequence). Chimeric receptor
constructs contain the N-terminal 500 amino acids of the human
glucocorticoid receptor (termed DBD) fused to C-terminal
segments of the dioxin receptor or Arnt. B, fibroblasts
(100 × 106 cells) were transfected with 5 µg of
pT109(GRE)2Luc as a reporter and 5 µg of expression
constructs encoding the indicated receptor derivatives. HepG2 cells (10 cm2) were transfected with 1 µg of reporter plasmid and 1 µg of expression constructs. Bars represent average values
of four to five experiments and have been normalized to activity
obtained from transfections with pT109Luc only and to protein content
of cellular extracts. ZN-F, zinc-finger; aa,
amino acid(s); TK, thymidine kinase.
|
|
Endogenous Arnt Is Functional as a Partner Factor to HIF-1
in
Fibroblasts--
Low oxygen tension, CoCl2, and
desferrioxamine induce expression of different genes such as
erythropoietin, vascular endothelial growth factor, and some glycolytic
enzymes (17). This effect is mediated by a heteromeric complex
consisting of HIF-1
and Arnt (16). Fibroblasts and HepG2 cells were
treated with various concentrations of CoCl2 for 24 h
and subsequently analyzed for mRNA levels of erythropoietin and
aldolase A. mRNA levels for both of these HIF-1
response genes
were strikingly increased, from barely detectable levels to abundant
ones in fibroblasts, whereas induction in HepG2 cells was only
~2-fold (Fig. 4). Moreover, HIF-1
mRNA levels were not affected in either of the cell lines. These
results indicate that both endogenous Arnt and HIF-1
are functional
in fibroblasts since the HIF-1
/Arnt heteromer is able to regulate
hypoxia-inducible genes.

View larger version (78K):
[in this window]
[in a new window]
|
Fig. 4.
RNA blot analysis of hypoxia-inducible
genes. Total RNA was prepared from cultures of fibroblasts and
HepG2 cells that were treated with increasing concentrations of
CoCl2 (50, 100, and 150 µM) for 24 h.
Subsequently, cells were analyzed for expression of HIF-1 ,
erythropoietin (EPO), aldolase A, and -actin mRNAs by
RNA blot analysis. Five micrograms of RNA was loaded in each
lane.
|
|
Identification of a Novel Partner Factor for Arnt in
Fibroblasts--
Nuclear extracts from fibroblasts and HepG2 cells
were analyzed by EMSA to characterize proteins binding to a
32P-labeled XRE oligonucleotide probe. Fibroblast extracts
gave rise to two constitutive, distinct, and specific protein-XRE
complexes (Fig. 5A) that were
not formed if nuclear extracts from HepG2 cells were used (Fig. 5,
compare A and B) (27). Formation of these two
complexes was not affected by cycloheximide treatment (Fig.
5A, compare lanes 1 and 3). Thus, two
critical parameters distinguish these complexes from a labile factor(s)
that has been implicated in negative regulation of CYP1A1
expression in a number of cell lines (39, 40): (i) cell-type
specificity and (ii) insensitivity to cycloheximide treatment.

View larger version (73K):
[in this window]
[in a new window]
|
Fig. 5.
EMSA of nuclear extracts from fibroblasts and
HepG2 cells. A, fibroblasts were treated for 4 h
with vehicle alone (lanes 1 and 2) or 10 µg/ml
cycloheximide (CHX; lanes 3 and 4),
and nuclear extracts were prepared. The protein extracts were analyzed
for the presence of specific XRE-binding activity. Competition was
performed by addition of a 100-fold molar excess of unlabeled XRE
oligonucleotide (lanes 2 and 4). B,
EMSA was performed with nuclear proteins extracted from fibroblasts or
HepG2 cells treated for 1 h with 50 nM TCDF. Protein
extracts were analyzed for the presence of specific XRE-binding
activity by competition experiments with a 100-fold molar excess of an
unrelated sequence motif (lanes 2 and 7) or the
XRE sequence motif (lanes 3 and 8). To identify
XRE-binding proteins, nuclear extracts were preincubated with diluted
(1:10) anti-dioxin receptor antiserum ( DR; lanes
4 and 9) or anti-Arnt antiserum ( A;
lanes 5 and 10) prior to the DNA-binding
reaction. Note that anti-dioxin receptor and anti-Arnt antisera
recognized a common single complex generated by both fibroblasts and
HepG2 cell extracts (denoted Rec), whereas formation of
fibroblast-specific complexes 1 and 2 was only
affected by anti-Arnt antiserum. In both A and B,
the asterisk indicates a nonspecific protein-XRE
complex.
|
|
Three protein-XRE complexes were detected by EMSA using extracts from
TCDF-treated fibroblasts (Fig. 5B). These were specific as
assessed by competition with unlabeled specific and nonspecific oligonucleotides (Fig. 5, A, compare lanes 1 and
2; and B, compare lanes 1-3). We have
presently no indications that the smaller fibroblast-specific complex
detected represents a degradation product of the larger, and thus, it
cannot formally be excluded that the small species (complex
2 in Fig. 5, A and B) represents a distinct
endogenous form. The dioxin receptor-containing complex was identified
by preincubating extracts with anti-dioxin receptor antiserum. As shown
in Fig. 5B (compare lanes 4 and 9),
this antiserum inhibited formation of the dioxin-inducible complex by
extracts from fibroblasts while leaving the two additional constitutive XRE-specific complexes unaffected. Interestingly, these two
constitutive fibroblast-specific complexes were supershifted to a
slower mobility in EMSA following incubation of extracts with anti-Arnt
antiserum (Fig. 5B, compare lanes 5 and
10).
In parallel experiments, UV-induced cross-linking of proteins binding
to XRE was performed. To cross-link nuclear proteins to the XRE, we
generated a double-stranded, bromodeoxyuridine-substituted XRE probe.
Nuclear extracts from TCDF-treated fibroblasts and HepG2 cells were
incubated with this oligonucleotide for 20 min prior to UV irradiation
for 5 min. Analysis of covalently cross-linked proteins from HepG2
cells by SDS-polyacrylamide gel electrophoresis revealed a single
radiolabeled band of ~120 kDa. Formation of this complex was
inhibited by preincubation with anti-dioxin receptor antiserum (Fig.
6, compare lanes 1 and
2). Moreover, 120 kDa was consistent with the size of the
human dioxin receptor as determined by immunoblot analyses (27),
whereas Arnt immunoreactivity was observed as a 90-kDa species using
extracts from either HepG2 cells or fibroblasts (Fig.
2B).

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 6.
UV cross-linking of nuclear proteins to a
bromodeoxyuridine-substituted XRE. Nuclear extracts were prepared
from fibroblasts and HepG2 cells treated with 50 nM TCDF
for 1 h. Extracts were then incubated with a
5-bromo-2'-deoxyuridine triphosphate-containing, double-stranded,
32P-labeled oligonucleotide encompassing the XRE1 motif,
and proteins were UV-cross-linked to the DNA in solution. Competition
experiments were performed by including a 100-fold excess of unlabeled
specific (lane 5) or nonspecific (lane 4)
oligonucleotide. The results of preincubating fibroblast nuclear
proteins with diluted (1:10) antiserum against the dioxin receptor
( DR) or Arnt ( A) for 20 min prior to the
DNA-binding reaction are shown in lanes 2, 6, and
7, respectively. Note that anti-dioxin receptor antiserum
recognized a single complex both in HepG2 and fibroblast extracts
(denoted Rec.), whereas formation of fibroblast-specific
complexes 1 and 2 was not affected by this
antibody. The fact that Arnt did not, under these experimental
conditions, cross-link to the XRE using HepG2 (lane 1) or
rodent (37) cell extracts makes it unlikely that Arnt would cross-link
when present in fibroblast extracts.
|
|
In contrast to results obtained with extracts from HepG2 cells, three
specific protein-XRE complexes of ~125, 120, and 90 kDa were detected
upon UV cross-linking using nuclear extracts from fibroblasts (Fig. 6,
lane 3). Consistently, the appearance of the ~120-kDa
protein-DNA complex was abolished when fibroblast extracts were
preincubated with anti-dioxin receptor antiserum (Fig. 6, compare
lanes 3 and 6). The ~125- and ~90-kDa
UV-cross-linked protein-DNA complexes were specific for the XRE (Fig.
6, compare lanes 3 and 5) as assessed by DNA
competition experiments. Formation of these two complexes was, however,
not affected either by antiserum against the dioxin receptor or by
antiserum against the Arnt protein (Fig. 6, compare lanes 3,
6, and 7). It is conceivable that these two novel
cross-linked proteins are part of the two complexes detected by EMSA
when using fibroblast extracts. In conclusion, these data indicate that
the constitutive XRE-binding factor(s) detected in fibroblasts are
heteromeric complexes containing, in addition to an as yet unidentified
partner factor(s), the bHLH/PAS factor Arnt.
Treatment with Trichostatin A Derepresses P4501A1 Induction by
Dioxin--
It was recently shown that transcriptional repression by
sequence-specific DNA-binding factors can involve recruitment of an
HDAC to the promoter region (reviewed in Ref. 20). Important tools to
study the role of histone acetylation are specific inhibitors of HDAC
activity such as sodium butyrate and the more potent and specific
inhibitor TSA (28). Fibroblasts and HepG2 cells were transfected with a
minimal XRE-luciferase reporter construct, and as a control, the
reporter construct without XRE was used. Cells were then treated with
dioxin in the absence or presence of TSA for 36-40 h. As shown in Fig.
7A, dioxin did not induce the
reporter gene in fibroblasts. However, a 3-fold induction of luciferase
activity was observed when fibroblasts were treated with both dioxin
and TSA. In control experiments, high ligand-dependent inducibility of the XRE-luciferase reporter construct was only marginally reduced upon exposure to TSA.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 7.
Inducibility of cytochrome P4501A1 is
derepressed by treatment with trichostatin A. A,
fibroblasts and HepG2 cells were transfected with 2 and 1 µg of
pT81Luc and pTX.Dir, respectively. Luciferase activity after treatment
with 10 nM TCDD in the presence or absence of either 0.3 or
3 µM TSA was determined. Results are representative of
several independent experiments, and values have been normalized to
protein content of cellular extracts. B, fibroblasts and
HepG2 cells were cotreated with 10 nM TCDD and increasing
concentrations of TSA (1.0, 1.5, 2.0, 2.5, and 3.0 µM) or
25 mM sodium butyrate (NaBu) for 24 h.
Cytochrome P4501A1 and GAPDH mRNA levels were assayed by RNA blot
analysis of 20 µg of total cellular RNA/lane. TK,
thymidine kinase.
|
|
To further clarify that TSA could relieve a repressive effect on dioxin
inducibility in fibroblasts, P4501A1 mRNA levels were determined by
RNA blot analysis. Fibroblasts and HepG2 cells were treated with dioxin
and various concentrations of TSA. As expected, dioxin alone did not
induce P4501A1 mRNA in fibroblasts. Cotreating fibroblasts with
dioxin and increasing concentrations of TSA, however, resulted in
induction of CYP1A1 mRNA levels as compared with those
detected in cells treated with TSA alone (Fig. 7B, compare
lanes 3-8). In addition, when fibroblasts were treated with
both TCDD and sodium butyrate, an induction of CYP1A1
mRNA levels was detected (Fig. 7B, lanes 9 and 10). However, HepG2 cells showed no significant
difference in dioxin inducibility after cotreatment with TSA (Fig.
7B, compare lanes 12-14).
 |
DISCUSSION |
Normal fibroblasts do not respond to dioxin with increases in
target gene expression, and an XRE sequence is not capable of mediating
ligand-dependent activation of a minimal promoter in this
cell type (27). This lack of response is seen despite the fact that
both the dioxin receptor and its partner factor Arnt are expressed in
these cells. The data presented here suggest that nonresponsiveness is
not due to lack of transactivating capacity of these two proteins.
Instead, we have identified in fibroblasts a novel Arnt-interacting
partner factor(s) that, in association with Arnt, directly contacts
DNA. The expression of this factor correlates with the phenomenon that
inducibility can be restored by inhibition of HDAC activity. Taken
together, these data have interesting implications for the negative and
positive regulation of bHLH/PAS proteins exerted at the level of
recruitment of corepressors/activators.
Although the endogenous dioxin receptor can be activated to its
DNA-binding form upon exposure to dioxin receptor ligands, this does
not lead to induction of transcription of either CYP1A1 or
CYP1B1 target genes or to increased expression of
XRE-containing minimal promoter constructs in fibroblasts (27). Thus,
it was important to establish that the functional properties of the
dioxin receptor and Arnt, other than their DNA-binding activity, were not inhibited by post-translational mechanisms. By fusing the dioxin
receptor and Arnt, devoid of their bHLH motifs, to a heterologous DNA-binding domain, the independent function of these two factors could
be studied in transfected cells. More important, these experiments show
that the chimeric dioxin receptor is conditionally regulated by dioxin
and that the constitutive transcriptional activation function of Arnt
is similar in both fibroblasts and dioxin-responsive HepG2 cells. In
addition, endogenous Arnt appears to be functional when targeting a
different response element together with another partner factor,
HIF-1
, since the hypoxia response genes erythropoietin and aldolase
A are induced under hypoxic conditions also in fibroblasts.
Using fibroblast protein extracts, three distinct XRE-binding complexes
can be visualized after UV cross-linking to an XRE probe. One of these
complexes represents the activated dioxin receptor. The other two
complexes are detected only when using fibroblast extracts, and these
are not related to either the dioxin receptor or the Arnt protein.
Notably, analyses of protein-XRE complexes in vitro by EMSA
show that the fibroblast-specific protein-XRE complexes harbor Arnt.
Thus, these results strongly indicate the presence of a
fibroblast-specific, DNA-contacting factor interacting with Arnt, the
expression of which correlates with the nonresponsive phenotype of the cells.
This putative repressor protein is clearly distinct from a previously
described P4501A1-repressing activity in a mutant hepatoma cell line
(41) in that the complex described here is a novel DNA-binding species.
Furthermore, repression by a heteromeric DNA-binding factor containing
the bHLH/PAS factor Arnt represents a mechanism of negative regulation
that is distinct from the action of the negative regulatory HLH factor
Id that forms abortive, non-DNA-binding heteromers with bHLH partner
factors of the myogenic family (42).
The identification of mechanisms underlying nonresponsiveness of cells
in the presence of a functional activator is interesting from a general
perspective in that less is known about negative than positive
regulation of gene expression. Several models have been proposed for
how repressors mediate their negative regulatory activity. For example,
the Ying-Yang-1 transcription factor can either repress or activate
transcription depending on promoter context (43, 44), whereas Max can
heterodimerize with either Myc or Mad and then function as a
transcriptional activator or repressor, respectively (45). Furthermore,
nuclear hormone receptors such as thyroid hormone receptors alternate
in their function as repressors or activators depending on occupation
of the hormone-binding domain (46). When bound to DNA, these
heterodimers recruit the corepressors mouse Sin3 and/or NCoR, which are
associated with an HDAC (22). This complex is believed to deacetylate
adjacent chromatin structures, which results in transcriptional
silencing. Using the HDAC inhibitor TSA, we show that repression of
CYP1A1 mRNA induction was abrogated, resulting in
ligand-dependent induction responses. This mechanism most
likely is mediated through the XRE since a minimal XRE construct also
was induced when fibroblasts were cotreated with dioxin and TSA. These
results indicate that the fibroblast-specific XRE-binding factor
recruits an HDAC activity, which subsequently silences transcription of
CYP1A1. Recently, it has been shown that only MeCP2
(methylcytosine-binding
protein-2) interacts with mouse Sin3 and
maintains transcriptional repression (47). To exclude the
possibility that cell type-specific inhibition of CYP1A1 is
caused by DNA methylation, we used the demethylating agent
5-aza-2'-deoxycytidine. However, we could not detect any induction of
CYP1A1 following cotreatment of fibroblasts with 5-aza-2'-deoxycytidine and dioxin (data not
shown).2
If Arnt is repressed in dioxin signal transduction, why is Arnt
functional in partnership with HIF-1
in mediating hypoxia-inducible gene expression in fibroblasts? At present, we have no mechanistic data
to explain this phenomenon. Interestingly, however, it has been shown
that both dimerization with distinct partner factors (48) and binding
to specific DNA response elements (49) may allosterically alter
functional properties of certain steroid receptors. It is therefore
possible that the conformation of the Arnt·HIF-1
complex in
hypoxic cells is not permissive for interaction with corepressors
and/or the fibroblast-specific factor. Recently, it has been reported
that CBP/p300 plays a critical role in signal transduction by HIF-1
(50, 51). Moreover, CBP and the CBP-associated factors P/CAF and SRC-1
have been demonstrated to harbor histone acetyltransferase activity.
Thus, when Arnt is interacting with HIF-1
, histone acetyltransferase
activity may therefore modulate the acetylation pattern of histones,
which in turn will alter chromatin structure. However, when Arnt
constitutively binds to the XRE together with the fibroblast-specific
factor, the complex seems to function as a repressor, which indicates
that, in the context of XRE binding, Arnt is involved in recruitment of
corepressors. Thus, exchange of HIF-1
for the fibroblast-specific
factor switches the Arnt heterodimer from transcriptional activator to
repressor, suggesting that the cell type-specific fibroblast factor is
needed for the recruitment of conceivable corepressors. Moreover, if the repressor in fibroblasts associates with a corepressor that harbors
HDAC activity, do positive transcriptional effects of the dioxin
receptor depend on recruitment of histone acetyltransferases? Although
this issue has not been conclusively elucidated, it has been shown that
induction of CYP1A1 transcription is dependent on the
C-terminal region of the dioxin receptor, which has transactivating capacity and mediates functional promoter communication with the transcription machinery (52). In addition, Kobayashi et al. (53) have observed that the histone acetyltransferase-containing coactivator CBP/p300 interacts with the C-terminal domain of Arnt. Together with our results, these observations are consistent with the
model that the Arnt partner in fibroblasts may determine
dominant-negative regulation of dioxin responsiveness by recruitment of
a corepressor harboring HDAC activity. To further elucidate this
mechanism of repression of dioxin signal transduction in fibroblasts,
it will now be important to identify the Arnt-associated factor and to examine whether it is associated with a corepressor harboring HDAC activity.
 |
FOOTNOTES |
*
This work was supported by the Swedish Cancer Society, the
Swedish Environmental Protection Board, and Swedish Medical Research Council Grant 11818 (to A. B.).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.
§
To whom correspondence should be addressed: Dept. of Cell and
Molecular Biology, Medical Nobel Institute, Karolinska Institute, Box
285, S-171 77 Stockholm, Sweden. Tel.: 46-8-728-7331; Fax: 46-8-34-88-19; E-mail: katarina.gradin{at}cmb.ki.se.
2
K. Gradin, unpublished observation.
 |
ABBREVIATIONS |
The abbreviations used are:
TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin;
XRE, xenobiotic
response element;
bHLH, basic helix-loop-helix;
HIF-1
, hypoxia-inducible factor-1
;
HDAC, histone deacetylase;
CBP, CREB
binding protein;
TSA, trichostatin A;
TCDF, 2,3,7,8-tetrachlorodibenzofuran;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
EMSA, electrophoretic mobility shift assay;
DBD, DNA-binding domain.
 |
REFERENCES |
-
Poland, A.,
and Knutson, J. C.
(1982)
Annu. Rev. Pharmacol. Toxicol.
22,
517-554[CrossRef][Medline]
[Order article via Infotrieve]
-
Campbell, S. J.,
Carlotti, F.,
Hall, P. A.,
Clark, J.,
and Wolf, C. R.
(1996)
J. Cell Sci.
109,
2619-2625[Abstract/Free Full Text]
-
Fujisawa-Sehara, A.,
Sogawa, K.,
Yamane, M.,
and Fujii-Kuriyama, Y.
(1987)
Nucleic Acids Res.
15,
4179-4191[Abstract]
-
Sutter, T. R.,
Tang, Y. M.,
Hayes, C. L.,
Wo, Y. Y.,
Jabs, E. W.,
Li, X.,
Yin, H.,
Cody, C. W.,
and Greenlee, W. F.
(1994)
J. Biol. Chem.
269,
13092-13099[Abstract/Free Full Text]
-
Morgan, J. E.,
and Whitlock, J. P., Jr.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
11622-11626[Abstract]
-
Wilhelmsson, A.,
Cuthill, S.,
Denis, M.,
Wikström, A.-C.,
Gustafsson, J.-Å.,
and Poellinger, L.
(1990)
EMBO J.
9,
69-76[Abstract]
-
Burbach, K. M.,
Poland, A.,
and Bradfield, C. A.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
8185-8189[Abstract]
-
Ema, M.,
Sogawa, K.,
Watanabe, Y.,
Chujoh, Y.,
Matsushita, N.,
Gotoh, O.,
Funae, Y.,
and Fujii-Kuriyama, Y.
(1992)
Biochem. Biophys. Res. Commun.
184,
246-253[Medline]
[Order article via Infotrieve]
-
Hoffman, E. C.,
Reyes, H.,
Chu, F.-F.,
Sander, F.,
Conley, L. H.,
Brooks, B. A.,
and Hankinson, O.
(1991)
Science
252,
954-958[Medline]
[Order article via Infotrieve]
-
Hankinson, O.
(1995)
Annu. Rev. Pharmacol. Toxicol.
35,
307-340[CrossRef][Medline]
[Order article via Infotrieve]
-
Schmidt, J. V.,
and Bradfield, C. A.
(1996)
Annu. Rev. Cell Dev. Biol.
12,
55-89[CrossRef][Medline]
[Order article via Infotrieve]
-
Ema, M.,
Morita, M.,
Ikawa, S.,
Tanaka, M.,
Matsuda, Y.,
Gotoh, O.,
Saijoh, Y.,
Fujii, H.,
Hamada, H.,
and Kikuchi, Y.
(1996)
Mol. Cell. Biol.
16,
5865-5875[Abstract]
-
Isaac, D. D.,
and Andrew, D. J.
(1996)
Genes Dev.
10,
103-117[Abstract]
-
Wilk, R.,
Weizman, I.,
and Shilo, B.-Z.
(1996)
Genes Dev.
10,
93-102[Abstract]
-
Chen, H.,
Chrast, R.,
Rossier, C.,
Antonarakis, S. E.,
Kudoh, J.,
Yamaki, A.,
Shindoh, N.,
Maeda, H.,
and Minoshima, S.
(1995)
Nat. Genet.
10,
9-10[Medline]
[Order article via Infotrieve]
-
Wang, G. L.,
Jiang, B. H.,
Rue, E. A.,
and Semenza, G. L.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
5510-5514[Abstract]
-
Wang, G. L.,
and Semenza, G. L.
(1996)
Curr. Opin. Hematol.
3,
156-162[Medline]
[Order article via Infotrieve]
-
Grunstein, M.
(1997)
Nature
389,
349-352[CrossRef][Medline]
[Order article via Infotrieve]
-
Wade, P. A.,
Pruss, D.,
and Wolffe, A. P.
(1997)
Trends Biochem. Sci.
22,
128-132[CrossRef][Medline]
[Order article via Infotrieve]
-
Struhl, K.
(1998)
Genes Dev.
12,
599-606[Free Full Text]
-
Torchia, J.,
Glass, C.,
and Rosenfeld, M. G.
(1998)
Curr. Opin. Cell Biol.
10,
373-383[CrossRef][Medline]
[Order article via Infotrieve]
-
Nagy, L.,
Kao, H.-Y.,
Chakravarti, D.,
Lin, R. J.,
Hassig, C. A.,
Ayer, D. E.,
Schreiber, S. L.,
and Evans, R. M.
(1997)
Cell
89,
373-380[Medline]
[Order article via Infotrieve]
-
Ayer, D. E.,
Lawrence, Q. A.,
and Eisenman, R. N.
(1995)
Cell
80,
767-776[Medline]
[Order article via Infotrieve]
-
Hassig, C. A.,
Fleischer, T. C.,
Billin, A. N.,
Schreiber, S. L.,
and Ayer, D. E.
(1997)
Cell
89,
341-347[Medline]
[Order article via Infotrieve]
-
Ioannides, C.,
and Parke, D. V.
(1990)
Drug Metab. Rev.
22,
1-85[Medline]
[Order article via Infotrieve]
-
Omiecinski, C. J.
(1990)
Cancer Res.
50,
4315-4321[Abstract]
-
Gradin, K.,
Wilhelmsson, A.,
Poellinger, L.,
and Berghard, A.
(1993)
J. Biol. Chem.
268,
4061-4068[Abstract/Free Full Text], and references therein
-
Yoshida, M.,
Horinouchi, S.,
and Beppu, T.
(1995)
Bioessays
17,
423-430[Medline]
[Order article via Infotrieve]
-
Berghard, A.,
Gradin, K.,
Pongratz, I.,
Whitelaw, M.,
and Poellinger, L.
(1993)
Mol. Cell. Biol.
13,
677-689[Abstract]
-
Gradin, K.,
Whitelaw, M. L.,
Toftgård, R.,
Poellinger, L.,
and Berghard, A.
(1994)
J. Biol. Chem.
269,
23800-23807[Abstract/Free Full Text]
-
Jantzen, H. M.,
Strahle, U.,
Gloss, B.,
Stewart, F.,
Schmid, W.,
Boshart, M.,
Miksicek, R.,
and Schutz, G.
(1987)
Cell
49,
29-38[Medline]
[Order article via Infotrieve]
-
Nordeen, S. K.
(1988)
BioTechniques
6,
454-457[Medline]
[Order article via Infotrieve]
-
Chomczynski, P.,
and Sacchi, N.
(1987)
Anal. Biochem.
162,
156-159[CrossRef][Medline]
[Order article via Infotrieve]
-
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)
Molecular Cloning: A laboratory Manual, 2nd Ed., pp. 5.70 and 7.52, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Gradin, K.,
McGuire, J.,
Wenger, R. H.,
Kvietikova, I.,
Whitelaw, M. L.,
Toftgård, R.,
Tora, L.,
Gassman, M.,
and Poellinger, L.
(1996)
Mol. Cell. Biol.
16,
5221-5231[Abstract], and references therein
-
Whitelaw, M. L.,
Pongratz, I.,
Wilhelmsson, A.,
Gustafsson, J.-Å.,
and Poellinger, L.
(1993)
Mol. Cell. Biol.
13,
2504-2514[Abstract]
-
Mason, G. G.,
Witte, A. M.,
Whitelaw, M. L.,
Antonsson, C.,
McGuire, J.,
Wilhelmsson, A.,
Poellinger, L.,
and Gustafsson, J.-Å.
(1994)
J. Biol. Chem.
269,
4438-4449[Abstract/Free Full Text]
-
Struhl, K. (1990) in Current Protocols in Molecular Biology
(Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) Suppl.
11, p. 12.2.1, John Wiley & Sons, Inc., New York
-
Lusska, A.,
Wu, L.,
and Whitlock, J. P., Jr.
(1992)
J. Biol. Chem.
267,
15146-15151[Abstract/Free Full Text], and references therein
-
Reick, M.,
Robertson, R. W.,
Pasco, D. S.,
and Fagan, J. B.
(1994)
Mol. Cell. Biol.
14,
5653-5660[Abstract], and references therein
-
Watson, A. J.,
Weirbrown, K. I.,
Bannister, R. M.,
Chu, F.-F.,
Reiszporszasz, S.,
Fujii-Kuriyama, Y.,
Sogawa, K.,
and Hankinson, O.
(1992)
Mol. Cell. Biol.
12,
2115-2123[Abstract]
-
Benezra, R.,
Davis, R. L.,
Lockshon, D.,
Turner, D. L.,
and Weintraub, H.
(1990)
Cell
61,
49-59[Medline]
[Order article via Infotrieve]
-
Johansson, E.,
Hjortsberg, K.,
and Thelander, L.
(1998)
J. Biol. Chem.
273,
29816-29821[Abstract/Free Full Text]
-
Basu, A.,
Lenka, N.,
Mullick, J.,
and Avadhani, N. G.
(1997)
J. Biol. Chem.
272,
5899-5908[Abstract/Free Full Text]
-
Ayer, D. E.,
and Eisenman, R. N.
(1993)
Genes Dev.
7,
2110-2119[Abstract]
-
Hörlein, A. J.,
Naar, A. M.,
Heinzel, T.,
Torchia, J.,
Gloss, B.,
Kurokawa, R.,
Ryan, A.,
Kamei, Y.,
Söderströ, M.,
and Glass, C. K.
(1995)
Nature
377,
397-404[CrossRef][Medline]
[Order article via Infotrieve]
-
Nan, X.,
Ng, H.-H.,
Johnson, C. A.,
Laherty, C. D.,
Turner, B. M.,
Eisenman, R. N.,
and Bird, A.
(1998)
Nature
393,
386-389[CrossRef][Medline]
[Order article via Infotrieve]
-
Schulman, I. G.,
Li, C.,
Schwabe, J. W.,
and Evans, R. M.
(1997)
Genes Dev.
11,
299-308[Abstract]
-
Lefstin, J. A.,
and Yamamoto, K. R.
(1998)
Nature
392,
885-888[CrossRef][Medline]
[Order article via Infotrieve]
-
Ebert, B. L.,
and Bunn, H. F.
(1998)
Mol. Cell. Biol.
118,
4089-4096
-
Kallio, J. K.,
Okamoto, K.,
O'Brien, S.,
Carrero, P.,
Makino, Y.,
Tanaka, H.,
and Poellinger, L.
(1998)
EMBO J.
17,
6573-6586[Abstract/Free Full Text]
-
Ko, H. P.,
Okino, S. T.,
Ma, Q.,
and Whitlock, J. P. J.
(1997)
Mol. Cell. Biol.
17,
3497-3507[Abstract]
-
Kobayashi, A.,
Numayama-Tsuruta, K.,
Sogawa, K.,
and Fujii-Kuriyama, Y.
(1997)
J. Biochem. (Tokyo)
122,
703-710[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.