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J. Biol. Chem., Vol. 277, Issue 2, 1619-1627, January 11, 2002
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From the Department of Psychiatry and Behavioral Science, State University of New York, Stony Brook, New York 11794-8101
Received for publication, October 9, 2001, and in revised form, November 7, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Transcription from the amyloid precursor protein
(APP) promoter is largely dependent on a nuclear factor binding site
designated as APB A defining neuropathological manifestation of Alzheimer's disease
and Down's syndrome is the extracellular deposition of amyloid The promoter of the APP gene is a necessary element in
regulating APP transcription, and it has been shown to
confer some degree of cell type-specific expression in transgenic mice
(11, 12). The proximal APP promoter is devoid of CCAAT and
TATA boxes but contains a prominent initiator element associated with
the main transcriptional start site (+1). The integrity of this
initiator element is essential for both start site selection and
optimal transcriptional activity (13). In addition, an intact nuclear factor binding site designated APB
. The protein that binds to this site is the
multifunctional transcription factor CTCF, which consists of 727 amino
acids and contains a domain of 11 zinc finger motifs that is flanked by 267 amino acids on the N-terminal side and 150 amino acids on the
C-terminal side. Depleting HeLa cell nuclear extract of endogenous CTCF
specifically reduced transcriptional activity from the APP promoter. However, transcriptional activity was restored by
replenishing the depleted extract with recombinant CTCF. Deleting 201 amino acids from the C-terminal end of CTCF had no detrimental effect on transcriptional activation, whereas deleting either 248 or 284 amino
acids from the N-terminal end abolished transcriptional activation.
Competing endogenous CTCF in vivo was accomplished by
cotransfecting COS-1 cells with a plasmid overexpressing CTCF constructs and a reporter plasmid containing the APP
promoter. Under these conditions, an N-terminal deletion of CTCF
reduced expression from the APP promoter, whereas the
C-terminal deletion had no effect. These results demonstrate that CTCF
activates transcription from the APP promoter and that the
activation domain is located on the N-terminal side of the zinc finger domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-protein (1-3). The amyloid -protein peptide is derived by proteolytic cleavage from a group of proteins designated amyloid -protein precursors
(APP),1 and these are derived
from the same gene by differential splicing (4). The APP
gene is differentially expressed in all major tissues, with the highest
levels observed in kidney and brain (5, 6). Increased levels of
APP gene transcript have been observed in Down's syndrome
and in certain areas of the brain in Alzheimer's disease (6-9).
Overexpression of APP also leads to amyloid deposition in transplanted
murine hippocampal tissue with trisomy 16, the mouse equivalent of
Down's syndrome (10). These observations suggest that overexpression
of APP may be one of several contributing factors in the formation of
amyloid depositions and in the neuropathology associated with
Alzheimer's disease.
is essential for effective transcription from the APP promoter (13, 14). The core
recognition sequence for this binding site is located between positions
82 and 93 (Fig. 1C), and
its elimination reduces transcriptional activity by ~70-90% (13,
14). The nuclear factor that activates transcription from APB was
identified as CTCF (15), a nuclear regulatory protein comprising 727 amino acids (16). It contains a centrally located DNA binding domain
with 11 zinc finger motifs that is flanked by 267 amino acids on the
N-terminal side and 150 amino acids on the C-terminal side (Fig. 1,
A and B). Selective deletions from the N- and
C-terminal sides of the zinc finger domain showed that the N-terminal
end of the zinc finger domain was aligned toward the transcriptional
start site (15).
View larger version (37K):
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Fig. 1.
CTCF and the proximal APP
promoter. A, amino acid sequence of human CTCF
(16). The N-terminal methionine is designated as M1. All
other amino acids designating the positions of specific deletions are
indicated by the specific amino acid followed by its position in the
sequence. Zinc fingers are indicated by brackets. B,
schematic representation of CTCF indicating the N-terminal, C-terminal,
and zinc finger domain. The approximate positions of N- and C-terminal
deletions are indicated by arrows. C, sequence of the
proximal APP promoter from position 120 to +15. The
primary transcriptional start site is designated as position
+1. Boxed sequences include the core recognition
sequences for transcription factors CTCF (APB) (14, 15), sp1 (37),
USF (APB
) (14, 38), and the initiator sequence surrounding
the transcriptional start sites from position 4 to +1 (13).
Black bar indicates the DNase I-protected domain produced by
full-length CTCF (FLCTCF) together with the orientation of
the N- and C-terminal end of the zinc finger domain (27).
CTCF was first described as a factor that binds to the chicken
c-MYC promoter (17) and to the silencer element of
the chicken lysozyme gene (18, 19). CTCF binds to diverse sequences by utilizing different combinations of essential zinc fingers (16, 19).
Consequently, a defined DNA recognition sequence cannot readily be
recognized. The function of CTCF in gene regulation is also diverse.
For example, CTCF binds to the chicken lysozyme silencer 2.4 kilobase
pairs upstream from the transcriptional start site. Here it
synergistically represses transcription in conjunction with the thyroid
hormone receptor or the retinoic acid receptor (18, 20). Another
example of synergistic repression is provided by the coordinate action
of CTCF and the thyroid hormone receptor on the TRE-containing rat
genomic element 144 (21, 22). Furthermore, CTCF has been found to
directionally block enhancer activation by binding to the insulator
element at the 5' end of the chicken -globin gene locus (23).
Similar CTCF -binding sequences were identified in a variety of
insulators from diverse vertebrate species, suggesting a widespread
role for CTCF in the regulation of enhancer-activated genes (23). CTCF
also binds to the proximal promoter of the chicken c-MYC gene where it acts either as a transcriptional repressor or activator (17, 24, 25). In the human and mouse c-myc, genes CTCF binds to divergent sequences that coincide with RNA polymerase pausing sites
within the transcribed region of the genes (16).
The above examples illustrate that CTCF primarily acts as a negative
regulator of transcription. In contrast, some of our previous studies
(13-15, 26) have provided indirect evidence implicating CTCF as a
transcriptional activator of the APP promoter. The mechanism
by which CTCF exerts its diverse regulatory effects remains unclear.
However, it is likely to involve interactions with specific secondary
factors. We provide here direct evidence both in
vitro and in vivo that CTCF activates
transcription from the APP promoter and that this
activation requires the N-terminal end of the molecule.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Expression of Recombinant CTCF Constructs in Pichia pastoris-- The cDNA encoding human CTCF was amplified by the polymerase chain reaction from a cDNA library derived from the human retinoblastoma cell line Y79 (CLONTECH) (27). Deletions from the 5' end were introduced at restriction sites AccI (position 965) and Bsu36I (position 1216), resulting in primary translation products with N-terminal transcriptional start sites at positions Met-249 and Met-285, respectively. Deletions from the 3' end were introduced at restriction sites BglII (position 2142) and PstI (position 1867), yielding translation products with C-terminal deletions at positions Asp-617 and Cys-525, respectively (Fig. 1, A and B).
Expression of CTCF and its deletion constructs in yeast was
accomplished with the Pichia Expression Kit (Invitrogen)
according to the manufacturer's instructions. Briefly, cDNA
encoding full-length CTCF or its N- and C-terminal deletions was
excised from plasmid pGEM 7Z() (Promega) and cloned either into the
EcoRI site of plasmid pHIL-D2 or between the
BamHI and NotI sites of plasmid pCIP3.5
(Invitrogen). Both are vectors that direct intracellular recombinant
protein expression in P. pastoris. The vectors containing the CTCF cDNA constructs in the correct orientation were
transformed into Pichia strain KM71 by electroporation.
Positive clones were amplified, induced for recombinant protein
expression, and screened for the presence of CTCF by mobility shift electrophoresis.
Purification of CTCF-- Recombinant CTCF proteins were extracted and purified from P. pastoris as described (27) with some modifications. Pichia cells (10-15 g) were mixed with 25 ml of 0.5-mm glass beads, adjusted to a total volume of 50 ml with buffer F (40 mM HEPES, pH 7.6, 2 mM MgCl2, 1 mM EDTA, 2 mM DTT, 100 mM KCl, 1 M urea, and 10 µM ZnSO4), and homogenized with the Bead Beater apparatus (Biospec Products, Inc., Bartensville, OK). Cell debris was pelleted at 5,000 × g for 10 min, and the supernatant was supplemented with 3 M KCl to a final concentration of 200 mM. The lysate was further clarified by centrifugation at 100,000 × g for 60 min.
CTCF was purified from the extracts by single step SP cation exchange chromatography. Briefly, a 1-ml HiTrap SP-Sepharose column (Amersham Biosciences) was equilibrated with buffer F containing 200 mM KCl. Cleared extract was loaded on the column, and proteins were eluted with a linear concentration gradient of KCl (0.2-1 M) in buffer F. Fractions of 0.5 ml were collected, and the CTCF binding activity was monitored by mobility shift electrophoresis. Fractions containing the peak activity were concentrated ~10-fold with a Microcon-30 centrifugal device (Millipore). It must be noted that the presence of 1 M urea is necessary for successful concentration. In the absence of urea, CTCF and especially some truncated forms of the protein are prone to bind to the concentrator's membrane.
Antibodies-- Rabbit polyclonal antibodies against the N-terminal part of CTCF were prepared as described elsewhere (26). The antibodies were affinity-purified on recombinant CTCF attached to a Ni2+-chelating column (28). Specifically, recombinant full-length CTCF containing a His tag at its N terminus was expressed in P. pastoris and purified as described above. The protein was loaded on a 1-ml HiTrap chelating column (Amersham Biosciences, Inc.) charged with NiSO4. The column was first washed with 3 volumes of buffer E (40 mM HEPES, pH 7.6, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT) containing 500 mM KCl and then with 3 volumes buffer E containing 300 mM KCl. Rabbit anti-CTCF serum (10 ml) was diluted with an equal volume of buffer E containing 400 mM KCl and loaded on the column. The column was washed with 5 volumes of buffer E containing 200 mM KCl. Antibody was eluted with 3 ml of 4 M MgCl2. The eluate was dialyzed overnight against 500 ml of buffer D (40 mM HEPES, pH 7.6, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 100 mM KCl, and 10% glycerol) with one change of dialysis buffer. Antibody was concentrated with a Centriplus-30 centrifugal device (Millipore) to a final volume of 0.4 ml. The resulting antibody recognized a single CTCF band on Western blots of whole HeLa cell nuclear extract. Antibody titer was determined by mobility supershift electrophoresis, which was performed as described elsewhere (26) using 2 µl of HeLa cell nuclear extract and increasing amounts of the affinity purified antibody. Under these conditions 0.3 µl of the antibody was sufficient to completely shift the band corresponding to the oligonucleotide-CTCF binding complex.
Immunodepletion of CTCF from Nuclear Extracts--
Nuclear
extract from HeLa cells was prepared as described elsewhere (13, 29).
To deplete CTCF from nuclear extract, 60 µl of the affinity-purified
antibodies were added to 400 µl of the extract and incubated for 30 min at 25 °C. The extract was cleared by centrifugation at
10,000 × g for 10 min and mixed with 80 µl of
protein G-Sepharose (Amersham Biosciences), which had been equilibrated
with buffer D. The extract was incubated for 30 min at 25 °C and
gently stirred periodically to keep the resin in suspension. The resin
was removed by centrifugation at 10,000 × g for 10 min. The extract was aliquoted and stored at 80 °C.
A control sample of native HeLa cell nuclear extract was subjected to exactly the same procedure except that in the initial incubation buffer D was used in place of the antibodies. This control preparation is referred to as whole nuclear extract.
Mobility Shift Electrophoresis--
The double-stranded
oligonucleotide APB-80WT (15) was 5' end-labeled with
[
-32P]ATP using T4 polynucleotide kinase (30). This
oligonucleotide contained the APP promoter sequence from
position 110 to 64, which includes the APB core recognition
sequence for CTCF (Fig. 1C). Recombinant CTCF was diluted
with buffer F containing 500 mM KCl. The binding reaction
was assembled by first mixing 0.5 µl of diluted CTCF and 1 µl of
CTCF-depleted nuclear extract with binding buffer (buffer D),
supplemented with 5 µg of yeast tRNA, 2 µg of
poly(dI-dC)·poly(dI-dC), and CHAPS to a final concentration of 2.5%
in a total volume of 16 µl. Alternatively, the control reaction
mixtures contained a total of 1 µl of HeLa nuclear extract (whole or
depleted) with no CTCF added. The mixture was preincubated for 5 min at
25 °C followed by the addition of 1 µl of labeled double-stranded
oligonucleotide (20 ng per binding reaction; 50,000-200,000 cpm) in
binding buffer. The final binding reactions were incubated for 30 min
at 25 °C and then supplemented with 1.5 µl of fetal calf serum and
electrophoresed in 6% polyacrylamide gels containing 0.5× Tris
borate/EDTA (30) at 100-150 constant voltage. The gels were dried, and
the amount of bound and free oligonucleotide was quantitated with a
GS-250 PhosphorImager (Bio-Rad). Binding activity was expressed in
mobility shift units (msu). One msu was designated as the amount of
protein that completely binds 1 ng of 80-mer oligonucleotide
APB-80WT during mobility shift electrophoresis.
Special conditions were employed in the binding reactions for mobility
shift electrophoresis in experiments dealing with in vitro
competition of native CTCF with purified recombinant CTCF proteins. In
this case the binding conditions were adjusted to reproduce exactly the
conditions of the corresponding in vitro transcription
reaction (see below). Recombinant CTCF was diluted with buffer F
containing 500 mM KCl, and 0.7 µl was mixed with 2 µl
of depleted nuclear extract, 5 µg of yeast tRNA, and 2 µg of
poly(dI-dC)·poly(dI-dC). The volume was adjusted to 8 µl with buffer E bringing the final KCl concentration to 100 mM,
and the mixture was incubated at 25 °C for 1 min. Subsequently, 25 ng of labeled 80-mer oligonucleotide APB-80WT was added to this mixture. This represented approximately the same molar amount of APB
CTCF-binding sequence present in 2 µg of the plasmid DNA that was
used in the in vitro transcription reaction (see below). This mixture was preincubated for 10 min at 25 °C. During this time,
in a separate tube, 2 µl of whole nuclear extract was mixed with 5 µg of yeast tRNA and 2 µg of poly(dI-dC)·poly(dI-dC) and adjusted
to a total volume of 8 µl with buffer E. After an additional minute
at 25 °C the mixtures were combined, and the binding reaction was
continued for 30 min at 30 °C. The samples were analyzed by mobility
shift electrophoresis as described above.
In Vitro Transcription--
We optimized our in vitro
transcription protocol (13) to better suit the aims of this study. The
reaction buffer contained 40 mM HEPES, pH 7.6, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 75 mM KCl, and 10% glycerol. Our
preliminary tests indicated that the highest in vitro
transcriptional activity from the APP promoter was achieved at a 75 mM KCl concentration. We also found that up to 0.75 µl of 1 M urea could be added to a 16-µl reaction
mixture without significantly affecting the activity of either the
APP or -actin promoters. The in vitro
transcription reaction mixture contained 4 µl of nuclear extract, 2 µg of the plasmid APP(488) (13), which is based on reporter plasmid
pCAT2bGAL (31), and 600 µM of each NTP in a total volume
of 16 µl. Recombinant CTCF was diluted with buffer F containing 500 mM KCl. The same volume of 0.5 µl of diluted CTCF or
buffer F was added to all the reaction samples to maintain a constant
concentration of urea. The sample volume was balanced with buffers D
and E to bring the final KCl concentration to 75 mM. After
incubation for 30 min at 30 °C, 0.4 µg of poly(A) was added to the
samples as a carrier, and RNA was purified with the RNeasy Mini Kit
(Qiagen). The RNA was eluted with 30 µl of RNase-free
H2O, which yielded ~23 µl of eluate. The eluate was combined with 6 µl of 5× annealing buffer containing 75 mM Tris, pH 7.6, 1.5 M NaCl, 50 mM
MgCl2, 60 mM DTT, and 1 µl (3 pmol, 50,000-200,000 cpm) of each of the primers specific for APP
and -actin promoter transcripts (13). The mixture was incubated at
99 °C for 5 min and at 75 °C for 20 min and then transferred to
48 °C. To the annealing mixture was added 10 µl of a reverse transcriptase reaction mixture containing 12.5 mM Tris, pH
7.6, 10 mM MgCl2, 12.5 mM DTT, and
6 mM of each of the dNTPs, supplemented with 20 units of
avian myoblastosis virus-reverse transcriptase and 20 units of RNasin
(both Roche Molecular Biochemicals). The reaction was continued at
48 °C for 90 min and stopped by phenol/chloroform extraction
followed by ethanol precipitation. The primer extension products were
separated on a 6% sequencing gel and quantitated with a GS-250
PhosphorImager (Bio-Rad).
In experiments where native CTCF was competed with purified recombinant
CTCF proteins, the reaction mixture was assembled in a modified way to
allow for preliminary binding of the recombinant CTCF to the APB
site. Briefly, 0.7 µl of buffer F containing 500 mM KCl
and recombinant CTCF at the desired dilution were combined with 2 µl
of depleted HeLa cell nuclear extract and 2 µg of reporter plasmid.
The volume of the sample was adjusted to 8 µl with buffer E. After
incubation at 25 °C for 10 min, 2 µl of whole HeLa cell nuclear
extract, 3 µl of buffer D, and 2 µl of buffer E were added to each
sample. The transcription reaction was carried out at 30 °C, and the
products were analyzed as described above.
In Vivo Competition--
The rationale for these competition
experiments was to analyze the effect of intracellular recombinant CTCF
expression on the expression from a cotransfected APP
promoter construct. The APP promoter from position 488 to
+100 was cloned into the polycloning site of plasmid pCAT2bGAL (31),
which is located immediately upstream of the bacterial chloramphenicol
acetyltransferase (CAT) gene. The plasmid also contains the
-galactosidase gene, which is transcribed from the -actin
promoter and serves as an internal control for experimental variations.
For the expression of CTCF by transient transfection, full-length CTCF,
N-terminal deletion Met-285, and C-terminal deletion Cys-525 were
provided with a green fluorescent protein (GFP) tag on the 5' end of
the respective CTCF cDNA constructs. The reading frame for GFP was
obtained from plasmid pEGFP-N1 (CLONTECH), which
was cut at restriction site BsrG1 and blunt-ended with T4
DNA polymerase. This operation eliminated the stop codon and the codon
for the C-terminal amino acid of the GFP reading frame. The blunt-ended
fragment was then cloned in-frame to the 5' end of the three CTCF
constructs. The resulting CTCF constructs, GFP-FL, GFP-M285, and
GFP-C525 were subcloned into the polycloning site of plasmid
pcDNA3.1 (Invitrogen), which is located downstream from the CMV
promoter. As a control, the entire GFP reading frame alone was also
cloned into the same position of plasmid pcDNA3.1.
The cell line COS-1 was used for the in vivo competition of endogenous CTCF with recombinant CTCF expressed from transiently transfected pcDNA3.1 plasmid constructs. This cell line displays an ~10-fold higher transfection efficiency (about 50%) than HeLa cells. In addition it has been transformed to express the SV40 large T antigen. This allows for the episomal replication of plasmid pcDNA3.1, which contains the SV40 origin of replication. These properties allow for a consistently high level of expression of recombinant CTCF from transfected plasmids.
COS-1 cells were grown to about 70% confluence in 25-cm2
flasks and were then transfected with 2 µg of plasmid mixture per flask using the FuGENE reagent according to manufacturer's
instructions (Roche Molecular Biochemicals). The plasmid mixture
contained the APP reporter plasmid (APP(488)) and the CTCF
expression vector (pcDNA3.1) at a molar ratio of 4:1. This ratio
was found to be optimal both for CTCF expression and APP
promoter activity. The FuGENE/plasmid mixture was left on the cells for
about 16 h. The cells were then examined by fluorescence
microscopy and harvested. CAT and -galactosidase assays were
performed as described elsewhere (14). The CAT activities resulting
from the APP promoter construct was normalized to identical
-galactosidase activities. Subsequently, in each experiment the
APP promoter activity obtained with the cotransfected
control GFP expression plasmid was assigned the value 100%.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Depleting Nuclear Extract of CTCF Reduces Transcriptional Activity
from the APP Promoter--
HeLa cell nuclear extract supports in
vitro transcription from both the -actin and APP
promoters. Transcription from the -actin promoter depends on a CCAAT
and TATA box as well as a serum-response element (32, 33). In contrast,
the APP promoter is TATA-less, which indicates that the two
promoters are transcribed by different mechanisms. Transcription from
the -actin promoter is initiated at one primary start site, whereas
the APP promoter is initiated at multiple sites from
position +1 to 4 (Fig. 2B, lane
1). When HeLa cell nuclear extract was depleted of CTCF by immunoprecipitation, transcription from the APP promoter was
drastically reduced to a level corresponding to 23% of the activity
observed with the non-depleted extract (Fig. 2B, lanes 1 and
5). When whole nuclear extract was mixed with increasing
amounts of CTCF-depleted extract, there was a gradual decline in the
transcriptional activity from the APP promoter (Fig.
2B, lanes 2-4). In all cases transcriptional activity from
the -actin promoter remained unaffected (Fig. 2B, lanes
2-4). The decrease in transcriptional activity was paralleled by
a comparable decrease in binding activity to the APB site (Fig.
2A, lanes 1-5).
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Replenishing CTCF-depleted Nuclear Extract with Purified
Recombinant CTCF Restores Transcriptional Activity from the APP
Promoter--
To determine whether purified recombinant CTCF expressed
in the yeast strain P. pastoris supports transcriptional
activity from the APP promoter, depleted nuclear extract was
replenished with increasing amounts of full-length recombinant CTCF.
Mobility shift electrophoresis showed that binding to the APB site
increased proportionally to the amount of CTCF added (Fig.
3A, lanes 1-5). In a similar
manner, transcriptional activity from the APP promoter was
restored to levels approximating those of whole nuclear extract, whereas the transcriptional activity from the -actin promoter remained unaffected. Incidentally, no CTCF binding activity can be
detected in control Pichia extracts, and such extracts do
not support transcription from the APP promoter (data not
shown). These results demonstrate that CTCF activates transcription
from the APP promoter in vitro and that the
recombinant version of the protein expressed in P. pastoris
can replace endogenous CTCF depleted from HeLa cell nuclear
extract.
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The N-terminal Domain of CTCF Is Essential for Restoring Transcriptional Activity from the APP Promoter in Depleted Nuclear Extracts-- The domains of CTCF that are located to the N- and C-terminal side of the zinc finger domain are not essential for DNA binding (27). However, they may contribute to functional activity. To address this issue, we analyzed CTCF deletions from the N- and C-terminal ends that had been purified from P. pastoris (Fig. 1, A and B). The deletions were used to supplement CTCF-depleted HeLa cell nuclear extract, and their effect on transcriptional activity was assessed.
Two N-terminal deletions were constructed that resulted in primary
translational start sites at methionine residues 249 and 285 (Fig.
1A) (32). Deletion Met-249 removed much of the
N-terminal domain, whereas deletion Met-285 extended into the first
zinc finger. The two C-terminal deletions terminated at cysteine
residue 525 and aspartic acid residue 617. C-terminal deletion Asp-617 removed 109 amino acids of the C-terminal end of CTCF without extending
into the zinc finger domain, whereas in C-terminal deletion Cys-525,
201 amino acids were deleted, which additionally eliminated peripheral
zinc fingers 10 and 11 (Fig. 1, A and B). All N-
and C-terminal deletions displayed DNA binding activity to the APB sequence (Fig. 3A, lanes 6-12). However, only the
C-terminal deletions restored transcriptional activity from the
APP promoter proportionally to their binding activity. In
contrast, transcriptional activity was not supported with the
N-terminal deletions despite adequate binding activity to the APB
sequence (Fig. 3B, lanes 6-12). This observation was
confirmed by combining data from several independent experiments (Fig.
3C). CTCF-depleted extract was supplemented with different
amounts of recombinant full-length CTCF, N-terminal deletion Met-249,
or C-terminal deletion Cys-525. In each case only full-length CTCF and
C-terminal deletion Cys-525 provided a dose-dependent
transcriptional activation, whereas N-terminal deletion Met-249 showed
little or no activation.
Competition of Endogenous CTCF with N-terminal Deletions of
Purified Recombinant CTCF Reduces in Vitro Transcriptional Activity
from the APP Promoter--
Because both endogenous CTCF and
recombinant N- and C-terminal deletions bind to the APB site of the
APP promoter, it should be possible to compete the binding
of endogenous CTCF by supplementing the native nuclear extract with an
excess of N- and C-terminal deletions. Indeed, mobility shift
competition showed that adding increasing amounts of recombinant
N-terminal deletion Met-249 to HeLa cell nuclear extract gradually
shifted the binding equilibrium from the endogenous CTCF toward the
N-terminal deletion (Fig. 4A, lanes
1-5). Similar shifts were observed with N-terminal deletion Met-285 and C-terminal deletion Cys-525 (Fig. 4A, lanes 5 and 7). When full-length recombinant CTCF was added, the
resulting mobility shift complex was indistinguishable from the
endogenous CTCF binding complex, and the total amount of binding
complex formed was merely enhanced (Fig. 4A, lane 6).
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However, in vitro transcription from the APP promoter was only reduced by the addition of N-terminal deletions of Met-249 and Met-285, and this reduction was somewhat more pronounced with deletion Met-285 (Fig. 4B, lanes 1-6). In contrast, the addition of recombinant full-length CTCF and C-terminal deletion Cys-525 increased transcriptional activity. These results confirm that CTCF activates transcription from the APP promoter and that the N-terminal domain is essential for this activation.
In Vitro Transcription from the APP Promoter Is Unaffected by Adding a GFP Tag to the N-terminal End of CTCF-- The results described above provide convincing evidence that CTCF activates transcription from the APP promoter in vitro. Furthermore, the activation is mediated by a portion of the CTCF molecule that is located close to the N-terminal side of the zinc finger domain. To complement this observation, it was of interest to investigate whether similar results could be obtained in cultured cells. Because CTCF is essential for cell survival, its elimination from the cells was precluded. We therefore devised an in vivo competition experiment to examine how the expression from a transfected APP promoter construct was affected by the concomitant expression of selected cotransfected CTCF constructs. For the purpose of this assay, issues relating to the effect of CTCF deletions on intracellular expression and nuclear translocation had to be resolved. Specifically, in the in vitro assay the transcripts derived from the N-terminal deletions contained different translational start sites than those derived from full-length CTCF and the C-terminal deletions. This was not an issue when the CTCF constructs were purified from P. pastoris, because the amount of CTCF added for extract complementation was normalized to DNA binding activity (mobility shift units). However, the utilization of inappropriate translational start sites in vivo could potentially result in variable translation efficiencies between different CTCF deletion constructs. We therefore decided to add the transcriptional unit of the GFP in frame with the N-terminal ends of the CTCF constructs. This addition of an N-terminal GFP tag served two purposes. First, all constructs contained the same translational start site, which minimized the possibility that the expression of the CTCF constructs was influenced by differential utilization of translational start sites. In addition, both the level of expression and the subcellular localization of the CTCF constructs could be evaluated by fluorescence microscopy.
For the purpose of the in vivo competition assay, we
selected full-length CTCF, N-terminal deletion Met-285, and C-terminal deletion Cys-525. Initially, all constructs were provided with an
N-terminal GFP tag, expressed in P. pastoris, and purified by chromatography. The purified constructs were analyzed by mobility shift electrophoresis. All GFP-tagged constructs supported binding to
the APB sequence of the APP promoter in a similar manner
as their non-tagged counterparts. However, the presence of the GFP moiety resulted in a slightly slower migration of the binding complex
when compared with full-length CTCF (Fig.
5A). When these GFP-tagged
CTCF constructs were used to supplement CTCF-depleted nuclear extract
for in vitro transcription, transcriptional activation of
the APP promoter was unaffected by the presence of the
N-terminal GFP tag. Specifically, full-length CTCF, full-length
GFP-CTCF, and GFP-C525 all activated transcription in proportion to the number of mobility shift units added. In contrast, the N-terminal deletion GFP-M285 did not activate transcription (Fig. 5B).
These results demonstrate that the N-terminal addition of the GFP tag did not alter the property of the CTCF constructs in the sense that
their ability to bind to the APB site and their ability to activate
transcription from the APP promoter remained unaffected.
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Nuclear Translocation of CTCF Is Unaffected by N- or C-terminal Deletions-- As a regulator of numerous gene functions CTCF unfolds its activity within the nucleus. However, following synthesis in the cytoplasm the mechanism of nuclear translocation has not been adequately examined to date. Consequently, it is conceivable that the extensive deletions of the N- and C-terminal ends might interfere with or eliminate nuclear translocation. To investigate this possibility, full-length GFP-CTCF, GFP-M285, and GFP-C525 were cloned into the polycloning site of vector pcDNA3.1 (Invitrogen). In this position the three constructs were transcribed from the upstream CMV promoter. As a negative control, the GFP transcriptional unit alone was cloned into the same site of plasmid pcDNA3.1. As a cell background for transfection, we chose the cell line COS-1 because it can be transfected at a high level of efficiency that approximates 50%. Furthermore, the expression from the APP promoter is regulated in a manner identical to the previously used HeLa cells.2
Cells were transfected under the same conditions as those used for
subsequent competition experiments (see below). Specifically, the
reporter plasmid (APP(488)) was mixed at a molar ratio of 4:1 with a
pcDNA3.1 plasmid containing one of the three GFP-tagged CTCF
constructs or the GFP reading frame alone. By using the Fugene reagent
(Roche Molecular Biochemicals), cells were exposed to the plasmid
mixture for 16 h and then examined by fluorescence microscopy. The
results show that a vast majority of GFP-tagged CTCF constructs were
localized to the nucleus (Fig. 6,
B-D). In contrast, the distribution of the GFP protein
alone was primarily cytoplasmic (Fig. 6A). These results
demonstrate that removing the N- or C-terminal domains of CTCF does not
affect the nuclear translocation of the molecule.
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In Vivo Competition Confirms that the N-terminal Domain of CTCF Is
Essential for Transcriptional Activation--
The above experiments
established that the N-terminal GFP tag had no discernible effect on
the binding of the CTCF constructs to their APB target sequence or
on their properties associated with transcriptional activation in
vitro. In addition, the N- and C-terminal deletions did not affect
nuclear translocation. We therefore proceeded to examine whether the
transfected CTCF constructs were able to compete with endogenous CTCF
for the expression from the cotransfected APP promoter
construct. The reporter plasmid (APP(488)) contained the
APP promoter from position 488 to +100, which activated
expression of the chloramphenicol acetyltransferase gene. In addition,
as an internal control, the same plasmid also contained the -actin
promoter driving the expression of the -galactosidase gene. The
reporter plasmid was cotransfected with a plasmid containing either one
of the GFP-tagged CTCF constructs or the GFP reading frame alone. These
CTCF constructs were transcribed from the constitutive CMV promoter.
The resulting CAT activities were normalized to identical
-galactosidase activities, and the CAT activity obtained with the
cotransfected GFP open reading frame alone was set at 100%. The
results show that cotransfection of either the full-length GFP-CTCF or
C-terminal deletion GFP-C525 had no significant effect on the
expression from the APP promoter. In contrast,
cotransfection of N-terminal deletion GFP-M285 resulted in a reduction
in APP expression to ~30% of the control value (Fig.
7). Under the applied conditions these
results were highly reproducible, and they can be interpreted as
demonstrating that the cotransfected N-terminal deletion competes with
endogenous CTCF for the binding to the APP promoter.
However, because the binding of the N-terminal deletion is
non-productive in terms of transcriptional activation, a reduction in
expression from the APP promoter occurred. Similarly, the
observation that the full-length CTCF construct and the C-terminal
deletion did not increase APP promoter expression suggests
that endogenous CTCF is not a rate-limiting factor in this cell
background. In general, these results are consistent with those
observed in vitro, and they support the notion that
transcriptional activation by CTCF is dependent upon the N-terminal
domain of the molecule.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We have previously presented extensive indirect evidence that the
nuclear factor binding site APB is essential for high levels of
expression from the APP promoter. Specifically, progressive deletions from the 5' end resulted in a 70-90% reduction in
expression from the APP promoter upon removal of the APB
site. This was observed both by transient transfection in HeLa cells
and by in vitro transcription (13, 14). In addition, block
mutations within the APB core recognition sequence that abolish CTCF
binding have a similar effect as 5' deletions that eliminate the site (13, 14). Furthermore, competition for endogenous CTCF in HeLa cell
nuclear extract with double-stranded oligonucleotides containing two
independent CTCF-binding sites specifically reduced in vitro
transcription from the APP promoter (15). Investigations on
embryonic mouse hippocampal neurons showed a time- and
differentiation-dependent up-regulation of expression of
both endogenous APP transcript and transiently transfected
APP promoter constructs (26). This increase in
APP expression was largely dependent on the presence of an
intact CTCF-binding site (APB). An increase in the level of CTCF in
hippocampal neurons as a function of differentiation was also observed.
A plausible interpretation of these results is that the level of CTCF
is a limiting factor in the expression of APP during
differentiation of mouse hippocampal neurons.
CTCF is a multifunctional protein that regulates the expression of a
wide variety of genes. However, in most of these cases CTCF acts as a
selective repressor of transcription, as exemplified by the binding to
gene silencers and insulators (see Introduction) (34). CTCF also binds
to the chicken c-MYC gene between positions 180 and 210 upstream from the transcriptional start site. It may act either as a
transcriptional repressor or activator depending on which cell
background the promoter is analyzed (17, 24, 25). In view of these
divergent functions, a demonstration that CTCF activates transcription
from the APP promoter was essential. We have here provided
several lines of direct evidence that this is the case. A large portion
of this evidence has been provided by in vitro
transcription, because in this system it is easier to control
experimental parameters. In contrast, experimental perturbations
in vivo are inherently more difficult to interpret because
of the probability of secondary intracellular responses and
interactions that are often beyond investigative control.
As a first step in demonstrating the requirement for CTCF in
transcriptional activation of the APP promoter, the HeLa
cell nuclear extract was selectively depleted by antibody adsorption. The depletion of CTCF was confirmed by mobility shift electrophoresis, and in vitro transcription from the APP promoter
was reduced to 23% compared with the undepleted extract (Fig.
2B). This residual level of transcription from the
APP promoter is consistent with transcriptional levels
observed with APP promoter constructs devoid of the APB
site both in vivo and in vitro (13, 14). Mixing CTCF-depleted extract with increasing ratios of native extract restored
the transcriptional activity from the APP promoter. The restoration of transcriptional activity was largely proportional to the
increase in CTCF binding activity to the APB site. Although these
results strongly suggest a direct role of CTCF in APP
promoter activation, it could not be excluded that additional factors
that interact with CTCF were also removed by the antibody depletion.
This concern was addressed by replenishing the depleted extract with CTCF from a heterologous source. CTCF was expressed in the yeast strain P. pastoris and was purified to near-homogeneity. Interestingly, recombinant CTCF was able to reactivate transcription from the APP promoter in depleted extract in the same manner as the native CTCF (Fig. 3B). This effect cannot necessarily be taken for granted because some functions of CTCF have been shown to depend on posttranslational modifications. For example, phosphorylation of CTCF is correlated with specific differentiation pathways of human myeloid cells, and this modification may alter the affinity for specific DNA-binding sites or alter the binding of cofactors (35). Furthermore, dephosphorylation of casein kinase II-dependent serine phosphorylation sites was associated with enhanced repression of c-myc promoters. However, these phosphorylation sites were located on the C terminus of the protein (36). We have shown previously that the binding properties of recombinant CTCF derived from Pichia is indistinguishable from their native counterparts (27). The observation that recombinant CTCF activates transcription indicates that either CTCF is correctly modified in Pichia or that no specific posttranslational modifications are necessary. However, the role of posttranslational modifications in transcriptional activation from the APP promoter has not yet been determined.
Because recombinant CTCF activates transcription from the APP promoter, specific deletions can be used to define the domain that is essential for activation. Similar deletions were employed to determine that the N-terminal end of the zinc finger domain is aligned toward the transcriptional start site of the APP promoter (27). In this study we investigated two deletions each from the N- (Met-249 and Met-285) and C-terminal (Cys-525 and Asp-617) ends of CTCF (Fig. 1, A and B). Of these, deletion Met-285 extended into zinc finger 2 from the N-terminal side and deletion Cys-525 extended into zinc finger 11 from the C-terminal side. Although these peripheral zinc fingers are not essential for binding to the APP promoter per se, their removal may reduce the stability of the binding complex (27). In this case the absolute amount of CTCF construct present in the reaction would not necessarily reflect its relative binding activity. In addition, it is conceivable that during the purification procedure of recombinant CTCF, a portion of the molecules might be rendered functionally inactive. It therefore became necessary to normalize the binding activity of the CTCF deletions to their potential to activate transcription. For this purpose we introduced the concept of the mobility shift unit (msu) (Fig. 3). This allowed for a direct comparison between the transcriptional activation and the binding activity achieved with each CTCF construct. The results show that the N-terminal domain is essential for transcriptional activation, whereas the C-terminal domain is dispensable. This conclusion was further corroborated by competing endogenous CTCF with the deletion fragments. However, it may be pointed out that N-terminal deletion Met-285 inhibited transcription somewhat more effectively than deletion Met-249 (Fig. 4). Although this phenomenon was not further investigated systematically, it suggested that the transcriptional activation domain of CTCF is located in close proximity to the N-terminal end of the zinc finger domain.
Because it was feasible to compete endogenous CTCF with recombinant CTCF deletions in nuclear extract, we investigated whether it was possible to achieve a similar competition in vivo. Indeed, cotransfection of an APP promoter construct with CTCF deletion Met-285 showed a dramatic decrease in APP promoter activity. In contrast, APP promoter activity was largely unaffected by cotransfection with recombinant full-length CTCF and C-terminal deletion Cys-525. These results support the in vitro observations that the N-terminal end of CTCF is essential for transcriptional activation of the APP promoter. They also suggest that in COS-1 cells the availability of endogenous CTCF is not a limiting factor in transcriptional activation, because APP promoter activity was unaffected by the expression of additional recombinant CTCF. In contrast, nuclear extract in vitro can be adjusted so that the amount of endogenous CTCF becomes a limiting factor. In such a case, providing additional recombinant CTCF results in an increase in transcription from the APP promoter (Fig. 4).
In summary, these results provide the first direct evidence that CTCF
is required for APP promoter activation and that the activation domain is located on the N-terminal side of CTCF, presumably close to the zinc finger domain. The mechanism by which CTCF activates transcription is currently under further investigation. However, preliminary evidence suggests that CTCF acts by recruiting additional factors to the transcription complex. An essential step in this process
would then be the binding of such a factor to the N-terminal domain of CTCF.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant NINDS NS30994 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.
To whom correspondence should be addressed. Tel.: 631-444-8025;
Fax: 631-444-7534; E-mail:
wquitschke@mail.psychiatry.sunysb.edu.
Published, JBC Papers in Press, November 8, 2001, DOI 10.1074/jbc.M109748200
2 A. A. Vostrov, M. J. Taheny, and W. W. Quitschke, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
APP, amyloid
-protein precursor;
DTT, dithiothreitol;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate;
CAT, chloramphenicol acetyltransferase;
GFP, green fluorescent protein;
msu, mobility shift units;
CMV, cytomegalovirus.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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