Identification of NF1 as a silencer protein of the human adenine
nucleotide translocase-2 gene
Peter Barath
1,2
, Daniela Poliakova
1,2
, Katarina Luciakova
1,2
and B. Dean Nelson
1
1
Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Sweden;
2
Cancer Research Institute,
Slovak Academy of Sciences, Bratislava, Slovak Republic
The human adenine nucleotide translocase-2 (ANT2) pro-
moter contains a silencer region that confers partial repres-
sion on the heterologous herpes simplex virus thymidine
kinase (HSVtk) promoter [Barath, P., Albert-Fournier, B.,
Luciakova, K., Nelson, B.D. (1999) J. Biol. Chem. 274,
3378–3384]. Two sequences in the silencer (Site-2 and Site-3)
are protected in the DNase I assay in vitro, and one of these
is a repeated GTCCTG element previously shown to act as
the active repressor element. We have now purified the DNA
binding protein, and identified it using MALDI-TOF MS
as a 33-kDa member of the nuclear factor 1 (NF1) family of
transcription factors. NF1 purified from rat liver and HeLa
cell nuclei bind to both silencer Site-2 and Site-3, resulting in
a DNase I footprint identical to that obtained with purified
recombinant NF1. Furthermore, transient transfection
experiments with reporter constructs containing mutated

separate repressor regions. One of these is an Sp1 binding
element (C box) juxtaposed to the transcriptional start site,
which, when occupied, decreases ANT2 expression several-
fold [11]. Repression appears to involve a direct interaction
between Sp1 bound to the AB and C boxes [12]. A second
repressor region, that is responsible for ANT2 down
regulation in growth-arrested cells, has recently been
identified in the distal promoter [10]. This region contains
two DNA elements, Go-1 and Go-2, that bind nuclear
factor 1 (NF1) in growth-arrested cells, but not in growth-
activated cells [13]. NF1 binding is associated with growth
arrest repression of ANT2 transcription.
A third repressor (silencer) region, which does not
participate in growth arrest repression of the gene [13],
but confers repression on a heterologous herpes simplex
virus thymidine kinase (HSVtk) reporter gene, has also
been located in the ANT2 promoter between the AB
activation boxes and the Go repressor region [14]. Two
DNA sequences in the silencer region (Site-2 and Site-3)
are strongly protected in the DNase I assay. One of these
(Site-2) is a repeated element (GTCCTG) shown to have a
role in repressing the HSVtk promoter [14]. In the present
study, we have purified the silencer element binding
protein, and identified it as a member of the NF1 family
of proteins (see [15] for review). NF1 binds to Site-2 and
Site-3 both in vitro and in vivo, and repression of the
heterologous HSVtk promoter is relieved by mutating
either element. Thus, NF1 plays a major role in regulating
ANT2 expression by modulating (repressing) the efficiency
with which Sp1 functions as a constitutive activator.

CAT plasmid. All clones were checked for fragment size
and orientation by digestion with restriction enzymes, and
mutations were verified by sequencing.
A DNase I footprint probe containing Site-2, 3 and Go-2
elements was made by adding XbaI sites to both ends of
ANT2 )825/)795 [13]. This oligonucleotide, which includes
Go-2, was ligated into the XbaI site of the pCAT-ANT2
()546/)235)wt [14]. The clones were checked by restriction
enzyme digestion and sequencing.
Cell culture and transfection
Growth and transfection of HeLa cells experiments were
performed as by Li et al. [11]. Five micrograms of reporter
plasmid DNA containing the CAT gene and 2 lg of control
luciferase plasmid DNA (pGL3, Promega) were used for
transfection. CAT and luciferase activities were measured as
described [17].
DNase I protection assay
Rat liver and HeLa nuclear extracts were prepared as
described previously [18,19]. The DNase I protection assay
was performed as described [17]. Radioactive probes were
prepared by PCR using 5¢ [
32
P]-labelled CAT primer, the
M13 primer and pCAT-ANT2-()546/)235)wt [14] or
pCAT-Go-2-ANT2()546/)235)wt [13] as the template.
Electrophoretic mobility shift (EMSA) and supershift
assay
EMSA analyses were performed as by Li et al.[11]. For
supershift experiments, 2 lL of antibody raised against
human NF1-C (rabbit polyclonal antiserum, 8199, kindly

followed by 30 s of annealing at 60 °C and 30 s of extension
at 72 °C. The last step included extension for 10 min at
72 °C.
NF1 purification from nuclear extracts
Rat liver nuclei were purified as described by Kadonaga
[20]. Nuclei from HeLa cells were prepared according to
Dignam et al. [18]. Proteins were extracted from nuclei by
addition of an equal volume of extraction buffer (20 m
M
Hepes pH 7.9, 820 m
M
NaCl, 5 m
M
MgCl
2
,1m
M
EDTA,
1m
M
EGTA, 0.5 m
M
phenylmethanesulfonyl fluoride,
1m
M
benzamidin, 0.5 m
M
dithiothreitol). The various
fractionation steps are described by Luciakova et al. [13].
Site-2 and Site-3 binding activity in all fractions was

M
MgCl
2
)was
loaded with 0.25 mL of sample. The column was eluted at
0.1 mLÆmin
)1
. Fractions of 0.5 mL were collected after the
void volume of the column.
SDS/PAGE and protein identification by MALDI-TOF MS
Samples from the DNA affinity column were precipitated
for 20 min on ice in 10% trichloroacetic acid, followed by a
15 min centrifugation at 10 000 g and two washes with ice-
cold acetone. Samples were air dried, dissolved in sample
buffer, and separated in 10% SDS/PAGE. Proteins were
visualized by silver staining and bands of interest were cut
1782 P. Barath et al. (Eur. J. Biochem. 271) Ó FEBS 2004
out. In-gel tryptic digestion and sample preparation was
carried out as described by Luciakova et al.[13].MALDI-
TOF analysis was performed in reflector mode using a
Voyager-DE STR MALDI-TOF mass spectrometer
(Applied Biosystems). Internal calibration was performed
with autodigested trypsin. Data were analysed using
MOVERZ
software (Proteometrics, LLC, Winnipeg, Canada),
and database searches were done with
MASCOT
(http://
www.matrixscience.com).
Results

tion factors [15] by trypsin fragment mass analysis using
MALDI-TOF MS. Out of 13 peptide masses, nine (69%)
were matched to different isoforms of NF1 from different
species covering up to 58% of the total protein sequence. The
matched peptides originate from a conserved, 240-residue
N-terminal DNA binding domain [21] (Fig. 2D), thus
excluding the possibility of determining the specific isoform
of NF1 (for review see [15]) involved. No other transcription
factors were detected in the purified preparations.
Identification of a 33 kDa NF1 polypeptide is consistent
with our earlier studies in which a 33–38 kDa silencer-
binding protein was suggested based on crosslinking and
South-western analysis with HeLa cell nuclear extracts [14].
However, gel filtration of the rat liver Heparin Sepharose
fraction shows that Site-2/Site-3 binding activity (33 kDa
NF1) is present only in fractions 12 and 13 (Fig. 3A) which
contain polypeptides of a greater mass. These data suggest
that the Site-2/Site-3 binding protein exists as a complex,
most likely as a dimer.
Proteins that bind the Site-2/Site-3 elements and the
upstream growth arrest (Go) elements copurify. NF1
also binds two upstream growth arrest elements (Go-1 and
Go-2) in the ANT2 promoter [13]. To determine if the
Site-2/Site-3 and Go-element binding activities are identical,
we constructed a DNAse I protection probe that included
both the Go-2 and the Site-2/Site-3 elements (see Methods).
As seen in Fig. 3A, Site-2/Site-3 and Go-2 binding proteins
comigrate during gel filtration, suggesting that the same or
similar proteins bind to both sets of elements. As a further
test, Go element binding-proteins were purified from rat

repressor element [14]. The arrow in the Site-3 indicates a NF1 protein-
binding half site described in the present study. The mutations intro-
duced into the Site-2 and Site-3 elements are indicated above the
sequence.
Ó FEBS 2004 NF1 is the silencer protein of ANT2 (Eur. J. Biochem. 271) 1783
hypersensitive sites produced by recombinant NF1 are
similar, if not identical, to those observed with rat liver
nuclear extracts (Figs 2 and 3A), affinity purified NF1 from
rat liver (Figs 2B and 3B and [13]) and HeLa nuclear
extracts [14]. NF1 did not footprint Site 1 [14] of the silencer
region, which is also present in the probe used in Fig. 4A.
Fig. 2. Purification and identification of the silencer Site-2 and Site-3 binding-protein. Rat liver nuclear extracts were purified as described. (A)
Fractions eluted from Heparin Sepharose in a 100 m
M
to 300 m
M
NaCl linear gradient, or (B) fractions eluted from the DNA affinity column in 200
or 500 m
M
NaCl were monitored for DNase I protection activity using the ANT2–546/)235 silencer region as a probe. Activity was also
determined in samples loaded onto the affinity column (S) and in flow through (F) fractions. Location of Site-2 and Site-3 elements are indicated on
the right. Asterisks mark hypersensitive sites, open circles mark protected nucleotides. (C) SDS/PAGE analysis of polypeptide eluted from the
DNA affinity column. The dot beside lane 1 indicates the polypeptide band identified as NF1 by MALDI-TOF MS. (D) Peptide mass alignment.
Sequences in bold match the trypsin fragment masses from the SDS band in (C).
1784 P. Barath et al. (Eur. J. Biochem. 271) Ó FEBS 2004
A direct DNase I protection analysis comparing HeLa cell
nuclear extract, DNA-affinity purified proteins from HeLa
cells and purified recombinant human NF1 show the same
pattern of protected nucleotides (Fig. 4B), thus confirming
that NF1 binds to the silencer elements.

human ANT2 promoter that conferred repression on the
heterologous HSVtk promoter [14]. Three protein-binding
sites were found by in vitro DNase I footprinting, one of
which contained a GTCCTG repeat required for repres-
sion. A 33–38 kDa DNA binding protein was purified
using the GTCCTG element (silencer Site-2) as an affinity
probe. In the present study, we identify the GTCCTG
binding protein as a member of the NF1 family of
transcription factors. We also show that NF1 binds to a
second silencer element (Site-3, Fig. 1) upstream of the
GTCCTG repeat, and that both elements contribute to
repression of the HSVtk promoter. Furthermore, NF1
occupies both silencer elements in HeLa cells in vivo,
implicating NF1 as a possible repressor even under
endogenous conditions.
The NF1 polypeptide identified in the present experi-
ments exhibits a mass of 33 kDa, which is slightly less
than that reported in our previous study [14], but fits well
with the mass of the protein that cross-linked to the
GTCCTG element [14]. We also reported the copurifica-
tion of p33 together with a 49-kDa polypeptide [14]. p49
appeared not to bind DNA, but was loosely associated
with p33, perhaps enhancing p33 binding [14]. Using the
present purification scheme for the Site-2/Site-3 binding
proteins, which is substantially modified from that used in
[14], p49 does not appear as a major polypeptide in the
purified fractions. This result is consistent with the loose
association to p33 observed earlier. However, partially
purified Site-2/Site-3 binding activity (NF1) moves on a
gel filtration column with an apparent molecular mass

remains to be resolved. In any event, the results of ChIP
analysis demonstrating the in vivo occupation of Site-2 and
Site-3 by NF1 strongly suggest that these interactions are of
physiological relevance.
Fig. 4. Identification of the Site-2 and Site-3
binding protein as a member of the NF1 family
of transcription factors. (A) Binding of
purified, recombinant NF1 to the Site-2 and
Site-3 elements was monitored by the DNase I
protection assay of ANT2 oligonucleotide
()546/)235). Only the coding strand is shown.
Competitor oligonucleotides contained either
a wt or mutated (mut) Site-2, Site-3 or NF1
element. Asterisks denote hypersensitive
nucleotides, open circles denote protected
nucleotides. (B) Proteins from HeLa cells and
purified recombinant NF1 protect the same
nucleotides in the ANT2 silencer region.
Asterisks denote hypersensitive nucleotides
and open circles denote protected nucleotides
in the Site-2/Site-3 region. (C) EMSA and
supershift analysis was performed with 10 lg
of HeLa nuclear extract or with purified
recombinant NF1 (as indicated) and the
32
P-labelled oligonucleotide probe containing
a NF1 bipartite consensus binding sequence
(probe NF1 wt, Santa Cruz Biotechnology).
Wild-type (wt) or mutated (mut) competitor
oligonucleotides were added. The NF1

located within the protein [30,31]. Furthermore, repression/
activation by individual NF1 isoforms can also depend on
cell context [13,30–32], suggesting that the molecular action
of NF1 depends on cell-specific factors. Indeed, a variety of
factors is reported to interact directly with NF1; including
coactivators p300/CBP and SRC-1 [33], histone H3 [34] and
the general transcription factor Sp1 [35], TFIIB [36], and
TAFII155 [37]. Thus, the molecular mechanisms of NF1
repression on ANT2 remain to be elucidated.
The physiological role of the Site-2/Site-3 repressor
elements remains to be investigated. Deletion of these
elements has no apparent influence on growth-arrest
repression of ANT2 exerted via the Go-2/Go-3 growth-
arrest elements [13]. Thus, we speculate that NF1 bound to
Site-2/Site-3 elements most probably has a role in adjusting
the tissue-specific constitutive expression of ANT2, similar
to that proposed for the unique Sp1 repressor element
(C box) juxtaposed to transcription start [11].
Acknowledgements
This study was supported by the Swedish Research Council (to B. D.
N.), the Slovak Science and Technology Assistance Agency (APVT)
Grant 26-002102 and the Slovak Grant Agency (VEGA) 2/3087/23 (to
K. L.). The authors thank O
¨
. Wrange for recombinant human NF1 and
N. Tanese for the generous gift of antihuman NF1-C serum.
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