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INTRODUCTION |
The basic-zipper (bZip)1
transcription factor NF-E2 plays a critical role in erythroid and
megakaryocytic gene expression (for review see Ref. 1). NF-E2 binds to
an extended AP-1-like element, TGCTGA(G/C)TCA, which is found in the
locus control regions (LCRs) of the
- and
-globin genes and in
the promoters of several heme biosynthetic enzyme genes (for review see
Refs. 2 and 3)). NF-E2 binding sites in the DNase I hypersensitive site
2 (HS2) of the
-globin LCR are essential for its enhancer activity
(4-6).
NF-E2 is a heterodimer consisting of a hematopoietic-specific subunit
p45, which is a member of the cap and collar (CNC) family, and a more
widely expressed small subunit, which is a member of the small Maf
protein family (MafG, MafK, and MafF) (for review see Refs. 2 and 3)).
MafG and MafK are the predominant small Maf molecules in erythroid
cells and megakaryocytes (7). p45 and a small Maf protein dimerize
through their leucine zipper domains to generate a composite DNA
binding domain that consists of the basic regions of both molecules.
Other members of the CNC family, including Nrf1 (8), Nrf2 (9),
Nrf3 (10), Bach1, and Bach2 (11) can also dimerize with small Maf
proteins. Despite the high levels of p45 expression in erythroid cells,
mice that are null for p45 displayed a surprisingly mild defect in
globin gene expression, suggesting that other members of the CNC
protein family can substitute for p45 function in vivo
(12).
The N terminus of p45 contains an activation domain that is important
for the biological activity of p45 (13, 14). Several molecules interact
with this domain and are candidate mediators of p45 activity. These
include TAFII130 (a component of the TFIID complex) (15),
cAMP-response element-binding protein (CREB)-binding protein (CBP)
(16), and several ubiquitin ligases (17, 18). The small Maf proteins
lack a typical activation domain and are believed to activate
transcription as heterodimers with members of the CNC family of
proteins. Small Maf proteins can also form homodimers and repress
transcription (19).
CBP and its close relative p300 serve as coactivators for a large and
diverse set of nuclear factors (20, 21). CBP and p300 possess intrinsic
histone acetyltransferase (AT) activity (21-23). Histone acetylation
is associated with a relaxed chromatin configuration, suggesting that
coactivators act in part through modifying chromatin structure.
Consistent with this idea, at the chicken
-globin gene locus, the
area of general DNaseI sensitivity coincides well with the region of
elevated histone acetylation (24). Interestingly, hyper-acetylation of
histones H3 and H4 was observed at the human
-globin LCR and at the
transcribed
-globin gene when compared with the acetylation status
of the inactive
-like globin genes (25, 26). Together, these
findings suggest that erythroid transcription factors might recruit
histone-modifying enzymes such as CBP/p300 to the LCR and globin gene
locus, thereby altering chromatin structure (27). This idea is
supported by our observation that E1A-mediated inactivation of CBP/p300
in erythroid cells leads to a block in cell differentiation and globin gene induction (28). CBP binds to several hematopoietic-restricted transcription factors involved in globin gene expression and enhances their activity, including GATA-1 (28) and the erythroid
Krüppel-like factor EKLF (29).
A new layer of complexity in the function of acetyltransferases emerged
with the discovery that CBP/p300 also acetylates a variety of
transcription factors. Acetylation can alter transcriptional activity
through several mechanisms. For example, acetylation of the tumor
suppressor protein p53 strongly increases its affinity for DNA
(30-32). Both GATA-1 and EKLF are also acetylated by CBP (29, 33, 34).
Mutations in the acetylation sites in GATA-1 compromise its function in
erythroid cells, suggesting that GATA-1 acetylation is biologically
relevant (34).
Recent studies implicated CBP/p300 in the regulation of NF-E2 activity
by showing that E1A, which inhibits CBP/p300 function, reduced the
enhancer activity of HS2 and that NF-E2 was an important target of
E1A-mediated inhibition (35). In addition, glutathione S-transferase (GST) pull-down experiments showed that p45
binds CBP in vitro (16).
Here we report that both subunits of NF-E2 interact with CBP in
vitro. In addition, NF-E2 can recruit CBP to a DNA template containing NF-E2 binding sites. Immunoprecipitation experiments demonstrate in vivo association between MafG and CBP in
erythroid cells. CBP acetylates MafG predominantly in the basic region, thereby stimulating DNA binding of NF-E2. Mutations at the major acetylation sites reduce DNA binding and transcriptional activation by
NF-E2. Thus, recruitment of CBP by NF-E2 might serve two functions, regulation of chromatin structure and transcription factor activity.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Full-length and truncated variants of GST murine
p45 and GST murine MafG were generated by polymerase chain reaction and
subcloned in the BamHI and the EcoRI sites of
pGEX2TK (Amersham Pharmacia Biotech). GST-tethered-NF-E2 contains
murine p45 and human MafG physically tethered by a flexible peptide
linker as described (36). pXM-Tethered NF-E2 was described
previously (36). Mutagenesis replacing the four lysine residues in the
basic region of MafG (see Fig. 4) was performed using the
QuikChangeTM method as instructed by the manufacturer
(Stratagene), and the resulting construct was verified by sequencing.
GST pull-down experiments in Fig. 1 were performed as previously
described (34) using 5 µg of GST protein and 10 µl of programmed reticulocyte lysates to map the interaction domains of NF-E2 and CBP.
Experiments in Fig. 6A were modified to determine the effect of acetylation on p45-MafG heterodimerization. Two µg of GST-MafG were acetylated by 0.3 µg of GST-CBPH-AT in the presence of
cold acetyl coenzyme A (0.6 mM) at 30 °C for 90 min and
incubated with 35S-labeled p45 in the presence or absence
of 1 µg of double-stranded oligonucleotide containing the NF-E2
binding site from the porphobilinogen deaminase (PBGD) promoter
(5'-GATCCTGGGGAACCTGTGCTGAGTCACTGGAGG-3') (37). GST-MafG quickly
associates with p45 in the presence of the NF-E2 binding site, and
equilibrium was reached within 30 min.
Coimmunoprecipitation of CBP and MafG--
EF1
-neo-HA-MafG
was transfected into murine erythroid leukemia cells (MEL) by
DMRIE-C (Life Technologies, Inc.) and selected in the presence
of G418. Western blots using anti-HA antibodies (12CA5, Roche Molecular
Biochemicals) were used to identify HA-MafG-expressing clones. A line
expressing high levels of HA-MafG was expanded and used in the
experiments. Coimmunoprecipitation of CBP and HA-MafG was carried out
essentially as described using anti-CBP (A-22, Santa Cruz
Biotechnology) as the precipitating antibody (28).
Recruitment of CBP to HS2--
A
HindIIII-XbaI fragment (374 base pairs)
containing HS2 from the human
-globin LCR (a gift from R. Hardison)
(38) was biotinylated and coupled to M-280 streptavidin magnetic beads (Dynal) according to the manufacturer's instructions. The tandem NF-E2
sites from 8661 to 8677 (GenBankTM HUMHBB) was replaced
with a SalI site in HS2
NF-E2 fragment (35). One pmol of
HS2 coupled to 100 µg of Dynal beads was resuspended in 100 µl of
1× DNA-protein binding buffer (10 mM Tris (pH 7.5), 50 mM KCl, 1 mM MgCl2, 1 mM EDTA, 5.5 mM dithiothreitol, 5% glycerol, 0.03% Igepal, 5 mg/ml bovine serum albumin, protease inhibitor mixtures) and incubated with 5 pmol of GST-p45 plus 5 pmol of GST-MafG
at room temperature for 20 min. To remove unbound recombinant proteins,
beads were washed twice with 1 ml of DNA-protein binding buffer and
resuspended in 1 ml of DNA-protein binding buffer supplemented with 2.5 µg of poly(dI-dC). One hundred µl of MEL cell nuclear extracts
corresponding to 107 cells were added to the beads and
incubated for 1 h at 4 °C. Beads were washed twice in 1 ml of
0.5× DNA-protein binding buffer supplemented with 100 mM
NaCl and analyzed on SDS-PAGE followed by Western blotting for CBP.
Acetyltransferase assays were performed as described (34). All
substrates and enzymes were expressed as recombinant GST fusion
proteins in Escherichia coli (DH5
) except for the
full-length GST-MafG and the full-length His-tagged CBP, which were
produced in baculovirus. GST proteins were prepared as described (30). Reactions were carried out with 50 pmol of substrate and 5 pmol of
enzyme in the presence of 0.06 µCi of 14C-labeled acetyl
coenzyme A (55 mCi/mmol, PerkinElmer Life Sciences) at 30 °C
for 90 min. Tethered NF-E2 protein used in gel shift experiments was
acetylated in the presence of 0.6 mM unlabeled acetyl
coenzyme A.
Anti-acetyl Lysine (AK) Immunoprecipitations--
Anti-acetyl
lysine antibodies (New England Biolabs) were used to immunoprecipitate
acetylated MafG from MEL cells expressing HA-MafG, transfected NIH3T3
cells, and transfected COS-7 cells. Preliminary experiments showed that
these antibodies recognize recombinant MafG acetylated by CBP. For Fig.
5B, 3 µg each of EF1
-HA-MafG (hemagglutinin-tagged
MafG), pCMV5CBP, and EF1
-E1A (28) were transfected into 50%
confluent NIH3T3 cells in 10-cm dishes using LipofectAMINE (Life
Technologies, Inc.). For Fig. 5C, 8 µg of
pXM-tethered NF-E2 was transfected into 40% confluent COS-7
cells in 10-cm dishes using LipofectAMINE. COS cells express high
levels of endogenous CBP and, therefore, were chosen to evaluate the
in vivo acetylation of wild type and mutant NF-E2. High salt whole cell lysates were prepared 48 h after transfections and immunoprecipitated using anti-AK antibodies (0.5 µg/sample) as described (34). Rabbit IgG was used in the control precipitation. Immunoprecipitates were analyzed by Western blotting using anti-HA antibodies to detect HA-MafG and anti-p45 (a gift from E. Bresnick) to
detect tethered NF-E2.
Gel mobility shift assays were performed as described previously (28,
34). The oligonucleotide probes used in gel shift assays contain the
NF-E2 sites from the PBGD promoter
(5'-GATCCTGGGGAACCTGTGCTGAGTCACTGGAGG-3') (37) and human HS2
(5'-GCAGTGCTGAGTCATGCTGAGTCATGCTG-3') (19). Tethered NF-E2 protein
(1.2 µg) was acetylated by full-length His-tagged CBP (100 ng)
before use in the gel shift reactions. Incubation of DNA complex
proceeds for 15 min on ice before loading on a 5% nondenaturing
acrylamide gel in 0.5× Tris borate EDTA.
Reporter Gene Assays--
A PBGD-GH reporter containing
310 to
+78 of the PBGD promoter (39) linked to the human growth hormone gene
was used to assay NF-E2 activity. 30% confluent NIH3T3 cells were
transfected using LipofectAMINE with 0.2 µg of PBGD-GH and with
increasing amounts (0.4, 0.8, and 1.6 µg) of pXM plasmids
expressing wild type or acetylation-defective (4A) NF-E2. The amounts
of transfected plasmid were kept constant by adding empty pXM.
To account for variability in transfection efficiency, a control
plasmid (0.1 µg) expressing the firefly luciferase gene SV40-GL3
(Promega) was included in the transfections. Growth hormone levels were determined using a radio-immunoassay (Nichols Diagnostic). Whole cell
lysates were prepared to determinate luciferase activity (Promega) and
to monitor NF-E2 expression by Western blot.
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RESULTS |
Both Subunits of NF-E2 Interact with CBP--
Previous work
demonstrated a functional link between NF-E2 and CBP (35). In addition,
it has been shown that p45 interacts with CBP in vitro (16).
To examine whether MafG also associates with CBP, GST pull-down
experiments were performed. Both full-length p45 and MafG proteins
fused to GST interact with in vitro translated CBP (Fig.
1A). We next mapped the
domains in p45 and MafG that mediate the association with CBP. The
results show that the N terminus (aa 1-144) of p45 is necessary and
sufficient for CBP binding (Fig. 1B). The domain in MafG
that contacts CBP was mapped to the bZip domain (aa 51-162) (Fig.
1C). Deletion of the basic region (construct 77-162)
resulted in complete loss of CBP binding. These results are summarized
in Fig. 1D. To map the domain of CBP that mediates binding
to MafG and p45, various CBP fragments were generated by in
vitro translation. The results in Fig. 1E show that a
C-terminal fragment of CBP containing the CH3 domain (aa 1626-2260)
(20) binds strongly to both p45 and MafG (Fig. 1E).
Consistent with a previous report (16), a fragment of CBP containing
the CREB binding domain (aa 117-737) also bound to p45, although with
much lower affinity. In summary, both subunits of NF-E2 bind to CBP,
suggesting that the NF-E2-coactivator complex might be stabilized by
multiple protein contacts.

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Fig. 1.
In vitro binding of NF-E2 to
CBP. A, both p45 and MafG associate with CBP in
vitro. GST, GST-p45, and GST-MafG (5 µg of each) were assayed
for binding to in vitro translated,
[35S]methionine-labeled CBP. Input: 10% of in
vitro translated material. B, mapping of CBP binding
sites in p45. C, mapping of CBP binding sites in MafG.
D, summary of mapping experiments. EHR, extended
homology region. E, mapping of CBP domains that bind p45 and
MafG.
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We next examined whether NF-E2 and CBP associate in vivo.
Due to the lack of an appropriate MafG antibody, we generated MEL cells
stably expressing HA-tagged MafG. There was no detectable difference in
the growth, differentiation, and hemoglobinization of
HA-MafG-expressing cells when compared with parental MEL cells (data
not shown). This indicates that HA-MafG expression did not occur at
levels sufficient to perturb cellular functions, as might have been
expected from studies in which small Maf proteins were over-expressed
in MEL cells (40). Nuclear extracts from these cells were
immunoprecipitated with anti-CBP antibodies followed by Western
analysis using anti-HA antibodies. HA-MafG was detected in
immunoprecipitates with anti-CBP antibodies but not with control antibodies (Fig. 2A). These
results suggest that NF-E2 associates with CBP in erythroid cells.

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Fig. 2.
NF-E2 interacts with CBP in vivo
and recruits CBP to a NF-E2 binding site-containing DNA
template. A, nuclear extracts from MEL cells expressing
HA-MafG were immunoprecipitated (I.P.) with anti-CBP
antibodies or nonimmune (n.i.) rabbit IgG followed by
anti-HA Western blotting. B, a DNA fragment containing HS2
from the human -globin LCR was immobilized on magnetic beads and
incubated with recombinant GST-p45 and GST-MafG followed by incubation
with MEL cell nuclear extracts (NE). After extensive
washing, the presence of CBP on the HS2 template was determined by
Western blot. Input: 10% of input MEL nuclear extracts. WT,
wild type.
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We next determined whether NF-E2 can recruit CBP to an enhancer
containing NF-E2 binding sites. A DNA fragment containing HS2 from the
human
-globin LCR was used as DNA template since HS2 contains two
functionally important NF-E2 binding sites that are positioned in a
tandem configuration (5). After biotinylation and immobilization on
streptavidin-coupled magnetic beads, the DNA template was incubated
with recombinant GST-MafG and GST-p45 proteins. After several washes to
remove free protein, DNA-bound NF-E2 was incubated with nuclear
extracts from MEL cells, washed, and analyzed for the presence of CBP
by Western blotting. In the presence of NF-E2, CBP derived from MEL
nuclear extracts bound to the NF-E2-DNA complex (Fig. 2B).
In the absence of NF-E2 protein (GST alone) or when a HS2 fragment that
lacked functional NF-E2 binding sites (HS2
NF-E2) was used, little or
no CBP was retained (Fig. 2B), indicating that recruitment
of CBP is mediated by DNA-bound NF-E2. These results suggest that NF-E2
might contribute to the strong enhancer activity of HS2 by recruiting
CBP, consistent with experiments that functionally linked NF-E2
elements of HS2 with CBP action (35).
Acetylation of MafG by CBP--
Since several CBP-associated
nuclear factors are regulated by protein acetylation (21), we examined
whether either subunit of NF-E2 is a substrate for CBP. In
vitro acetylation assays were performed using recombinant purified
GST fusion proteins containing the acetyltransferase domain of CBP
(CBP-AT), p45, and MafG in the presence of [14C]acetyl
coenzyme A. Products were analyzed by SDS-polyacrylamide gel
electrophoresis and visualized by autoradiography. The results show
that CBP-AT acetylates full-length MafG (Fig.
3A) but not p45 (Fig.
3B). Deletion analysis revealed that CBP-AT strongly acetylates MafG at the bZip domain (aa 51-162) but only very weakly at
the N terminus (aa 1-50) (Fig. 3B). Deletion of the basic
region (aa 51-77) significantly reduced acetylation, suggesting that it is the predominant CBP-acetylated site (Fig.
4A). The basic region contains
4 lysine residues at positions 53, 60, 71, and 76 (Fig. 4B).
Deletion of lysines 53 and 60 (construct 61-162) results in decreased
acetylation, whereas loss of all four lysines further reduces
acetylation (construct 77-162), suggesting that all 4 lysines are
targets for acetylation (Fig. 4A). Of note, we observed
residual acetylation of a construct that contained the leucine zipper
but lacked the basic region (77).

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Fig. 3.
MafG is acetylated by CBP. GST-p45 and
GST-MafG were acetylated by GST-CBP-AT in the presence of
14C-labeled acetyl coenzyme A and resolved on
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
GST-GATA-1 served as a positive control (34).
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Fig. 4.
Acetylation of MafG by CBP occurs primarily
at the basic region. A, upper panel,
acetylation of MafG truncation constructs. Lower panel,
Coomassie staining. B, acetylation by full-length CBP of
tethered wild type (WT) NF-E2 and NF-E2-4A.
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Since both small Maf proteins and p45 can form homodimers, we examined
whether lysines 53, 60, 71, and 76 of MafG are the major acetylation
sites in the context of heterodimeric NF-E2. For this purpose we used a
tethered form of NF-E2 that contains p45 linked to MafG by a flexible
peptide. Importantly, tethered NF-E2 binds DNA with high efficiency and
is fully active in restoring NF-E2 function in p45 null erythroid
cells, demonstrating that the linker peptide does not adversely affect
NF-E2 function (13, 36). The advantage of using tethered NF-E2 over
both subunits prepared separately is the absence of homodimeric forms
of MafG and p45, respectively (see also below). To examine the
acetylation of lysines 53, 60, 71, and 76 of MafG, we generated a form
of tethered NF-E2 in which all 4 lysines were substituted with alanines (NF-E2-4A). GST-CBP-AT acetylated tethered NF-E2 somewhat less efficiently than MafG (data not shown). However, when full-length baculovirus-expressed CBP was used, tethered NF-E2 was acetylated with
high efficiency (Fig. 4B). In contrast, acetylation of
NF-E2-4A was substantially reduced when compared with wild type NF-E2. This indicates that the four lysines in the basic region of MafG are
the major acetylation sites of the NF-E2 heterodimer. Since the basic
region is directly involved in DNA binding, these results raised the
possibility that acetylation of MafG might modulate its ability to bind
DNA (see below).
MafG Is Acetylated in Vivo--
The use of anti-acetyl lysine
(anti-AK) antibodies allowed monitoring of protein acetylation in
vivo. Broad-specificity anti-AK antibodies reacted well with MafG
acetylated by CBP in vitro but not with nonacetylated MafG
(data not shown). Using these antibodies, we determined whether
acetylation of MafG occurs in vivo. Extracts of MEL cells
stably expressing HA-MafG were immunoprecipitated by anti-AK antibodies
followed by Western blotting with anti-HA antibodies. The results show
that anti-AK, but not nonimmune rabbit IgG, precipitated significant
amounts of HA-MafG (Fig.
5A), indicating that MafG is
acetylated in erythroid cells. To determine whether MafG acetylation is
regulated by CBP, NIH 3T3 cells were transfected with HA-MafG alone or
together with CBP. As shown in Fig. 5B, MafG was acetylated,
consistent with the results obtained in MEL cells. Coexpression of CBP
enhanced acetylation, which in turn was inhibited when an
E1A-expressing plasmid was included in the transfections (Fig.
5B). Control Western analysis showed that coexpression of
E1A and CBP did not alter MafG protein levels. These results
demonstrate that CBP stimulates MafG acetylation in an E1A-sensitive
manner in intact cells, similar to what we observed for GATA-1 (34). To
determine whether acetylation of MafG occurs at the same sites in
vitro and in vivo when bound to p45, wild-type tethered
NF-E2 and NF-E2-4A were transfected into COS cells, which contain high
levels of endogenous acetyltransferase activity (34), and analyzed by
anti-AK immunoprecipitation. Anti-AK precipitated substantial amounts
of wild type NF-E2 but not NF-E2-4A (Fig. 5C), suggesting
that the four lysine residues in the basic region of MafG are the major
acetylation sites in vivo.

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Fig. 5.
In vivo acetylation of MafG.
A, acetylation of MafG occurs in erythroid cells. Whole cell
lysates from MEL cells expressing HA-MafG were immunoprecipitated
(I.P.) with anti-AK antibodies followed by Western blotting
using anti-HA antibodies. n.i., nonimmune rabbit IgG. As
control, 10% of the lysates were analyzed directly (input).
B, in vivo acetylation of MafG is enhanced by
over-expression of CBP, which in turn is inhibited by coexpressed E1A.
HA-MafG, CBP, and E1A constructs were transiently transfected in NIH
3T3 cells. Whole cell lysates were immunoprecipitated with anti-AK
antibodies (Ab) as in A. C,
acetylation of MafG occurs at the same sites in vivo and
in vitro. Whole cell lysates from COS cells transfected with
wild type (WT) NF-E2 or NF-E2-4A were immunoprecipitated
(I.P.) with anti-AK antibodies (Ab) followed by
Western analysis with anti-p45 antibodies.
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Acetylation by CBP Stimulates DNA Binding of NF-E2--
Since
CBP-mediated acetylation of MafG occurs in the bZip region, we examined
whether acetylation affects the interaction between the p45 and MafG
subunits or their ability to bind DNA. To test whether acetylation
affects heterodimerization of MafG and p45, GST pull-down experiments
were carried out using acetylated or mock-acetylated GST-MafG and
in vitro translated, 35S-labeled p45.
Acetylated and nonacetylated GST-MafG bound equal amounts of p45 (Fig.
6A, left panel).
However, since in both cases p45 binding was very inefficient, we
examined whether heterodimer formation might be facilitated or
stabilized in the presence of DNA. As shown in Fig. 6A
(right panel), inclusion in the binding reaction of an
oligonucleotide containing a single NF-E2 binding site substantially
increased binding between GST-MafG and p45. p45 associated very rapidly
with GST-MafG, reaching a maximum within 30 min. Acetylation of
GST-MafG did not alter the association with p45, even at a time point
before saturation was reached (10 min, Fig. 6A, right
panel). Together, these results suggest that acetylation does not
significantly affect association between MafG and p45.

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Fig. 6.
Dimerization and DNA binding of NF-E2.
A, dimerization between acetylated or mock-acetylated
GST-MafG and in vitro translated p45 in the presence
(right panel) or absence (left panel) of an
oligonucleotide containing the NF-E2 element derived from the PBGD
gene. Two µg of acetylated or mock-acetylated GST-MafG were incubated
with in vitro translated, 35S-labeled p45 for
1 h (without oligonucleotide) or 10 min (with oligonucleotide).
Input: 10% of the in vitro translation reaction.
B, DNA binding of in vitro acetylated NF-E2.
Tethered NF-E2 was acetylated by His-tagged full-length CBP, and
increasing amounts of protein (3, 6, 12 ng) were used in gel shift
assays. Mock acetylation reactions lacked acetyl coenzyme A. The
oligonucleotides used in the gel shift reactions contained the NF-E2
sites of the PBGD promoter (left panel) and HS2 from the
human -globin LCR (right panel), respectively. The
experiments were done in duplicate with two independent acetylation
reactions, one of which is shown. The averages of fold-increases in DNA
binding upon acetylation of NF-E2 were 2.8-, 3.4-, and 2.5-fold (when
3, 6, and 12 ng of NF-E2 were used) for the PBGD probe and 1.9-, 1.9-, and 2.1-fold for the HS2 probe. C, DNA binding of NF-E2
constructs expressed in COS cells. Wild type (WT) NF-E2 and
NF-E2-4A were expressed in COS cells, and nuclear extracts were used in
gel shift experiments using the oligonucleotide containing the
PBGD-derived NF-E2 element. Western blotting confirmed that wild type
NF-E2 and NF-E2-4A proteins were expressed at comparable levels.
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Since the predominant acetylation sites reside in the basic region of
MafG, we tested whether acetylation by CBP affects DNA binding of
NF-E2. Initial gel shift experiments with recombinant GST-p45 and
GST-MafG showed that GST-p45 bound DNA relatively well as homodimer and
that the presence of GST-MafG led to an only moderate increase in DNA
binding (data not shown). Furthermore, p45 homodimers migrated with a
mobility very similar to that of p45-MafG heterodimers in gel shift
experiments. These results contrast with previous reports where maltose
binding protein (MBP) fusion proteins of p45 and MafG bound DNA
significantly better as heterodimers than as homodimers (19, 41). We
suspect that the difference between these observations might be related
to the use of different tags (GST versus MBP) or to
differences in protein preparation. In mammalian nuclear extracts, p45
is found in a complex with small Maf proteins (42, 43), and the p45-Maf heterodimer is the predominant DNA-bound form of NF-E2 (13, 42). To
test DNA binding of the NF-E2 heterodimer in the absence of confounding
homodimeric complexes, we used the tethered form of NF-E2, which binds
DNA as an obligate heterodimer. As shown in Fig. 6B
(left panel), acetylation by CBP significantly increased DNA
binding of NF-E2 at various protein concentrations. Phosphorimaging analysis revealed that acetylated NF-E2 binds to the NF-E2 binding site
derived from the PBGD promoter with up to 3.4-fold higher efficiency
when compared with nonacetylated NF-E2. We also observed stimulation of
DNA binding when the NF-E2 binding site from HS2 of the human
-globin LCR was used (Fig. 6B, right
panel).
If acetylation of the basic region in MafG enhances DNA binding,
mutations of the acetylated residues would be expected to reduce DNA
binding of mammalian-expressed NF-E2. To test this possibility, NF-E2
constructs were expressed in COS cells where wild type NF-E2 but not
NF-E2-4A is acetylated with high efficiency (Fig. 5C). When
nuclear extracts were examined by gel shift analysis, strong DNA
binding was observed with wild type NF-E2, whereas NF-E2-4A failed to
bind DNA very efficiently (Fig. 6C). Western analysis
confirmed the presence of equal amounts of proteins in the reactions.
These results indicate that the acetylated residues are important for
DNA binding of mammalian-expressed NF-E2 and establish a correlation
between acetylation and DNA binding of NF-E2 (Figs. 5C and
6C).
Intact Acetylation Sites Are Required for Transcriptional
Activation by NF-E2--
To determine whether the major acetylation
sites are important for the function of NF-E2, transient transfection
assays were performed in NIH 3T3 cells using the
NF-E2-dependent promoter of the PBGD gene fused to the
human growth hormone gene as a reporter. As shown in Fig.
7, NF-E2-4A displayed significantly lower
activity when compared with wild type NF-E2. Control Western analysis
showed that NF-E2-4A was consistently expressed at about 2-fold higher levels than wild type NF-E2 (Fig. 7, lower panel), making
the observed reduction in activity even more significant. These results indicate that the acetylation sites of NF-E2 are important for NF-E2 as
a transcriptional activator.

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Fig. 7.
Acetylation-deficient NF-E2 has reduced
transcriptional activity. Increasing amounts of plasmid expressing
wild-type (WT) NF-E2 and NF-E2-4A were cotransfected into
NIH3T3 cells with a PBGD-GH reporter. The transcriptional activity of
NF-E2 was determined by measuring the levels of secreted growth
hormone. Results represent the averages of three independent
experiments. Whole cell lysates were used in Western blot to determine
the levels of NF-E2.
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DISCUSSION |
In this report we demonstrate that both subunits of NF-E2 interact
with CBP. We further show that MafG and CBP associate in erythroid
cells. CBP and acetylates MafG in vitro and in
vivo mainly in the basic region, thereby increasing DNA binding
and transcriptional activation of NF-E2.
Our observation that both subunits of NF-E2 bind to CBP suggests that
the complex might be stabilized by multiple protein contacts in
vivo. It is possible that by binding to both subunits, CBP might
stimulate heterodimer formation, leading to enhanced DNA binding
independent of protein acetylation. Recruitment of CBP to the
-globin LCR might be aided further by the presence of additional
erythroid transcription factors that interact with CBP. Both GATA-1 and
EKLF fulfill these criteria since GATA-1 and EKLF binding sites are
important elements at the LCR, and both factors have been shown to bind
CBP (28, 29). GATA-1 and EKLF are also acetylated by CBP (29, 33, 34),
indicating that a common theme underlies the regulation of structurally
diverse nuclear factors involved in globin gene expression.
In agreement with a requirement of CBP for LCR function, interference
with CBP/p300 activity leads to a complete block in globin gene
expression and erythroid differentiation (28). In the context of HS2,
NF-E2 binding sites are the predominant E1A-sensitive cis-acting elements, implicating a functional link between
NF-E2 and CBP (35). Therefore, it is possible that the effects of E1A
on globin gene expression are the combined result of inhibition of
GATA-1, EKLF, and NF-E2 function.
In vitro acetylation of MafG by CBP occurs predominantly at
a fragment in the basic region containing four lysine residues. Further
deletion analysis showed that each of two lysine pairs (residues 53 and
60, and 71 and 76, respectively) contributes to MafG acetylation.
Although all four lysines account for the majority of MafG acetylation
when assayed alone or in a complex with p45, the relative contribution
of each single lysine to the total acetylation of MafG remains to be
determined. All four sites are conserved among all small Maf proteins
and across all species examined (44), suggesting that they are
functionally important. Minor acetylation was also observed at the N
terminus and the leucine zipper. The functional significance of these
acetylation sites is unknown. In contrast, in vivo
acetylation as determined by anti-AK immunoprecipitation experiments
showed virtually no acetylation outside the basic region (Fig.
5C). This suggests that the basic region is the major
acetylation site in vivo. However, it is possible that the
anti-AK antibodies used may have selectivity toward the acetylated
residues in the basic region.
Although the molecular consequences of GATA-1 and EKLF acetylation are
not yet established (29, 33, 34), this work shows that acetylation by
CBP augments DNA binding of NF-E2. Although acetylation of MafG might
increase DNA binding by multiple mechanisms, the observation that
acetylation occurs predominantly in the basic region suggests that it
might directly increase the affinity of MafG for DNA. Although the
residues in MafG that contact DNA have not been determined, the crystal
structure of the yeast bZip protein GCN4 complexed with DNA provides
some insight into how bZip proteins contact DNA (45). In GCN4, the
underlined residues of the
NTEAARRSR motif in the basic region
contact the central 7 base pairs in the GCN4 binding site. Two of the
four major acetylation sites in MafG (aa 60 and aa 71, in
bold) directly flank the
KNXXYAXXCRYK core motif
(Fig. 4B), supporting a possible role for acetylation in the
formation of DNA contacts. However, it remains possible that
acetylation might stimulate DNA binding by triggering allosteric changes in NF-E2, similar to what has been described for p53 (30). Some
acetylation of MafG was also observed in the leucine zipper domain,
suggesting that this might stimulate heterodimerization with p45,
thereby indirectly increasing DNA binding. However, two observations
suggest that this is unlikely. First, in vitro protein
binding studies failed to yield any significant differences in
dimerization of acetylated and nonacetylated MafG with p45. Second,
acetylation in the zipper domain both in vitro and in vivo is minimal when compared with the acetylation observed in the
basic region. Although an acetylation-induced increase in DNA binding
was observed on two distinct NF-E2 elements derived from the PBGD
promoter and human HS2, respectively, it remains possible that
acetylation might lead to subtle changes in DNA binding site
preferences between variant NF-E2 sites. Comparable increases in DNA
binding upon acetylation have been observed in several transcription
factors (21). One can envision at least two scenarios regarding the
order of events. First, DNA binding of NF-E2 might occur before
recruitment of CBP and NF-E2 acetylation. In this case, acetylation
might stabilize the NF-E2 complex on DNA. Second, CBP and NF-E2 might
be bound to each other in solution before DNA binding. In this event,
acetylation might increase the rate of association of NF-E2 with its
cognate binding element. Our observation that CBP can bind and
acetylate NF-E2 in solution in the absence of DNA is consistent with
the latter possibility.
We showed that acetylation-defective NF-E2 (NF-E2-4A) has diminished
transcriptional activity in transient reporter gene assays, further
suggesting that acetylation of NF-E2 is important for its function.
However, NF-E2-4A still retained significant activity, especially at
higher amounts of transfected DNA. This suggests that the reduced
affinity of NF-E2-4A for DNA can be overcome by increased protein concentrations.
The interaction between NF-E2 and CBP suggests that CBP regulates
transcription by modulating chromatin structure as well as
transcription factor activity. Although protein acetylation is an
attractive mechanism by which CBP acts at the LCR or other erythroid or
megakaryocytic genes, additional mechanisms have to be considered as
well. For example, since CBP also contacts certain components of the
basal transcription machinery (for review see Refs. 20 and 21), it
might mediate enhancer activity of the LCR in an
acetylation-independent manner, for example by bridging to the globin
gene core promoters. Furthermore, NF-E2 has been shown to associate
with a chromatin remodeling activity (46, 47), suggesting that
CBP-independent activities might contribute to NF-E2 function.
Analogously, EKLF associates with E-RC1, an SNF/SWI-related protein
complex with ATP-dependent chromatin-remodeling activity
(48). It is possible that ATP-dependent remodeling complexes and AT complexes act in different promoter contexts. Alternatively, it is conceivable that ATP-dependent and
AT-containing complexes act sequentially at the same genes, similar to
what has been described for the yeast HO gene (49, 50).