Regulation of E2A Activities by Histone Acetyltransferases in
B Lymphocyte Development*
Curtis
Bradney
,
Mark
Hjelmeland
,
Yasuhiko
Komatsu§,
Minoru
Yoshida¶,
Tso-Pang
Yao
, and
Yuan
Zhuang
**
From the Departments of
Immunology and
Pharmacology and Cancer Biology, Duke University Medical Center,
Durham, North Carolina 27710, § Advanced Life Science
Institute, Inc., 2-10-23 Maruyamadai, Wako-shi, Saitama 351-0112, Japan, and ¶ Chemical Genetics Laboratory, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
Received for publication, November 11, 2002
 |
ABSTRACT |
Genetic studies have demonstrated that the basic
helix-loop-helix protein E2A is an essential transcription factor in B
lymphocyte lineage commitment and differentiation. However, the
mechanism underlying E2A-mediated transcription regulation is not fully understood. Here, we investigated the physical and genetic interactions between E2A and co-activators histone acetyltransferases (HATs) in B
cells. Gel filtration analysis of human pre-B cell nuclear extract
showed that E2A co-elutes with the HATs p300, CBP, and PCAF. A
co-immunoprecipitation assay further demonstrated that a fraction of
endogenous E2A proteins is associated with each of the three HATs. We
show that these HATs acetylate E2A in vitro, enhance
E2A-mediated transcription activity, and promote nuclear retention of
E2A proteins. A catalytic mutation of p300 completely abrogates the
ability of p300 to acetylate E2A and to promote E2A nuclear retention
in 293T cells. A breeding test between E2A heterozygous mice and p300
heterozygous mice demonstrated that these two genes interact for proper
B cell development. Collectively, these results suggest that E2A
and HATs collaboratively regulate B cell development.
 |
INTRODUCTION |
The development of B lymphocytes in the bone marrow is
initiated and tightly regulated by at least three transcription
factors, E2A, EBF, and Pax5 (1). Mice missing any one of these
transcription factors show complete block in B cell development at the
pro-B cell stage (2-5). Although the expression of EBF and Pax5 are relatively restricted to the B cell lineage, E2A is found to be much
broadly expressed. Both biochemical and genetic analyses have indicated
that E2A is the most upstream regulator among the three transcription
factors and is continuously involved in regulating the expression of B
cell-specific genes through the later stages of B cell development (6,
7). It is not clear how E2A controls the broad array of tissue-specific
and stage-specific gene expression during B cell development.
E2A is a founding member of the basic helix-loop-helix
(bHLH)1 transcription factor
family, which plays an evolutionarily conserved role in regulating the
differentiation events in various tissue types including B lymphocytes
in mammals (8). The E2A gene encodes two bHLH transcription
factors, E12 and E47, which are generated through differential splicing
to two adjacent exons that encode the bHLH domains (9). The bHLH
domains are required for protein dimerization and DNA binding (10). Two
transactivation domains (AD) are mapped to the amino terminus of the
E2A proteins (11, 12). E2A proteins form homodimers or heterodimers
through HLH interactions with other broadly expressed bHLH
transcription factors such as HEB (13) or with tissue-restricted
bHLH transcription factors such as MyoD (14). These bHLH protein dimers
bind to DNA at the consensus sequence CANNTG, designated as the E-box. Functional E-box sites are found in the promoters and enhancers of a
wide variety of tissue-specific genes including immunoglobulin genes in
B cells (15).
The ubiquitously expressed histone acetyltransferases (HATs) p300, CBP,
and PCAF are transcription co-activators that interact with a broad
spectrum of tissue-specific and nonspecific transcription factors (16).
Transfection experiments have demonstrated that p300 can associate with
the bHLH region (17) and the amino-terminal region (18) of E2A
proteins. Studies in yeast further postulate that E2A mediates
transcriptional activation through a recruitment of the SAGA
(Spt-Ada-Gen5-acetyltransferase)-like chromatin remodeling complex,
which contains a HAT (19). However, no study thus far has addressed
directly the functional significance of HAT-E2A interaction in B cell
development. Here, we provide biochemical and genetic evidence to show
that the HATs p300, CBP, and PCAF are regulators of E2A activity in B
lymphocytes and the regulation is important for proper B cell development.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
The B cell lines REH and
NALM6 were maintained in RPMI 1640 supplemented with 10% fetal bovine
serum, 100 units/ml penicillin and streptomycin, and 5 mM
HEPES. The cell line 293T was maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and 100 units/ml penicillin and streptomycin. Cells were transfected using the
calcium phosphate transfection method. For transfections, a Myc-tagged
E2-5 was generated by adding six repeats of Myc tag to the amino
terminus of E2-5. E2-5 is a version of human E2A cDNA that
contains the E47 bHLH domain (20). Individual plasmids encoding
FLAG-tagged PCAF, Myc-tagged p300, Myc-tagged p300DY, and FLAG-tagged
CBP were generated as described (21).
Gel Filtration--
High and low molecular weight standards
(Amersham Biosciences) were resuspended in AG buffer (20 mM HEPES, pH 7.0, 100 mM KCl, 0.1 mM EDTA, 20% glycerol) and run through the Superose 6 HR
10/30 column (Amersham Biosciences) using an FPLC apparatus (Amersham
Biosciences) at 4 °C at a flow rate of 0.2 ml/min to generate a
standard curve. For each cell line, nuclear extract amounts of 2.5-5
mg were loaded onto the column in a total volume of 250 µl and run at
a flow rate of 0.2 ml/min. Fractions were collected at 500-µl
intervals and assigned numbers from 1 to 40. Upon fraction collection,
proteins present in each fraction were precipitated by adding 4-8
volumes of HPLC grade acetone to each fraction. Recovered protein
samples were run on SDS-PAGE and analyzed by Western blotting.
Immunoprecipitations and Western Blot
Analysis--
Immunoprecipitations were carried out with protein
G-Sepharose under conditions described previously (22). Antibodies used were monoclonal anti-E2A (13641A, Pharmingen), polyclonal anti-p300 (sc-585, Santa Cruz Biotechnology), polyclonal anti-CBP (sc-583, Santa
Cruz Biotechnology), rabbit polyclonal anti-PCAF (sc-6300, Santa Cruz
Biotechnology), and monoclonal anti-acetylated lysine (AKL5C1 (23)).
Proteins were separated by SDS-PAGE followed by liquid transfer to
nitrocellulose and immunostaining of the blot according to standard
procedure (24). Primary antibodies for Western blot analysis as
described previously were used at a 1:4000 dilution.
In Vitro Acetylation Assay--
Each HAT expression plasmid (5 µg) was transfected into 293T cells via the calcium phosphate
method. Cells were harvested after 48 h, and HAT proteins were
purified from transfected 293T cells using either FLAG (FLAG-CBP,
FLAG-PCAF) or Myc (Myc-p300, Myc-p300DY)-conjugated agarose. HAT
recovery was verified by colloidal staining and Western blotting of
purified protein. Equivalent amounts (1 µg) of each HAT protein were
used for each experiment. GST-tagged E2-5 and the deletion mutants
were cloned into the PGEX-1 vector and were expressed in
Escherichia coli per the instructions of the supplier
(Amersham Biosciences). The N' truncation was generated by
deleting sequence upstream from the internal NotI site at
the position corresponding to amino acid 401 of E2-5. The
C' truncation was generated by deleting sequence downstream of the bHLH domain at position corresponding to amino acid 513 of
E2-5. Acetyltransferase assays were carried out as described (22).
Briefly, 1 µg of GST-E2-5 was added to 1 µg of HAT protein purified from transfected 293T cells in 1× HAT buffer with the addition of [1-14C]acetyl coenzyme A (Amersham
Biosciences, catalog no. CFA729, 10 µCi (370 kBq)). Equivalent
loading of GST proteins was established by running samples on a
SDS-PAGE and visualizing the protein bands by colloidal staining (Invitrogen).
In Vivo Acetylation Assay--
DNA (2-5 µg) coding for
Myc-E2A and each individual HAT was transfected into 293T cells via the
calcium phosphate method. Cells were harvested after 48 h, and
Myc-E2A protein was purified from transfected 293T cell extract by
Myc-conjugated agarose using the standard immunoprecipitation protocol
with the addition of Trichostatin H (5 µm). Immunoprecipitated
samples were washed and run on an SDS-PAGE for separation. After
protein transfer to a nitrocellulose membrane, the presence of
acetylation was detect with the aid of an anti-acetylated lysine
antibody. All blots were stripped and reprobed for the presence of E2A.
The expression of HAT protein was verified by Western blot.
Reporter Assays--
Both the dual luciferase and
chloramphenicol reporter assays were conducted according to
manufacturer's specifications (Promega). For each assay, 1-3 µg of
E2A was co-transfected with 2 µg of HAT and 1 µg of each reporter
into 3T3 fibroblast cells. Both reporter constructs contained four
repeats of E-box sequence from the muscle creatine kinase gene and the
minimal TK promoter, also known as 4R-TK (17). A Renilla
constitutive expression vector and a constitutive
-galactosidase
expression vector were used as transfection controls for the luciferase
and chloramphenicol reporter assays, respectively.
In Situ Immunofluorescence Assay--
Human 293T cells were
transferred to glass coverslips and transfected as described previously
(25). Once fixed and blocked, the cells were immunostained with
anti-E2A antibodies followed by a FITC-conjugated secondary antibody.
Anti-fade mounting medium (Molecular Probes) was used to mount a
coverslip, and slides were examined for FITC fluorescence under a
fluorescent microscope.
Mouse Breeding and Fluorescence-activated Cell Sorter
Analysis--
Mice heterozygous for E2A (2) or p300 (26) were
maintained on a 129/sv and BL/6 mixed background. Compound heterozygous mice were obtained through intercrosses of these single heterozygous mice. Phenotypic analysis was conducted on 126 neonates and 26 adult
mice. Bone marrow and thymus samples were prepared by suspending the
macerated tissue in phosphate-buffered saline containing 5% fetal
bovine serum. For each sample, 20,000 cells were scored on a BD
Pharmingen FACScan analyzer and analyzed by CellQuest (BD Pharmingen).
The following antibodies were used in the experiment: B220-APC,
CD19-PE, IgM-FITC, CD8-APC, CD4-PE, CD5-FITC, and CD43-PE (Caltag).
Cell viability was determined by the exclusion of 7AAD staining
(Molecular Probes).
 |
RESULTS |
E2A Forms High Molecular Weight Complexes in Transfected HeLa Cells
and in Nuclear Extract from the Human Pre-B Cell Line, NALM6--
We
first used a biochemical approach to evaluate E2A-containing protein
complexes in HeLa cells transfected with a Myc-tagged E2-5, a fully
functional human E2A cDNA (20). Size exclusion gel filtration of
nuclear extract from E2A-transfected HeLa cells eluted E2A-containing
complexes in fractions corresponding to molecular sizes greater than
the molecular mass of an E2A dimer, ~160 kDa. Monomeric E2A
protein in whole cell extract was detected by the anti-Myc antibody
9E10 to have a molecular mass of ~80 kDa (Fig.
1A). Western blot analysis
indicated that E2A proteins were detected in eluted fractions
corresponding to molecular masses as large as 1 MDa and remained
present in fractions that eluted down to the monomeric protein
molecular weight. These results indicate that E2A is present in
complexes consisting of more than just a dimer.

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Fig. 1.
Gel filtration analysis of E2A
complexes. A, Western analysis using the anti-Myc
antibody 9E10 detected Myc-E2-5 in fractions collected after FPLC size
exclusion filtration of HeLa nuclear extract. WCE, whole
cell extract. The sizes of the molecular weight standards are indicated
at the top. B, Western analysis detection of E2A,
p300, CBP, and PCAF in fractions collected after FPLC size exclusion
filtration of NALM6 nuclear extracts.
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This result prompted us to evaluate further the endogenous E2A proteins
present in pre-B cells. Fractions eluted from size exclusion analysis
of the human pre-B cell line NALM6 nuclear extract were analyzed by
Western blotting for E2A and the histone acetyltransferases p300, CBP,
and PCAF. The results indicated that a significant percentage of
endogenous E2A is found in a high molecular weight range. The HATs
p300, CBP, and PCAF were found co-eluted with E2A in a broad spectrum
of molecular mass ranging up to 1 MDa in size (Fig. 1B). On
denaturing gels, monomeric E2A, p300, CBP, and a subunit of PCAF were
detected with molecular masses of ~70, 300, 300, and 95 kDa,
respectively. The detection of E2A and HATs in the same eluted
fractions is consistent with previous transfection studies in which E2A
was shown to interact with p300 (17, 18).
Endogenous HATs and E2A Interact in Pre-B Cells--
Fractionation
data suggest that E2A has the potential to interact specifically with
each of the three HATs, p300, CBP, and PCAF, in B cells. To investigate
this possibility, E2A was immunoprecipitated from the nuclear extract
of two pre-B cell lines, NALM6 and REH, using a monoclonal antibody
against E2A. After SDS-PAGE analysis of the pull-down products,
polyclonal antibodies detected p300, CBP, or PCAF in NALM6 nuclear
extract pull-downs (Fig. 2, lane 3) and CBP or PCAF in REH nuclear extract pull-downs (Fig. 2, lane 4). A direct comparison of the band intensities derived
from the pull-down products (Fig. 2, lanes 3 and
4) and crude nuclear extracts (Fig. 2, lanes 1 and 2) indicated that approximately one-fiftieth (1/50) of
the HAT protein present in the NALM6 nuclear extract was associated
with E2A. To confirm the presence of E2A in the pull-down samples, all
blots were stripped and reprobed with an anti-E2A antibody to verify
the presence of E2A (data not shown). As a negative control, an
antibody against the GST protein was not able to pull down each HAT
from the two cell lines (Fig. 2, lanes 5 and 6).
These results indicate that fractions of E2A are associated with p300,
CBP, or, PCAF in pre-B cells.

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Fig. 2.
E2A co-immunoprecipitation with p300, CBP, or
PCAF in nuclear extract from human B cell lines. Endogenous CBP
and PCAF were detected in 20 µg of nuclear extract from NALM6
(lane 1) and REH cells (lane 2). E2A
immunoprecipitation of p300, CBP, or PCAF was detected using 1 mg of
nuclear extract from NALM6 (lane 3) and REH cells
(lane 4). To show specificity, an antibody against GST did
not immunoprecipitate p300, CBP, or PCAF in 1 mg of nuclear extract
from NALM6 (lane 5) or REH cells (lane 6).
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E2A Is Acetylated by p300, CBP, and PCAF in Vitro--
Because
HATs are known to acetylate lysine residues present in histone and
non-histone proteins (16), in vitro acetylation assays were
employed to determine whether an enzymatic interaction occurs between
E2A and each HAT. Three GST-E2A fusion proteins, the full-length E2-5,
a carboxyl-terminal truncation, and an amino-terminal truncation, were
used in the acetylation assay (Fig.
3A). GST and GST-p53 were used
as a negative and a positive control, respectively. The size and
equivalence of loading for each GST fusion protein were demonstrated by
colloidal blue staining (Fig. 3B, lanes 1-3). Western analysis further confirmed that an anti-E2A antibody could recognize all three versions of GST-E2A proteins (Fig. 3B,
lanes 5-7). The acetylation assay shows that p300, CBP, and
PCAF can acetylate bacterially expressed E2-5 (Fig. 3C,
lanes 2, 8, and 14) and the
carboxyl-terminal deleted E2-5 (Fig. 3C, lanes
4, 10, and 16) but not the amino-terminal
deleted E2-5 (Fig. 3C, lanes 3, 9,
and 15). The ability of each HAT to autoacetylate itself
demonstrates that the HAT proteins are fully functional and are equally
loaded based on band intensities (Fig. 3C, upper panel).
Acetylation was also found in several protein degradation products from
the E2-5 full-length and carboxyl-terminal deletion constructs (Fig.
3C, bands marked with *). It is consistent with the notion
that the amino terminus of the E2-5 is acetylated because these
protein fragments were recovered from the GST pull-down and thus
contain the amino part of the E2-5 proteins. In the same tests, we
show that GST-p53 was acetylated (Fig. 3C, lanes
5 and 11) and that GST alone (Fig. 3C,
lanes 1, 7, and 13) or E2A without the
presence of HAT (Fig. 3C, lanes 6 and
12) was not acetylated. Altogether, this assay demonstrates
that the amino half of E2A is a direct target for the
acetylation by HATs in vitro.

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Fig. 3.
In vitro acetylation of E2A by
p300, CBP, and PCAF. A, diagram of GST E2-5 fusion
proteins. The GST fusion proteins contain either the full-length E2-5,
E2-5 with the N' truncation, or E2-5 with the
C' truncation. The relative positions for the AD2 and bHLH
domains are indicated. B, colloidal stain representation of
full-length GST-E2-5 (lane 1), C'-deleted
GST-E2-5 (lane 2), N'-deleted GST-E2-5
(lane 3), and GST protein (lane 4). Western
analysis of GST E2-5 proteins using an anti-E2A antibody detected
full-length GST-E2-5 (lane 7), N'-deleted
GST-E2-5 (lane 6), and C'-deleted GST-E2-5
(lane 5). The anti-E2A antibody did not detect GST protein
(lane 8). FL, full-length E2-5.
C, full-length GST-E2-5 was acetylated by p300 (lane
2), CBP (lane 7), and PCAF (lane 12). The
C'-deleted GST-E2-5 protein was also acetylated by p300
(lane 4), CBP (lane 9), and PCAF (lane
14). The N'-terminal deleted GST-E2-5 mutant was not
acetylated by p300 (lane 3), CBP (lane 8), or
PCAF (lane 13). P53 serves as a positive control for
acetylation by p300 (lane 5) and CBP (lane 10).
GST alone was not acetylated by p300 (lane 1), CBP
(lane 6), or PCAF (lane 11). Protein degradation
bands are indicated by an asterisk.
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E2A Is Acetylated by p300, CBP, and PCAF in Vivo--
To further
investigate HAT-mediated E2A acetylation, we preformed an in
vivo analysis to determine whether each HAT could acetylate E2A.
Antibodies against acetylated lysine were used to determine the
acetylation status of E2A in a transfection assay. Myc-tagged E2A
proteins were immunoprecipitated with anti-Myc antibodies before being
used in the Western analysis (Fig. 4). We
show that E2A co-transfection with wild-type p300 and CBP in human 293T
cells significantly increased E2A acetylation in comparison with E2A
alone (Fig. 4A). In addition, the acetylation of E2A by p300
was dependent upon the acetyltransferase activity of p300, as an
acetylase-deficient mutation (DY) derived from a human tumor mutation
(21) failed to induce E2A acetylation (Fig. 4A, lane 3). Under this assay condition, we did not detect a significant level of acetylation by PCAF even though PCAF was expressed in the
transfected cells (Fig. 4B). This experiment did not rule out the possibility that each HAT may interact with E2A in a cell type-specific manner.

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Fig. 4.
In vivo acetylation of E2A by
p300, CBP, or PCAF. A, Myc-E2A was immunoprecipitated
from transfected 293T cells. Anti-acetylated lysine antibody
(upper panel) and anti-E2A antibody (lower panel)
were used to detect the acetylated and total E2A proteins present in
the immunoprecipitation products loaded on duplicated gels.
Samples loaded to the Western gel are E2-5 transfection alone
(lane 1) or co-transfection with p300 (lane 2),
p300DY mutant (lane 3), CBP (lane 4), or PCAF
(lane 5). B, Western blot analysis of HAT
expression in transfected cells. Antibodies against p300
(top), CBP (middle), and PCAF (bottom)
were used in detecting individual HAT proteins. Small amounts of
endogenous p300 (lane 1) and CBP (lane 4) were
also detected in the untransfected controls. No endogenous PCAF could
be detected in 293T cells (lane 6).
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Co-transfection of E2A and p300, CBP, or PCAF Increases
Transcription Activity--
A role for p300 in stimulating the
transcription activity of E2A has been shown previously (17, 18). We
wanted to evaluate further whether CBP and PCAF could similarly enhance
E2A-mediated transcription. Reporter constructs containing four repeats
of the E2A binding site (17) were used in two separate transient transfection assays. Consistent with the earlier findings, figure 5 shows that co-transfection of E2A with
p300 (lanes 6 and 14) in 3T3 fibroblast cells
increases reporter gene expression over E2A alone (lanes 5 and 13). Similar to p300, CBP (lanes 7 and 15) and PCAF (lanes 8 and 16) can also
enhance E2A-mediated transactivation of the reporter genes. These
results indicated that p300, CBP, and PCAF may play similar roles in
modulating E2A activity.

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Fig. 5.
p300, CBP, or PCAF enhance E2A-mediated
transcription. Luciferase and chloramphenicol
(CAT) reporter constructs were used in transient
transfection assays for E2A activity. 1 µg of reporter construct was
included in each transfection. E2-5 (1-3 µg) and HAT cDNA
expression vectors (2 µg) were added in the co-transfection as
indicated. The results are displayed as -fold increase over the
transfection of reporter alone and represent an average of four
experiments for each assay.
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Co-transfection of E2A and p300, CBP, or PCAF Increases the Amount
of Nuclear Retained E2A--
Studies indicate that acetylation can
affect nuclear retention of transcription factors (25). We examined the
expression patterns of E2A proteins in 293T cells co-transfected with
p300, CBP, or PCAF. Low levels of endogenous E2A expression in 293T cells produced an undetectable background of E2A staining (Fig. 6, A1). In contrast, nuclear
and cytoplasmic dispersal of E2A are clearly visible after transfection
with E2-5 expression vector (Fig. 6, A2). Co-expression of
p300 (Fig. 6, A3) or CBP (Fig. 6, A5)
significantly increased the amount of E2A retained in the nucleus.
Co-transfection of PCAF mildly increased nuclear retention of the
transiently expressed E2A protein (Fig. 6, A6). Enhanced nuclear retention of E2A was not observed in 293T cells co-transfected with the enzymatic inactive mutant p300DY (Fig. 6, A4). The
greatest increase in nuclear retention was observed in cells
co-transfected with either E2A and CBP or E2A and p300, which accounted
for ~65 and 55%, respectively, of E2A-positive cells (Fig.
6B). Cells co-transfected with E2A and PCAF demonstrated
enhanced nuclear retention in ~35% of the E2A-positive cells. In
contrast, only 1-2% of the cells showed nuclear retention in
transfections with E2A alone or E2A and p300DY, respectively.
Expression of individual HAT proteins in transfected cells was verified
independently by Western blot assay (Fig. 4B). These results
suggest that acetylation of E2A or acetylation of a protein that
associates with E2A increases nuclear retention.

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Fig. 6.
Assay for nuclear retention of E2A in
transfected 293T cells. A, immunofluorescence
microscopy analysis for E2A expression and localization in human 293T
cells transfected with E2-5 (panel 2), E20-5 and p300
(panel 3), E2-5 and p300DY (panel 4), E2-5 and
CBP (panel 5), and E2-5 and PCAF (panel 6). The
endogenous E2A could not be detected in untransfected cells under the
same assay conditions (panel 1). B, the
percentage of cells displaying nuclear retention of E2A among E2A
positive-staining cells was quantified for 293T cells transfected with
E2-5, E2-5 and p300, E2-5 and p300DY, E2-5 and CBP, and E2-5 and
PCAF. Four independent sets of transfection were performed, and a total
of 50 cells were counted from each transfection.
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E2A/p300 Double Heterozygous Mice Exhibit a Defect in B
Cell Development--
It has been shown that mice heterozygous for E2A
produce fewer B cells than wild-type controls (2). This
phenotype can be enhanced further by mutations in functionally related
genes when tested in the compound heterozygous mice (26, 27). A study
of p300 heterozygous mice has also revealed a gene dosage effect of
p300 on embryonic development (28). To determine whether a functional
interaction between E2A and the histone acetyltransferase p300 is
required for normal B cell development, we analyzed bone marrow B cells
from littermates of a cross between mice that were heterozygous for
either E2A or p300. Phenotypic analysis of bone marrow cells of
6-10-day-old littermates demonstrated that E2A and p300 double
heterozygous mice had a greater reduction of total B lineage cells
(CD19+B220+) and mature B cells
(IgM+B220+) than either of the
single heterozygous littermates (Fig. 7, A and B, left
panel (Neonates)). This additive effect on bone marrow B cell development seems restricted to neonates. Analysis of
3-16-month-old adult mice shows a consistent reduction of B cell
numbers in E2A heterozygous mice, but the phenotype is not exacerbated
in the double heterozygous mice (Fig. 7B, right
panel (Adults)). Both neonates and adults show no
defect in T cell development (Fig. 7A and data not shown),
indicating that the genetic interaction between E2A and p300 is
somewhat restricted to the B cell lineage.

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Fig. 7.
Genetic test for E2A and p300
interaction. A, B cell analysis of neonatal littermates
from a cross between an E2A and a p300 heterozygous mouse. The relative
numbers of B cells derived from bone marrow were compared between
littermates. B cells were analyzed for expression of B220, CD19, and
IgM. Thymic T cells were analyzed with CD4 and CD8 markers.
B, bar graph representation of the B cell analysis of
neonatal and adult littermates from breeding between E2A heterozygous
mice and p300 heterozygous mice. Relative numbers of total B lineage
cells (B220+CD19+) and IgM+ B cells
from bone marrow were assessed for neonates (left) and adult
mice (right). The wild-type mice within each litter were
used as the base line in calculating the relative B cell percentage for
the other genotype groups. A paired t test was used to
evaluate the significance of variations between different genotype
groups. An asterisk indicates that a significant difference
was observed between compared groups.
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DISCUSSION |
Previous studies have indicated that the HAT p300 may interact
with E2A at multiple, independent sites including AD1 (18, 19), AD2
(18), and the bHLH domain (17). It is generally hypothesized that these
interactions allow the enhancer-bound E2A to recruit p300, which
subsequently facilitates transcription through acetylation of either
histones or the general transcription apparatus (17-19). However, the
significance of this interaction in regulating E2A function is still
undetermined for normal tissues under physiological conditions. In a
series of biochemical studies we have shown that the endogenous E2A
proteins interact with p300, CBP, and PCAF acetyltransferases in pre-B
cells. We have shown further that E2A is also a target of the enzymatic
activity of these HATs. Although the effect of acetylation on E2A
function remains to be determined fully, our data suggest that this
modification can increase E2A-mediated transcription, which may
in part be the result of increased nuclear retention of E2A. Finally,
we have provided genetic evidence to show that E2A and p300 interaction is important for normal B lymphocyte development in the bone marrow.
The work presented here is the first to show that E2A is acetylated by
three functionally related HATs: p300, CBP, and PCAF. This acetylation
of E2A is apparently independent of AD1 because the E2-5 cDNA used
in this study lacks the AD1 domain. Using deletion mutants of
GST-E2-5, we have determined that the amino half of E2-5 is
acetylated by p300, CBP, or PCAF. The amino terminus of E2-5 contains
multiple, isolated lysine residues and a cluster of lysine residues at
position 80-84 (KKVRK). This lysine cluster along with the nearby
sequences is highly conserved between Caenorhabditis elegans
and man. However, mutagenesis conversion of these lysine residues to
arginine did not prevent E2-5 from been acetylated by HATs (data not
shown). Although acetylation of a protein frequently occurs at a site
of multiple lysine residues (24, 29, 30), the potential for individual
lysine residue involvement can not be ruled out. Further mutagenesis
combinations involving individual lysine residues might aid in
determining the site of acetylation.
HAT-mediated acetylation of a transcription factor may alter its DNA
binding activity, ability to interact with other proteins, nuclear
localization, and protein stability. For example, the acetylation of
p53 and GATA1 leads to an increase in sequence-specific DNA binding and
transcription activity (31, 32); acetylation of MyoD leads to a
conformational change affecting its ability to recruit coactivators
(29); acetylation of HNF-4 leads to increased nuclear localization
(25); and acetylation of E2F1 leads to an increase in DNA binding and
protein stability (30). We found that acetylation of E2A did not alter
its ability to bind to E box-specific DNA (data not shown). Through
transfection experiments we provided two points of evidence that
HAT-E2A interaction affects E2A function. First, co-transfection of E2A
with each HAT increases E2A-mediated transcription of a reporter gene.
Second, transfection experiments have shown that E2A nuclear retention is substantially increased in the presence of HATs. For p300, this
increased nuclear retention is dependent on the acetylation activity of
p300. This result is consistent with the notion that nuclear retention
of E2A may be affected by acetylation. However, our study did not
formally rule out the possibility that nuclear retention of E2A is
mediated by an unknown protein that is the target of acetylation by
HAT. In fact, a moderate increase in E2A nuclear retention was observed
with PCAF, which does not show significant acetylation activity toward
E2A in transfected 293T cells. It is also possible that E2A may be
acetylated at several sites by multiple HATs. Each acetylation
may affect E2A function in a different way and in a cell
type-dependent manner. Although the current work provided
important clues, further investigations are required to determine which
interaction and modification of E2A is directly relevant to B cell development.
Genetic studies have shown that bone marrow B cells in E2A heterozygous
mice are reduced to half the number maintained in wild-type littermates
(2). Mice heterozygous for p300 demonstrate a similar gene dosage
effect in embryogenesis, differentiation, and cell proliferation (28).
Our study has now shown a genetic interaction between these two
genes in bone marrow B cell development. A similar type of genetic test
has been used previously to confirm functional interaction between E2A
and several other transcription factors present in B cells, including
HEB, E2-2, and EBF (26, 27). In all cases, a strong correlation
between protein interaction and genetic interaction is observed.
Although the dosage-dependent phenotype persists from
neonates to adult for E2A heterozygous mice, the genetic interaction
between E2A and p300 in the compound heterozygous mice was observed
only in neonates and not in adults. One explanation for the recovery of
B cell numbers in adult compound heterozygotes is the possible
compensation for the loss of p300 by other histone acetyltransferases
such as CBP, which shares considerable functional and sequence homology
with p300 (33). It has been recognized that lymphopoiesis in fetal and
neonatal life is regulated differently than in the adult (34).
Our data suggest that although E2A is required for B cell development
in both young and adult mice, its activity and specificity in
regulating B cell-specific gene expression may be modulated by
association with different HATs.
The data provided in our study suggest that E2A interact with multiple
HATs in B cells. It is conceivable that an individual HAT may be
preferentially required for a certain aspect of E2A function at a
certain stage of B cell development. It remains a challenge to
determine the specificity of these HATs in regulating E2A activities in
the context of specific E2A target genes during B cell
development. The genetic data presented in this report provide not only
the first physiological evidence that E2A and p300 collaboratively
regulate bone marrow B cell development but also, more importantly,
clues for where and when E2A and p300 interaction might have occurred
in live animals.
 |
ACKNOWLEDGEMENTS |
We thank Aaron Goldstrohm for advice and
assistance on FPLC, Dr. Akihiro Ito for reagents in acetylation assay,
Dr. Meifang Dai for assistance in mouse breeding and genotyping, and
all members of the Zhuang laboratory for comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from the Leukemia and
Lymphoma Society of America and the National Institutes of Health (to
Y. Z.).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.: 919-613-7824;
Fax: 919-613-7853; E-mail: yzhuang@acpub.duke.edu.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M211464200
 |
ABBREVIATIONS |
The abbreviations used are:
bHLH, basic
helix-loop-helix;
AD, activation domain;
HAT, histone
acetyltransferase;
CBP, CREB-binding protein;
PCAF, p300/CBP-associated factor;
GST, glutathione S-transferase;
FITC, fluorescein isothiocyanate;
FPLC, fast protein liquid
chromatography.
 |
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