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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 745-755
REVIEW ARTICLE
CREB-binding protein and p300: molecular integrators of
hematopoietic transcription
Gerd A. Blobel
From the Division of Hematology, Children's Hospital of
Philadelphia, and the University of Pennsylvania School of Medicine,
Philadelphia, PA.
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Introduction |
Differentiation of pluripotent hematopoietic stem cells into mature
circulating blood cells is coordinated by a complex series of
transcriptional events. During the last decade, numerous
transcription factors have been identified whose expression is highly
lineage-restricted within the hematopoietic system. These include
the GATA family of transcription factors, NF-E2, EKLF, the C/EBP
family of proteins, EKLF, and AML-1.1,2 However,
tissue-specific and developmentally correct expression of a given gene
is not achieved by a single transcription factor. Rather, unique
combinations of cell-type specific and widely expressed nuclear factors
account for the enormous specificity and diversity in gene expression
profiles. Recently, 2 highly related and widely expressed molecules,
CREB-binding protein (CBP) and p300, have emerged as important
cofactors for a broad number of transcription factors both within
and outside the hematopoietic system. Haploinsufficiency of CBP
results in Rubinstein-Taybi Syndrome (RTS) in humans, a
disease characterized by mental retardation, craniofacial
abnormalities, broad toes and thumbs, and an increased propensity
for malignancies, including those derived from the hematopoietic
system.3 Mice heterozygous for a disrupted CBP gene display
a phenotype similar to RTS,4 and have an increased
incidence of leukemias and histiocytic sarcomas.5 Mice
lacking both CBP alleles die during embryonic development and display
severe defects in primitive and definitive hematopoiesis, and in
vasculo-angiogenesis.6 Chromosomal translocations involving the CBP and p300 genes are associated with certain forms of leukemia, underscoring the importance of these genes in the regulation of hematopoietic cell differentiation and proliferation.
A series of recent reviews 7-9 serve as excellent guides
through the large number of factors interacting with CBP and p300. This
review will focus on the role of CBP and p300 in the transcriptional control of hematopoietic cell differentiation.
After a general overview of CBP and p300, the hematopoietic
transcription factors regulated by CBP and p300 are described in a
systematic fashion. Subsequently, human diseases involving the
CBP and p300 genes and animal models related to these diseases are
described. This is followed by an attempt to conceptualize our
knowledge by discussing mechanistic aspects of CBP and p300 function.
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Overview |
CBP was originally discovered based on its ability to
interact with the cAMP response element-binding protein
(CREB),10 whereas p300 was isolated as a cellular target of
the adenoviral oncoprotein E1A.11 Although E1A binds to
various cellular proteins, including the Rb family of tumor suppressor
proteins, its ability to block cell differentiation and to induce cell
cycle progression in many cell types depends, at least in part, on its
interaction with CBP and p300. The functions of CBP and p300 appear
interchangeable in many published reports, yet both molecules also
fulfill unique roles as revealed by gene inactivation
studies.5,12,13
During the last 5 years, numerous transcriptional regulators have been
found to interact with CBP and p300 (see Figure
1 for examples; for review see Shikama et
al8). CBP and p300 are widely expressed and are believed to
regulate gene expression in most cell types. Consistent with a function
in a wide range of tissues, CBP-1, a C elegans factor closely
related to CBP and p300, acts at an early stage in development and is
essential for all non-neuronal differentiation pathways.14
In mammals, the situation is more complex because of the existence of
at least 2 such molecules, CBP and p300.
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| Fig 1.
Structure of CBP adapted from Shikama et al.8
Not all known CBP interacting proteins are shown. Amino acid numbers
are approximate. HAT, histone acetyltransferase domain; CH,
cysteine/histidine-rich region; BROMO, Bromodomain. Bromodomains are
found in most histone acetyltransferases and in many
chromatin-associated factors. Bromodomains specifically bind to
acetylated lysine.167 CBP and p300 interact with
tissue-specific (eg, MyoD), broadly expressed (eg, nuclear receptors),
and general (eg, TFIIB and TBP) transcription factors. In addition, CBP
and p300 interact with oncoproteins, including c-Jun and c-Fos, and
tumor supressor proteins such as p53. CBP and p300 also interact with
other HAT-containing molecules, such as p/CAF, SRC-1, and ACTR.
Finally, CBP and p300 regulate the activity of signal-dependent
transcriptional activators such as CREB and the STATs.
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The complexity of protein-protein interactions surrounding CBP and p300
has led to their description as molecular integrators. Their ability to
integrate multiple transcriptional signals is illustrated by the
observation that many nuclear factors that interact with CBP and p300
can synergize with each other when bound to the same promoter in
cis. On the other hand, inhibition between these factors might
occur if they are bound to different promoters. Inhibition has been
proposed to result at least in some cases from competition
between these factors for limiting amounts of CBP and p300 in the
nucleus.15,16 Genetic evidence for the idea that CBP and
p300 are limiting stems from the discovery that patients who lack one
allele of CBP suffer from RTS. Finally, normal development of
Drosophila embryos is highly dependent on CBP gene
dosage.17,18
Many of the protein interactions surrounding CBP and p300 are regulated
by cellular signals. For example, phosphorylation of the transcription
factor CREB regulates its interaction with CBP and p300, and hormones
such as estrogens, glucocorticoids, and retinoic acid stimulate CBP and
p300 binding to nuclear hormone receptors.
To add to the complexity, CBP and p300 can stimulate both the
activating and repressive functions of certain nuclear factors. For
example, although CBP and p300 increase p53 activity on certain p53-dependent promoters,19-21 they can also augment
p53-mediated transcriptional repression on others.22
Moreover, CBP and p300 support cellular differentiation, but can also
cooperate with gene products that interfere with it. Thus, promoter and
cellular context are critical determinants of CBP and p300 function.
A breakthrough in the understanding of CBP and p300 function was the
discovery that they act not only in a stoichiometric fashion, as is the
case for most transcriptional cofactors, but that they also possess
enzymatic activity. The laboratories of Bannister23 and
Ogryzko Nakatani24 found that CBP and p300 possess
intrinsic histone acetyltransferase (HAT) activity. Acetylation of
histones is associated with a relaxed chromatin configuration, which is
thought to facilitate transcription factor access to DNA. For example,
work by Hebbes and colleagues25 demonstrated a strong
correlation between the presence of acetylated core histones and DNase
I sensitivity at the chicken  -globin locus. DNase I sensitivity
occurs before transcription is initiated and might reflect a state
poised for transcriptional activation. The importance of a balance
between the acetylated and nonacetylated state of histones in
transcriptional regulation is supported by the discovery that certain
transcriptional repressors are associated with histone deacetylases
(for review see Pazin and Kadonaga26).
More recently, CBP and p300 have been shown to acetylate nonhistone
nuclear proteins, including the tumor suppressor protein p53,27-29 dTCF,30 EKLF,31
GATA-1,32,33 NF-Y,34 the basal transcription
factors TFIIE and TFIIF,35,36 and the architectural transcription factor HMG I(Y).37 In the case of p53,
acetylation strongly increases DNA binding in vitro, providing a
potential mechanisms for CBP and p300-mediated transcriptional
control.27-29 Given the large number of factors that
interact by CBP and p300, it is likely that some of these are also
regulated by acetylation. Additional mechanisms by which CBP and p300
might operate are discussed later.
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Roles of CBP and p300 in hematopoiesis |
The viral oncoprotein E1A has been an invaluable tool for examining
the requirements of CBP and p300 in gene expression and differentiation
in various cell types. The N-terminus of E1A binds to dedicated domains
within CBP and p300 and blocks their function.38,39 Indeed,
in numerous studies, the first clues suggesting a requirement for CBP
and p300 during gene regulation derived from experiments showing that
forced expression of E1A, but not mutant forms of E1A defective for CBP
and p300 binding, interfered with expression of certain myeloid,
erythroid, and B-lymphocytic genes (Figure 3).
The following recurring themes are found in many of the studies
summarized here. First, the activities of most transcription factors
that interact with CBP and p300 are sensitive to coexpressed E1A.
Inhibition by E1A is possible even if transcription factor binding
occurs outside the E1A-binding domain of CBP and p300, suggesting that
simple competition for CBP and p300 binding cannot account for all the
effects of E1A. Second, stimulation of transcription factor activity by
CBP or p300 usually ranges between 2-fold and 10-fold in transient
transfection assays, indicating that CBP and p300 are limiting under
these conditions. Third, various combinations of nuclear factors
regulated by CBP and p300 synergize with each other when bound to the
same promoter.
The following section is divided according to classes of CBP and
p300-regulated hematopoietic transcription factors (summarized in
Figure 2)
rather than according to hematopoietic cell lineages, because most
transcription factors are expressed in multiple cell types. Moreover,
the biological functions of CBP and p300 in hematopoiesis are linked to
the functions of the transcription factors with which they interact.
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| Fig 2.
Structure of CBP indicating docking sites for
hematopoietic transcription factors.
See text for a detailed description of the listed factors. The
domain(s) of CBP responsible for EKLF binding has not yet been
determined. The observation that different factors interact with
distinct domains of CBP might explain the transcriptional synergy
observed between many of these factors.
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| Fig 3.
Interference with CBP and p300 function in erythroid
cells leads to a block in differentiation.
MEL cells stably expressing a conditional, estradiol-dependent form of
E1A were left untreated (U), or were treated with the
differentiation-inducing agent DMSO (D), estradiol (E), or both (D/E).
To monitor differentiation, cells were stained with benzidine, which
stains hemoglobin (brown), and counterstained with May-Grunwald. Note
the absence of benzidine-positive cells following estradiol-induced E1A
activation (D/E). Control cell lines expressing mutant forms of E1A
defective for CBP and p300 binding had no effect (not shown). For
details, see Blobel et al.68
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c-Myb
c-Myb is among the first hematopoietic transcription factors found
to be regulated by CBP. c-Myb is the cellular counterpart of the v-Myb
oncoprotein identified in the avian myeloblastosis virus (AMV). In the
E26 virus, which causes mixed leukemia in chickens, v-Myb is part of a
Gag-Myb-Ets fusion protein. Interestingly, Ets itself is regulated by
CBP and p300 (see below). c-Myb expression is highest in progenitor
cells of the myeloid, erythroid and lymphoid lineages and is
downregulated during maturation/differentiation of these cells (for
review see Weston40). Forced expression of c-Myb blocks
differentiation of erythroid and myeloid cell lines.41-45
Expression of a dominant interfering form of c-Myb results in enhanced
erythroid differentiation,46 whereas treatment with
antisense oligonucleotides directed against c-Myb reduces proliferation
of immature cells of the erythroid, myeloid and T-lymphoid
lineages.47-49 Disruption of the c-Myb gene in mice leads
to lethal anemia during fetal liver hematopoiesis.50 Along with the leukemogenic potential of c-Myb, the previously mentioned studies suggest that c-Myb functions in maintaining hematopoietic precursor cells in a proliferative state.
CBP was found to stimulate both c-Myb and v-Myb transcriptional
activity in transient transfection experiments.51,52 c-Myb binds CBP in vivo and in vitro in a phosphorylation-independent manner
at a site that overlaps with the CREB-binding domain of CBP. Expression
of E1A, of antisense CBP RNA, or of dominant-negative CBP interferes
with c-Myb-dependent transactivation.51,52 Although CBP
moderately enhances c-Myb activity (approximately 3-fold), the presence
of another CBP-regulated DNA binding protein such as NF-M, strongly
increases the effects of CBP in a synergistic fashion.52
Given the requirement for CBP and p300 during differentiation of
various cell types, it seems paradoxical that CBP would cooperate with
gene products such as c-Myb or v-Myb that block differentiation. A
possible explanation is that factors inducing differentiation and those
stimulating proliferation compete for the action of CBP, depending on
their expression levels during cellular maturation, or depending on
cellular signals that regulate their interaction with CBP and p300.
The E2A proteins
Work from more than a decade ago demonstrated that E1A can repress
the activity of the gamma 2b heavy chain (IgH) and the kappa light
chain genes in lymphoid cells.53,54 However, at that time,
the identity of transcription factors inhibited by E1A was unknown.
Recent studies suggest that the basic helix-loop-helix (bHLH) proteins
E47 and E12 might present critical targets for inhibition by E1A. E12
and E47, which are both encoded by the E2A gene (not to be confused
with E1A), are essential regulators of B-cell gene expression. In most
cell types, E12 and E47 proteins bind to DNA and regulate transcription
as heterodimers with tissue-specific bHLH proteins, such as the
hematopoietic transcription factor tal-1/SCL or the muscle-determining
factors of the MyoD family. Remarkably, despite its broad distribution,
only in B-cells can E47 bind DNA and activate gene expression as a
homodimer.55 Targeted disruption of the E2A gene in mice
leads to perinatal death and a selective ablation of mature
B-cells.56,57 The cause of death is uncertain, but
surprisingly, there are no obvious abnormalities present in other
hematopoietic and nonhematopoietic tissues.56-58
Work by Eckner and colleagues59 demonstrated that p300
forms a stable complex with E47 on DNA. In addition, p300 stimulates E47 activity in transient transfection experiments by using a reporter
gene driven by an intact IgH enhancer or by isolated E47-binding sites.
p300 also interacts with bHLH proteins involved in
myogenesis,59 suggesting that it has the capacity to target various members of bHLH protein superfamily that might include those
involved in hematopoiesis. Along with the findings outlined later, this
suggests a role for CBP and p300 in B-lymphoid gene expression.
GATA-1
GATA-1, one of the best studied hematopoietic transcription factors,
is a zinc finger protein involved in the regulation of virtually all
erythroid and megakaryocytic genes. GATA-1 is required for survival and
maturation of primitive and definitive erythroid precursor
cells.60-64 In addition, GATA-1 plays a critical role during megakaryocytic proliferation and
differentiation.61,65 GATA-1 can trigger terminal
differentiation and cell cycle arrest when reintroduced into a
GATA-1-deficient immortalized proerythroblastic cell
line.66
Among the genes regulated by GATA-1 are the globin genes, which, in
turn, are under the influence of the locus control regions (LCRs). The
LCRs, which contain multiple functionally important GATA-binding sites,
are thought to act in part by regulating the chromatin structure at the
globin gene loci.67 Given that CBP has histone
acetyltransferase activity, it is noteworthy that GATA-1 interacts with
CBP in vivo and in vitro.68 This interaction involves the
zinc finger region of GATA-1 and the E1A-binding domain of CBP. CBP
strongly augments GATA-1 activity in transient expression
assays.68 Expression of E1A in the erythroid cell line MEL
leads to a complete block in differentiation and to reduced expression
of GATA-1-dependent genes, including the  - and -globin genes
(Figure 2).68 These findings are consistent with a
mechanism by which CBP and p300 mediate at least some functions of
GATA-1 in intact erythroid cells.
Other GATA factors, including GATA-2 and GATA-3, which have distinct
expression patterns in hematopoietic cells, are also stimulated by CBP
(G. A. Blobel, unpublished). GATA-2 levels are high in progenitor cells
and decline during erythroid maturation.69,70 In contrast,
GATA-1 levels increase as cells mature.69,70 Thus, it is
possible that as its levels rise, GATA-1 recruits CBP away from factors
required for proliferation of precursor cells such as
GATA-271 and c-Myb,50 using them for the
activation of differentiation-specific genes.
One mechanism by which CBP regulates GATA-1 activity appears to involve
direct acetylation of GATA-1 itself. Two reports showed that CBP and
p300 acetylate GATA-1 at 2 highly conserved lysine rich motifs near the
zinc fingers.32,33 In addition, CBP stimulates acetylation
of GATA-1 in vivo at the same sites acetylated by CBP in
vitro.33 In vivo acetylation of GATA-1 by CBP is inhibited by E1A but not by mutant E1A defective for CBP and p300
binding,33 establishing a correlation between acetylation
of GATA-1 and its transcriptional activity. Although Boyes et
al32 reported that acetylation by p300 stimulates DNA
binding of chicken GATA-1 in vitro, no change in DNA binding upon
acetylation was observed by Hung et al.33 This discrepancy
may be the result of using chicken GATA-1/p300 versus murine
GATA-1/CBP, respectively. However, several lines of evidence suggest
that changes in DNA binding might not be the mechanism by which
acetylation regulates GATA-1 activity in vivo. First, mutations in the
acetylation sites do not affect DNA binding of mammalian expressed
GATA-1 molecules but do affect the transcriptional response to CBP and
p300.32,33 Second, although CBP and p300 stimulate GATA-1
activity in transient transfection assays, no evidence exists showing
that this stimulation is associated with an increase in DNA binding of
GATA-1. Third, when assayed in the context of differentiating erythroid
cells, mutations in either of the 2 acetylation motifs impair the
ability of murine GATA-1 to trigger erythroid differentiation without affecting its ability to bind DNA.33 This indicates that
the biological activity of the acetylation sites can be uncoupled from
their putative role in DNA binding.
Although acetylation of GATA-1 is likely to be important for GATA-1
function in vivo, the underlying molecular mechanism remains to be
determined. Acetylation of GATA-1 does not affect its interaction with
Fog, CBP, or GATA-1 itself.33 However, it is possible that acetylation leads to changes in the conformation of GATA-1 or affects
interaction with other as yet unidentified cofactors. The acetylation
motifs of GATA-1 might serve as docking sites for interaction with such cofactors.
NF-E2
Given the large number of CBP-interacting proteins, it is likely
that the strong inhibitory effects of E1A on MEL cell differentiation and globin gene expression might involve multiple CBP-interacting factors. Indeed, a very recent report showed that NF-E2 binding sites
in the LCR are important in mediating E1A sensitivity of the -globin
LCR.72 Moreover, both NF-E2 and EKLF (see below), have been
reported to physically interact with CBP. The basic zipper (bZip)
transcription factor NF-E2 is composed of a hematopoietic-restricted p45 subunit and a widely expressed p18 subunit, which is a member of
the maf family of proteins73-75 (for review see Blank and
Andrews76). Other p45-related molecules capable of
interacting with maf family members include Nrf1, Nrf2, Nrf3, Bach 1, and Bach 2 (for references see Kobayashi et al77). Multiple
functionally important NF-E2-binding sites are present in the
- and -globin LCRs. Loss of a functional p45 gene leads
to a pronounced defect in platelet formation,78 whereas
globin gene expression and erythroid development are only mildly
affected.79 This suggests that other members of the p45 family might substitute for p45 function in erythroid cells.
In vitro binding experiments showed that the p45 subunit of NF-E2 binds
directly to CBP.80 This study further suggests that CBP
might participate in mediating the ligand-dependent stimulation of the
thyroid hormone receptor by p45. This is of biologic interest given the
role of thyroid hormone during erythropoiesis.81 Although the functional and molecular consequences of the p45-CBP interaction have not been studied in detail, it is conceivable that NF-E2 cooperates with GATA-1 and EKLF in the formation at the LCR of a high
molecular weight transcription factor complex (enhanceosome) surrounding CBP and p300.
It is important to point out that NF-E2 activity on chromatinized
templates cannot be attributed solely to the recruitment of histone
acetyltransferases. A report by Armstrong and Emerson82 demonstrated that NF-E2 can disrupt chromatin structure on templates containing regulatory regions of the -globin locus, and that the
NF-E2-associated chromatin modifying activity is ATP-dependent.
EKLF
Another transcription factor regulated by CBP is the zinc
finger-containing erythroid Krüppel-like factor
EKLF.83 EKLF is specifically required for the expression of
adult -globin but not -globin genes, and loss of EKLF function
leads to lethal -thalassemia in mice.84,85 Moreover,
EKLF  / mice carrying a human globin gene locus display a
delayed  - to -globin switch that normally occurs at the onset of
adult bone marrow erythropoiesis.86,87 Interestingly,
absence of EKLF also results in a loss of DNase 1 hypersensitive site
formation at both the transgenic and endogenous -globin
promoters,87 consistent with a role of EKLF in remodeling chromatin at these promoters.
EKLF can interact with both CBP and p300, and the CBP- and
p300-associated acetyltransferase p/CAF in transfected cells. However, CBP and p300, but not p/CAF, acetylate EKLF in vitro.31
Acetylation most likely occurs at 2 residues that are part of an
inhibitory domain adjacent to the zinc finger region. Metabolic
labeling experiments that used [3H]acetate further
suggest that EKLF is acetylated in vivo.31 CBP and p300,
but not p/CAF, stimulate EKLF activity in transient transfection
experiments that used the erythroleukemia cell line K562.31
It will be interesting to determine whether acetyltransferase activity
of CBP and p300 is required for stimulation of EKLF activity. Acetylation did not affect DNA binding of EKLF, and the molecular consequences of acetylation are not yet known.31
Together, the above reports suggest that erythroid transcription
factors controlling globin gene expression might cooperate in the
formation of a high molecular weight complex in which GATA-1, NF-E2,
and EKLF are linked through CBP and p300 (Figure
4). Consistent with such a model is the
observed synergy between GATA-1 and EKLF in transactivation
experiments.88
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| Fig 4.
Hypothetical model in which NF-E2, GATA-1, and EKLF
cooperate to recruit CBP and p300 to the locus control region of the
-globin gene cluster.
This could lead to acetylation of nearby histones and transcription
factors. Acetylation of histones leads to changes in chromatin
structure, and acetylation of transcription factors might stabilize
their interaction with DNA or alter their transcriptional activity. It
is conceivable that this high molecular weight complex also connects to
the promoters of the globin genes through a looping mechanism.
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C/EBP
CCAAT-box/enhancer binding proteins (C/EBPs) belong to the basic
region/leucine zipper class of transcription factors and play a role in
the differentiation of a broad range of tissues. In the hematopoietic
system, C/EBP family members are expressed mostly in the
myelomonocyctic lineage and participate in the regulation of macrophage
and granulocyte-restricted genes, such as the M-CSF receptor, G-CSF
receptor, and GM-CSF receptor genes (for review see Lekstrom-Himes and
Xanthopoulos, and Yamanaka et al89,90). Targeted disruption
of the C/EBPd, C/EBP, or C/EBP genes resulted in defects
predominantly affecting the granulocytic lineage,91-94 whereas other hematopoietic lineages remained intact. C/EBP
transcription factors are also critical mediators of inflammatory and
native immune functions (for review see Poli95).
Studies by Mink et al96 showed that C/EBP-dependent
transcription is E1A-sensitive and that overexpressed p300 stimulates C/EBP activity on the macrophage/granulocyte-specific mim-1 promoter and, importantly, also on an endogenous C/EBP-regulated gene, called
126. Moreover, p300 increases the synergy between c-Myb and C/EBP.
C/EBP binds to the E1A-binding region of p300 through its
N-terminus. Overexpression of the minimal C/EBP-binding domain of
p300 reduced the activity of C/EBP presumably by interfering with
the C/EBP-p300 interaction.96 The N-terminus of C/EBP contains stretches of amino acids conserved among C/EBP family members
suggesting that other C/EBP molecules might also be regulated by CBP
and p300.96 Together, these results implicate CBP and p300
as important cofactors during granulocytic gene expression.
Ets
The Ets family of transcription factors is a diverse group of
approximately 30 proteins that share a conserved DNA binding domain.97 The c-ets-1 proto-oncogene is transduced
by the E26 avian acute leukemia virus to form part of the Gag-Myb-Ets
gene fusion. This virus induces both erythroid and myelomonocytic
leukemias. Full transforming activity of E26 requires the presence of
both the Myb and Ets portions of the fusion protein.98,99
Ets-1 is expressed predominantly in lymphoid cells and regulates a
number of lymphocyte-specific genes. Gene knockout studies demonstrated a role for Ets-1 in T-cell proliferation and
survival.100,101 Effects on B-cell differentiation were
also observed.100,101 Ets-1 and some of its relatives
synergize with a number of transcriptional regulators known to interact
with CBP and p300, such as AP-1,102 and
Myb.103-106 Especially striking is the frequently observed
cooperativity between Ets-like factors and GATA-1 during the
expression of several megakaryocyte-restricted genes, including the
IIb,107 GPIX,108 GP1b,109 the
thrombopoietin receptor (c-mpl),110 and PF4
genes.111 The synergy of Ets proteins with CBP and
p300-regulated factors led to the hypothesis that they too are
regulated by CBP. Indeed, Yang et al112 showed that the
Myb- and Ets-dependent promoter of the myeloid-expressed gene CD13/APN
is sensitive to the expression of E1A but not mutant E1A defective for
CBP and p300 binding. Ets-1 activity is stimulated by coexpressed CBP,
and Ets-1 associates with CBP in nuclear extracts. In vitro, the
N-terminus of Ets-1 can form 2 contacts with CBP involving the CH1 and
CH3 domains of CBP. In support of the functional importance of the
physical interaction between Ets-1 and CBP, the authors demonstrated a good correlation between binding of Ets-1 to the CH1 region and its
ability to transactivate. In addition, Ets-1 coprecipitates with
histone acetyltransferase activity, consistent with its association with CBP and p300 and/or other acetyltransferases in
vivo.112
Of note, another Ets family transcription factor, PU.1, was recently
found to interact with CBP through the activation domain of PU.1 in a
yeast 2-hybrid assay.113 CBP stimulates PU.1
transcriptional activity in transient transfection assays. PU.1 is
specifically expressed in hematopoietic organs with the highest levels
detected in myeloid and lymphoid cells.114 Thus, CBP and
very likely p300 target a broad range of myeloid and lymphoid expressed
transcription factors.
AML1
Another leukemogenic transcription factor controlled by p300 is
AML1.115 The AML1 gene is rearranged in several distinct chromosomal translocations associated with acute myeloid leukemia (AML;
t[8;21]), acute lymphatic leukemia (ALL; t[12;21]), and myelodysplastic syndrome (t[3;21]) (for review see
Look116). The AML1 gene is the most frequent target for
chromosomal translocations in human leukemias. AML1 constitutes a
family of at least 3 factors derived from the same gene by alternative
splicing. The AML1 gene products bind to DNA as heterodimeric complexes
with CBF. Of note, the CBF gene itself is involved in chromosomal
rearrangements found in cases of AML.116 Consistent with
its broad expression pattern and the presence of functionally important
AML1 binding sites in the promoters and enhancers of myeloid and
lymphoid expressed genes, knock-out studies revealed that both AML1 and
CBF genes are essential for the formation of all definitive blood
lineages.117-121
AML1b, one of the AML1 isoforms containing an activation domain, and
p300 associate in vivo and in Far Western blots, and p300 stimulates
AML1b activity on the myeloperoxidase promoter in transient
transfection experiments.115 Overexpression of the t(8;21)
translocation product AML1-ETO in the IL-3-dependent myeloid cell line
L-G interferes with G-CSF-induced differentiation along the
neutrophilic lineage. Forced expression of wild-type AML1b can overcome
the effects of AML1-ETO and restore differentiation.115 In
contrast, AML1a, which lacks an activation domain, is inactive in this
assay. The potential of various AML1b constructs to induce differentiation is further enhanced by coexpression of p300 and correlates well with their ability to interact with
p300.115 This indicates that p300 plays a role in myeloid
cell differentiation and suggests that the rearranged AML genes found
in chromosomal translocations act as dominant negative alleles. The
latter notion is consistent with the recent finding that AML-ETO
associates with a transcriptional repressor complex containing histone
deacetylases and that this deactylase complex is required for blocking
differentiation of myeloid cells.122-124 This raises the
interesting possibility that the intrinsic (or associated)
acetyltransferase activity of p300 might be required to overcome the
repressive effects of AML-ETO. Indeed, a truncated form of p300 lacking
the acetyltransferase domain was impaired in its ability to synergize
with AML-1b. However, a more detailed mutagenesis of p300 will be
required to establish a correlation between its HAT activity and its
ability to cooperate with AML1b.
Finally, AML-1 synergizes with c-Myb and with C/EBP on myeloid and
lymphoid promoters.125-127 This synergy is apparently not the result of cooperative DNA-binding,127,128 suggesting
that it is instead mediated through recruitment of a common cofactor such as CBP and p300, similar to what has been proposed for other CBP
and p300 regulated factors.
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CBP and p300 in leukemia-associated chromosomal translocations |
Both CBP and p300 bind the viral oncoproteins E1A and SV40 T. This
raised the possibility that alterations in the functions of CBP and
p300 might play a role in the development of malignancies in humans.
This suspicion was supported by the finding that 1 copy of the CBP gene
is inactivated in the rare disease Rubinstein-Taybi syndrome,3 which is manifested by an increased propensity
for tumors (mostly of the nervous system), craniofacial malformations, and mental retardation.129,130
The involvement of CBP and p300 in hematologic malignancies was
realized through the discovery of leukemia-associated chromosomal translocations involving the CBP and p300 genes. These translocations generally result in fusion products that preserve most of the CBP and
p300 molecules, suggesting that the disease mechanism does not simply
involve loss of function of CBP, as is the case in Rubinstein-Taybi
syndrome. Instead, they suggest altered function (dominant positive or
dominant negative) through fusion to another molecule. For example,
AML-derived leukemic blast cells containing the t(8;16)(p11;p13)
translocation, which is often associated with acute myelogenous
leukemia subtype M4/M5, have the CBP gene fused to the MOZ
(monocytic leukemia zinc finger)
gene.131 This fusion results in a small deletion of the
N-terminal 266 amino acids of CBP leaving the rest of the molecule
intact.131 Interestingly, the MOZ gene also has a putative
acetyltransferase domain that is retained in the MOZ-CBP fusion.
In principle, any translocation event could lead to gain or loss of
function of either fusion partner, to the formation of dominant
interfering alleles, or to entirely new activities. Fusion of CBP to a
given transcription factor might result in aberrant recruitment of CBP
to certain promoters, leaving less free CBP available for other
transcription factors involved in balancing proliferation and
differentiation. In addition, it is possible that misdirected or
deregulated acetyltransferase activity by CBP and p300 fusion products
causes changes in gene expression profiles that contribute to the
transformed state. One likely mechanism by which the MOZ-CBP fusion
contributes to malignant transformation involves constitutive
recruitment of CBP to MOZ-regulated genes. The MOZ gene contains 2 C4HC3 zinc finger regions, also found in CBP and p300, and a C2HC zinc
finger. These regions might serve as protein-protein interaction
domains and might target MOZ to chromatin-associated proteins and
DNA.131 The MOZ-CBP fusion protein contains the CBP-derived
and the putative MOZ acetyltransferase domain that together could be
powerful regulators of chromatin structure and transcriptional activity
at MOZ-regulated genes.
Since their initial discovery, additional cases of AML with t(8;16)
translocations resulting in CBP and MOZ gene arrangements have been
reported.132 However, in these cases no MOZ-CBP fusion transcripts were detected, raising the possibility that CBP or MOZ gene
rearrangements might contribute to leukemogenesis by alternative mechanisms.
Another clinically relevant example of the importance of balancing
histone acetylation and deacetylation comes from studies of acute
promyelocytic leukemia (APL)-associated translocations that fuse the
retinoid acid receptor alpha (RAR) to the PLZF or PML genes.
PML-RAR and PLZF-RAR fusion proteins have a high affinity for a
transcriptional repressor complex containing histone deacetylases.
Although normal RAR responds to retinoic acid (RA) by shedding the
deacetylase complex, followed by association with an acetyltransferase
complex (which contains CBP), PML-RAR responds only to very high
concentrations of RA, and PLZF-RAR is RA
resistant.133-135 The ability of leukemic cells to
differentiate upon RA treatment correlates with the ability of their
translocation fusion proteins to displace the repressor complex in
response to RA. In fact, patients with PML-RAR APL typically achieve
remission upon treatment with high doses of RA, whereas PLZF-RAR APL
patients do not.
The chromosomal translocation, t(11;16), which is associated with
therapy-induced acute myeloid leukemia, therapy-induced chronic
myelomonocytic leukemia, and myelodysplastic syndrome, fuses the MLL
and CBP genes such that most of the CBP molecule stays
intact.136-139 The MLL gene was also found to be fused to
the p300 gene in an AML patient carrying a t(11;22)
translocation.140 The MLL gene encodes a large multidomain
protein containing zinc fingers and AT-hook motifs,141-143
and is involved in translocations with at least 40 different fusion
partners (for references see Sobulo et al137). This raises
the question whether the structural alterations of MLL itself or of its
fusion partners are critical for leukemogenesis. Together, these
findings underscore the importance of CBP and p300 function in
balancing growth and differentiation of hematopoietic cells.
| |
Mechanisms of CBP and p300 function |
Clues from studies of intact animals.
Some unexpected insights into the function of CBP and p300 have come
from gene knock out studies. The CBP and p300 null mice display similar
phenotypes.13 The p300/ embryos die between days 9 and 11.5. Their main defects are severe developmental
retardation, reduced size, failed neural tube closure, and altered
cardiac ventricular trabeculation. A fraction of the p300 +/ mice
die early, displaying neural tube closure defects similar to the
p300/ mice, indicating a requirement for full p300 gene
dosage during neural development. Mice heterozygous for CBP deficiency
suffer from skeletal abnormalities and growth retardation, a phenotype resembling RTS in humans.4 CBP and p300 compound
heterozygous mice die early and display a phenotype very similar
to the individual homozygous knock outs.13
More extensive analysis of mice heterozygous for CBP deficiency
revealed defects in the hematopoietic system that only became apparent
in newborn pups beginning at 3 months of age.5 The CBP
+/ animals have extramedullary myelopoiesis and erythropoiesis, and
display enlarged, hypercellular spleens. In the peripheral blood, the
most striking defect is a decrease in the number of B-lymphocytes,
whereas in the bone marrow, cells of the erythroid, myeloid, and
B-lymphocytic lineage were significantly reduced. No overt malignancies
were observed in the CBP +/ mice until the mice reached at least 1 year of age. Then, 4 of the 18 mice analyzed had overt tumors, 2 had
histiocytic sarcomas, 1 had myelomonocytic leukemia, and 1 had
lymphocytic leukemia. In light of the small number of cases studied, it
is conceivable that other types of hematologic neoplasms might occur at
an increased rate in CBP +/ mice. When splenocytes or bone marrow
cells from apparently tumor-free CBP+/ donors were engrafted into
sublethally irradiated wild-type mice, the recipients developed
histiocytic sarcomas at a high rate with latency periods of 3 to 5 months. Grafts derived from 1 CBP +/ donor resulted in the formation
of plasmacytomas with monoclonal gammopathy and renal amyloid
deposition. DNA analysis of 1 plasmacytoma and 1 histiocytic sarcoma
from bone marrow-transplanted mice revealed the specific loss of the
wild-type CBP allele with retention of the targeted one. Loss of
heterozygosity in these cases suggests that CBP is a tumor supressor
gene, similar to the RB family of proteins that are also targeted by
the E1A oncoprotein. Surprisingly, no hematologic defect or cancer
predisposition was observed in age- and strain-matched p300 targeted
mice.5 This suggests that, despite their similarity, CBP
and p300 might play distinct roles in certain cell types.
The tumors observed in CBP +/ in mice appear to be restricted to the
hematopoietic system, although additional types of neoplasms might be
found as more mice are analyzed. In contrast, patients with RTS have an
increased risk for tumors of various origins, the most common tumors
being neurally derived. Hematologic malignancies observed in RTS
patients occur less frequently and include acute lymphocytic leukemia,
acute myelogenous leukemia, and non-Hodgkin lymphoma.130
A very recent report describes the hematologic consequences of
homozygous CBP-deficiency in mice.6 In this study,
disruption of the CBP gene resulted in the formation of a truncated
form of CBP that retains the N-terminal 1084 amino acids (of 2441) but
lacks the HAT domain. Mice homozygous for this defect die between day
9.5 and 10.5 of embryogenesis similar to the CBP knock-out mice. Before
their deaths, embryos are anemic, and their yolk sacs contain fewer
erythroid cells and display a defective vascular network. Although the
number of yolk sac-derived erythroid colony forming units is reduced,
a few mature erythroid cells are found, suggesting that CBP is not
absolutely required for erythroid maturation and that p300 might be
able to partially compensate for the CBP defect. To examine definitive
hematopoiesis in the CBP / mice, organ culture was
performed from E9.5 embryos with tissue from the
aorta-gonad-mesonephros (AGM) region, followed by colony forming assays. These experiments revealed dramatically reduced numbers of
definitive erythroid and granulocyte/macrophage progenitor cells. Organ
cultures from these embryos also revealed a strong reduction in
vasculo-angiogenesis.
The mechanisms by which CBP deficiency cause RTS in humans and the
severe hematologic and nonhematologic defects in mice are entirely
unknown. The answer to this question is complicated by the enormous
complexity of protein interactions surrounding CBP and the multitude of
mechanisms by which CBP regulates gene expression. Analysis of gene
expression profiles in tissues from CBP-deficient mice, as well as gene
complementation experiments with mutant CBP gene constructs, could be
used to tackle this question. Progress in the understanding of the
phenotypic defects that result from CBP deficiency requires a
reductionistic approach involving the study of individual CBP- and
p300-binding transcription factors and the genes that they control. For
example, it is conceivable that the reduced number of B lymphocytes in
CBP +/ mice results from reduced activity of the E47 transcription
factor that interacts with CBP and p300, and that is required for
B-cell development.59
Strength in numbers.
CBP and p300 interact with numerous transcription factors. Many of
these interactions might take place simultaneously because they are
mediated by distinct domains. This could account for the observed
synergy between factors regulated by CBP. Thus, CBP might provide a
platform for the assembly of high molecular weight complexes
(enhanceosomes; for review see Carey144) containing multiple DNA-binding proteins that position the complex in a sterically correct fashion at promoters and enhancers. Because this complex is
likely to include non-DNA-binding proteins such as p/CAF, ACTR, or
SRC-1, which also possess acetyltransferase activity, it would constitute a powerful regulator of chromatin
structure.145-147 For example, a high molecular weight
complex centered on CBP and p300 could form at the LCR, which
participates in regulating chromatin structure at the -globin locus
(Figure 4). The LCR contains binding sites for GATA-1, EKLF, and NF-E2
all of which bind to CBP and p300.31,33,68,80 Thus, CBP and
p300 might integrate signals from multiple transcriptional regulators
and perhaps even present targets for global regulators of gene
expression, such as signaling cascades used by growth/differentiation
factors. The latter notion is supported by the observation that CBP and
p300 are acetylated and phosphorylated.
CBP and p300 are also thought to mediate negative cross-talk between
transcription factors. Competition for limiting amounts of CBP and p300
has been invoked to account for mutual inhibition of CBP- and
p300-regulated transcription factors when bound to separate DNA
templates.15 This might explain the inhibition of GATA
factors by ligand-activated nuclear hormone receptors (NR).148-150 The observation that overexpression of CBP
alleviates NR-mediated repression of GATA-1, and that ligand-bound NR
reduce the stimulation of GATA-1 activity by CBP (G. A. Blobel,
unpublished) are consistent with such a model. Together, these findings
support a role of CBP and p300 as molecular integrators of positive and negative transcriptional signals that govern hematopoietic gene expression.
Building a bridge.
The large number and diversity of genes and transcription factors
regulated by CBP and p300 could be explained if CBP and p300 were
components of the basal transcription apparatus. In support of such a
model, CBP and p300 have been found to interact with
TFIIB,151 TBP,152-155 and RNA polymerase
II.156-160 Thus, recruitment of CBP by a DNA-bound
transcription factor could facilitate the formation of a preinitiation
complex at relevant promoters (Figure 5).
Such a mechanism would imply that CBP and p300 act in a stoichiometric fashion. Although this might be true on some promoters, additional evidence suggest that CBP and p300 also act catalytically (see next
paragraph).
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| Fig 5.
Hypothetical model in which CBP and p300 link DNA-bound
nuclear factors to components of the basal transcription machinery.
GTFs, general transcription factors; TBP, TATA-binding protein, Pol II,
RNA polymerase II.
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Action by catalysis.
The observation that CBP, p300, and some of its associated factors
possess acetyltransferase activity suggests an enzymatic mechanism of
gene regulation. Targeting of CBP and p300 to the appropriate sites
could lead to local increases in histone acetylation, followed by
rearrangement of chromatin structure (Figure 4). This in turn could
favor access of other transcriptional regulators. Again, the LCR
provides an example where such a mechanism might be operating. As
previously mentioned, histone acetylation and open chromatin correlate
well at the chicken -globin gene locus.25 However,
depending on transcription factor/promoter context, CBP and p300 can
also act in a HAT-independent fashion.161
If some nuclear factors act by recruiting a histone-modifying enzyme to
trigger chromatin opening, how do they find access to DNA in the first
place? One possibility is that other transcription factors might pave
their way by opening chromatin structure in an acetylation-independent
fashion. An example for such a scenario is provided by the observation
that NF-E2 disrupts chromatin structure in a ATP-dependent manner on a
chromatinized template containing DNase1 hypersensitive site 2 of the
-globin LCR.82 This leads to increased access of GATA-1
to adjacent GATA sites.
Alternatively, GATA-1 might find access to chromatin without the
assistance of other factors. A recent report162
demonstrated that chicken GATA-1, or a peptide comprising just its
DNA-binding domain, can bind to DNA packaged into a nucleosome. This
leads to a reversible breakage of histone/DNA contacts, thus perturbing nucleosome structure.162 Once bound to DNA, the
GATA-1-associated acetyltransferase complex might modify adjacent
histones, thus facilitating access of other transcription factors to DNA.
It is important to keep in mind that modification of chromatin is not
restricted to acetylation, and that numerous regulated chromatin
modifying complexes have been identified (for review see
Kadonaga163). For example, an elegant study by Armstrong et
al164 demonstrated that EKLF interacts with a complex,
called E-RC1, which contains components of the mammalian SWI/SNF
complex, an ATP-dependent chromatin remodeling machine.163
However, E-RC1 does not appear to contain histone acetyltransferases
(Beverly Emerson, personal communication).
Acetylation of nonhistone proteins, including transcription factors,
might turn out to be of equal importance for CBP and p300 function. For
example, acetylation of p53 leads to an increase in DNA binding
activity.27-29 It is likely that acetylation regulates transcription factor activity by a variety of mechanisms. In the case
of the drosophila transcription factor dTCF, acetylation by CBP
decreased its affinity for its cofactor -catenin/Armadillo, leading
to transcriptional inhibition.30 An interesting variation of this theme is the finding that acetylation of the architectural transcription factor HMG)-I(Y) by CBP leads to destabilization of an
enhanceosome complex at the interferon gene promoter, resulting in
termination of transcription.37
It is conceivable that acetylation might be a widely used mechanism to
trigger allosteric changes in proteins, thereby regulating protein-protein and protein-DNA interactions, similar to what has been
observed upon protein phosphorylation. In both cases, the modification
results in a change of charge, addition of a negative charge in the
case of phosphorylation, and neutralization of a positive charge in the
case of acetylation. Moreover, acetylation changes the size of the
lysine side chain, which could be important in protein folding.
| |
Summary and perspective |
CBP and p300 are large, multifunctional molecules that can exert
both positive and negative effects on transcription and cell differentiation. It is likely that additional factors will be discovered to interact with CBP and p300, and that a subset of these
might be regulated by acetylation. The challenge that lies ahead will
be to determine the significance of such interactions in
physiologically relevant settings. Given that CBP and p300 share many
functions this will not be an easy task, especially because it has not
been possible so far to generate CBP and p300 double knock-out cell
lines. The mechanisms by which CBP and p300 act likely depend on
promoter and cellular context as well as the chromatin configuration in
which a given target gene is embedded. One approach that would allow
dissection of CBP and p300 functions in a physiologic context would be
to knock in mutant CBP and p300 alleles bearing mutations in domains
associated with specific functions such as the HAT domain or important
protein docking sites. Such experiments might also yield insights into
the mechanism by which loss of CBP leads to RTS.
Given the broad variety of CBP and p300 regulated factors, an important
and challenging task will be the identification of the relevant
downstream target genes that mediate their function in vivo.
Subtractive hybridization and microarray technologies might be useful
approaches to identify genes most sensitive to changes in CBP and p300 levels.
Although CBP and p300 are expressed in most tissues, their importance
in regulating gene expression and differentiation in hematopoietic
cells is illustrated by their involvement in leukemia-associated chromosomal translocations. It remains to be determined why these chromosomal translocations result in leukemias mostly of the
myeloid/monocytic lineage.
Because CBP and p300 have intrinsic and associated acetylase activity,
they might present targets for pharmacological intervention. It can be
envisioned that novel drugs might be developed that alter their
specific activity or substrate specificity, thereby allowing for
modulation of gene expression and cell differentiation. For example, in
cases in which CBP acetyltransferase activity mig |
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Introduction
Overview