Download Chromatin modifying activity of leukaemia associated fusion proteins

Human Molecular Genetics, 2005, Vol. 14, Review Issue 1
doi:10.1093/hmg/ddi109
R77–R84
Chromatin modifying activity of leukaemia
associated fusion proteins
Luciano Di Croce*
ICREA and Centre de Regulació Genòmica (CRG), Passeig Maritim 37-49, 08003 Barcelona, Spain
Received January 12, 2005; Revised and Accepted February 24, 2005
The leukaemias, which are divided into chronic and acute forms, are malignant diseases of haematopoietic cells
in which the proper balance between proliferation, differentiation and apoptosis is no longer operative. Genes,
such as those of mixed-lineage leukaemia, AML1 and retinoic acid receptor alpha, have been found to be aberrantly fused to different partners, which often encode transcription factors or other chromatin modifying
enzymes, in numerous types of acute lymphoid and myeloid leukaemias. These chimeric fusion oncoproteins,
generated by reciprocal chromosomal translocations, are responsible for chromatin alterations on target genes
whose expression is critical to stem cell development or lineage specification in haematopoiesis. Alterations in
the ‘histone code’ or in the DNA methylation content occur as consequence of aberrant targeting of the corresponding enzymatic activities. Here, the author will review the most recent progress in the field, focusing on
how fusion proteins generated by chromosomal translocation are responsible for chromatin alterations,
gene deregulation and haematopoietic differentiation block and their implication for clinical treatment.
INTRODUCTION
The packaging of DNA into chromatin fibres provides the cell
with the means to compact and to store its 2 –3 billion base
pairs of DNA, but it also regulates the accessibility of transcription factors and general transcription machinery to gene
promoters (1). The chromatin fibre is composed of repetitive
units, known as nucleosomes, which are comprised of an
octamer of core histones (two of each H2A, H2B, H3 and
H4), around which 146 bp of DNA are wrapped. In addition,
higher eukaryotes possess linker histones (e.g. H1), which are
thought to bind to the exit and entry points of DNA as it winds
around the nucleosome, thereby facilitating higher-order
packaging of chromatin (2,3). Within chromatin, the Nterminal of core histones protrude out of the core structure
and can contact adjacent nucleosomes. These ‘tail’ interactions are crucial for the regulation of chromatin structure
and are subjected to multiple post-translational modifications
(Fig. 1). These modifications form the basis of the ‘histone
code’ hypothesis, where combination of modifications forms
the recognition surfaces for chromatin regulatory factors (4).
Lysine acetylation
Acetylation of lysine residues within the histone tails is associated with a relaxed chromatin configuration, which is believed
to decrease the histone – DNA interaction and facilitate transcription factor access to DNA (5). Consistent with this, many
transcriptional co-activators possess histone acetyltransferase
(HAT) activity. Conversely, many transcriptional co-repressors
are enzymes with histone deacetylase (HDAC) activity, which
contribute to maintain chromatin in the condensed state (6).
Lysine and arginine methylation
Histone lysines can be mono-, di- and trimethylated by a group
of enzymes called histone methyltransferases (HMTase) (7).
Members of this enzyme family contain a conserved SET
domain, which is flanked by cysteine-rich regions. Methylated
lysines are recognized by the conserved chromodomain
modules found in chromatin-associated proteins related to heterochromatin proteins HP1 and Pc (8,9). Although methylation of histone H3 lysine 9 (H3-K9), H3-K27 and H4-K20
functions as a repressive mark, not all lysine methylation
appears to be a signal for repression of transcription. It has
been recently demonstrated that methylation of histones
H3-K4, H3-K36 and H3-K79 are associated with transcriptionally competent euchromatin. Similarly, histone arginine
methylation correlates with gene activation (10). An enzyme
with histone demethylase activity has been recently identified (11), suggesting that dynamic regulation of histone
*To whom correspondence should be addressed. Email: [email protected]
# The Author 2005. Published by Oxford University Press. All rights reserved.
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Figure 1. Major modifications of the N-terminal tail of histones.
methylation by both histone methylases and demethylases
occurs, in a mechanism similar to acetylation/deacetylation.
Serine phosphorylation
Phosphorylation at histone 3 serine 10 (H3-S10), H3-S28 and
H4-S1 has been documented. Phosphorylation at H3-S10 and
H3-S28 coincides with the induction of immediate-early gene
expression and with the onset of mitosis. Phosphorylation is
reversed by the protein phosphatase 1 family. The kinases that
can phosphorylate H3 are Aurora-B/Ipl1, PKA, Rsk-2 and
Msk1, which tend to target Ser/Thr sites that are surrounded
by basic residues (12). Phosphorylation of a site adjacent to
(or nearby) a methyl mark that engages an effector module
could lead to consecutive loss of binding to that factor, a mechanism that has been proposed as ‘methyl/phos switching’ (13).
DNA methylation
Modification of the DNA itself can likewise lead to remodelling of chromatin and gene regulation. DNA methyltransferase
(DNMT) enzymes catalyze the addition of a methyl group to
cytosine residues at CpG dinucleotides, which if located
within a gene’s regulatory regions can lead to transcriptional
silencing (14). The process of DNA methylation in
mammals is carried out by at least three catalytically active
DNMT enzymes (15).
DNA methylation represses gene transcription by creating
docking site for methyl-CpG binding domain (MBD) proteins,
which selectively recognize methylated CpG dinucleotides
(16). The presence of MBD proteins within methylated
promoters could prevent gene activation by precluding
binding of positive transcription factors. Recent data demonstrated that in addition to this ‘passive’ mechanism, MBD proteins act also by recruiting other repressive enzymes, such as
HDACs and HMTs, to hypermethylated promoters (17). Aberrant methylation underlies susceptibilities to several forms of
cancer and is likely to be involved in numerous other human
diseases (18,19).
BLOOD CELL DIFFERENTIATION
All blood cell types are derived from haematopoietic stem
cells (HSCs) (20). Two properties define these cells. First,
they can generate more HSCs (self-renewal), and second,
they have the potential to differentiate in a stepwise process
of binary decisions into various progenitor cells (common
lymphoid and myeloid progenitors), which in turn differentiate
into additional intermediate progenitors. Self-renewal is
required for HSCs to maintain haematopoiesis over the lifetime of the host. Differentiation defines the sequence of
events by which cells undergo orderly changes into mature
cells, which is regulated by several lineage-restricted transcription factors (Fig. 2). Mis-expression, disruption or,
more often, chromosomal translocation of one of these
‘master’ genes required for haematopoiesis is usually a key
event in leukaemias (21). In leukaemia, chimeric proteins
created through chromosomal translocations are believed in
several instances to function in a dominant-negative fashion
on gene regulation, thus preventing proper cell differentiation.
The fusion proteins exert their action via direct or indirect
interactions with transcription co-factors or chromatin modifying enzymes. In contrast, tyrosine kinases, rather than transcription factors, are targeted by chromosomal translocation
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Figure 2. Schematic representation of adult haematopoiesis. Haematopoietic differentiation pathway according to the model proposed by Weismann et al. (20).
Leukaemogenic fusion proteins could arise either in multipotent progenitors (including HSC) prior to lineage commitment or alternatively in committed cells,
which are permissive for fusion expression, as indicated by the red arrows. The target cells in which the translocations can occur differ for the various fusion
proteins. HSC, haematopoietic stem cell; CLP, common lymphoid progenitor; CMP, common myeloid progenitor.
in chronic myeloid leukaemias (CML). CML are characterized
by leukocytosis with normal differentiation and cell function.
This suggests that alterations in tyrosine kinases provide
mainly a proliferative and survival advantage, while chimeric
transcription factors impair cellular differentiation (22). Why
are solid tumours so rarely characterized by the presence of
chimeric fusion proteins? The answer could be linked to the
high frequency of recombination events that occurs in lymphocytes and to the requirement of a ‘permissive’ cellular
context, which may be present in haematopoietic cells.
MIXED-LINEAGE LEUKAEMIA FUSION PROTEINS
Mixed-lineage leukaemia (MLL) protein (also known as ALL1
or HRX), the mammalian orthologue of the epigenetic transcriptional regulator trithorax in Drosophila, has been implicated in fusions with more than 60 other partners in both
acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML) (23). The MLL protein (Fig. 3) was shown to
possess HMTase activity toward H3-K4 via its SET domain,
leading to transcriptional activation at Hox gene promoters
(24,25), the expression of which is high in progenitor cells
and downregulated during differentiation and maturation.
MLL, additionally, functions in a large super-complex of at
least 29 proteins, which is involved in the remodelling, acetylation, deacetylation and methylation of nucleosomes and histones through association with SWI/SNF, TFIID, NuRD,
MBDs, HDACs and HAT CBP (24). MLL fusion proteins are
predicted to disrupt critical patterns of HOX gene expression
in haematopoietic progenitor cells. Indeed, the Hoxa9 gene is
upregulated in MLL-dependent leukaemia and together with
Hoxa7 is required for MLL-ENV-dependent transformation
(26). It has been recently shown that the CXXC domain of
MLL, which is also found in MBD1 and DNMT1 proteins, is
essential for myeloid transformation (27).
Because MLL fusion proteins lack the SET domain, the
CBP-binding domain, and the PHD domain (involved in
HDAC binding), the proper balance between activation and
repression is likely compromised in MLL fusions. Fusion products of MLL with CBP t(11;16) or with p300 t(11;22) retain
the bromodomain and HAT domain and might lead to leukaemia by increasing histone acetylation of genomic regions targeted by MLL (23). Another fusion partner of MLL is ENL, a
subunit of the SWI/SNF complex. MLL –ENL fusion protein,
t(11;19), associate and co-operate with SWI/SNF complexes
to activate transcription of the Hoxa7 promoter (28). Moreover, partial tandem duplications of exons 2– 6 or 2 – 8 are
present in 10% of AML.
Although, at present, it is unclear whether a loss or gain of
MLL function is responsible for oncogenesis, these data
suggest that MLL can activate genes at inappropriate times by
mis-targeting enzymes involved in epigenetic decisions, consequently modifying the chromatin to allow gene activation
(Fig. 4).
A similar scenario has been postulated for the fusion proteins
generated by the t(5;11) translocation, NUP98 –NSD1 (29), and
by the t(8;11) translocation, NUP98 –NSD3 (30), which are
specifically associated with AML. The NUP98 gene encodes
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Figure 3. Structure of wild-type and aberrant MLL proteins. Various putative functional domains are represented by coloured boxes. AT hooks, AT hook DNA
binding domain; SNL, speckled nuclear localization signal; CxxC, cysteine-rich motif homologous to DNMT1 and MBD1; RD, repressor domain; PHD, plant
homeodomain; Br, bromodomain; TAD, transactivation domain; SET, SET domain. Black arrows indicate protein–protein interactions between MLL and
transcriptional repressors or activators. The breakpoint region and cleavage sites are indicated.
for the 98 kDa subunit of the nuclear pore complex and has been
already identified as partner of recurrent translocations in leukaemia with several homeobox genes (e.g. HOXA9, HOXD13,
PMX1 ). NSD1 and NSD3, which interact with nuclear receptors, are members of a family of proteins with a conserved architecture: multiple PHD fingers, a PWWP domain and a SAC/SET
domain. The SAC/SET domain possesses HMTase activity
towards H3-K36 and H4-K20, thus suggesting a potential involvement of these fusion proteins in chromatin remodelling and
regulation of transcription.
MECHANISM OF REPRESSION OF AML1-FUSION
ONCOPROTEINS
CBF is a heterodimeric transcription factor composed of a
DNA-binding component, AML1 (RUNX1/CBFA2) and the
CBFb subunit. CBF heterodimer transactivates expression of
a broad spectrum of genes that are critical for normal haematopoietic development. Thus, chromosomal translocations that
resulted in loss-of-function of CBF would be predicted to
impair haematopoietic development.
The AML1 protein binds to the co-repressors Groucho/
transducin-like enhancer and Sin3 and also to CBP. Therefore,
depending on the specific target gene, AML1 is capable of functioning as both a transcriptional activator and a repressor (Fig. 4).
Molecular mechanisms of AML1 –ETO
translocation in AML
ETO, the partner of AML1 in t(8; 21), is the mammalian homologue of Drosophila nervy and is expressed in haematopoietic
progenitors (31). ETO interacts with HDACs, Sin3, N-CoR
and SMRT co-repressors (32). The AML1– ETO fusion
protein localises to AML1 target genes, where, in contrast to
the wild-type AML1 protein, it actively suppresses transcription via the co-repressors N-CoR/Sin3/HDAC1. Because
AML1 is required for differentiation of haematopoietic cells,
AML1 –ETO may be directly responsible for the block in
myeloid development and for the leukaemic transformation,
in part, by dominantly interfering with the function of the
residual normal allele, resulting in a complete loss-of-function
phenotype for CBF (33). However, murine model demonstrates that AML1 – ETO expression alone is not sufficient to
cause leukaemia, suggesting that additional event(s) may be
necessary for AML1 – ETO-associated leukaemogenesis, at
least in murine cancer model. Roeder and co-workers (34)
have recently suggested that the ability of AML1– ETO to
interfere with E protein transcriptional activation could
provide the ‘second hit’ necessary for leukaemia progression.
TEL-AML1 and childhood ALL
TEL –AML1, the most prevalent fusion gene in pediatric
cancer, acts as a transcriptional repressor in ALL (35). TEL
is a member of the ETS family transcription of factors,
which mediates the interaction with several co-repressors
including mSin3A, N-CoR and HDAC3. Recruitment of
mSin3A and/or N-CoR through the N-terminal part of the
TEL moiety possibly converts the fusion protein into an
HDAC-dependent constitutive repressor, thus contributing to
leukaemogenesis. Recent evidence suggests that TEL –
AML1 usually arises prenatally as an early or initiating
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Figure 4. Model of gene repression mediated by leukaemia associated fusion proteins. (A) MLL regulates haematopoietic cell differentiation by activating or
repressing target genes critical for normal haematopoietic development (such as Hox cluster, see text). In leukaemic cells, MLL fusion protein acts as a constitutive activator of target genes, thus preventing proper cell differentiation. (B) Haematopoietic transcription factors (HTF) regulate cell differentiation via
interaction with either co-activator (HAT) or co-repressor (HDAC). Chimeric proteins (such as PML– RARa and AML1–ETO) constitutively repress gene
transcription because of the stronger affinity for co-repressor proteins.
mutation (36) and been the ‘second hit’ in most cases deletion
of the non-rearranged TEL allele. Because recent data have
identified SUV39H1 as a co-factor for AML1 (37), a therapeutic intervention direct against HMTase should be
considered.
Role of lysine acetyltransferases in leukaemia
As discussed earlier, fusion products of MLL with CBP
t(11;16) or with p300 t(11;22) are associated with the pathogenesis of leukaemia. The involvement of alteration of HAT
activity in haematological malignancies is further supported
by the discovery of chromosomal translocations where CBP
(38) or p300 (39) is fused to monocytic leukaemia zinc
finger (MOZ). Interestingly, MOZ is also a lysine acetyltransferase enzyme. One likely mechanism by which MOZ –CBP
and MOZ –p300 contribute to malignant transformation is by
chromatin decondensation and constitutive activation of
MOZ-regulated genes. Besides p300 and CBP, other partners
are involved in the fusion with the MOZ gene including
TIF2, a member of the p160 family of nuclear receptor coactivators known to interact with p300 and CBP (40).
Finally, MOZ-related factor (MORF) is rearranged in a
manner similar to the MOZ gene. Indeed, the MORF gene
was recently found to be rearranged and fused to the CBP
gene in t(10;16) translocation (41). The resulting MORF –
CBP fusion protein is structurally similar to the MOZ – CBP/
MOZ– p300 as described earlier and may cause the development of leukaemia by mis-targeting HAT activity and/or
deregulating AML1-dependent gene expression.
EPIGENETIC TRANSCRIPTIONAL SILENCING
IN ACUTE PROMYELOCYTIC LEUKAEMIAS
Acute promyelocytic leukaemias (APLs) are phenotypically
characterized by the accumulation of clonal haematopoietic
precursors blocked at the stage of promyelocytic cells (42).
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Genetically, they are consistently associated with chromosomal translocations that involve the retinoic acid (RA) receptor
alpha (RARa) locus on chromosome 17 and one of five different partner genes (PML, PLZF, NUMA, NPM or STAT5b ). In
the absence of ligand, RAR behaves as a transcription repressor of target genes through binding to specific DNA sequences
(so-called RA responsive elements or RARE) and recruitment
of co-repressors such as the NCoR – HDAC complex. RA dissociates the NCoR –HDAC complex and leads to recruitment
of HATs and other co-activators, thus resulting in chromatin
decondensation and transcriptional activation. The fusion proteins cause a block in differentiation by interfering with the
RAR signalling pathway, which is important for cellular proliferation and differentiation of the haematopoietic myeloid
cellular compartment (43). The most extensively studied,
and the most common fusion gene in APL (.95%) is
PML – RARa, t(15;17), which involves the promyelocytic leukaemia (PML ) gene (16). When compared with RARa,
PML – RARa has increased binding efficiency to transcriptional co-repressors, in a manner similar to the AML1 –ETO
and TEL –AML1 fusions. Furthermore, at physiological concentration of RA (1029 to 1028 M), PML –RARa remains
associated with co-repressors; pharmacological doses of RA
(1026 M ) are needed to dissociate the NCoR –HDAC
complex and to recruit HAT enzymes. The stronger affinity
for co-repressors could also be responsible for the reduced
intranuclear mobility of the fusion protein when compared
with that of wild-type RAR (44).
Our group has recently demonstrated that the transcriptional
repression of PML –RARa target genes is further reinforced
by recruitment of DNMTs, leading to methylation of CpG
islands of key promoters (e.g. RARb2) (45). Once established,
the PML –RARa-induced epigenetic chromatin modifications
and the resulting gene repression are stable and maintained
throughout cell divisions. Importantly, these epigenetic modifications were found to contribute to the leukaemogenic potential of PML –RARa, prompting similar analyses also in other
leukaemias bearing oncogenic transcription factors. Although
pharmacological doses of RA dissociate the NCoR –HDAC
complex and restore terminal differentiation of AML blasts
in vitro, clinical evidence indicates that RA per se is unable
to cure this disease. Similarly, combination of RA and chemotherapeutic agents induce clinical remission, but often
patients experiencing relapse. Our hypothesis is that epigenetic modifications persist under these conditions and that
combinatorial treatment of RA with HDAC inhibitors and/or
DNA demethylating drugs might be required for removal of
epigenetic repressor markers.
Several genes have been identified as PML – RARa targets,
including members of the C/EBP family of transcription
factors (46), Hox genes (47), CL2 and TNFR2 (48) and
genes involved in stem cell maintenance and DNA repair
(49). Not all of the identified PML – RARa target genes
contain a RARE. As recently demonstrated, PML – RARa
homodimers are also able to bind to non-consensus RAREs,
thus deregulating a much wider network of target genes than
previously believed (50).
Like AML1 – ETO, expression of PML –RARa is not sufficient to induce leukaemia (51) and additional genetic events
might provide ‘second-hits’ for progression to leukaemia.
A potential ‘second-hit’ could be provided by the reciprocal
fusion protein (RARa– PML) (52). Alternatively, inhibition
of the p53 pathway (53,54), activating mutations of FLT3
receptor tyrosine kinase (55) or additional cytogenetic
abnormalities could contribute to APL pathogenesis (56).
The nature of the fusion partner has an important impact on
the disease characteristics. In fact, PLZF – RARa fusion
protein binds to co-repressors not only through the RAR
moiety, but also through the PLZF moiety. Thus, RA cannot
induce the release of the co-repressor complex bound to the
PLZF moiety. Consequently, PLZF – RARa behaves as a constitutively transcriptional repressor even in presence of
pharmacological doses of RA (43). It has been recently
demonstrated that Bmi-1 member of the Polycomb group
(PcG) of proteins repress the HOXd genes via direct interaction with PLFZ (57). Thus, similar to MLL, the HOX
genes may be the major targets of the PLZF – RARa fusion
protein. Polycomb group (PcG) proteins form multimeric
chromatin-associated protein complexes (58), which are not
only involved in repression of gene activity, but also contribute to the cellular memory system responsible for maintaining
the epigenetic status of target genes throughout cell divisions.
Therefore, the PLZF – RARa fusion protein could repress gene
activity in a hereditable manner. In fact, RA-induced degradation of the fusion protein in leukaemic blast does not
restore cell differentiation, suggesting that PLZF –RARa
may leave a permanent epigenetic mark on the leukaemic
cell and cause heterochromatinization of otherwise transcriptionally active region of the chromatin. As a consequence of
the initial repression, the spreading of inactivation into flanking regions may occur, unless boundary elements maintain and
protect active domains from this process.
PERSPECTIVES
Much of the pathological gene silencing that occurs in cancer
is a consequence of the mis-targeting of enzymes involved in
chromatin regulation (59,60). This is often accompanied by an
enforced oligomerization of the fusion proteins that may alter
the binding capacity to DNA or their affinity to co-factors (61),
usually co-repressors. Moreover, sequestration and/or mislocalization of co-repressors, such as HDACs and DNMTs,
may result in unwanted gene activation of otherwise silenced
genes. Although much has been learned, there are still several
unanswered questions: how DNA methylation and histone
modification influence each other, how transcriptional
memory is propagated through cell division, how non-coding
RNAs (including micro-RNAs) affect gene regulation and,
most importantly, how to re-set the epigenetic equilibrium in
tumour cells. Pathologically, silenced genes are uniquely susceptible to drugs that inhibit these enzymes. Indeed, experiments performed using cell lines demonstrated that HDAC
inhibitors and demethylating agents synergize in upregulation
of silenced genes (62). Unfortunately, the use of HDAC
inhibitors (alone or in combination with RA) has been somewhat disappointing (63,64) in APL clinical trials, because of
the appearance of blasts resistant to differentiation. Because
epigenetic modifications are so intricately interconnected,
novel approaches that include demethylating agent and
Human Molecular Genetics, 2005, Vol. 14, Review Issue 1
HMTase inhibitors should be considered to completely re-set
the epigenetic alterations of both undifferentiated cells and
in those rare cells that posses a stem-like neoplastic phenotype. Such combinatorial therapies may also more effectively
target the pre-malignant clone, thus reducing the likelihood of
a relapsed disease.
ACKNOWLEDGEMENTS
The work from Di Croce laboratory described in this review
was supported by grants from the Ministry of Science and
Technology (BFU2004-03862/BMC) and the Fundació ‘La
Caixa’ (04/054-00). We thank Veronica Raker and all
members of the Di Croce laboratory for stimulating
discussions.
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