From the Laboratory of Molecular Aspects of Hematopoiesis, Sloan Kettering Institute for Cancer Research, New York, New York 10021
Received for publication, January 19, 2003, and in revised form, February 7, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
H-L(3)MBT, the human homolog of the
Drosophila lethal(3)malignant brain tumor protein, is a
member of the polycomb group (PcG) of proteins, which function
as transcriptional regulators in large protein complexes. Homozygous
mutations in the l(3)mbt gene cause brain tumors in
Drosophila, identifying l(3)mbt as a tumor
suppressor gene. The h-l(3)mbt gene maps to
chromosome 20q12, within a common deleted region associated with
myeloid hematopoietic malignancies. H-L(3)MBT contains three repeats of
100 residues called MBT repeats, whose function is unknown, and a
C-terminal The human lethal(3)malignant brain tumor
(h-l(3)mbt)1 gene,
a recently cloned human homolog of the Drosophila l(3)mbt
gene, encodes a member of the polycomb group (PcG) of proteins (1). Drosophila l(3)mbt functions as a tumor suppressor gene
that, in the homozygous mutated state, leads to malignant
transformation of adult optic neuroblasts and ganglion mother cells in
the larval brain (2, 3). Because these mutant alleles have never been characterized, the biochemical role of the Drosophila
L(3)MBT protein in suppressing tumorigenesis is not known.
The h-l(3)mbt gene is located on chromosome 20q12 (1),
within the common deleted region identified in patients with
deletions of the long arm of chromosome 20. 20q abnormalities are found in hematological disorders including the myeloproliferative disorders, especially polycythemia vera where it is identified in ~10% of the
patients (4), the myelodysplastic syndromes, and in a fraction of acute
myeloid leukemias cases (5-8).
H-L(3)MBT is a member of the PcG of chromatin-associated proteins
originally defined by their ability to maintain long term repression of
the homeotic genes that govern axial patterning during
Drosophila embryogenesis (9-12). More than 15 PcG genes have been characterized to date, and subfamilies can be identified based on the presence of common protein domains. The human L(3)MBT protein contains three MBT repeats, making it a member of a subfamily that includes at least three other genes: h-l(3)mbt-like
(13), SCMH1 (14), and SCML2 (15). PcG
proteins function in large multiprotein complexes to maintain long term
repression of gene expression thereby permitting stable and heritable
transmission of gene activity. This function is crucial to maintaining
differentiated identity of cells over subsequent generations. The
mechanism by which the PcG proteins maintain repression is unknown;
however, recent evidence indicates that an important component of this repression occurs via chromatin remodeling. Another set of proteins, the trithorax group, is thought to antagonize PcG-mediated repression and maintain active gene expression. The trithorax group complex include proteins with diverse activities including histone
acetyltransferase (16) and ATP-dependent nucleosome
remodeling (SWI/SNF) activities (17).
The h-l(3)mbt gene gives rise to three major mRNA
species that encode protein isoforms that differ at their C-terminal
region and contain respectively, 772, 752, and 738 amino acids.
H-L(3)MBT protein contains three MBT repeats, which are 95-105-amino
acid motifs of unknown function. Although analysis of the MBT motif has
not shown homology with any catalytic domains or sequence-specific DNA
binding domains (13), these repeats are highly conserved between the
Drosophila and human homologs, suggesting that they have an
important function.
The SPM domain present at the H-L(3)MBT C terminus is an
Even though the PcG-SPM and the TEL-SAM domains share very little
sequence identity, the polyhomeotic SPM domain has been recently
reported to form a helical polymer structure similar to the one formed
by TEL-SAM, with both domains displaying closely related structural
architecture (33). This homology prompted us to screen several
SAM-containing ETS proteins for their ability to interact with
H-L(3)MBT.
We report that H-L(3)MBT is an HDAC-independent transcriptional
repressor, and we have characterized its in vivo and
in vitro physical association with TEL; this interaction
requires their respective SPM and SAM domains. Using both the
stromelysin-1 (matrix metalloproteinase-3) promoter, which is
physiologically regulated by TEL, and an artificial TEL-regulatable
promoter, we show that H-L(3)MBT is targeted to these promoters through
its interaction with TEL, enhancing the repressive effect of TEL on
transcription. We believe that this is the first report of a functional
interaction between a PcG protein and a mammalian DNA sequence-specific
binding transcription factor. Given the pivotal role that ETS factors (and TEL in particular) play in human malignancies, the functional interaction between H-L(3)MBT and TEL provides a clue as to the potential role of PcG proteins such as L(3)MBT in cancer.
Plasmid Construction--
To generate a full-length
h-l(3)mbt cDNA clone, total RNA from placenta (Research
Genetics) was reverse transcribed using a GeneAmp RNA PCR Core kit
(Roche Diagnostics). cDNA products were PCR-amplified with the
Expand High Fidelity PCR System (Roche Diagnostics) in 5%
dimethyl sulfoxide. using forward (5'-GAGTCTTGAGGCCTGCTGAG-3') and
reverse (5'-GGATCCTTGCAGTAAATCACAA-3') primers for the entire open reading frame of the type I h-l(3)mbt transcript (1). Full-length h-l(3)mbt cDNA was FLAG- or HA-tagged at the
5'-end using PCR methodology.
To generate an h-l(3)mbt cDNA lacking the SPM domain,
the pCR2.1-FLAG/HA-h-l(3)mbt construct was digested with
StuI and SspI to liberate a 525-bp fragment
containing the SPM domain sequences. Blunt end ligation of the larger
fragment generated an h-l(3)mbt clone with an intact reading
frame, lacking the SPM domain. To generate an h-l(3)mbt
cDNA lacking the MBT repeats, the splicing by overlap extension
(SOEing) method was used (34). The SOEing process involves
amplification of two products (P1/P2 and P3/P4) in which a tail is
added to one product (P1/P2) that is complementary to the end of the
second sequence. The two fragments are then able to anneal by virtue of
the complementary terminal tails and can self-prime with subsequent
amplification of the full-length product by flanking primers (P1 and
P4). Hence a template pCR2.1-FLAG-h-l(3)mbt construct
DNA was PCR amplified using primer P1 (5'-TCGTTCGACCCCGCCTCGAT-3') and
P2 (5'-TGAGAGGGGGACAGCCCCCAGCAGGCTGGCTGCTGCTCCA-3') to generate a P1/P2
fragment, and P3 (5'-TGGGGGCTGTCCCCCTCTCA-3', which is complementary to
the end of P2) and P4 (5'-TCCAAGCGGCTTCGGCCAGT-3') to generate a P3/P4
fragment. Equimolar amounts of P1/P2 and P3/P4 were mixed
and PCR amplified using P1 and P4. The resulting P1/P4 fragment encodes
an in-frame H-L(3)MBT polypeptide lacking 330 amino acids corresponding
to the MBT repeats. The GAL4-DBD-H-L(3)MBT construct was generated by
cloning the h-l(3)mbt cDNA in-frame into pFA vector
(Stratagene). For creation of the MBT mutant version of this GAL4
fusion construct, SOEing was applied using the P2 and P3 primers
mentioned above, with P1 (5'-TGCGACATCATCATCGGAAGAGAG-3') and P4
(5'-GGGCCTGAAATGAGCCTTGG-3') amplifying within the pFA vector.
For the GAL4-H-L(3)MBT-C2HC deletion mutant
5'-CTTCTTGCGGGCTGAGGCCCCAGGGGAGGCAGAGC-3' was used for P2, and
5'-GCCTCAGCCCGCAAGAAGA-3' was used for P3. For the SPM mutant the
pFA-h-l(3)mbt DNA was digested with BamH1 and
StuI, and the resulting fragment lacking SPM domain
sequences was subcloned into the BamHI and SmaI
sites of pFA.
Cell Culture, Transient Transfection, and Luciferase
Assay--
NIH3T3, U2OS, and 293 cells were cultured in Iscove's
modified Dulbecco's medium with 10% fetal bovine serum, 2 mM glutamine, and antibiotics and were trypsinized and
replated to the needed density the day before transfection. NIH3T3
cells were transfected with LipofectAMINE Plus reagent (Invitrogen);
293 cells were transfected using a calcium phosphate DNA precipitation
technique (ProFection, Promega, Madison, WI). Unless otherwise
specified, 1 µg of expression construct (or empty vector), 2 µg of
the reporter construct, and 0.2 µg of the cytomegalovirus-driven GFP
expression plasmid (pEGFP-C3) control plasmid (Stratagene) were used
for each transfection; the empty pcDNA3 expression vector was
added, as needed, to keep the amount of transfected DNA the same for
each sample. For the luciferase assays, cells were collected 48 h
after transfection and lysed; luciferase activity was assessed using
Luciferase Assay Substrate (Promega) on a LUMAT LB9501 (Berthold)
luminometer. Luciferase activity was normalized according to the
percent GFP-positive cells (as assessed by flow cytometry) and the
total protein concentration. To rule out general effects of the
transfected proteins on the cytomegalovirus promoter the mean
fluorescence of the GFP peaks was determined for the samples and found
to be comparable.
Immunoprecipitation Assays--
After collection, cells were
washed with PBS once, lysed by brief sonication in NET-N lysis buffer
(1% Nonidet P-40, 150 mM NaCl, 50 mM Tris pH
8, 1 mM EDTA) in the presence of proteinase inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5 mM
phenylmethylsulfonyl fluoride) and phosphatase inhibitors (10 mM NaF, 20 mM
Nuclear extracts were prepared in 0.4 M NaCl, according to
the protocol of Dignam and co-workers (35), and the immunoprecipitates from nuclear extracts were washed with a buffer containing 0.4 M NaCl.
Expression of GST Fusion Proteins in Bacteria and GST Pulldown
Assays--
The GST fusion protein plasmids were transformed into the
Escherichia coli BL21 strain (Novagen, Inc., Madison, WI).
After overnight culture, protein expression was induced with 0.1 mM isopropyl- Preparation of Anti-H-L(3)MBT Antisera and Coupling to Protein A
Gel--
Anti-H-L(3)MBT antisera were generated by immunizing rabbits
with either a 174-amino acid fragment encoding the second half of the
MBT repeats (MBT1) or a protein fragment containing the three MBT
repeats (MBT2). Each fragment was bacterially expressed as a GST fusion
protein using pGEX-4T expression plasmid (Amersham Biosciences) and
purified using glutathione-Sepharose 4B beads. The antisera were
affinity purified using
N-hydroxysuccinimide-activated Sepharose columns
(Amersham Biosciences) coupled to GST alone (first passage for negative
selection) and then GST-MBT-coupled beads (second passage for positive
selection). The affinity-purified MBT2 antibody (suitable for
immunoprecipitation) was then coupled to recombinant protein A gel,
using the Immunopure rProtein A IgG Plus Orientation kit from Pierce.
The SPM Domain in H-L(3)MBT Mediates Homotypic
Interactions--
PcG proteins form large multiprotein complexes, thus
we examined the dimerization properties of H-L(3)MBT. First, we
determined whether H-L(3)MBT protein could homodimerize using FLAG or
HA epitope-tagged H-L(3)MBT cDNAs (shown in Fig.
1a), which we transfected into
293T cells. As shown in Fig. 1b (the 6th
lane) the H-L(3)MBT protein clearly homodimerizes; to
determine whether the self-binding ability resides in the SPM domain,
epitope-tagged versions of mutant H-L(3)MBT proteins lacking either
the SPM domain or the three MBT repeats were cotransfected with the
full-length HA-H-L(3)MBT. Removal of the SPM domain is sufficient to
abrogate homodimerization of H-L(3)MBT (Fig. 1b, 10th
lane), whereas removal of the three MBT repeats had no effect on
homodimerization (Fig. 1c, 4th lane). The protein
membrane was stripped and probed for HA antibody to confirm the
presence of equal amount of the HA-tagged proteins (not shown). We
conclude that H-L(3)MBT self-associates in vivo and that
self-association requires the SPM domain.
H-L(3)MBT Has General Transcriptional Repressor Activity--
PcG
proteins maintain a chromatin-repressed state, but unlike classic
transcription factors they do not recognize specific DNA target
sequences. Thus far, the only PcG protein shown to have
sequence-specific DNA binding activity is pleiohomeotic, the
Drosophila homolog of mammalian YY1 (36). Although PcG
proteins act on chromatin, their repressive function can also be
detected in transcriptional assays using transiently transfected
reporter gene plasmids (which are believed to form less organized
chromatin-like structures) (37).
To evaluate the transcriptional regulatory activity associated with
H-L(3)MBT, we fused it to the DNA-binding domain (DBD, amino acids
1-147) of GAL4. The GAL4-H-L(3)MBT construct was tested using a
luciferase reporter plasmid that contains four copies of the GAL4 DNA
recognition sequence positioned immediately upstream of a thymidine
kinase (tk80) minimal promoter (diagrammed in Fig. 2a, upper panel).
Increasing amounts of GAL4-H-L(3)MBT were cotransfected into 293 cells
together with a constant amount of luciferase reporter plasmid; a
GAL4-TEL fusion protein was included as a positive control for
repression. The GAL4-H-L(3)MBT repressed transcription in a
dose-dependent manner (Fig. 2a), and the
magnitude of this trans-repression effect was similar to that observed
with TEL. (The correct expression of the GAL4 fused protein was
verified with an anti-GAL4 DBD antibody on the protein lysates (Western blot not shown).) Cotransfection of full-length H-L(3)MBT without the
GAL4 moiety (pcDNA3-H-L(3)MBT) did not repress the promoter activity,
excluding a general effect on transcription (Fig. 2a). Thus,
H-L(3)MBT can function as a transcriptional repressor. Notably, repression of the reporter by GAL4-H-L(3)MBT was not influenced by the
addition of a range of concentrations of trichostatin A, whereas
repression by GAL4-TEL was partially reversed by it (Fig. 2b). This suggests that transcriptional repression by
H-L(3)MBT does not depend on HDAC activity.
Repression by H-L(3)MBT Requires the MBT Repeats--
To determine
the regions of H-L(3)MBT involved in transcriptional repression, we
generated constructs fusing the GAL4-DBD to the N terminus of H-L(3)MBT
mutants in which either the three MBT repeats, the SPM domain, or the
zinc finger region was deleted. These mutants (diagrammed in Fig.
3a) were tested for expression by Western blot analysis (Fig. 3c) and were shown to
localize in the cell nucleus by immunofluorescence staining (data not
shown). Mutants lacking either the zinc finger or the SPM domain
retained the ability to repress the GAL4-reporter gene (Fig.
3b). In contrast, the mutant missing the three MBT repeats
had a minimal capacity to repress the GAL4-TK-LUC reporter (30%
repression compared with ~90% repression for the wild type protein).
Thus, the trans-repressive ability of H-L(3)MBT requires the presence
of the MBT repeats for full activity, whereas the zinc finger and SPM
domains appear to be dispensable for this function. Repression by
H-L(3)MBT- H-L(3)MBT and TEL Physically Interact through Their
SPM/SAM Domains--
The SPM domain contained in several
PcG proteins shares structural homology with the SAM domain found in a
subset of members of the ETS family of transcriptional factors
(e.g. TEL) (33, 38, 39). To determine whether H-L(3)MBT and
TEL interact in vivo we performed immunoprecipitation
assays, using a rabbit polyclonal antibody (MBT2) to precipitate
H-L(3)MBT followed by Western blotting for TEL. To avoid interference
from the heavy chain of the antibody used to pull down H-L(3)MBT, it
was necessary to cross-link covalently both the H-L(3)MBT antibody and
the normal rabbit polyclonal IgG to protein A-agarose gel (see
"Materials and Methods"). Using the osteosarcoma-derived U2OS
cells, which express readily detectable levels of both proteins, we
showed that the MBT2 anti-H-L(3)MBT antibody, but not the preimmune
antiserum, could coprecipitate H-L(3)MBT and TEL (Fig.
4). The intensity of the bands in the immunoblot suggests that more TEL protein than H-L(3)MBT protein is
present in the immunoprecipitate, however, potential differences in the
affinities of the two antisera may account for some of the differences
seen. Nonetheless H-L(3)MBT and TEL clearly interact in
vivo.
To map the regions of H-L(3)MBT and TEL involved in their interaction,
we expressed full-length FLAG-H-L(3)MBT and HA-TEL in 293T cells and
subjected the cell lysates to immunoprecipitations, using either an
anti-HA monoclonal antibody or mouse IgG as a control. HA-TEL
coprecipitates with FLAG-H-L(3)MBT (Fig.
5a, lane 1); and
using intact H-L(3)MBT or TEL, or deletion mutants lacking the MBT
repeats or the SAM/SPM domains, we show (in lane 5) that the
MBT repeats of H-L(3)MBT are not required for its interaction with TEL.
In contrast, deletion of the SAM domain of TEL or the SPM domain of
H-L(3)MBT completely abrogates heterodimerization (lanes 3,
7, and 9). Thus, these domains are involved in
the physical association of TEL and H-L(3)MBT.
Recently, TEL2 (or TELB), a new member of the ETS family of
transcription factors which is very similar to TEL, was cloned (39,
40). TEL2 is predicted to be structurally similar to TEL and has been
shown to form homodimers as well as heterodimers with TEL (41). We
tested whether H-L(3)MBT could also interact with TEL2 and found that
HA-TEL2 coimmunoprecipitates with FLAG-H-L(3)MBT when the two proteins
are coexpressed (Fig. 5b).
To further study the interaction between TEL and H-L(3)MBT, we used
GST-based in vitro protein binding assays.
35S-Radiolabeled H-L(3)MBT (and deletion-mutant proteins)
were produced by in vitro transcription/translation (IVT)
and tested for their ability to bind to a bacterially expressed GST-TEL
fusion protein. The self-association of TEL with GST-TEL was used as a
positive control. These studies determined that GST-TEL specifically
binds H-L(3)MBT but not H-L(3)MBT-
TEL and FLI-1 are known to bind to each other via their SAM domains,
but they do not interact with ETS-1 (42). We investigated whether
in vitro translated H-L(3)MBT can bind to either of these SAM domain-containing ETS proteins. As shown in Fig. 5d,
neither GST-FLI-1 nor GST-ETS-1 binds H-L(3)MBT in this assay. The
association of in vitro translated TEL with GST-FLI-1 served
as the positive control.
Cooperation of H-L(3)MBT and TEL in the Repression of
Transcriptional Targets--
To assess the functional impact of the
physical interaction between H-L(3)MBT and TEL on transcriptional
regulation, we generated a reporter gene construct whose luciferase
expression is regulated by a minimal tk80 promoter element, and three
concatemerized ETS binding sites (43). This artificial
promoter/enhancer sequence has been shown to drive high levels of
luciferase activity in all of the cell lines tested (compared with the
control tk80Luc reporter plasmid), probably reflecting the binding of
endogenous transactivating ETS proteins (44). Transfection of the
pcDNA3-H-L(3)MBT construct had no effect on the activity of the
3xEBStk80Luc reporter plasmid, whereas pcDNA3-TEL somewhat repressed
its activity. However, the combination of H-L(3)MBT and TEL
significantly repressed luciferase expression (Fig.
6a), indicating that TEL
binding its consensus binding site can recruit H-L(3)MBT to a promoter
or enhancer regulatory region. Although previous studies have shown
that the Drosophila PcG proteins can interact with the basic
transcriptional apparatus (e.g. transcription factor IID)
(45), we did not find any appreciable effect of H-L(3)MBT on the
activity of either an SV40 promoter or a herpes simplex virus TK
promoter-driven reporter construct (Fig. 6a).
It was shown recently that TEL can repress transcription of
stromelysin-1 (matrix metalloproteinase-3) gene by direct binding to
its promoter (46). To study the functional relevance of the H-L(3)MBT-TEL interaction on a natural target of TEL, we cotransfected pcDNA3-h-l(3)mbt, pcDNA3-TEL, and
pcDNA3- TEL- Koga et al. (1) and our group (6) have previously
isolated the h-l(3)mbt gene, which we showed is located
within the commonly deleted region of 20q- seen in patients with
myeloid hematologic malignancies. We now report that H-L(3)MBT is a
transcriptional repressor and has the ability to direct repression of
specific promoters by binding to the DNA sequence-specific
transcription factor TEL. We show that two protein motifs in H-L(3)MBT,
the MBT repeats and the SPM domain, which are conserved in other PcG family members, are required for its repression and protein binding activities.
The Trans-repressing Ability of H-L(3)MBT Requires Mainly the
Presence of the MBT Repeats--
Removal of the MBT repeats from
H-L(3)MBT eliminated much of its trans-repressing ability,
demonstrating the role for these repeats in establishing repression,
likely through protein-protein contacts. Future studies will determine
whether all three MBT repeats are required for H-L(3)MBT function;
however, the known MBT-containing PcG proteins possess two, three, or
four MBT repeats, suggesting that the functional unit may require a
minimum of two repeats. Little is known about the biological relevance
of the MBT repeats; however, these repeats appear to be indispensable to the function of the SCM protein in Drosophila (47). Three hypomorphic SCM alleles, which are mutated in the MBT repeats, interact
genetically with PcG mutations more strongly than SCM null alleles, and
the strongest interactions produce partial syntenic lethality (47).
These SCM mutant proteins can still associate with polytene
chromosomes, suggesting that although the MBT repeats have a critical
function in the PcG complex, they may not be essential for protein localization.
The SPM Domain of H-L(3)MBT Mediates Homotypic and Heterotypic
Interactions--
We have demonstrated homodimerization of H-L(3)MBT
and have determined that the SPM domain of H-L(3)MBT mediates both
homotypic and heterotypic interactions. Structural studies of the
TEL-SAM domain have shown this to be an oligomerization motif (41), yet
other ETS proteins that contain a SAM domain, such as FLI-1, ERG, ETS1,
ETS2, or GABP
In addition to binding H-L(3)MBT, the TEL-SAM domain is involved in
several other interactions, such as the binding of TEL to TEL2 (39) or
to FLI-1 (42). TEL is conjugated to SUMO-1 after interacting with UBC9,
and this modification requires the SAM domain and largely involves a
specific lysine residue (Lys-99) in the TEL-SAM domain (49, 50).
Finally, the TEL-SAM domain is required to target TEL to specific
subnuclear structures called TEL bodies (50).
Functional Interaction of H-L(3)MBT and TEL--
We have
demonstrated that H-L(3)MBT potentiates the repression established by
TEL on the stromelysin-1 promoter and that H-L(3)MBT increases the
efficiency of silencing of an artificial 3xETSBS enhancer by TEL.
Augmentation of TEL-mediated repression requires the direct physical
association of H-L(3)MBT and TEL through their SPM and SAM domains,
respectively. The available literature suggests that the role of the
SAM domain in TEL-mediated repression may be highly
context-dependent. For instance, TEL-mediated repression still occurs when the SAM domain is replaced by a leucine zipper dimerization motif (44), suggesting that dimerization but not corepressor binding to the SAM domain is essential. Yet TEL point mutants that fail to dimerize can retain their ability to bind mSin3A
and maintain partial trans-repressor activity (51). An explanation for
these apparently conflicting results could be that the SAM domain
constitutes an interface for numerous protein contacts, including the
H-L(3)MBT polycomb protein, which provide TEL with several mechanisms
of repression. Transcriptional repression by TEL has been shown to
involve at least two distinct regions and mechanisms of action.
Repression by the central region of TEL involves the recruitment of a
complex that includes N-CoR, SMRT, and mSin3A (which is also contacted
by the SAM domain (51)); these corepressors recruit HDAC activity to
DNA-binding proteins to actively repress transcription (52). The SAM
domain of TEL has been shown to repress gene transcription through a
different mechanism, which appears to be HDAC-independent (53).
We have shown that H-L(3)MBT functions as an HDAC-independent repressor
and interacts with the TEL-SAM domain, which supports a model in which
TEL recruits components of two separate repressor complexes through
different regions of the protein. In an analogous manner, the
retinoblastoma protein (pRB) represses cyclin E expression via an
HDAC-dependent mechanism but utilizes PcG complexes for the
HDAC-independent, long term silencing of cyclin A and cdc2 expression,
which leads to arrest in G2 (54). In fact, this G2 block is maintained even if p16 is depleted and pRB
becomes hyperphosphorylated, which suggests that PcG complexes help
mediate irreversible repression by pRB. Short term inhibition of
transcription might therefore be achieved through the recruitment of
corepressors and HDACs to the promoter. Later, long term silencing
might be established by an H-L(3)MBT-containing PcG protein complex
that can maintain repression and would make HDACs available to other target genes in the cell. Additional work aimed at identifying other
components of this complex is under way, which will help address the
respective contributions of HDAC-dependent and -independent transcriptional repression to the regulation of TEL target genes.
PcG Protein Recruitment to DNA through Canonical DNA-binding
Factors--
With the exception of YY1 (36), none of the PcG proteins
has been shown to bind DNA in a sequence-specific fashion (55). Notwithstanding, PcG complexes are found in stable association with
chromatin on polytene chromosome in Drosophila (18) and with
core promoter regions in chromatin immunoprecipitation assays (45).
Although formation of a multiprotein complex could generate a DNA
binding activity, more likely, PcG complex formation at DNA target
sites is dependent on a mosaic of interactions of different PcG
proteins with multiple DNA-binding proteins that act as recruiters (55). The physical and functional interaction of H-L(3)MBT with TEL
presents some analogy to the interaction of PcG proteins in the
Drosophila polycomb repressive complex 1 (PRC1) with the
Zeste protein (56). Zeste is a sequence-specific DNA-binding factor, which has consensus binding sites in the promoter and regulatory regions of several homeotic genes (57); it has been proposed that the
interaction of Zeste with PRC1 aids in the targeting of PcG proteins to
repressed gene loci. TEL is a sequence-specific DNA-binding protein
that is directly involved in establishing the repression of specific
target genes. By binding both H-L(3)MBT and specific promoter
sequences, TEL may bridge the PcG complex with specific regulatory elements.
Significance of H-L(3)MBT/TEL Interaction--
Silencing by PcG
proteins seems to depend on the state of transcriptional activity of
the target gene because PcG complexes generally require a silenced gene
as template (58). When the target is transcriptionally active,
silencing is not established (59); whereas once established, the
repressed state persists throughout embryonic development. Thus, the
PcG complex has been proposed to function as a memory system that can
stabilize previous regulatory events by "locking" them in. However,
this model may be too simplistic, given that the vertebrate EED·EZH2
complex possesses HDAC activity (60), which could be involved in
initiating gene repression.
We found that substituting a thymidine kinase minimal promoter with the
much stronger SV40 promoter in the 3xEBStkLuc construct led to a
loss of repression by TEL (and, as a consequence, by TEL plus
H-L(3)MBT) (data not shown). This is consistent with the principles of
gene silencing observed in Drosophila, where high doses of
trans-activators can antagonize PcG-mediated silencing (61).
The molecular mechanisms through which PcG proteins achieve and
maintain repression are largely unknown. Purification of two different
PcG protein complexes has identified proteins that could interact
functionally with PcG proteins to repress gene expression, such as
HDAC2, which is found in the EED·EZH2 complex (60) and the
corepressor molecules SMRT and Sin3A, which are found in the Drosophila PRC1 (56). Although repression mediated through
the EED·EZH2 complex is relieved by trichostatin A (a potent HDAC inhibitor), the PRC1 complex blocks chromatin remodeling by the trithorax group-related SWI·SNF complex in vitro through a
mechanism that appear to be HDAC-independent (62). Trichostatin A is
unable to relieve the repression brought about by H-L(3)MBT; this
suggests that the mechanism of repression of H-L(3)MBT may be more
similar to that of the PRC1 complex.
PcG Genes in Hematopoiesis--
Several lines of evidence support
a critical role for PcG proteins in regulating the early and late
stages of hematopoiesis. Mice lacking bmi-1 display a
progressive marrow hypoplasia similar to aplastic anemia and have a
hypoplastic spleen and thymus (63). On the contrary,
eed+/
The stage-specific expression of PcG genes in the human bone marrow
compartment suggests that the regulation of homeobox, cell cycle, and other critical regulatory genes by PcG proteins is
important in hematopoietic processes (66). H-L(3)MBT is expressed in
CD34+ blood progenitor
cells,2 and TEL is absolutely
required for the establishment of definitive hematopoiesis in the
murine bone marrow (67). The contemporaneous expression of TEL and
H-L(3)MBT in the hematopoietic stem cell compartment may signify that
their physical and functional interactions play a critical role in the
transcriptional regulation of the commitment and differentiation processes.
-helical structure, the SPM (SCM,
PH, MBT domain, which is structurally
similar to the SAM (sterile alpha
motif) protein-protein interaction domain, found in several
ETS transcription factors, including TEL (translocation Ets leukemia). We report that H-L(3)MBT is a
transcriptional repressor and that its activity is largely dependent on
the presence of a region containing the three MBT repeats. H-L(3)MBT
acts as a histone deacetylase-independent transcriptional repressor,
based on its lack of sensitivity to trichostatin A. We found that
H-L(3)MBT binds in vivo to TEL, and we have mapped the
region of interaction to their respective SPM/SAM domains. We show that
the ability of TEL to repress TEL-responsive promoters is enhanced by
the presence of H-L(3)MBT, an effect dependent on the H-L(3)MBT and the
TEL interacting domains. These experiments suggest that histone deacetylase-independent transcriptional repression by TEL depends on
the recruitment of PcG proteins. We speculate that the interaction of
TEL with H-L(3)MBT can direct a PcG complex to genes repressed by TEL,
stabilizing their repressed state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical structure of ~60 amino acids. The SPM domains in the
Drosophila Sex Comb on Midleg (SCM) and polyhomeotic (PH)
proteins have been found to mediate protein-protein interactions (18).
The predicted
-helical secondary structure and conservation of
hydrophobic residues prompt comparison of the SPM domain with the
helix-loop-helix type motifs used for homotypic and heterotypic protein
interactions in other transcriptional regulators. The SPM domain
belongs to the extended family of SAM (sterile
alpha motif) domains (also known as HLH or
pointed domains) which are found in several regulatory proteins
including kinases, adaptor proteins, and transcription factors (19,
20). These more distantly related proteins include several members of
the ETS family of transcription factors (for review, see Ref.
21), such as TEL. The tel (translocation
Ets leukemia) gene was identified and located
at the chromosomal breakpoints of several leukemia-associated
translocations (22-25). The fusion proteins derived from these
translocations contain the N-terminal portion of TEL, including the SAM
domain, fused to a variety of tyrosine kinase domains, such as the
platelet-derived growth factor receptor (PDGFR)
, ABL, Janus kinase
(JAK) 2, and NTRK3, or to transcription factors such as AML1
(22, 26, 27). Dimerization of the TEL-SAM domain results in the
constitutive activation of the TEL-ABL (25), TEL-PDGFR (28), and
TEL-JAK2 (29) tyrosine kinase activity and is essential for
transcriptional repression by TEL-AML1 (30-32).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate), and then
clarified by centrifugation. The protein concentration was adjusted to
1 mg/ml, and the lysates were incubated with 2 µg of specific
antibodies for 1 h at 4 °C. Protein A+G- agarose beads (Santa
Cruz) were added to the extracts and mixed for at least 1 h at
4 °C. The immune complexes were washed four times with NET-N lysis
buffer, once with high salt buffer (1 M NaCl, 20 mM Tris-HCl, pH 8), and once with low salt buffer (10 mM MgCl2, 50 mM HEPES-KOH, pH 7.5);
they were released by adding SDS sample buffer and boiled for 4 min.
Samples were subjected to SDS-PAGE with 7.5% gel, transferred to a
polyvinylidene difluoride membrane, and Western blotted with the
following antibodies: FLAG-horseradish peroxidase-conjugated (M2,
Sigma), HA mouse monoclonal antibody (12CA5, Roche Molecular
Biochemicals), GAL4-DBD (RK5C1, Santa Cruz), anti-TEL (gift of P. Marynen), and anti-H-L(3)MBT (MBT1) generated in our laboratory (see
below). In each experiment, the membranes were stripped and reprobed
with the appropriate antibody to assess the correct expression of the
cotransfected expression plasmids.
-D-thiogalactopyranoside for
2-4 h. The bacterial pellets were then lysed in 1 ml of PBS-T buffer
(140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM KH2
PO4, pH 7.3, 1% Triton X-100), with the aid of sonication.
5 µg of GST-TEL fusion protein (or GST) was incubated with 20 µl of
50% slurry glutathione-Sepharose 4B beads (Amersham Biosciences) in a
total volume of 1 ml of PBS-T for 45 min at 4 °C. The beads were
washed three times with 1 ml PBS-T and then incubated with 2 µl of
in vitro transcribed and translated 35S-labeled
protein in 1 ml of NET-N buffer for 2-4 h at 4 °C. The beads were
then washed with 1 ml of NET-N six times. The bound proteins were
released from the beads by boiling in SDS-gel loading buffer for 4 min.
Proteins were analyzed by SDS-PAGE with 7.5% gel and autoradiography.
The TNT-coupled reticulocyte lysate system (Promega)
was used to generate in vitro translated proteins, following the procedures specified by the manufacturer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
H-L(3)MBT homodimerizes through the SPM
domain. a, schematic representation of type I H-L(3)MBT
(772 amino acids) protein and the H-L(3)MBT- -MBT and
H-L(3)MBT-
-SPM deletion mutants. b and c,
H-L(3)MBT and its mutant expression vectors were coexpressed in 293T
cells (as indicated). Protein-protein interactions were detected by
coimmunoprecipitation (IP) from cell lysates using an
anti-HA monoclonal antibody (12CA5) and Western blotting
(WB) with FLAG-horseradish peroxidase-conjugated monoclonal
antibody (M2). Input protein lysates are shown in the left
parts of the panels. The right parts of the
panels show the coimmunoprecipitated proteins.
View larger version (20K):
[in a new window]
Fig. 2.
L(3)MBT has transcriptional repressor
activity. Repression of the reporter 4xGAL-TK-LUC by
GAL4-H-L(3)MBT fusion protein was assayed in 293 cells. a,
the expression constructs and the reporter gene plasmid are diagrammed
in the top panel. Cells were collected 48 h after
transfection and lysed; luciferase activity was normalized to the
percent GFP-positive cells as assessed by flow cytometry and to the
total protein concentration and is expressed as RLU (relative
luciferase units). b, trichostatin A was added to the cells
24 h after the transfection at the indicated final concentrations,
of the GAL4-L(3)MBT, GAL4-TEL, or empty vector plasmids. The reporter
activity in the presence of GAL4-L(3)MBT and GAL4-TEL is plotted as a
percentage of the activity of the empty vector in cells treated with
the same concentration of trichostatin A. Values shown are the
average ± S.E. of three independent experiments.
-SPM suggests that self-dimerization is not necessary for
trans-repression; however, a role for protein dimerization cannot be
completely excluded because the GAL4-DBD itself can direct the
oligomerization of GAL4 and GAL4-derived proteins.
View larger version (21K):
[in a new window]
Fig. 3.
Mapping the region required for H-L(3)MBT
repressor activity. Repression by GAL4-H-L(3)MBT fusion proteins
on the 4xGAL-TK-LUC reporter was assayed using transient transfection
assays in 293 cells. The expression constructs and the reporter plasmid
are diagrammed in a; promoter activity is shown in
b. Relative luciferase units (RLU) were calculated as the
luciferase activity of the sample divided by the luciferase activity of
the GAL4 empty vector. Values shown are the average ± S.E. of
three experiments. c, protein lysates were assayed for the
expression of the fusion protein using an anti-GAL4-DBD monoclonal
antibody and Western blot (WB) analysis.
View larger version (46K):
[in a new window]
Fig. 4.
In vivo interaction of the
endogenous H-L(3)MBT and TEL proteins. Affinity-purified
anti-H-L(3)MBT rabbit polyclonal antibody (MBT2 Ab) or normal rabbit
IgG covalently coupled to protein A-agarose matrix was used to
immunoprecipitate H-L(3)MBT from 1 mg of U2OS cell nuclear extract. The
immunoprecipitated protein complexes were run on SDS-PAGE (7.5% gel)
and subjected to Western blot (WB) analysis. Inputs in the
first lanes of both panels represent 20 µg of
nuclear extract. Either the anti-H-L(3)MBT rabbit polyclonal antibody
(MBT1) (left panel) or the anti-TEL rabbit antiserum
(right panel) was used for immunodetection.
View larger version (23K):
[in a new window]
Fig. 5.
H-L(3)MBT interacts with TEL and TEL2 but not
with ETS1 or FLI-1. This interaction requires the SAM/SPM
domain(s). a, the H-L(3)MBT and the TEL constructs were
expressed in 293T cells in the indicated combinations
(A-E). Protein lysates were subjected to
immunoprecipitation (IP) using either a mouse anti-HA
monoclonal antibody or mouse normal IgG as a control (for nonspecific
pulldown). The anti-FLAG-horseradish peroxidase-conjugated antibody
(M2) was used for Western blot (WB) analysis. Western blot
of input proteins is shown on the right side of the
panel (lanes 11-15) for H-L(3)MBT and on the
top right panel for TEL. b, FLAG-H-L(3)MBT and
HA-TEL2 cDNAs were expressed alone or in combination. Protein
lysates were subjected to immunoprecipitation using a mouse anti-HA
monoclonal antibody. Anti-FLAG-horseradish peroxidase conjugated
antibody (M2) was used for Western blot analysis. Western blot of input
protein is shown in the right three lanes of the
panel. c, results of GST-TEL pulldown assays. The
in vitro translated proteins are indicated on the
top of each group of lanes. I, input;
G, GST pulldown control; T, GST-TEL pulldown.
d, results of GST-ETS-1 (lanes 1 and
5) and GST-FLI-1 (lanes 2 and 6)
pulldown assays. In vitro translated TEL and H-L(3)MBT were
used for lanes 1-4 and 5-8, respectively.
[35S]Methionine-radiolabeled in vitro
translated proteins were incubated with 5 µg of bacterially produced
GST or GST fusion proteins immobilized on GSH-Sepharose. After
incubation and washing, the bound proteins were separated by SDS-PAGE
(7.5% gel), fixed, and stained with Coomassie to show the presence and
purity of the purified GST fusion proteins. Input represents 5% of the
total protein used in the pulldown assay.
-SPM (Fig. 5c).
The interaction of TEL with
H-L(3)MBT-
-MBT and with H-L(3)MBT-
-C2HC appears to be much less
efficient than its interaction with the intact H-L(3)MBT. This is shown
in Table I, where the interactions between GST-TEL and the IVT
proteins are assigned a +, +/
, or a
based on the relative
amounts of input IVT protein detected after the pulldown.
The interaction of H-L(3)MBT with TEL requires the SPM domain
View larger version (20K):
[in a new window]
Fig. 6.
Coexpression of H-L(3)MBT potentiates the
repression by TEL of the stromelysin-1 and 3xETS BS-TK promoter
activities. a, NIH3T3 cells were cotransfected with the
pcDNA3 constructs indicated at the bottom of the
graph and either the SV40 luciferase or the three ETS
binding sites-tk80Luc construct (diagrammed above the
graphs). Cells were collected at 48 h after
transfection; luciferase activity was normalized to the percentage of
GFP-positive cells as assessed by flow cytometry and the total protein
concentration. The -fold repression was calculated as the -fold
decrease in luciferase activity compared with the empty vector. Values
shown are the average ± S.E. of two experiments performed in
triplicate. b and c, NIH3T3 cells were
cotransfected with the pcDNA3 constructs indicated on the
bottom of the graphs and the diagrammed rat
stromelysin-1 promoter sequence (1-754 bp) luciferase construct, which
contains at least two TEL binding sites (TBS), (shown at the
top). Cells were collected 48 h after transfection;
luciferase activity was normalized to a percentage of GFP-positive
cells as assessed by flow cytometry and total protein concentration.
The -fold repression was calculated as the -fold decrease in luciferase
activity compared with the empty vector. Values shown are the
average ± S.E. of two experiments performed in triplicate.
-SAM alone, or in combination, with a rat
stromelysin-1 promoter-regulated luciferase reporter gene plasmid
(shown schematically in Fig. 6b). This promoter contains 754 bp of 5'-flanking sequence and at least two TEL binding sites (46);
overexpression of TEL represses the stromelysin-1 promoter 4-5-fold,
whereas the TEL-
-SAM mutant has no effect on promoter activity (Fig.
6b). H-L(3)MBT, itself, was able to repress transcription
from the stromelysin-1 promoter 3-fold (Fig. 6b), and
coexpression of TEL with H-L(3)MBT caused further repression of the
stromelysin-1 reporter (8.5-fold), suggesting an additive rather than
synergistic activity on this promoter (Fig. 6b). No additive
repression was seen when TEL-
-SAM was coexpressed with H-L(3)MBT
(Fig. 6b). Similarly, when H-L(3)MBT mutants lacking either
the SPM domain or the MBT repeat region were coexpressed with TEL, they
had no effect on repression (Fig. 6c). Thus, the combined
effects of TEL and H-L(3)MBT on the stromelysin-1 promoter require the
presence of their respective SAM and SPM domains, and the MBT repeats
(the region required for repression by the GAL4-H-L(3)MBT fusion
protein (see Fig. 3b)).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, lack the ability to form polymers. ETS1 is monomeric
even at high concentrations (48), and in our in vitro
experiments, we were unable to coimmunoprecipitate H-L(3)MBT with
either ETS-1 or FLI-1. Rather, we found that TEL and TEL2 interact with
H-L(3)MBT through their
-helical SAM/SPM domains. Further testing
would define whether these proteins are unique among ETS proteins in
their ability to interact with H-L(3)MBT. Kim et al. (41)
defined a subset of ETS-SAM domains which maintain hydrophobic residues
at the interacting surface, and their model predicts that TEL2, and the
Drosophila YAN protein, would in fact oligomerize similarly
to TEL.
mice have a propensity to develop both
myeloproliferative and lymphoproliferative diseases later in life (64).
Mice lacking mel-18 have a severe combined immunodeficiency,
and mel-18
/
lymphocyte precursors respond
poorly to interleukin-7 stimulation (65). PcG genes regulate Hox gene
expression, and there are numerous examples where dysregulated Hox gene
expression profoundly perturbs hematopoiesis or leads to acute myeloid
leukemia, underscoring the importance of maintaining the timely and
tight down-regulation of homeobox gene expression.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. P. Marynen for providing the TEL antibody, Dr. G. Nucifora for the TEL cDNA, Dr. J. Liu for the GAL4 vector and the GAL4-BS-TK-LUC construct, Dr. G. Grosveld for the HA-TEL2 construct, Dr. L. Matrisian for the rat stromelysin-1 promoter construct, and Magdalena Wodnar-Filipowicz for technical assistance in generating deletion mutants.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health RO1 grant DK52208, the Sunshine Lady Foundation & the Gabrielle Rich Leukemia Research Foundation.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: Memorial Sloan Kettering Cancer Center, 1275 York Ave., Box 575, New York, NY 10021. E-mail: nimers@mskcc.org.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M300592200
2 D. MacGrogan, N. Kalakonda, P. Boccuni, S. Alvarez, and S. D. Nimer, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: l(3)mbt and L(3)MBT, lethal(3)malignant brain tumor gene and protein, respectively; DBD, DNA binding domain; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; HDAC, histone deacetylase; IVT, in vitro transcription/translation; LUC/Luc, luciferase; PBS, phosphate-buffered saline; PcG, polycomb group; PRC, polycomb repressive complex; SAM, sterile alpha motif; SOEing, splicing by overlap extension; TEL, translocation Ets leukemia; TK/tk, thymidine kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Koga, H., Matsui, S., Hirota, T., Takebayashi, S., Okumura, K., and Saya, H. (1999) Oncogene 18, 3799-3809[CrossRef][Medline] [Order article via Infotrieve] |
2. | Gateff, E., Loffler, T., and Wismar, J. (1993) Mech. Dev. 41, 15-31[CrossRef][Medline] [Order article via Infotrieve] |
3. | Wismar, J., Loffler, T., Habtemichael, N., Vef, O., Geissen, M., Zirwes, R., Altmeyer, W., Sass, H., and Gateff, E. (1995) Mech. Dev. 53, 141-154[CrossRef][Medline] [Order article via Infotrieve] |
4. | Bench, A. J., Cross, N. C., Huntly, B. J., Nacheva, E. P., and Green, A. R. (2001) Best Pract. Res. Clin. Haematol. 14, 531-551[CrossRef][Medline] [Order article via Infotrieve] |
5. | Kurtin, P. J., Dewald, G. W., Shields, D. J., and Hanson, C. A. (1996) Am. J. Clin. Pathol. 106, 680-688[Medline] [Order article via Infotrieve] |
6. | MacGrogan, D., Alvarez, S., DeBlasio, T., Jhanwar, S. C., and Nimer, S. D. (2001) Oncogene 20, 4150-4160[CrossRef][Medline] [Order article via Infotrieve] |
7. | Bench, A. J., Nacheva, E. P., Hood, T. L., Holden, J. L., French, L., Swanton, S., Champion, K. M., Li, J., Whittaker, P., Stavrides, G., Hunt, A. R., Huntly, B. J., Campbell, L. J., Bentley, D. R., Deloukas, P., and Green, A. R. (2000) Oncogene 19, 3902-3913[CrossRef][Medline] [Order article via Infotrieve] |
8. | Wang, P. W., Eisenbart, J. D., Espinosa, R., III, Davis, E. M., Larson, R. A., and Le Beau, M. M. (2000) Genomics 67, 28-39[CrossRef][Medline] [Order article via Infotrieve] |
9. | Pirrotta, V. (1997) Trends Genet. 13, 314-318[CrossRef][Medline] [Order article via Infotrieve] |
10. | Simon, J. (1995) Curr. Opin. Cell Biol. 7, 376-385[CrossRef][Medline] [Order article via Infotrieve] |
11. | Pirrotta, V., and Rastelli, L. (1994) Bioessays 16, 549-556[Medline] [Order article via Infotrieve] |
12. | Pirrotta, V. (1997) Curr. Opin. Genet. Dev. 7, 249-258[CrossRef][Medline] [Order article via Infotrieve] |
13. | Wismar, J. (2001) FEBS Lett. 507, 119-121[CrossRef][Medline] [Order article via Infotrieve] |
14. | Berger, J., Kurahashi, H., Takihara, Y., Shimada, K., Brock, H. W., and Randazzo, F. (1999) Gene (Amst.) 237, 185-191[CrossRef][Medline] [Order article via Infotrieve] |
15. | Montini, E., Buchner, G., Spalluto, C., Andolfi, G., Caruso, A., den Dunnen, J. T., Trump, D., Rocchi, M., Ballabio, A., and Franco, B. (1999) Genomics 58, 65-72[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Bantignies, F.,
Goodman, R. H.,
and Smolik, S. M.
(2000)
Mol. Cell. Biol.
20,
9317-9330 |
17. | Tamkun, J. W., Deuring, R., Scott, M. P., Kissinger, M., Pattatucci, A. M., Kaufman, T. C., and Kennison, J. A. (1992) Cell 68, 561-572[Medline] [Order article via Infotrieve] |
18. | Peterson, A. J., Kyba, M., Bornemann, D., Morgan, K., Brock, H. W., and Simon, J. (1997) Mol. Cell. Biol. 17, 6683-6692[Abstract] |
19. |
Ponting, C. P.
(1995)
Protein Sci.
4,
1928-1930 |
20. |
Schultz, J.,
Ponting, C. P.,
Hofmann, K.,
and Bork, P.
(1997)
Protein Sci.
6,
249-253 |
21. | Sharrocks, A. D. (2001) Nat. Rev. Mol. Cell. Biol. 2, 827-837[CrossRef][Medline] [Order article via Infotrieve] |
22. | Golub, T. R., Barker, G. F., Lovett, M., and Gilliland, D. G. (1994) Cell 77, 307-316[Medline] [Order article via Infotrieve] |
23. | Buijs, A., Sherr, S., van Baal, S., van Bezouw, S., van der Plas, D., Geurts van Kessel, A., Riegman, P., Lekanne Deprez, R., Zwarthoff, E., Hagemeijer, A., and Grosveld, G. (1995) Oncogene 10, 1511-1519[Medline] [Order article via Infotrieve] |
24. |
Wlodarska, I.,
Mecucci, C.,
Marynen, P.,
Guo, C.,
Franckx, D.,
La Starza, R.,
Aventin, A.,
Bosly, A.,
Martelli, M. F.,
Cassiman, J. J.,
and Van Den Berghe, A.
(1995)
Blood
85,
2848-2852 |
25. | Golub, T. R., Goga, A., Barker, G. F., Afar, D. E., McLaughlin, J., Bohlander, S. K., Rowley, J. D., Witte, O. N., and Gilliland, D. G. (1996) Mol. Cell. Biol. 16, 4107-4116[Abstract] |
26. | Knezevich, S. R., Garnett, M. J., Pysher, T. J., Beckwith, J. B., Grundy, P. E., and Sorensen, P. H. (1998) Cancer Res. 58, 5046-5048[Abstract] |
27. | Golub, T. R., Barker, G. F., Stegmaier, K., and Gilliland, D. G. (1997) Curr. Top. Microbiol. Immunol. 220, 67-79[Medline] [Order article via Infotrieve] |
28. |
Jousset, C.,
Carron, C.,
Boureux, A.,
Quang, C. T.,
Oury, C.,
Dusanter-Fourt, I.,
Charon, M.,
Levin, J.,
Bernard, O.,
and Ghysdael, J.
(1997)
EMBO J.
16,
69-82 |
29. |
Lacronique, V.,
Boureux, A.,
Valle, V. D.,
Poirel, H.,
Quang, C. T.,
Mauchauffe, M.,
Berthou, C.,
Lessard, M.,
Berger, R.,
Ghysdael, J.,
and Bernard, O. A.
(1997)
Science
278,
1309-1312 |
30. | Hiebert, S. W., Sun, W., Davis, J. N., Golub, T., Shurtleff, S., Buijs, A., Downing, J. R., Grosveld, G., Roussell, M. F., Gilliland, D. G., Lenny, N., and Meyers, S. (1996) Mol. Cell. Biol. 16, 1349-1355[Abstract] |
31. |
Fears, S.,
Gavin, M.,
Zhang, D. E.,
Hetherington, C.,
Ben-David, Y.,
Rowley, J. D.,
and Nucifora, G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1949-1954 |
32. | Uchida, H., Downing, J. R., Miyazaki, Y., Frank, R., Zhang, J., and Nimer, S. D. (1999) Oncogene 18, 1015-1022[CrossRef][Medline] [Order article via Infotrieve] |
33. | Kim, C. A., Gingery, M., Pilpa, R. M., and Bowie, J. U. (2002) Nat. Struct. Biol. 9, 453-457[Medline] [Order article via Infotrieve] |
34. | Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 61-68[CrossRef][Medline] [Order article via Infotrieve] |
35. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
36. | Brown, J. L., Mucci, D., Whiteley, M., Dirksen, M. L., and Kassis, J. A. (1998) Mol. Cell 1, 1057-1064[Medline] [Order article via Infotrieve] |
37. | Bunker, C. A., and Kingston, R. E. (1994) Mol. Cell. Biol. 14, 1721-1732[Abstract] |
38. |
Bornemann, D.,
Miller, E.,
and Simon, J.
(1996)
Development
122,
1621-1630 |
39. |
Potter, M. D.,
Buijs, A.,
Kreider, B.,
van Rompaey, L.,
and Grosveld, G. C.
(2000)
Blood
95,
3341-3348 |
40. |
Gu, X.,
Shin, B. H.,
Akbarali, Y.,
Weiss, A.,
Boltax, J.,
Oettgen, P.,
and Libermann, T. A.
(2001)
J. Biol. Chem.
276,
9421-9436 |
41. |
Kim, C. A.,
Phillips, M. L.,
Kim, W.,
Gingery, M.,
Tran, H. H.,
Robinson, M. A.,
Faham, S.,
and Bowie, J. U.
(2001)
EMBO J.
20,
4173-4182 |
42. |
Kwiatkowski, B. A.,
Bastian, L. S.,
Bauer, T. R., Jr.,
Tsai, S.,
Zielinska-Kwiatkowska, A. G.,
and Hickstein, D. D.
(1998)
J. Biol. Chem.
273,
17525-17530 |
43. | Janknecht, R., Ernst, W. H., Pingoud, V., and Nordheim, A. (1993) EMBO J. 12, 5097-5104[Abstract] |
44. |
Lopez, R. G.,
Carron, C.,
Oury, C.,
Gardellin, P.,
Bernard, O.,
and Ghysdael, J.
(1999)
J. Biol. Chem.
274,
30132-30138 |
45. | Breiling, A., Turner, B. M., Bianchi, M. E., and Orlando, V. (2001) Nature 412, 651-655[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Fenrick, R.,
Wang, L.,
Nip, J.,
Amann, J. M.,
Rooney, R. J.,
Walker-Daniels, J.,
Crawford, H. C.,
Hulboy, D. L.,
Kinch, M. S.,
Matrisian, L. M.,
and Hiebert, S. W.
(2000)
Mol. Cell. Biol.
20,
5828-5839 |
47. |
Bornemann, D.,
Miller, E.,
and Simon, J.
(1998)
Genetics
150,
675-686 |
48. |
Slupsky, C. M.,
Gentile, L. N.,
Donaldson, L. W.,
Mackereth, C. D.,
Seidel, J. J.,
Graves, B. J.,
and McIntosh, L. P.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12129-12134 |
49. |
Chakrabarti, S. R.,
Sood, R.,
Ganguly, S.,
Bohlander, S.,
Shen, Z.,
and Nucifora, G.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7467-7472 |
50. |
Chakrabarti, S. R.,
Sood, R.,
Nandi, S.,
and Nucifora, G.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
13281-13285 |
51. |
Fenrick, R.,
Amann, J. M.,
Lutterbach, B.,
Wang, L.,
Westendorf, J. J.,
Downing, J. R.,
and Hiebert, S. W.
(1999)
Mol. Cell. Biol.
19,
6566-6574 |
52. | Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J., Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., Seto, E., Eisenman, R. N., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1997) Nature 387, 43-48[CrossRef][Medline] [Order article via Infotrieve] |
53. | Chakrabarti, S. R., and Nucifora, G. (1999) Biochem. Biophys. Res. Commun. 264, 871-877[CrossRef][Medline] [Order article via Infotrieve] |
54. | Dahiya, A., Wong, S., Gonzalo, S., Gavin, M., and Dean, D. C. (2001) Mol. Cell 8, 557-569[Medline] [Order article via Infotrieve] |
55. | Pirrotta, V. (1998) Cell 93, 333-336[Medline] [Order article via Infotrieve] |
56. | Saurin, A. J., Shao, Z., Erdjument-Bromage, H., Tempst, P., and Kingston, R. E. (2001) Nature 412, 655-660[CrossRef][Medline] [Order article via Infotrieve] |
57. | Benson, M., and Pirrotta, V. (1988) EMBO J. 7, 3907-3915[Abstract] |
58. | Poux, S., Kostic, C., and Pirrotta, V. (1996) EMBO J. 15, 4713-4722[Abstract] |
59. |
Poux, S.,
McCabe, D.,
and Pirrotta, V.
(2001)
Development
128,
75-85 |
60. | van der Vlag, J., and Otte, A. P. (1999) Nat. Genet. 23, 474-478[CrossRef][Medline] [Order article via Infotrieve] |
61. | Zink, D., and Paro, R. (1995) EMBO J. 14, 5660-5671[Abstract] |
62. | Shao, Z., Raible, F., Mollaaghababa, R., Guyon, J. R., Wu, C. T., Bender, W., and Kingston, R. E. (1999) Cell 98, 37-46[Medline] [Order article via Infotrieve] |
63. | van der Lugt, N. M., Domen, J., Linders, K., van Roon, M., Robanus-Maandag, E., te Riele, H., van der Valk, M., Deschamps, J., Sofroniew, M., van Lohuizen, M., and Berns, A. (1994) Genes Dev. 8, 757-769[Abstract] |
64. |
Lessard, J.,
Schumacher, A.,
Thorsteinsdottir, U.,
van Lohuizen, M.,
Magnuson, T.,
and Sauvageau, G.
(1999)
Genes Dev.
13,
2691-2703 |
65. | Akasaka, T., Tsuji, K., Kawahira, H., Kanno, M., Harigaya, K., Hu, L., Ebihara, Y., Nakahata, T., Tetsu, O., Taniguchi, M., and Koseki, H. (1997) Immunity 7, 135-146[Medline] [Order article via Infotrieve] |
66. |
Lessard, J.,
Baban, S.,
and Sauvageau, G.
(1998)
Blood
91,
1216-1224 |
67. |
Wang, L. C.,
Swat, W.,
Fujiwara, Y.,
Davidson, L.,
Visvader, J.,
Kuo, F.,
Alt, F. W.,
Gilliland, D. G.,
Golub, T. R.,
and Orkin, S. H.
(1998)
Genes Dev.
12,
2392-2402 |