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NEOPLASIA
From the Department of Pathology and the Division of
Pediatric Hematology/Oncology, Stanford University School of Medicine,
Stanford, CA; the Department of Biochemistry, St Louis
University School of Medicine, St Louis, MO; and the
Department of Pathology and Laboratory Medicine, University of
Pennsylvania School of Medicine, Philadelphia, PA.
The t(11;19)(q23;p13.1) chromosomal translocation in acute myeloid
leukemias fuses the gene encoding transcriptional elongation factor ELL
to the MLL gene with consequent expression of an MLL-ELL chimeric protein. To identify potential mechanisms of leukemogenesis by
MLL-ELL, its transcriptional and oncogenic properties were investigated. Fusion with MLL preserves the transcriptional elongation activity of ELL but relocalizes it from a diffuse nuclear distribution to the nuclear bodies characteristic of MLL. Using a serial replating assay, it was demonstrated that the MLL-ELL chimeric protein is capable
of immortalizing clonogenic myeloid progenitors in vitro after its
retroviral transduction into primary murine hematopoietic cells.
However, a structure-function analysis indicates that the elongation
domain is not essential for myeloid transformation because mutants
lacking elongation activity retain a potent ability to immortalize
myeloid progenitors. Rather, the highly conserved carboxyl terminal R4
domain is both a necessary and a sufficient contribution from ELL for
the immortalizing activity associated with MLL-ELL. The R4 domain
demonstrates potent transcriptional activation properties and is
required for transactivation of a HoxA7 promoter by MLL-ELL
in a transient transcriptional assay. These data indicate that
neoplastic transformation by the MLL-ELL fusion protein is likely to
result from aberrant transcriptional activation of MLL
target genes. Thus, in spite of the extensive diversity of MLL fusion
partners, these data, in conjunction with previous studies of MLL-ENL,
suggest that conversion of MLL to a constitutive transcriptional
activator may be a general model for its oncogenic conversion in
myeloid leukemias.
(Blood. 2000;96:3887-3893) The development of more effective therapies for de
novo and therapy-related acute myeloid leukemia (AML) represents a
major challenge for oncology. Insights into the molecular pathogenesis of AML have come from the study of acquired chromosomal rearrangements specific to the leukemic clone. In particular, reciprocal
translocations fusing the MLL (ALL-1, HRX,
Htrx) gene on chromosome 11q23 with the ELL
(MEN) gene at chromosome band 19p13.1 are found in a large proportion of AML arising in cancer patients previously treated with
etoposide-based chemotherapy.1-3 The t(11;19)(q23;p13.1) translocation has also been identified in association with myeloid leukemias in infants and de novo AML and myelodysplastic syndromes in
children and adults, suggesting that this rearrangement is not limited
to chemotherapy-related mutagenesis.4,5 The consistent retention of a der(11) chromosome that encodes a translatable MLL fusion mRNA in the leukemic clone6,7 implies
a transforming function for the chimeric protein encoded by the
5'-MLL-ELL-3' transcript. However, the mechanism of
transformation by MLL-ELL remains unclear.
ELL is one of more than 30 genes fused to the MLL
gene in leukemia-associated translocations (reviewed in DiMartino and
Cleary8). MLL normally functions as an upstream regulator
of Hox gene expression9 and contains amino
terminal DNA-binding domains that are retained by the chimeric proteins
(Figure 1). Studies of leukemogenesis by
MLL-AF9 in vivo and MLL-ENL in vitro provide compelling evidence that
the amino terminal portion of MLL gains a transforming function from
its partner proteins.10-12 No obvious structural features shared among the broader group of fusion partners, however, have emerged to explain their function in transformation. The lack of
functional information about these proteins has made it difficult to
generate testable hypotheses regarding their contributions to
transformation as MLL fusion partners.
ELL is one of the few MLL fusion partners for which a substantial
amount is known about its biochemical role. This 620-amino acid protein
has been shown to facilitate transcriptional elongation by suppressing
transient pausing of RNA polymerase II along the DNA
template.13 In addition, the amino terminal 50 amino acids of ELL mediate negative regulation of promoter-specific transcription initiation by RNA polymerase II.14 ELL2, another member of
the ELL family of proteins, was identified by its homology with ELL. These 2 proteins share extensive similarity in their transcriptional elongation domains (R2) and their lysine-rich (R3) and
carboxyl-terminal (R4) domains (Figure 1A).15 The presence
of an RNA polymerase II inhibition (R1) domain, however, is specific to
ELL. Although highly conserved, the functions of the R3 and R4 domains
are unknown.
The well-characterized biochemical functions and structural features of
ELL provide an excellent opportunity to define its functional
contributions to transformation by MLL-ELL. Leukemia-associated translocations at 11q23 join the R2-R4 domains of ELL to the putative DNA-binding domains of MLL (Figure 1B). It is unclear whether the loss
of the RNA polymerase II inhibiting R1 motif contributes to the
oncogenic properties of the chimeric protein. Moreover, it is unknown
whether the elongation-promoting activity of ELL is retained after its
fusion to MLL. In addition to the unknown effect(s) that fusion to MLL
would have on the functional properties of ELL, it is unclear how its
subcellular distribution would be affected. Fusion of several partner
proteins to MLL results in their colocalization with
MLL.16-18 Thus, 11q23 translocations involving ELL may
create a state of haplo-insufficiency by sequestering ELL away from its
normal location. To address these questions, we have assayed the
transcriptional elongation activity of MLL-ELL fusion proteins,
characterized their subnuclear localization, and defined the domains of
ELL that are required for the immortalization of primary murine myeloid
progenitors. We report that a novel transcriptional transactivation
domain at the carboxyl terminus of ELL, rather than its elongation
function, is essential for transformation by MLL-ELL.
Plasmid constructs
Transcriptional elongation assays
Immunofluorescence staining Mouse embryonic fibroblasts were transfected with 500 ng of either FLAG-MLL, ELL, MLL-ELL, MLL-ELL 150-200, or
MLL-ELL374-620 expression vectors in 4-well chamber
slides using Lipofectamine (Life Technologies, Rockville, MD).
Forty-eight hours later, cells were fixed with cold 4%
paraformaldehyde in phosphate-buffered saline (PBS) and were
permeabilized with 0.2% Triton X-100/PBS. Slides were blocked with 2%
BSA/PBS and stained with the M2 FLAG monoclonal antibody (10 µg/mL)
(Sigma, St Louis, MO) or a monoclonal antibody directed against ELL
(1:400) in PBS/2% horse serum, followed by staining with goat
antimouse immunoglobulin G conjugated to Alexa 488 in PBS/2% horse
serum (1:400; Molecular Probes, Eugene, OR). Slides were washed 3 times
with PBS/0.05% Triton X100 after each incubation period and were
finally washed with 3 changes of PBS before they were cover-slipped.
Slides were photographed with an Olympus (Tokyo, Japan) BX40 microscope
equipped for immunofluorescence (original magnification,
1000 ×).
Myeloid immortalization assays Transduction of murine bone marrow (BM) cells was performed essentially as described in Lavau et al.11 Briefly, 4-week-old C57B/6 mice were injected with 5-FU (150 mg/kg) by tail vein, and bone marrow was harvested from both femurs after 5 days. Transient retroviral supernatants were produced by transfecting NX
cells with MSCV vectors as described by Pear et al.21 Bone
marrow cells were infected with recombinant virus by centrifugation at 2500×g for 2 hours at 32°C. Transduced cells were plated
in 0.9% methylcellulose (Stem Cell Technologies, Vancouver, BC,
Canada) supplemented with 20 ng/mL stem cell factor (SCF) and 10 ng/mL each of interleukin-3 (IL-3), IL-6, and granulocyte
macrophage-colony-stimulating factor (GM-CSF) (R&D Systems,
Minneapolis, MN) in the presence or absence of G418 (1 mg/mL). After 7 to 10 days, colonies were counted and retroviral transduction
efficiency was calculated for each construct as the ratio of
G418-resistant (G418R) colonies to unselected
colonies. Single-cell suspensions were made from pooled
G418R colonies, and 104 cells were plated in
secondary methylcellulose cultures without G418.
Cell surface phenotype analysis Flow cytometry was performed by staining cells with phycoerythrin and fluorescein isothiocyanate isotype controls and with monoclonal antibody (mAb) for Gr-1, Mac-1, c-Kit, and other markers (Pharmingen, San Diego, CA). Stained cells were analyzed on a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA).Western blotting A confluent 100-mm dish ofNX cells transiently transfected
with each construct was lysed in 400 µL 1× sample buffer.
Approximately 20µL lysate was loaded in each lane and electrophoresed
through a 6% sodium dodecyl sulfate-polyacrylamide gel. Proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) using
CAPS transfer buffer as described in Slany et al.12 After blocking with 5% milk, membranes were probed with monoclonal antibody N6.3 directed against an MLL amino terminal epitope between residues 161 and 356.
Transcriptional activation assays Four micrograms pGL3-HoxA7 (kind gift of Dr R. Slany) was cotransfected with 4 µg MSCVneo, MLL5', MLL-ELL, MLL-ELL374-620, or MLL-ELLC2 and 2 µg
pCMVsport gal (Gibco BRL, Grand Island, NY) into 293T cells by
calcium-phosphate precipitation. After 48 hours, lysates were assayed
for luciferase and -galactosidase activity using commercially
prepared reagents (Promega; Tropix, Bedford, MA) and a
Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Relative light units (RLU) from duplicate luciferase assays were corrected for transfection efficiency using RLU from their
respective -galactosidase controls.
MLL-ELL stimulates transcriptional elongation by RNA polymerase II To determine whether MLL-ELL retained the transcriptional elongation activity of ELL, the chimeric protein was evaluated for its ability to increase the catalytic rate of transcription elongation from the AdML promoter in the presence of general initiation factors. Analysis of the kinetics of accumulation of full-length runoff transcripts revealed that MLL-ELL stimulated the rate of transcription elongation of promoter-specific transcription by RNA polymerase II as effectively as native ELL (Figure 2). We previously demonstrated that the elongation stimulation domain of both ELL and ELL2 resides in their respective R2 domains and that deletion of amino acids 150 to 200 resulted in the loss of transcriptional elongation stimulatory activity of these proteins.14,15 Similarly, deletion of these amino acid residues from MLL-ELL significantly compromised its ability to stimulate the rate of transcriptional elongation (Figure 2). In contrast, the removal of C-terminal ELL amino acids 374 to 620 had no effect on elongation by either native ELL or its MLL chimera.
Subnuclear localization of ELL is altered by its fusion to MLL Because MLL-ELL retained an ability to stimulate transcriptional elongation, we determined whether this activity was present in the same nuclear compartment as that of native ELL. Mouse embryo fibroblasts were transfected with constructs expressing MLL-ELL fusion proteins or the respective wild-type proteins. Immunofluorescent staining with an ELL-specific mAb showed that ELL exhibited a diffuse nuclear distribution (Figure 3B). In contrast, MLL displayed a punctate and diffuse localization pattern as described16 (Figure 3A). Analysis of cells expressing either MLL-ELL or its mutants with an anti-ELL mAb showed a punctate and diffuse localization pattern identical to that of MLL (Figure 3C-E). This indicated that fusion of ELL with MLL resulted in a relocalization of ELL from its normal diffuse nuclear distribution to the nuclear bodies characteristic of MLL localization.
MLL-ELL immortalizes primitive myeloid progenitors To study the oncogenic properties of MLL-ELL, the MSCV retroviral vector22 was used to transduce BM cells whose growth properties were then evaluated in a serial replating assay (Figure 4A). Previously, we showed that retroviral transduction of primary BM cells with an MLL-ENL fusion cDNA under these conditions enhanced the self-renewal of a clonogenic myeloid progenitor that induced AML in transplanted mice.11,12 Using similar methodology, MLL-ELL-transduced BM cells were plated in methylcellulose cultures supplemented with SCF, IL-3, IL-6, and GM-CSF. Because the MSCV vector contained a pgkNeo cassette, G418 was also added to first-round cultures to select for transduced cells. In the first round of plating, numerous colonies were observed for cells transduced with empty MSCV vector (not shown) or MLL5' cDNA (Figure 4B) reflecting the normal clonogenic potential of progenitors harvested from 5-FU-treated BM. Moreover, mock-transduced BM plated in the absence of G418 gave similar numbers of colonies as retrovirally transduced BM, suggesting that retroviral infection per se was not overtly toxic to these cells. Fewer Neo-resistant colonies were observed in first-round cultures of MLL-ELL-transduced BM (Figure 4B), consistent with the lower retroviral transduction efficiency obtained with this construct compared to MLL5'. As expected, the colonies were primarily of GM morphology, and their appearance did not vary appreciably between MLL-ELL- and MLL5'-transduced cultures (not shown). First-round colonies were then harvested, and 104 cells were plated in secondary methylcellulose cultures. In contrast to MLL5'-transduced cells that had exhausted their self-renewal potential and produced only a few macrophage colonies, MLL-ELL-transduced cells produced hundreds of colonies with primitive GM morphology in second-round cultures. This effect was sustained through subsequent rounds of plating (Figure 4B and data not shown).
The ability to produce GM colonies in methylcellulose cultures is
a property of primitive myeloid progenitors.
May-Grünwald-Giemsa-stained cytospin preparations of these cells
showed a range of morphologies. Myeloblastic cells with large nuclei
and no obvious granules were seen along with cells showing morphologic
evidence of monocytic and granulocytic differentiation (Figure
5). Consistent with the degree of
heterogeneity seen in stained cytospins, analysis of surface marker
expression by flow cytometry revealed a range of myeloid
differentiation (Figure 5). Less than 10% of cells expressed low
levels of Mac-1, whereas 27% expressed substantial levels of c-Kit, a
marker associated with primitive myeloid cells. Twenty-two percent of
cells also expressed Gr-1, demonstrating a degree of granulocytic
differentiation. Expression of erythroid (Ter119) or lymphoid (B220,
Thy1.2) differentiation antigens was not observed (not shown). Thus,
MLL-ELL-transduced cells were immortalized at a primitive
stage of myeloid differentiation, consistent with their high clonogenic
potential. Similar to MLL-ENL-transformed cells, however,
MLL-ELL-transformed cells exhibited a range of myeloid differentiation
characteristics.11 This suggested that primitive myeloid
cells transduced with either of these MLL fusion proteins retained a
limited capacity for myeloid differentiation in vitro.
ELL elongation activity is dispensable, but its C-terminal domain is necessary and sufficient for immortalization of primitive myeloid progenitors To identify domains in ELL that were required for the observed effects of MLL-ELL on the clonogenic potential of myeloid cells, a series of mutants was tested for immortalizing potential. Consistent production of third-round colonies, regardless of number, was considered to be indicative of immortalization activity. Construct MLL-ELLR1 contained all of ELL, including the RNA polymerase II inhibition domain (R1) that is not included in translocation-generated MLL-ELL chimeras. The presence of R1 in MLL-ELL did not inhibit its ability to enhance the clonogenic potential of primary 5-FU-treated BM cells (Figure 6A). This observation correlated with our finding that the R1 domain no longer inhibited RNA polymerase II initiation as part of a chimeric MLL-ELLR1 protein (A. Shilatifard, unpublished observation). Deletion of ELL amino acids 150 to 200 (construct MLL-ELL150-200), which removed most of
the R2 domain and significantly diminished the transcriptional
elongation activity of both native ELL and MLL-ELL (Figure 2), had only
a modest effect on the number of third-round colonies produced (Figure
6A). In contrast, deletion of amino acids 374 to 620 at the
C-terminus of ELL (MLL- ELL374-620), which removed
both the lysine-rich R3 domain and the highly conserved R4 domain,
completely abrogated immortalization. Conversely, a construct
containing only the R3 and R4 domains of ELL fused to MLL
(MLL-ELLC1) resulted in the production of substantial
numbers of third-round colonies. A fusion construct in which the
lysine-rich region (R3) of ELL was removed, leaving only the R4 domain
fused to MLL (MLL-ELLC2), showed similarly efficient
immortalizing properties. Taken together, these data showed that the R4
domain was both necessary and sufficient for the immortalization of
primary murine myeloid progenitors.
The inability of MLL5' and MLL-ELL MLL-ELLC2 immortalizes cells in an IL-3-dependent manner To determine whether MLL-ELLC2-transduced cells were capable of sustained growth in suspension cultures, colonies from third-round platings were placed in liquid media supplemented with various combinations of cytokines. Cells initially grew in RPMI 1640 media with 10% fetal calf serum (FCS) and recombinant SCF, IL-3, and IL-6 with a doubling time of 24 to 48 hours (not shown). Subsequently, MLL-ELLC2-transduced cells were passaged in RPMI 1640 with 20% FCS and 20% WEHI-conditioned media (WCM) as a source of IL-3. These cells have been maintained in continuous culture for more than 3 months without any change in their doubling time. Seven to 10 days after the removal of WCM, MLL-ELLC2-transduced cells cease proliferating. These data suggest that, similar to MLL-ENL-transformed myeloid progenitors, cells immortalized by MLL-ELLC2 require exogenous IL-3 for sustained viability or growth in liquid culture. Unlike the former, however, the injection of 106 MLL-ELLC2-immortalized cells into SCID mice has not yet produced myeloid leukemias 90 days after injection.ELL R4 domain confers transcriptional activation activity to MLL-ELL The foregoing structure-function analysis identified the portion of ELL that was required for the immortalizing activity of MLL-ELL; however, the biochemical function of the R4 domain remained unknown. Our previous studies of MLL-ENL showed that the portion of ENL required for MLL-ENL-mediated transformation exhibited transcriptional activation properties when fused to a GAL4 DNA-binding domain.12 Subsequently, MLL-ENL itself was shown to be capable of activating transcription of a luciferase reporter gene under the control of HoxA7 upstream sequences (pGL3-HoxA7) in a transient transcriptional assay.23 To determine whether the R4 domain of ELL may confer similar properties on MLL-ELL, the transcriptional properties of various MLL-ELL proteins were evaluated in 293T cells cotransfected with the pGL3-HoxA7 reporter gene. Under these conditions, MLL-ELL displayed significant transactivation potential, inducing luciferase expression 10- to 15-fold above the levels observed for MLL5' or vector alone (Figure 7). This induction by MLL-ELL was not observed when a luciferase reporter gene under the control of the E1B promoter and GAL4 UAS was used (not shown), indicating that HoxA7 upstream sequences were required for MLL-ELL-mediated activation. The MLL-ELL374-620 mutant protein lacking the 247 C-terminal
residues of ELL was substantially compromised in its activation
potential, which was reduced to levels near background. In
contrast, MLL-ELLC2 containing the R4 domain of ELL alone
fused to MLL retained transcriptional activation potential equal to or
greater than the full-length fusion protein. The R4 domain of ELL was,
therefore, both necessary and sufficient for transcriptional
activation, and for immortalization of primary murine myeloid
progenitors, when fused with 5' MLL.
The studies in this report evaluated the functional properties of MLL-ELL to establish the oncogenic contributions of ELL, a known transcriptional elongator. Transcriptional elongation assays using MLL-ELL showed that ELL retains its elongation function in the context of an MLL chimera. However, fusion of ELL to MLL alters its distribution within the nucleus, localizing it to the distinct nuclear bodies in which MLL is also localized. The latter is consistent with previous observations18 showing that MLL fusion partners, including those whose normal localization is cytoplasmic, are relocalized within the nucleus after fusion with MLL. Taken together, these findings appear to support a mechanism by which aberrant elongation activity at MLL target loci by MLL-ELL accounts for deregulated gene expression in myeloid leukemias. Based on our data, however, it is highly unlikely that this mechanism is responsible for the transforming function of MLL-ELL. Rather, our data provide evidence for a mechanism in which MLL-ELL must function as a chimeric transcriptional activator as opposed to an elongator to transform myeloid progenitors. As the second MLL fusion protein to demonstrate such activity, our data suggest that constitutive activation may be a general model for the oncogenic role of MLL chimeric proteins. ELL has been shown in previous studies to be multifunctional, having both transcriptional elongation activity and inhibitory effects on transcriptional initiation by RNA polymerase II. The data in the current report demonstrate that the domains of ELL that confer these transcriptional activities are not necessary for the immortalization of primary murine myeloid progenitors by MLL-ELL. Rather, a new functional domain of ELL was defined by our studies. The carboxy-terminal R4 domain, which is highly conserved within the ELL family of proteins, was found to have potent transcriptional activation properties in transient transcriptional assays. The R4 domain does not display similarity with other known transcriptional activation domains, but its molecular role is likely to reflect an ability to interact with one or more components of the cellular machinery that regulates activated transcription. The fact that this activity did not read out in previous in vitro studies of ELL raises the possibility that it was masked by the inhibitory role of the amino-terminal R1 domain of ELL on RNA polymerase II initiation, which is not included in translocation-generated MLL-ELL fusion proteins. Alternatively, necessary cofactors may not have been present in the extracts used for previous in vitro studies, or the activation properties of ELL may be promoter-specific. Identification of the ELL R4 domain as critical for immortalization is likely to be highly relevant to leukemogenesis induced by MLL-ELL. Generation of colonies in the third round of plating in our cultures reflects the ability of MLL-ELL to enhance the self-renewal of primitive myeloid progenitors. Such immortalizing activity may parallel the first steps of leukemogenesis in cells having undergone an 11q23 rearrangement. Indeed, the primitive morphology and surface phenotype of MLL-ELL-immortalized cells resemble those of leukemic blasts in human AML, further substantiating the link between immortalization in our in vitro assay and de novo leukemic transformation. Moreover, others have shown that primary murine BM cells transduced with MLL-ELL induce AML after approximately 150 days in transplanted mice.24 The failure of MLL-ELLC2 immortalized cells to produce AML in SCID mice after 90 days may be due to an insufficient latency period. These animals will continue to be monitored for the development of myeloid leukemia. Our studies do not exclude the possibility that transcriptional
elongation by MLL-ELL, though not essential, may contribute to myeloid
transformation. We observed an approximately 50% decrease in the
numbers of third-round colonies between full-length MLL-ELL and
MLL-ELL We examined MLL-ELL for transcriptional activating function to address the general applicability of observations made with MLL-ENL, whose oncogenic contributions were shown previously to be intimately associated with its transcriptional activation function. The transactivation potential of ENL, when fused to a heterologous DNA-binding domain, and its contribution to the transformation activity of MLL-ENL co-localize to conserved motifs in the C-terminus of ENL.25,12 Recently, MLL-ENL has been shown to activate transcription in a more physiologic context with the HoxA7 promoter used for our current studies.23 MLL-ELL now also proves to have significant transactivation activity on this promoter. Interestingly, despite the lack of sequence homology between ENL and ELL, both proteins are predicted to exhibit helical secondary structure at their carboxyl termini based on PHDsec computer modeling26 (not shown). These findings support the hypothesis that fusion of MLL to at least a subset of partner proteins results in the creation of hybrid transcription factors. MLL is thought to function as an epigenetic regulator of transcription rather than as a classical transcriptional activator. It does not activate expression of its target genes but ensures that they remain in an activated state through cell division. The enhancer-like activity displayed in our transient transfection assays, therefore, reflects a true gain of function by MLL. Perhaps more important, the replacement of PHD finger and SET domains of MLL by a constitutive transcriptional activation domain may preclude its ability to down-regulate target genes in response to differentiation signals. Thus, both gain and loss of MLL function may lead to aberrant expression of target genes, resulting in leukemogenic transformation.
We thank C. Lavau for assistance in establishing the myeloid immortalization assay in our laboratory and Y. Jacobs and B.-T. Rouse for expert assistance in producing MLL monoclonal antibodies. We also thank M. Seto and R. Slany for providing the TK-882 and pGL3-HoxA7 constructs, respectively.
Submitted April 4, 2000; accepted July 26, 2000.
Supported in part by grants from the National Cancer Institute (CA55029 and 5T32CA09151), the National Institutes of Health (RO1CA78815-01), and the American Cancer Society (RP69921801). A.S. is an Edward Mallinckrodt Young Investigator.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jorge F. DiMartino, Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, L-216, Stanford, CA 94305; e-mail: jorged{at}leland.stanford.edu.
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  S. Lindsey, W. Huang, H. Wang, E. Horvath, C. Zhu, and E. A. Eklund Activation of SHP2 Protein-tyrosine Phosphatase Increases HoxA10-induced Repression of the Genes Encoding gp91PHOX and p67PHOX J. Biol. Chem., January 26, 2007; 282(4): 2237 - 2249. [Abstract] [Full Text] [PDF]
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 M. A. Gerber, A. Shilatifard, and J. C. Eissenberg Mutational Analysis of an RNA Polymerase II Elongation Factor in Drosophila melanogaster Mol. Cell. Biol., September 1, 2005; 25(17): 7803 - 7811. [Abstract] [Full Text] [PDF]
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 S. Hayette, P. Cornillet-Lefebvre, I. Tigaud, S. Struski, S. Forissier, A. Berchet, D. Doll, L. Gillot, W. Brahim, E. Delabesse, et al. AF4p12, a Human Homologue to the furry Gene of Drosophila, as a Novel MLL Fusion Partner Ca |
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Abstract
Introduction
-32P]-CTP. These short
promoter-specific transcripts were then elongated to full-length runoff
transcripts in the presence of MLL-ELL chimeras and an excess of
nonradioactive CTP. Transcripts were analyzed by electrophoresis
through a 6% polyacrylamide, 7.0 mol/L urea gel and visualized using a
Molecular Dynamics PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
150-200), 29% (