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AF4 Encodes a Ubiquitous Protein That in Both Native and MLL-AF4 Fusion
Types Localizes to Subnuclear Compartments
By Quanzhi Li, Joy L. Frestedt, and John H. Kersey
Acute leukemia with t(4;11)(q21,q23) translocation results
from the in-frame fusion of the MLL to the AF4/FEL gene.
In previous studies, we and others demonstrated that
AF4 transcripts are present in a variety of hematopoietic
and nonhematopoietic human cells. To further study the
wild-type and leukemia fusion AF4, we used glutathione
S-transferase (GST)-fusion proteins as immunogens to produce rabbit polyclonal antibodies that were specific for
normal and chimeric AF4 proteins. Using Western blotting
analysis, we demonstrated that the AF4 gene encodes
proteins with apparent molecular weight of 125 and 145 kD.
A 45-kD protein coprecipitated with AF4 protein in immunoprecipitation. Also, the anticipated MLL-AF4–encoded 240-kD
protein was detected in all cell lines with t(4;11) transloca-
tions; fusion proteins were present in lesser quantity than
the wild-type AF4. The proteins recognized by the antibodies
are of the predicted sizes of the AF4 and MLL-AF4–encoded
proteins based on previous DNA sequencing analysis. The
MLL-AF4 fusion protein had a similar subcellular distribution
as AF4. Both t(4;11) and non-t(4;11) leukemic cells showed a
similar pattern of punctate nuclear staining in all cell lines
tested using confocal immunofluorescence microscopy. AF4
antibodies should be useful for further elucidation of the
function of AF4 in normal cellular physiology, as well as the
function of MLL-AF4 in leukemogenesis. The antibodies
should also be helpful for the diagnosis of the MLL-AF4
fusion proteins in t(4;11) leukemias.
r 1998 by The American Society of Hematology.
A
specific for N-terminal epitopes of MLL protein has also been
reported. This antibody detected MLL-AF4 fusion protein in
Western blotting.14 Recently, Nilson et al15 reported rabbit
antibodies against human AF4 protein; an unexpected 116-kD
protein was detected in cell lysates by western blottings with no
detection of the MLL-AF4 fusion proteins. The major purpose
of our study was to identify and characterize the proteins
encoded by AF4 and MLL-AF4 genes by developing antibodies
specific for AF4 proteins using molecular and immunological
approaches.
F4, ALSO KNOWN AS FEL, is a gene which was first
described as a fusion partner with MLL in the t(4;11)
acute leukemia.1-3 Based on analysis by Northern blotting, AF-4
mRNAs were found to be widely expressed in hematopoietic
cells and normal human tissues.4,5 cDNA sequence analysis of
the AF-4 gene showed that it encodes a serine/proline-rich
protein with a predicted size of 130 to 140 kD containing
guanosine triphosphate (GTP)-binding and putative nuclearlocalization sequences (NLS).2 Studies have demonstrated that
a related gene, LAF4, isolated from Burkitt’s lymphoma, shares
high degree of sequence homology with AF4. LAF4 was shown
to possess DNA binding ability and transcriptional activation
potential.6 Both AF4 and LAF4 were shown to be evolutionary
conserved in vertebrates suggesting an important functional role
of the genes. Recently, a third gene (FMR2) has been recognized as a member of AF4/LAF4 gene family. FMR2 maps to X
chromosome at position Xq28. Mutations of FMR2 are associated with mild hereditary mental retardation.7,8 Members of the
homologous AF4/LAF4/FMR2 gene family are expected to
have transcriptional activation functions. We previously found
that another DNA sequence located on chromosome 5q31,
probably is a member of this gene family.9
Clinical studies have demonstrated that MLL (also known as
HRX and ALL-1) rearrangements in infant ALL are associated
with very poor prognosis.4 At the molecular level, important
sequence characteristics and putative functional motifs of MLL
and MLL-AF4 fusion genes have been defined. Molecular
analysis of the MLL gene indicates that it contains two central
zinc-fingers, a C-terminal region with homology to Drosophila
trithorax gene, three N-terminal A/T hook regions, a domain
with homology to methyltransferase, and a proline-rich region.3,10-12 The t(4;11) translocation separates the second and
third DNA binding domain of MLL from the zinc finger
region.10 The AT-hook and proline-rich regions of MLL are
fused in-frame to the region in AF-4, which contains the NLS
and GTP-binding activity, resulting in a chimeric mRNA of 12.5
kb that encodes a predicted fusion protein of 240 kD.2,4 Rabbit
polyclonal antibodies specific to N-terminal fragments of
MLL-encoded proteins expressed in Escherichia coli have been
recently reported.13 The antibodies recognized the MLL and
MLL/AF-4–encoded proteins in a leukemic cell line and cells
transfected with portions of MLL. A monoclonal antibody
Blood, Vol 92, No 10 (November 15), 1998: pp 3841-3847
MATERIALS AND METHODS
Cell lines and construction of expression vectors. The RS4;11 cell
line was established in our laboratory16 and is available from ATCC
(American Type Culture Collection, Rockville, MD). Two t(4;11) cell
lines, B117 and AN4;114 have been described. Another t(4;11) line,
Sem-k2, was a gift from Dr F.E. Cotter (Department of Haematology
and Oncology, Institute of Child Health, London, UK). Other cell lines
have been described and used in our laboratory for a number of years
including Nalm-6, KM3, BLIN-1 (B-cell lines), Raji (B-cell lymphoma), Molt-4, CEM (T-cell lines), M418 (human neuroblastoma),
MG-63 (human osteosarcoma), and K562 (myeloid cell line).4 The cells
were grown and maintained in RPMI 1640 tissue culture medium
supplemented with 10% fetal bovine serum (FBS) and penicillin/
streptomycin. A 3.3-kb cDNA designated PL12 that contained most of
AF4 gene open reading frame was isolated from a human placenta
cDNA library.9 Two regions of the PL12 clone, one from the 58 end
(base pair 612-1110, designated C9) and another from the 38 end (base
From the University of Minnesota Cancer Center and Departments of
Pediatrics and Laboratory Medicine/Pathology, University of Minnesota, Minneapolis, MN.
Submitted March 23, 1998; accepted July 10, 1998.
Supported in part by an Outstanding Investigator Grant Award No.
R35 CA 49721 from the National Cancer Institute (to J.H.K.).
Address reprint requests to John H. Kersey, MD, University of
Minnesota Cancer Center, Box 86 MAYO, 420 Delaware St, SE,
Minneapolis, MN 55455.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/92/9210-0002$3.00/0
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3842
pair 1942-2242, designated C15) of the RS4;11 breakpoint and other
breakpoints (Fig 1) were amplified by polymerase chain reaction (PCR)
using standard protocols. The C9 and C15 clones encode polypeptides
of 18 kD and 10 kD, respectively. The selection of these regions from
AF4 gene for construction of expression vectors was based on the
‘‘plotstructure analysis’’ using the GCG computer program (Genetics
Computer Group, Madison, WI). Both regions showed high surface
probability, low hydrophobicity, and high antigenic index, suggesting a
strong potential for stimulating immune responses. PCR primers were
selected using the oligos 4s computer software (National Bioscience,
Plymouth, MN) and engineered to include EcoR1 and BamH1 restriction sites for direct in-frame insertion into the GST expression vector
(Pharmacia, Piscataway, NJ). The PCR products were gel purified and
ligated into TA cloning vectors (Invitrogen, San Diego, CA). The inserts
were sequenced from both ends into the vector sequences to assure
proper orientation. The GST vectors with C9 and C15 inserts were
expected to encode a 44- and a 36-kD protein, respectively (GST, 26
kD; inserts, 18 kD and 10 kD).
Expression and purification of GST-fusion proteins. GST expression vectors with or without the inserts were inserted into DH5 alpha
bacteria (GIBCO/BRL, Grand Island, NY) using standard bacterial
transformation procedures. The transformed colonies were selected and
grown in ampicillin-containing (100 µg/mL) LB medium at 37°C
overnight with vigorous shaking. GST-fusion proteins were induced
with 0.1 mmol/L IPTG (Isopropyl-B-D-thiogalactopyranoside, Sigma
Chemical Co, St Louis, MO) for 3 to 4 hours. The bacteria were
harvested by centrifugation, washed and resuspended in PBST-100, pH
7.4 (phosphate-buffered saline, 1% Triton-X100, 1 mmol/L EDTA) and
sonicated (Branson Ultrasonic Corp, Brandury, CT) to release the
proteins. The presence of the fusion proteins in the cell lysates were
determined by analyzing the samples in 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). GST and GST-fusion
proteins were affinity purified by passing the bacterial lysates through a
glutathione sepharose 4B (Pharmacia) column and washed thoroughly
with PBST-100. The bound proteins were eluted with 100 mmol/L NaCl
solution containing 25 mmol/L reduced glutathione (Sigma). The
proteins were concentrated to 2 to 3 mg/mL using a Centricon-10
protein concentrator (Amicon, Bedford, MA) and buffer exchanged to
100 mmol/L HEPES buffer using a PD-10 desalting column (Pharmacia). The protein concentration was determined by a protein assay
reagent (Pierce, Rockford, IL). After affinity purification, GST-fusion
proteins were further purified by preparative electrophoresis. The
protein samples were separated in 10% SDS-PAGE gels and surfacestained with Commassie blue staining solution for 2 to 3 minutes. The
bands of interest were cut from the gels and eluted into SDS-PAGE
electrode buffer using an electro-elution device. SDS contained in the
protein solution was removed by acetone precipitation method of Hager
and Burgess.18
Production and purification of antibodies. Two hundred microliters
(1 mg/mL) of the 44-kD or 36-kD GST-fusion protein obtained from
preparative electrophoresis were emusified with an equal volume of
complete Freund’s adjuvant and injected into two rabbits at multiple
sites. Booster injections with the decreasing amounts of antigen
LI, FRESTEDT, AND KERSEY
emusified with incomplete Freund’s adjuvant were performed at a
2-week interval over a period of 84 days. The immune responses of the
animals were monitored by indirect enzyme-linked immunosorbent
assay (ELISA)19 and Western blottings. The rabbits were killed and the
sera collected when the ELISA titers of the immune sera against the
immunizing antigen reached 1:5,000 or greater. Our initial attempts to
immunize the rabbits with glutathione sepharose 4B purified GSTfusion proteins in denatured or native forms failed to generate useful
antibodies. This initial problem led us to further purify the proteins by
preparative electrophoresis to obtain a higher proportion of full-length
fusion protein for immunizations. Rabbits immunized with these highly
purified and denatured antigens stimulated strong antibody responses as
determined by immunoblotting and ELISA using native and denatured
proteins as coating antigens. Specific antibodies in the sera were
purified by affinity chromatography. Briefly, 2 mg of purified GST,
GST-C9, or GST-C15 fusion proteins were conjugated to 1 mL
Affigel-10 (Bio-Rad, Hercules, CA) following manufacturer’s recommendations. Protein-conjugated Affigel-10 was packed into a minicolumn and equilibrated with PBS. The antiserum diluted fourfold with
PBST (PBS containing 0.1% Tween-20) was passed through the column
containing Affigel-10 conjugated GST to remove antibodies against
GST. The eluent was then passed several times through the column
containing conjugated GST-C9 or GST-C15 fusion protein, washed
extensively with PBST, and eluted with 50 mmol/L glycine buffer (pH
2.8). The eluted antibody was neutralized by 1.89 mol/L Tris buffer (pH
8.9) in the collection tubes. Purified antibodies were designated
anti-C–9 and anti-C15 and stored at 4°C in a solution containing 0.5%
BSA, 0.4 mol/L L-arginine, and 0.02% sodium azide. IgG fractions of
preimmune sera were purified by a protein A column (Pierce).
Western blotting. Cultured cell lines in log phase were harvested by
low speed centrifugation (1,500g), washed twice with PBS, and
subjected to sonication at maximum intensity for 10 seconds. The cell
lysates were solubilized in SDS-PAGE sample buffer, heated in boiling
water for 2 minutes, and separated on SDS-PAGE gels under reducingconditions. After electrophoresis, the proteins in the gels were electrophoretically transferred onto hydrated nitrocellulose membranes for 2
hours. The membranes were blocked with 5% nonfat milk in PBS for 2
hours at 37°C or at 4°C overnight. The membranes were probed with
diluted primary antibodies in blocking buffer at room temperature for 1
hour. For detection of AF4 and MLL-AF4–encoded proteins, affinity
purified anti-C15 and anti-C9 antibodies (50 to 100 µg/mL) were diluted
1:100 to 1:200 and used in Western blotting. Mouse antihuman b
tubulin monoclonal antibody (Sigma) used as an internal control in
some experiments was diluted according to supplier’s recommendations. After incubating with primary antibodies, the blots were washed
either under high or low stringent conditions. Unless specified, all
Western blottings were performed under high stringent washing conditions. Under high stringent conditions, the blots were washed three
times (15 to 20 minutes/each) with PBS containing 0.05% Tween-20.
Low stringent wash consisted of three washes (5 to 8 minutes/each)
with PBS containing 0.01% Tween-20. After washing, the blots were
incubated with goat antirabbit or goat antimouse IgG conjugated to
horseradish peroxidase (HRP, Amersham, Alington, IL) for 45 to 60
Fig 1. Schematic representation of predicted AF4
gene structure. The relative location and size of C9,
C15, and PL12 as compared with AF4 gene are shown
as dark lines; alternate splicing sites, breakpoints of
t(4;11) translocations, nuclear localization sequences
(NSL), and GTP binding motifs of AF4 are indicated.
The figure was constructed based on the data from
Morrissey et al,2 Frestedt et al,9 Tkachuk et al,11 and
Hilden et al.22
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AF4 ENCODES A UBIQUITOUS NUCLEAR PROTEIN
minutes at room temperature. The blots were washed as before and
incubated with enhanced chemiluminescence (ECL) reagents (Amersham) for 1 minute. The blots were blotted dry with filter papers,
enclosed in transparent plastic sheets, and exposed to autoradiographic
film. Other antibodies used in this study were rabbit anti-LAF4 (A gift
from Dr Louis M. Staudt, Metabolism Branch, National Cancer
Institute, National Institutes of Health, Bethesda, MD) and monoclonal
anti-MLL antibody (a gift from Dr Lisa H. Butler, John Radcliffe
Hospital, Oxford, UK).
Blocking experiment. The specificity of purified anti-C15 and
anti-C9 were accessed by blocking with their corresponding immunizing antigens in Western blotting format. Total cell lysate of Km3 was
separated in 8% SDS-PAGE (20 to 40 µg/lane) and electrophoretically
transferred to nitrocellulose membrane, cut into strips, and incubated
with different preparations of primary antibodies. Affinity-purified stock
solutions (50 to 100 µg/mL) of anti-C15 and anti-C9 were diluted 1:100
with PBST containing 5% nonfat milk and 80 µg of corresponding
immunizing antigens in a final volume of 10 mL. Preimmune IgG and
the same amount of antibodies blocked with GST (80 µg/sample) were
used as controls. After 30 minutes incubation at room temperature, the
nitrocellulose strips were incubated with primary antibodies and the rest
of the Western blotting procedures were performed as above.
Immunoprecipitation and pulse chase analysis. A total of 1 3 107
cells were harvested by low speed centrifugation and washed twice with
cold Hank’s balanced salt solution. The cells were resuspended in 5 mL
RPMI medium containing 5% dialized FBS (Gibco) and 0.2 mCi 35S
(ICN, Costa Mesa, CA) and incubated at 37°C with gentle shaking for 3
hours. The labeled cells were washed twice with cold Hank’s balanced
salt solution and lysed with 600 µL of lysis buffer (PBS containing 1%
Triton X-100, 0.5% SDS, 0.5% deoxycholate, 1% BSA, leupeptin [10
µg/mL] and 0.2 mmol/L phenylmethyl sulfonyl fluoride [PMSF],
Sigma). The cell lysate was incubated in ice for 10 minutes and
centrifuged in a microcentrifuge at top speed for 10 minutes to remove
insoluble materials. Three hundred microliter cell lysate was precleared
by incubating with 1 µg rabbit IgG and 30 µL protein G plus/protein
A-agarose (CALBIOCHEM, Cambridge, MA) for 1 hour at room
temperature. The supernatant was incubated with 0.5 to 1 µg anti-C15
and 30 µL protein A/G agarose at 4°C for 3 hours. After incubation, the
agarose beads were washed three times with lysis buffer. The immunoprecipitates were solubilized in 40 µL SDS-PAGE sample buffer,
separated by 10% SDS-PAGE, and visualized by autoradiography.
Pulse chase labeling was performed based on the methods of Bonifacino.20 Briefly, 5 3 107 cells were incubated with 5 mL methioninefree RPMI medium with 5% dialized FBS (pulse medium) for 30
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minutes at 37°C to deplete intracellular methionine. The cells were
pelleted and incubated in 5 mL fresh warmed pulse medium containing
1 mCi/mL 35S for 0.5 to 1 hour. An aliquot of 1 3 107 cells for time 0
were collected, and the remaining cells were washed once with Hank’s
balanced salt solution, resuspended in 20 mL complete RPMI medium
supplemented with excessive L-methionine (15 mg/mL) and incubated
at 37°C for the chase time 1, 2, 3, and 6 hours. At each time point, equal
amount of cells were washed, lysed, precleared, and immunoprecipitated with anti-C15 as described above.
Confocal immunofluorescence microscopy. Cultured cells were
harvested by low speed centrifugation and resuspended in complete
RPMI 1640 medium. The cells (1 to 2 3 105/mL) were fixed onto
microscopic slides by cytospin. The slides were air dried and fixed in
absolute methanol solution at 220°C for 10 minutes. The fixed cells
were blocked in blocking solution (2% BSA, 10 mg/mL goat IgG in
PBS) for 30 minutes followed by incubation with affinity-purified
anti-C15 (1:40) at room temperature for 1 hour. The slides were washed
extensively with PBS and incubated with goat antirabbit IgG Fab2
conjugated to fluorescein isothiocyanate (FITC) (Sigma) for 1 hour and
washed extensively as described. Stained slides were mounted in
glycerol mounting buffer (PBS, 10%; p-phenylenediamine, 10 mg/mL;
and Glycerol, 90%)21 and examined by a Bio-Rad MR C600 confocal
microscope. The images were processed using Adobe photoshop
software (Adobe Systems Inc, San Jose, CA).
RESULTS
Specificity of anti-C15 and anti-C9 antibodies. The specificity of affinity-purified anti-AF4 antibodies was first evaluated
by Western blotting. As shown in a high stringency experiment
(see Materials and Methods), affinity-purified anti-C15 recognized a single protein band of about 145 kD (Fig 2A, lane 1) in
Western blotting against total Km3 cell lysate. The signal was
absent using preimmune serum (lane 2) and was completely
blocked by incubating the antibody with immunizing antigen
before Western blotting (lane 3). Lane 4 shows lack of blocking
with GST. Under low stringent washing conditions, anti-C15
also binds to a 80-kD protein in Western blotting; however, the
cross-reactivity could be removed by extensive washing. In
contrast to anti-C15, affinity-purified anti-C9 detected four
major proteins in Western blotting (Fig 2B, lane 1) with an
estimated molecular size of 184, 145, 75, and 50 kD. All four
signals were completely blocked by the immunizing antigen
Fig 2. Blocking of anti-C15 (A) and anti-C9 (B) by immnunizing antigens. Km3 cell lysate was separated in 8% SDS-PAGE and electrophoretically
transferred onto a nitrocellulose membrane. The blots were cut into strips and Western blotting was performed using various preparations of primary
antibodies. (A) Lane 1, anti-C15 versus cell lysate; lane 2, preimmune IgG; lane 3, anti-C15 blocked by immunizing antigen; lane 4, anti-C15 blocked with
GST. (B) Lane 1, anti-C9; lane 2, preimmune IgG; lane 3, anti-C9 blocked by immunizing antigen; lane 4, anti-C9 blocked by GST.
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3844
Fig 3. Immunoprecipitation using anti-C15. (A) Immunoprecipitation followed by Western blotting. Lane 1, Km3 cell lysate without
immunoprecipitation; lane 2, unlabeled Km3 lysate immunoprecipitated with preimmune IgG; lane 3, Km3 lysate immunoprecipitated
with anti-C15; lane 4, anti-C15 with no lysate. The protein samples
were separated in 8% SDS-PAGE, transferred to a nitrocellulose
membrane, and probed with anti-C15. (B) Immunoprecipitation of
radiolabeled proteins. 35S-labeled Km3 lysate (300 mL) was precleared
with preimmune IgG and incubated with 1 mg anti-C15 for 3 hours at
4°C. The immune complex was pelleted by protein A/G beads,
washed, separated by 10% SDS-PAGE, and autoradiographed.
(Fig 2B, lane 3). Preimmune IgG did not show any reactivities
against the cell lysate (lane 2) and GST failed to block
immunoreactivity of the antibody (lane 4).
Immunoprecipitation was performed using anti-C15 antibody. After immunoprecipitation of Km3 lysate with anti-C15,
a protein of about 145 kD was detected in the immunoprecipitates by Western blotting using the same antibody (Fig 3A,
lane 3). The protein band was not seen using preimmune serum
(lane 2) or without lysate (lane 4). The protein signal seen in Fig
3A, lane 3, was identical to that of nonprecipitated lysate (lane
1). We also conducted experiments to evaluate proteins that
were physically associated with AF4. Km3 cells were radiolabeled with 35S and the cell lysate was immunoprecipitated by
anti-C15. In results shown in Fig 3B, the expected protein of
about 145 kD and a second 45-kD protein were detected (Fig
3B). The 45-kD protein was not detected in Western blottings
suggesting that the 45-kD protein was coprecipitated with the
larger protein.
More than one AF4 protein is detected in Western blotting.
Because the immunizing antigens of anti-C9 and anti-C15 were
from the 58 and 38 end of AF4 gene, respectively, both
antibodies should recognize the full-length AF4 protein in total
cell lysate. As shown in Fig 4A, a side-by-side comparison of
anti-C15 and anti-C9 showed that anti-C15 recognized a single
protein band in the cell lysates and anti-C9 bound to several
proteins. One of the proteins detected by anti-C9 (lane A3)
showed the same migration as that detected by anti-C15 (lane
A1). When anti-C9 and anti-C15 were mixed (lane A2) and used
for Western blotting, the AF4 protein signal was stronger than
the signals detected by either antibody alone, while other
LI, FRESTEDT, AND KERSEY
protein bands detected by anti-C9 remained comparable. These
results strongly suggest that both antibodies recognized the
same AF4 protein. When the Western blottings were performed
using lower percentage gels (Fig 4B), the AF4 protein migrated
as doublets (145 kD and 125 kD), which were detected by both
anti-C15 (lane B1) and anti-C9 (lane B2). Repeated experiments showed that anti-C15 generally reacted stronger with the
125-kD band, which is most likely due to the results of alternate
splicing of AF4 mRNA. In previous studies, a 10.5-kb and a
12-kb mRNA transcript of AF4 gene was observed.2,4 An
alternate explanation for the doublets is the possibility of
precursor-product relationship of the doublets. To evaluate this
possibility, pulse chase analysis was conducted. Despite repeated attempts, we were able to show the doublets in autoradiographes using low percentage gels only after long periods of
incubation (data not shown). A time course study of cell lysates
labeled with 35S and precipitated by anti-C15 indicated that the
doublets were observed in autoradiographes only in cell lysates
that have been labeled for a longer period of time (.3 hours).
The extented labeling time required to show the doublets
prevented us from obtaining interpretable results of pulse chase
analysis, which requires a short labeling window of 30 minutes
to 1 hour.20 Low immunoprecipitation efficiency of anti-C15
may also be in part responsible for the observations.
Anti-C15 and anti-C9 recognize MLL-AF4 and AF4-MLL
reciprocal fusion proteins, respectively. When the cell lysates
were run on low percentage SDS-PAGE gels (4% to 5%), a
protein band with a calculated molecular weight of 240 kD was
detected by anti-C15 in cell lines containing MLL-AF4 fusion
gene (Fig 5). This protein was consistently detected in all cell
lines with known t(4;11) translocations (Fig 5A, lanes 2, 4, and
6), but not in those without the translocations (lanes 1, 3, and 5).
These data indicate that the 240-kD protein is the MLL-AF4–
encoded fusion protein and is consistent with the predicted
protein sizes.2 A side-by-side comparison of anti-C15 and the
anti-MLL/HRX antibody (HRX107)14 in Western blotting
showed that a 240-kD protein band were detected by both
antibodies (data not shown). In these low percentage gels,
anti-C15 also generally detected the AF4 doublet in cells used
Fig 4. Comparison of Western blotting patterns of anti-C15 and
anti-C9. (A) Km3 lysate (20 mg/lane) was separated in 8% SDS-PAGE,
transferred to nitrocellulose paper, and probed with anti-C15 (lane 1),
anti-C15 plus anti-C9 (lane 2), and anti-C9 (lane 3). (B) K562 lysate (20
mg/lane) was separated in 4% SDS-PAGE, transferred to nitrocellulose paper, and probed with anti-C15 (lane 1) and anti-C9 (lane 2).
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AF4 ENCODES A UBIQUITOUS NUCLEAR PROTEIN
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Fig 5. Western blotting patterns of anti-C15 and
anti-C9 in 4% SDS-PAGE. Cell lysates were separated
by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and probed with affinitypurified anti-C15 (A) or anti-C9 (B). Lane 1, Nalm-6;
lane 2, RS4;11; lane 3, Km3; lane 4, Sem-k2; lane 5,
K562; lane 6, B1.
(Fig 5A). Again, as shown in Fig 4B, the smaller protein species
showed stronger signals than the larger species.
We also evaluated anti-C9 antibody for study of the fusion
proteins. Figure 5B shows the immunoblotting patterns of
anti-C9 against total cell lysates in 4% gel. In addition to 180
kD and 145 kD proteins, the antibody also recognized a high
molecular weight protein (.240 kD) in cells with t(4;11)
translocations, RS4;11 and SEM-k2 (lanes 2 and 4, respectively). The B1 cells, which were previously shown to have low
to absent levels of 14 kb der4 RNA transcript,4 were found to
have a very weak band as shown in Fig 5B, lane 6. Although the
exact size of this high molecular weight protein cannot be
determined due to the lack of proper molecular weight markers,
by comparing its migration rate with that of the 240 kD
MLL-AF4 fusion protein, the size of the protein was estimated
in the range of 300 to 350 kD. Because the high molecular
weight protein signal is much weaker than that of AF4 protein,
the blot in Fig 5B was intentionally overexposed to show the
high molecular weight band. Because anti-C9 was raised using
N-terminal polypeptide of AF4 as immunogen, it might expect
to recognize AF4 and reciprocal AF4-MLL proteins, but not the
MLL-AF4 fusion proteins. In view of the observation that the
high molecular weight protein detected by anti-C9 was seen
only with t(4;11) lines and that the size of the protein falls in the
calculated size range of AF4-MLL fusion protein 5(430 (MLL) 1
140 (AF4) 2 240 (MLL-AF4) 5 335 kD (AF4-MLL)6, we
suspect that anti-C9 recognizes the AF4-MLL reciprocal fusion
protein in all t(4;11) lines tested.
AF4 and MLL-AF4 fusion proteins are localized in the
nucleus, but not in the cytoplasm. Purified anti-C15 was used
to evaluate localization of the AF4 and MLL-AF4–encoded
fusion proteins in immunofluorescence studies. All cell lines
stained with the antibody showed a strong punctate fluorescence
staining in nucleus, but not in cytoplasm (Fig 6). The immunostaining patterns were generally similar in all cells tested and
there was no significant difference in the patterns and distribution of the labeling for cell lines with or without t(4;11)
translocations. Further study will be required to establish
whether the AF4 and MLL-AF4 proteins colocalize in the same
subcellular compartment. It is also important to determine
whether wild-type AF4 and the fusion proteins function in
distinct cellular compartments.
DISCUSSION
In this study, we have produced and characterized specific
antibodies to AF4-encoded proteins. We have also provided
direct evidence that AF4-encoded proteins are present in a
variety of human leukemic and nonleukemic cells, as demonstrated by immunoblottings and immunofluorescence microscopy. In Western blottings, anti-C15 and anti-C9 antibodies
consistently recognized a protein of about 145 KD in all cell
lines tested. Under high stringency conditions of washing, the
anti-C15 antibody recognized a single 145-kD AF4 protein in
Western blottings, which migrated as doublets when lower
percentage gels (4%) were used, the antibody also detected a
240-kD protein in all cell lines with t(4;11) translocations. The
molecular weight of the 240- and 145-kD proteins are consistent with predicted sizes of MLL-AF4 and AF4-encoded
proteins based on previous DNA sequencing and Northern
blotting analysis.2,4,22 Previous studies demonstrated the presence of 12 kb and 10.5 kb AF4 mRNA transcripts in cell lines
tested. The 12-kb and 10.5-kb RNA transcripts identified in the
previous studies are most likely to be the results of alternate
splicing of AF4 mRNA, which subsequently encode the 145-kD
and 125-kD proteins. The 12.5-kb mRNA transcript previously
detected in all cell lines with t(4;11) translocations corresponds
to the 240-kD protein detected in this study. Coprecipitation of
the 45-kD protein with AF4 protein suggests the possibility that
it may exert its biological function through interaction with a
second protein.
The anti-C9 that was raised against N-terminal end of AF4
protein detected the protein of about 145 kD. However, it also
bound to a 184-kD, a 75-kD, and a 50-kD protein in Western
blottings. It is not clear whether these proteins are AF4-related.
Thus far, mRNA species corresponding to these proteins have
not been identified. It was of interest that anti-C9 reacted to a
high molecular weight protein (.240 kD) in RS4;11 and
Sem-k2 cells with t(4;11) translocations, while the protein was
absent from non-t(4;11) cells. These observations suggest that
anti-C9 is able to detect AF4-MLL reciprocal fusion protein if it
is present.
Molecular analysis of the MLL and AF4 genes by our group
and others3,10,11,12,22 indicates that the N-terminal portion of
MLL fuses to C-terminal portion of AF4. The C15 clone used
for expression and immunization was chosen to be downstream
of all known breakpoints. Thus, anti-C15 recognizes the
chimeric MLL-AF4 protein in all cell lines tested (B1, RS4;11,
and Sem-k2). The MLL-AF4 chimeric proteins in RS4;11,
AN4;11, and B1 cells are calculated to contain 2319, 2276, and
2234 amino acid residues, respectively. Fusion proteins seen in
the Western blottings migrated as a single band in 4% SDS-
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
3846
LI, FRESTEDT, AND KERSEY
Fig 6. Indirect immunofluorescence microscopy. Cultured cells in log phase were harvested and adjusted to 1 to 2 3 105/mL. The cells were
attached to microscope slides by cytospin. The cells were probed with anti-C15 followed by goat antirabbit IgG conjugated to FITC. The image of
the stained cells was obtained by a Bio-Rad MR C600 confocal microscope and processed using Adobe photoshop software. The cell lines used
are indicated.
PAGE gel (Fig 5) with apparent similar molecular weight,
which is consistent with the predicted sizes and showing that the
size difference of the proteins was too small to be resolved
under the experimental conditions used.
Recently, Joh et al13 have generated rabbit polyclonal antibodies specific for N-terminal epitopes of MLL. These antibodies
recognized a 240-kD protein in Western blotting. Butler et al14
produced a monoclonal antibody against a 15 amino acid
peptide at N-terminal of MLL gene and the antibody (HRX 107)
also recognized the 240-kD MLL-AF4 fusion protein in the
Sem-k2 cell. It is of some interest that the quantity of MLL-AF4
fusion protein detected by our Western blotting is significantly
less than that of the AF4 protein. The significance of these
differences is not clear at present.
A gene previously cloned by Ma and Staudt6 from a cDNA
library of a Burkitt’s lymphoma is closely related to the AF4
gene. Both genes share a high degree of homology (75%
homology at C-terminal and 62% at N-terminal), contained a
proline/serine-rich region and NLSs at virtually the same
position. The LAF4 gene encodes a major protein of 135 kD.
However, the expression of LAF4-encoded protein was shown
to be restricted to lymphoid cells. As predicted from DNA
sequence analysis, our Western blotting results indicate that
LAF4 encoded a smaller protein than that detected by AF4
antibodies (data not shown).
Results of our experiments indicate that anti-C15 detects the
AF4 protein in subcellular compartments in immunofluoresence
microscopy. The subcellular localization of AF4 with punctate
distribution in the nucleus is similar to that previously described
for transcription-associated proteins. Other proteins with similar distribution are LAF4, MLL,13 and several other MLL
partners including ENL/LT6,13,23 AF9/LT69,13 and ELL.24 ELL
is of interest because of the demonstration that the ELL gene
encodes a RNA polymerase II enlongation factor.25 Consistent
with a role for AF4 and LAF4 in transcriptional regulation is the
previous observation by Ma and Staudt that AF4 has domains
that activate transcription when fused to GAL4 DNA-binding
domain. Of note is the observation that the transactivation
domain of AF4 is retained within the MLL-AF4 fusion protein.6
Further studies will be necessary to determine the significance
of the punctate compartmentalized staining seen with anti-AF4
antibody and other antibodies to transcription-related molecules. Previous studies with another transcription factor have
demonstrated that phosphorylation of hepatic nuclear factor 4 is
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
AF4 ENCODES A UBIQUITOUS NUCLEAR PROTEIN
required for nuclear compartmentalization.26 It has been shown
that the compartmentalized nuclear staining of the protein of
Drosophila polycomb (related to Drosophila trithorax) developed homogeneous nuclear staining after the protein was
mutated.27
Finally, the observation that the anti-C15 antibody was able
to detect the MLL-AF4 fusion proteins in leukemia lines
studied demonstrates the potential use of this AF4-specifc
antibody as a diagnostic reagent using Western blotting.
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From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
1998 92: 3841-3847
AF4 Encodes a Ubiquitous Protein That in Both Native and MLL-AF4 Fusion
Types Localizes to Subnuclear Compartments
Quanzhi Li, Joy L. Frestedt and John H. Kersey
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