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A Serine/Proline-Rich Protein Is Fused To HRX in t ( 4 ; l l ) Acute Leukemias
By Joseph Morrissey, Douglas C. Tkachuk, Athena Milatovich, Uta Francke, Michael Link, and Michael L. Cleary
Translocations involving chromosome band 1 1q23 in acute
leukemias have recently been shown to involve the HRX
gene that codes for a protein with significant similarity to
Drosophilatrithorax. HRX gene alterations are consistently
observed in t(4;ll) (q21;q23)-carrying leukemias and cell
lines by Southern blot analyses and are accompanied by
HRXtranscriptsof anomalous size on Northern blots. HRXhomologous cDNAs were isolated from a library prepared
from t(4;1l)-carrying acute leukemia cells. cDNAs representative of transcription products from the derivative 11
chromosome were shown to containHRXsequences fused
to sequences derived from chromosome band 4q21. Fragments of the latter were used to clone and analyze cDNAs
for wild-type4q21 transcripts that predicted a 140-Kd basic
protein (named FEL) that is rich in prolines, serines, and
charged amino acids. FEL contains guanosine triphosphatebinding and nuclear localization consensus sequences and
uses one of two possible 5 exons encoding the first 1 2 or
5 amino acids. After t(4;ll) translocations, 913 C-terminal
amino acids of FEL are fused in frame to the N-terminal
portion of HRX containing its minor groove DNA binding
motifs. These features are similar to predicted t ( l l ; 1 9 ) fusion proteins, suggesting that HRX consistentlycontributes
a novel DNA-binding motif to at least two different chimeric
proteins in acute leukemias.
0 1993 by The American Society of Hematology.
T
chromosomes were shown to be transcriptionally active and
encode potential HRX fusion protein^.^.' The predominant
t( 1 1;19) product originated from the derivative 1 1 chromosome and encoded a fusion protein with the features of a
chimeric transcription factor consisting of an N-terminal
portion of HRX fused to a novel serine/proline-rich protein
from 1 9 ~ 1 3The
. ~ predicted t(4;ll) products have not been
completely characterized thus, it is unclear whether various
HRX fusion partners might share significant similarities to
suggest their respective contributions to the pathogenesis of
leukemias carrying 1 lq23 translocations.
In this report we describe the product of the chromosome
4 gene (named FEL) involved in the t(4;ll) chromosomal
translocation. The FEL gene is expressed normally in both
B- and T-lymphoid cell lines and encodes a predicted 140Kd protein that lacks significant homology to known proteins.
Following t(4;l l), the derivative 1 1 chromosome was shown
to encode a 240-Kd fusion protein containing a large C-termina1 portion of FEL fused to the N-terminal portion of
HRX containing its AT hook DNA binding motifs! These
features are comparable to t( 1 1;19)fusion proteins and suggest
that similar HRX chimeric nuclear factors may be consistently generated by 1 lq23 translocations in acute leukemias.
RANSLOCATIONS involving chromosome band 1 lq23
have been observed in several different types of hematolymphoid malignancies, including acute lymphoblastic
and nonlymphocytic leukemias and non-Hodgkin’s lymphoCytogenetic observations indicate that at least 10 different chromosomal loci may participate in translocationmediated exchanges with band 11q23.’ The most common
of this group is the t(4;ll) (q21;q23), which is frequently
associated with biphenotypic leukemias expressing both
lymphoid and myeloid surface antigens, and particularly
common in leukemias of infants.’-’
The gene involved in recurring 1 lq23 translocations has
recently been cloned and chara~terized~,~
following its localization within a yeast artificial chromosome that spanned
several 1 lq23 translocation breakpoint^.'.^ This gene (variously called HRX, MLL, or ALLI) codes for a potential zinc
finger protein of 431 Kd that shares significant but limited
similarity to the Drosophila trithorax
Several
1 lq23 translocation breakpoints have been localized to a
cluster region within the HRX gene6,7,9.10712-14
and fusion
transcripts have been isolated and characterized from t( 1 1;19)
and t(4;11)-carryingcell line^.^.^ In each case, both derivative
From the Laboratory of Experimental Oncology, the Departments
of Pathology, Pharmacology, Pediatrics, Genetics, and the Howard
Hughes Medical Institute, Stanford University School of Medicine.
Stanford, CA.
Submitted December 17, 1992; accepted December 18. 1992.
Supported in part by National Institutes of Health research grants
(CASSO29, CA496OS and HG00298) and training grants (T32
GM07149 and T32 GM08404 to J.M. and A.M., respectively), and
the Howard Hughes Medical Institute of which U.F.is an investigator.
D.C.T. was supported in part by a Centennial Fellowship from the
Canadian Medical Research Council. M.L.C. is a Scholar of the Leukemia Society of America.
Address reprint requests to Michael L . Cleary, MD, Department
of Pathology, Room L-224, Stanford University School of Medicine,
300 Pasteur Dr, Stanford, CA 94305-5324.
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.
0 I993 by The American Society of Hematology.
0006-4971/93/81OS-OO36$3.00/0
1124
MATERIALS AND METHODS
Patient materials and cell lines. Leukemia cells (ALL no. 1 and
ALL no. 2) were obtained from children with acute lymphoblastic
leukemia (ALL) and cryopreserved before their use in these studies.
Karyotype analyses at the time of diagnosis indicated that both leukemias camed balanced t(4;ll) (q21;q23) translocations. The t(4;II)carrying cell lines RS4:ll and MV4:11 were obtained from the
American Type Culture Collection (ATCC, Rockville, MD) and have
been described else~here.’~.’~
Molecular cloning of t(4;l I ) fusion cDNAs. Polyadenylated RNA
was prepared from leukemia cells (+I1 ALL no. 1)canying the t(4; 1 1)
(42 l;q23) using commercially prepared reagents (Invitrogen, San
Diego, CA). Two micrograms of poly(A+) RNA was converted to
double-stranded DNA using random hexamer or oligo-dT priming
and commercially prepared reagents (Superscript; BRL/GIBCO,
Gaithersburg, MD). After adapter addition and size-fractionation in
agarose gel, the cDNA products were cloned into XgtlO, packaged
in vitro, and plated for subsequent screening using previously described methods?*” The 4;11 library was screened with a probe from
the HRX cDNA that spanned the t( 1 1;19) fusion site (fragment B6).
Blood, Vol81, No 5 (March 1). 1993: pp 1124-1 131
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1125
HRX FUSION TRANSCRIPT IN t ( 4 ; l l ) LEUKEMIAS
I
L
FEL
.
I
BamH1
Twenty cDNAs were isolated and characterized by nucleotide sequencing using synthetic oligonucleotides homologous to HRX or
to sequences determined for the fusion partner.
Wild-type FEL cDNAs were isolated from cDNA librariesprepared
from cell lines (HBI I19 and HAL-OI) lacking the t(4;I I)?.'' For
library screening, fragments of t(4; I I ) fusion cDNAs that contained
only chromosome 4 sequences were used as probes under high-stringency hybridization conditions.
Southern and Northern blotting. Southern and Northern. blot
analyses were performed using high-stringency conditions described
previously.'*Chromosome I lq23 DNA rearrangements weredetected
with a 0.9-kb BamHl fragment of the HRX cDNA that spans the
I lq23 fusion region of HRY' Probes for Northern blot hybridizations
consisted of portions of the HRX cDNA (fragment B6) or portions
of the FEL cDNA (fragments 2 and 3, see Fig 2).
Chromosomal mapping by Southern blot and J'uorescence chromosomal in situ hybridization (FISH). Chromosomal localization
of the FEL locus was accomplished by using a 32PdCTPrandom
primer labeled 1.6-kb probe (probe 2, see Fig 2) on Southern blots
of Hindllldigested genomic DNA from normal human and Chinese
hamster controls and 13 human X Chinese hamster somatic cell
hybrids derived from seven independent fusion experiments'920using
methods described elsewhere.2'
For finer mapping of the FEL locus. FISH was performed with
two plasmids with cDNA inserts as probes (probes 2 and 3, see Fig
2). The plasmids were biotin- I I d U T P labeled by nick-translation
using commercially prepared reagents (Boehringer Mannheim, Indianapolis, IN). Hybridizations and washes were performed as previously described?' A biotin/avidin/fluorin isothyocyanate(FITC)
detection system was used. Chromosomes were counterstained with
propidium iodide ( I60 ng/mL, final concentration). Hybridization
solutions consisted of 0.1 to 0.2 ng/pL of each probe DNA (either
separately or together), 100 ng/pL each of human placental and
salmon sperm DNA as competitors, 50% formamide, 2X sodium
chloride sodium citrate (SSC), and 10%dextran sulfate. Metaphase
spreads were obtained by standard cytogeneticprocedures except that
the cells were synchronized by excess BrdU (200 pg/mL, final con-
I
T
S
- 28s
rRNA
Fig 1. (A) Southern blot
analysis of t(4;1l)-canying leukemias. DNA from t(4;11)-carwing leukemias (ALL nos. 1 and
2) and cell lines (RS4:ll and
M V 4 : l l ) were analyzed by
Southern blot analysis using an
HRX-specific probe. Germline
band of 8 kb is denoted with a
dash. (B) Northern blot analysis
of HRX and FEL transcripts in
various cell lines and leukemias.
Poly(A)+ RNAs from cell lines
lacking (6078 and Jurkat) and
containing ( R W 1 1 and MV4:
11) t ( 4 ; l l ) translocationswere
analyzed by Northern blot hybridization using FEL-specific
(upper panel) or HRX-specific
(lower panel) probes. Migration
of HRX-FEL fusion RNAs and
wild-type FEL and HRX transcripts are indicated on the right
of each panel. Residual signal
from prior FEL hybridization is
seen in lower panel and indicated by asterisk.
centration) for 17 hours and subsequently the block was released
with thymidine (10 pmol/L, final concentration) for 5 hours as described.22A total of 76 metaphase spreads were scored for specific
hybridization signals. Signals were considered specific only if two
fluorescent signalscould be seen lying side-by-side, one on each chromatid of a particular chromosome. All other single signals were assumed to represent random hybridization. Metaphase spreads were
examined in a Zeiss Axiophot microscope equipped with epifluorescence. Images were captured and enhanced using a cooled charge
coupled device (CCD) camera (PM512; Photometrin, Tucson, AZ)
and the Macintosh GeneJoin program developed by Tim Rand (Yale
University, New Haven, CT). Pseudocolored images stored as PlCT
files were converted to color slides, from which photographs were
prepared for publication.
Computer analyses. Compilation of nucleic acid sequences, restriction enzyme, and peptide analyses were performed using the
DNA Inspector IIE (Textco, West Lebanon, NH) application. Protein
similarity and consensus sequence analyses were performed using
lntelligenetics (Mountain View, CA) software. Similarities were determined using Quest and FastDB algorithms against keytools-I0
and SWISSPROT file banks, respectively.
RESULTS
HRX DNA rearrangements in t(4;11)-carryingleukemias.
DNA isolated from leukemias or cell lines carrying t(4;ll)
(92 l;q23) chromosomal translocations was analyzed by
Southem blot to determine the potential involvement of the
previously characterized I lq23 gene HRX. Using a 0.7-kb
fragment of HRX cDNA as a probe, all cases showed at least
one and in most cases two nongermline bands (Fig I A) demonstrating breakpoint clustering in a limited region of the
HRX gene. Detection of two rearranged HRX bands was
consistent with detection of both derivative translocation
products and suggested that the HRX cDNA probe used
spanned the breakpoint region in the HRX gene. Northem
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MORRISSEY ET AL
1126
blot analyses showed HRX RNAs with altered mobility (12
kb) slightly smaller than wild-type HRX RNAs in cell lines
carrying the t(4;l l), suggesting that the translocation resulted
in HRX fusion transcripts (Fig lB, lower panel).
Isolation of HRX fusion transcripts from a t(4;lI ) leukemia. To further characterize potential fusion transcripts, a
cDNA library was constructed from mRNA isolated from
t(4;11)-carrying leukemia cells (ALL no. 1, Fig 1A). The library was screened with an HRX cDNA probe that spanned
the potential fusion site (probe B6). Four types of clones were
obtained (Fig 2B) representing either wild-type HRX cDNAs
or three different candidate fusion cDNAs containing nonHRX sequences fused to HRX at nucleotide no. 4220.6 All
clones in the latter group showed identical sequences for 426
nucleotides beyond the site of fusion with HRX,but then
diverged from one another (Fig 2B and data not shown).
Types la and lb differed at this point by the inclusion or
exclusion of a single nucleotide and both open reading frames
diverged and ended shortly thereafter. The observed variations
suggested these clones were derived from RNAs that resulted
from aberrant splicing at this site particularly because they
were considerably shorter than the fusion RNA of 12 kb detected by Northern blotting. Type 2 clones contained an a p
parently normal splicing pattern and were analyzed further
as described below.
Chromosomal mapping of the t(4;Il)fusion partner. To
confirm that the isolated cDNAs represented 4; 1 1 fusions,
the non-HRX portions were regionally mapped by Southern
analysis of somatic cell hybrid lines that contained defined
A
regions of human chromosome 4 (Fig 3A). Three humanspecific fragments cosegregated and were concordant with
human chromosome 4q2 1-q31.2. All other human chromosomes were excluded by at least two discordant hybrids
(data not shown).
More specific localization was obtained by fluorescence in
situ hybridization to human metaphase chromosomes. Two
probes (2 and 3; Fig 2, B and C) were hybridized to the chromosomes simultaneously; 11 of 31 metaphases scored had
specific signal on at least one chromosome 4 homolog. Two
of these 1 1 metaphases had specific signal on both chromosome 4 homologs. Overall, using the probes separately or
together, in 26 of 76 metaphases examined, specific hybridization signals were seen at 4q2 1 on at least one chromosome
4 homolog. All specific signals seen were scored as located at
4q21; there were no other specific signals on any other chromosomes. These data demonstrated that the cDNAs isolated
from ALL no. 1 contained sequences derived from chromosome 4q21, in addition to HRX sequences from chromosome 1
Analysis of the 4q21 fusion partner. To further characterize the 4q2 1 fusion partner, cDNA clones corresponding
to the wild-type transcript were isolated from two different
libraries constructed from cell lines that lacked the t(4; 1 1).
Overlapping clones showed a cDNA contig of approximately
6 kb (Fig 2, A and C). When portions of these cDNAs were
used as probes on Northern blot analyses, a major transcript
of 11 kb was observed in all lymphoid cell lines examined
along with a minor transcript of 11.5 kb (Fig 1B and data
fu:t!til:b
1461
Xhol
1 .o
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2.0
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incll
4.0
5.0
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Type la
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41 07
Fig 2. Physical maps of t ( 4 ; l l ) fusion clones and
wild-type E L . Restriction sites in wild-type E L
cDNA are indicated at the top. (A) Schematic illustration of composite wild-type E L cDNAs. Open box
indicates open reading frame. Cross-hatched box
indicates alternative 5 exon. Lines indicate untranslated sequences. (B) Schematic diagram of
4;ll fusion transcripts. Heavy cross-hatchindicates
HRX sequences. Light cross-hatch denotes short
open reading frame 3 of atypical splice site in FEL.
(C) Schematic of overapping cDNA clones used to
establish composite wild-type FEL map. Horizontal
bars indicate positions of probes described in text.
Numbers refer to nucleotide positions as shown in
Fig 4.
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1127
HRX FUSION TRANSCRIPT IN t(4;ll) LEUKEMIAS
not shown). Nucleotide sequence analyses showed that the
isolated portion of the transcript contained a complete open
reading frame (Fig 4), suggesting that the remaining uncloned
portions ofthe 1 1-kb RNA contained untranslated sequences.
A
a
b c d
1
S(
4 + + + -
Two forms of the wild-type cDNAs were isolated and found
to differ at their 5’ ends, perhaps as a result of differential
splicing (Figs 2A and 4).
Analysis of the open reading frames for the wild-type FEL
transcript showed two predicted proteins (referredto as types
A and B in Fig 4) that initiated at one of two alternative
methionines in highly favorable contexts for translation initiation.*’ Types A and B differed in the composition of their
N terminal 12 and 5 amino acids, respectively, apparently
reflecting alternative 5’ exon usage. In-frame stop codons were
observed upstream of each initiating ATG, indicating that
the open reading frames could not extend further in the 5’
direction. Data base searches indicated that the predicted
proteins lacked significant similarity to previously reported
proteins. Motif searches showed a guanosine triphosphate
(GTP)-binding motif (residues 946 through 952), possible
nuclear localization sequences (residues 670 through 674 and
709 through 7 13), and numerous potential phosphorylation
sites possibly reflecting the high serine content of FEL. The
predicted protein is quite hydrophilic with a net positive
charge ($37 at normal pH) and also notable for its high serine
(14.7%) and proline ( 1 1.3%)content.
ChimericHRX-FELprotein in 4;ll leukemias. In all cells
examined, two wild-type FEL transcripts were observed, a
major RNA of approximately 11 kb and a minor, slower
migrating transcript of 11.5 kb (Fig 1B). In cell lines and
leukemias carrying the t(4;11) an additional transcript of 12
kb was observed (Fig 1B and data not shown). The 12-kb
RNA also hybridized with an HRX probe and migrated in a
position different than the wild type HRX transcripts of 13
and 15 kb (Fig IB). These data indicated that the 12-kb RNA
contained both HRXand FEL sequences constitutinga fusion
RNA that crossed the t(4;11) breakpoint. Hybridization with
5‘ HRX and 3’FEL probes indicated that this transcript originated from the derivative 11 translocated chromosome and
corresponded to the fusion cDNAs described above. Failure
to detect altered transcripts with FEL probes 5’ of the fusion
site (data not shown) suggested that the der(4) was not expressed at significant levels or comigrated with the wild type
FEL transcripts.
<
Fig 3. Mapping of FEL gene to chromosome band 4q21. (A) Gbanded chromosome 4 ideogram at the 850-band level** illustrates
the mapping of the t(4;ll) clone to region q21-q31.2 by Southern
blot analysis of somatic cell hybrids (SCH) and to bandq21 by FISH.
The bars to the right represent specific regions of human chromosome 4 contained in somatic cell hybrids. (a) Somatic cell hybrid
linesthat retained an intact human chromosome 4; (b) a hybrid that
underwent a spontaneous translocation between human chromosome 4 (witha breakpointat or near the centromere) and a Chinese
hamster chromosome, and retained only the long arm (cen-qter);
(c) a hybrid retaining only 4q21-qter (hybrids [b] and [c] described
ref 29). (d) A somatic cell hybrid line that retains the der(X)t(X:4)
(p21.2;q31.22) described in.zo Hybrids (a), (b), and (c) were scored
as positive for all the human-specific FEL Hindlll fragments, while
(d) was scored as negative. Bracket labeled SCH represents the
shortest region of overlap. Left-hand bracket represents the FISH
results that place the FEL locus at 4q21. (6)Representative partial
metaphase spread from the FISH experiment with specific signals
on chromosome 4 at band q21 (arrowhead).
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MORRISSEY ET AL
1128
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2C
3 7C5C C C C C C a 9
E K K K B K S S L P A P S K A L S G P E P A K O N V E D R T P E H
tqaqaagaagaaqcac~~gagccccccccccgccccccccaaggctcccccaggcccagdacccgcgaaggacaacgcggaqqacaggacccctqaqcac 2 4 7 5
F A L V P L T E S Q G P P H S G S G S R T S F C R S R G G P G G Q
rccg~r~ccgcc~~~~cgaccgagagccaggqcccaccccacagcggcagcggcagcaggaccagtccccgccgaagccgtggcqqccca~qaqgaca~c
2575
P Q R Q T P I A F E R H Q A A L T A Q D T P P P Q S L M V K I T L D
c g c a a a g a c ~ g a c C c c c ~ C C g c c c C C g a g a g a ~ c c a ~ g c C g c C c c c a c ~ c C c a g g a c ~ C t c c C c c C C C ~ C ~ ~ a g c C C g a C g g t g a a ~2a6C7 C
5 ~CcCt~9a
L L S R I P Q P P G K G A A R G K Q K I N S R P P K K H S S ~ K R
~ ~ c g ~ c c c c t ~ g g a c a ~ ~ ~ ~ ~ g c c c c c c g g g a a g g g a g c c g c c a g ~ g g ~ ~ ~ g c a g a a g a c a ~ ~ c ~ g c ~ c c c g c c g ~ a g a a q c a2c1a1 5g c c c C q a g a a 9 a 9 9
S
S
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~
~
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A
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D
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a g C C C a q ~ C d g C t C l d g C a a g t C g g C ~ ~ ~ ~ d g ~ g ~ ~ ~ ~ g C g A ~ g C ~ g ~ ~ ~ g ~ g ~ C C g C g * C ~ d C ~ ~ g ~ ~ ~2~8 C
1 5C ~ g ~ C C g g ~ g d d g 9 a ~ d C C d ~ d C C a C
Q
S
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a q t C a t C C C C a C C C C C a C C C C C c C d C a a a g a ~ C C t t C C 4 d ~ ~ C ~ ~ ~ g C C c t C C d g ~ C C C C C ~ C ~ C ~ ~ C C C C a d A g a a g g ~ ~ a ~2915
9C~CCCCCC~C=aCC
V
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A
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C g C q t C C C C q t C C t C C C a g a d g C C d ~ C C A ~ g C ~ g C ~ C ~ C ~ ~ g ~ ~ C C ~ d g g C g g g ~ ~ g C ~ g ~ C d C C C 9 C g 9 C ~ 9 g a C3C0 7C5C C C ~ ~ ~ ~ g ~ 9 C C ~ 9 C a 9 C a
P
R
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K
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A
D
K
G
S
S
G
D
ccaaqaqtcaaccacaaagaccccCcc~ttcccaa~cagagaagagCagaggggaagggcCccaqaagcCcct~gcagacraggggCCcCCccqqaqata
3115
T A N P F P V P S L P N G N S K P G K P Q V K F D K Q Q A D L H X R
crqcaaarcctccrccagcqccccccccqccaa~~gqc~ac~craaaccagggaaqccccaagtgaagt~~gac~aacaacaagcagacc~ccacacqaq
3275
E A K K ~ K Q K A E L M T D R V G K A F K Y L E A V L S F I E C G
qqaqqcaaaaaagarqaagc~gaaagcagagccaacgaerjgacagggccgg~aagqcccccaagC~ccCgga~qccgCcCCqCcctCcaCtqdqCqcq9a
3315
I A T E S E S Q S S K S A Y S V Y S E T V D L I K F X ~ S L K S F
actqccacagagccrqaaagcc~gccacccaagtcagcccaccccgccc~cccagaaacc~~a~accCca~~aaaccca~aatg~
3475
c a ~ c a a ~ aFig
~ c c ~4.
~ ~ ~ Nucleotide and preS D A T A P T Q E K I F A V L C M R C Q S I L N M A ~ F R C K K D I
dicted
amino
acid sequences for
caqacqccacagcqccaacacaagaga~~~catccgccqtcccacgcacgc~ccqccdgtccaCtccgaacaCqgcgacqccCcqccqcaaaaaaqacaC
3515
wild-type FEL. The nucleotide
A I K Y S R T L N K H F E S S S K D R P G T F S M H C K K H R H T
~qcaat~aaqcacc~t~gcacccctaataaacaccccgagaqttctcccaaagarerjccca~caccttcCccat~caCC~caa~
3675
aa~c~~a
sequence
~~cd~~~~
for wild-type FEL
I P S F P N A F S C Q L R R V P V K C Y Q C G E Q W G G C H Y Q H
a c ~ ~ ~ ~ t ~ t c t ~ ~ ~ ~ a ~ t g c c r c ~ ~ ~ c r g ~ ~ a q c t ~ ~ g t a g g g c ~ ~ c a g c ~ a ~ g t g ~ c q g c a g t g + g q g g a q c a q c g3g1g7 5q t g g ctranscrip~
t g c c ~ c C a Care
c a qshown
c a c c in
a 5 to
3 direction. Diierent 5 seP S H H P D M T S S Y V T I T S H V L T A F D L W E Q P R P S R G R
~ ~ a g t ~ a ~ ~ a c ~ ~ a g ~ c a t q a ~ a c c c c ~ ~ c a c g t ~ a ~ c a c c a ~ a t ~ ~ ~3 8 1a5 c g tquences
c ~ t c containing
a ~ ~ q ~ ~
alternative
f ~ ~ g ~ ~ ~ ~ ~ ~ ~ ~
I K N S L L G S D K C V H L G P Q Q Q F G G P G A L Y T T G F S A
~ ~ c a a a q a a t ~ c ~ ~ c q c t c q q c c ~ a g a c a a a t g t g t g ~ a ~ ~ ~ t q q c c c c ~ a a ~ a g ~ a ~ c c 9 q c g g a ~ ~ c q g c g c a c t a r a c a3975
c q a c a q qATG
q c ~ c ~stert
c a g c codons
a
are denoted
A T R I N Q N T L M E P Q V D S M P W E L F L H I G S L K N S P D
types A and B, respectively.
q ~ t a ~ a a q ~ ~ c c ~ a ~ ~ a a a a ~ ~ ~ ~ t c a a c q q ~ q c c c ~ a q g c c ~ a t t c a a t q c c c c g q g a a c r a r c r t t q c a c a c c g g a a q c c t c a a4015
a a a c a q ~~~i~~
c c a ~ a c qacids
are sbwn
insin-
V C F I R T P N S K K E A P R D C Q D I C H L N S Q Q Q C D H ~ L D
cctg~tt~at~aqqa~a~~aaacccca~aaaaqaaqcaccacqag~cqqccaggacatttqccactcaaacccccaac~acagcqcgatcaccgqcC~qa
4115
gle-leeer code. t(4;11) fusion
T V V M Q K Q R .
site is indicatedwith horizontal
caccg~ggcratqcaqaaqcaqaqatgaqqaggccqqc~ca~agarqaccccg~ccftcc~aactraaggacaqa~qrgcaacctaqcttaaacqq~CqC
4215
acgaacqgt~taqaaa~atctctatcccccctccaaaccaqc~ggacacaacqccacctcagtaqccacqccagtccactcccagaaggaaaCC:c~~:: 4375
bracket' Underlines
indicate nut t t a a ~ a a t ~ a ~ t t t t g g ~ a a a g g g c t c c q c g q a ~ q a c t t ~ t ~ c t c c c c c g ~ ~ c c t g g g a g a a a c a ~ ~ ~ g c c t a a c a a c c t c c a a c4q4g1 c
5 c a c ccleotide
cqca~a
(Kozak consensus23)
c c a c a a q t a a t q a a g q a c t c c a c c q t g c c c c a c c c c c t g c c a a c q a a c a ~ c q q c c c g a c a ~ ~ a c c a d ~ ~ a ~ t g t ~ ~ t a a ~ r t4575
a ~ a a a a ~and
t ~ a aprotein
~ ~ ~ ~ ~ ~(nuclearlocalization
C C C C ~ C C c C t g C C ~ C C c c C a a C c t C C C d C C ~ C ~ ~ ~ ~ q ~ C ~ ~ C a C ~ C C ~ ~ C C C C ~ g C C C C g ~ C C C C ~ C ~ C ~ d C4615
C~C~CC~d~~gCC~qCCC~CgC~9~~
and GTP-binding'
motifs
gcg~tqaqqaqgagccacagcaca~gqqq~gcacc~cg~ggCctgcacaggaggactcqcgctqc~~CC~Cacccrgcca~~tccc~cccctcc~tcaqc
4115
aacagqqactctctaacaqqgcagccaccqccgaccccaCC
481s
scribed in the text.
de-
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
HRX FUSION TRANSCRIPT IN t(4;ll) LEUKEMIAS
1129
t(4;l I ) translocations.’ Expression of both derivative products
is not unexpected because both wild-type HRX and FEL
promoters are active in lymphoid cells. Our inability to detect
or clone derivative 4 products from ALL no. 1 is consistent
with detection of only one rearranged HRX band on Southern
blots and may reflect deletion of chromosome 1 1 DNA 3’
(telomeric) of the breakpoint which has been observed previously.* These features underscore the pathogenetic importance of the derivative 1 I fusion product as proposed earliee
and supported by the cytogenetic features of three-way transIocation~.~~
The FEL gene appears to be expressed normally in lymphoid cells because transcripts were detected in all cell lines
examined (Fig I and data not shown). Characterization of
cDNA clones showed two alternative amino termini for FEL
that appeared to result from differential utilization of 5’ exons.
It is not clear from our data whether the two wild-type FEL
transcripts observed on Northern blots correlate with utilization of alternative 5’ exons or result from other differential
processing of the FEL transcript. Because it is unlikely that
the minor amino terminal variations in the predicted FEL
proteins confer functional differences, it is possible that the
5’ FEL exons are associated with alternative promoters, but
additional studies are required to address their significance
and possible differential regulation.
The predicted FEL protein shares several interesting features with ENL, the previously reported protein fused to HRX
after t( I 1;19) translocations in ALL.6 Both proteins are unusually serine- and proline-rich; in fact, serines and prolines
Nucleotide sequence analyses of 4; I I fusion transcripts
showed that the junction of HRX and FEL sequences occurred at or near amino acids I406 of HRX and 362 of FEL,
the precise point undetermined because of a 5-nucleotide
homology between HRXand FEL at the fusion site (Fig 5A).
Based on these observations, the HRX-FEL fusion protein
had a predicted length of 2,319 amino acids. The fusion site
in HRX is 34 amino acids upstream of that observed for the
fusion protein described in the t( I 1;19)-carrying cell line
HBl I 19.6 The fusion site in FEL is 15 amino acids downstream to that observed in a partial 4; I I clone reported prev i o ~ s l yThese
.~
observations suggested that translocation-associated fusion sites in HRX and FEL are not invariant but
are restricted in their distribution.
DISCUSSION
The studies presented here establish a complete structure
for the product of the chromosome 4 gene involved in t(4; 1 1)
translocations in acute leukemias. Fusion of FEL to HRX
appears to be a consistent feature of t(4; I 1) (q21;q23) translocations. Both t(4;l I)-carrying cell lines in this study contained FEL-HRX fusion transcripts of comparable size detectable with an FEL cDNA probe isolated from a t(4;l l)
leukemia. In addition, partial sequence analyses of fusion
cDNAs reported earlier further support the involvement of
FEL (AF-4) in the RS4;l I cell line.7
Although we detected and characterized the derivative 1 1
translocation product, other studies have reported the presence of both derivative 4 and 1 I fusion products following
A
Fusion site
n
ATC AGA GTG GAC TTT AAG GAG GAT TGT GAA GCA GAA AAT GTG
I
R
V
D
F
K
E
D
C
E
A
E
N
V
HRX
1401
I
I I I
1414
I
HRX-FEL
ATC AGA GTG GAC TTT AAG GAA ATG ACC CAT TCA TGG CCG CCT
I
R
V
D
F
K
E
M
T
H
S
W
P
P
FEL
GTT GAA GAG ATT CTG AAG GAA ATG ACC CAT TCA TGG CCG CCT
V
E
E
I
L
K
E
M
T
H
S
W
P
P
1 1 1 1 1 1 1 I I
356
B
A-T hooks
HRX
I Ill
A-T hodts
369
1O00
2060
3OoO
I
I
I
zinc finaen,
I
SP-rich
HRX-FEL
Fig 5. Structure of H R X 4 1 1
fusion products. (A) Nucleotide
and predictedpmtein sequences
at fusion sites betweenHRX and
E L RNAs. (B)Schematic representation of wild-type and fusion proteins involved in the
t ( 4 ; l l ) and t ( l l ; l 9 ) .
411 der(l1) product
SP-rich
FEL
A.T honks
SP-rich
11;19 der(l1) product
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
1130
MORRISSEY ET AL
constitute fully 26% of the residues in each protein. ENL and
FEL are both positively charged, hydrophilic proteins with
many potential phosphorylation sites. Both ENL and FEL
may be nuclear proteins as each contains sequences homologous to consensus nuclear localization sequences present in
known nuclear proteins.25In addition, both proteins contain
sequences that conform to a consensus GTP-binding site
(GXXXXGK) suggesting possible energy requiring functions
for ENL and E L . Despite these similarities, ENL and FEL
are not significantly homologous to one another beyond their
overall amino acid composition nor are they significantly
similar to previously reported proteins preventing further
functional comparisons.
The data suggest that ENL and FEL may be widely expressed, nuclear proteins without obvious DNA binding motifs. Because serine/proline-rich domains have been associated
with transcriptional activator proteins, ENL and FEL may
play an accessory role in some aspects of transcription regulation. Interestingly, as a result of translocations with the
1 lq23 HRYgene, large C-terminal portions of ENL and FEL
are fused to the amino terminal portion of HRX that previous
studies have demonstrated contains motifs (AT hooks and
'SPKK') implicated in minor groove DNA binding.6326,27
Therefore, a consistent feature of two 1 lq23 translocations
is the fusion of HRX DNA binding motifs with different
serine/proline-rich partners resulting in proteins that could
bind DNA inappropriately and function as chimeric transcription factors.
Comparison of the chimeric products resulting from
t( 1 1;19) and t(4; 1 1) translocations supports the previous
hypothesis6 that an important contribution of HRX to 1 lq23
translocation-associatedfusion proteins is its amino terminal
DNA binding domain. A common HRX DNA binding motif
in both the HRX-ENL and HRX-FEL fusion proteins suggests they may deregulate a similar set of subordinate genes,
although the sequence-specific DNA binding properties of
the HRX motifs remain to be determined. The importance
and specific contributions of each fusion partner in the t(4; 1 l),
t( 1 1;19), and other 1 lq23 translocations to different leukemia
phenotypes are not resolved by our studies. The compositional similarities of FEL and ENL may imply a similar leukemogenic role; however, their respective contributions and
potential transcriptional properties need to be addressed in
appropriate model systems.
ACKNOWLEDGMENT
We thank Cita Nicolas and Roxane Brown for technical assistance
and Phil Verzola and Judith Quenvold for photography.
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HRX FUSION TRANSCRIPT IN
t(4;ll) LEUKEMIAS
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From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
1993 81: 1124-1131
A serine/proline-rich protein is fused to HRX in t(4;11) acute leukemias
J Morrissey, DC Tkachuk, A Milatovich, U Francke, M Link and ML Cleary
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