Ikaros Isoform X Is Selectively Expressed in
Myeloid Differentiation
This information is current as
of June 18, 2017.
Kimberly J. Payne, Grace Huang, Eva Sahakian, Judy Y.
Zhu, Natasha S. Barteneva, Lora W. Barsky, Marvin A.
Payne and Gay M. Crooks
J Immunol 2003; 170:3091-3098; ;
doi: 10.4049/jimmunol.170.6.3091
http://www.jimmunol.org/content/170/6/3091
Subscription
Permissions
Email Alerts
This article cites 53 articles, 34 of which you can access for free at:
http://www.jimmunol.org/content/170/6/3091.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2003 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
References
The Journal of Immunology
Ikaros Isoform X Is Selectively Expressed in Myeloid
Differentiation1
Kimberly J. Payne,2* Grace Huang,* Eva Sahakian,† Judy Y. Zhu,* Natasha S. Barteneva,*
Lora W. Barsky,* Marvin A. Payne,† and Gay M. Crooks*
T
he Ikaros protein (also designated Lyf-1) (1) and gene (2)
were first identified using strategies designed to detect
transcription factors that regulate lymphoid-specific
genes, leading to the conclusion that Ikaros was a lymphoid-specific transcription factor. Subsequent studies provided evidence
that the role of Ikaros proteins in normal hematopoiesis extended
beyond the lymphoid lineages (3– 6). In addition, Ikaros proteins
were shown to act as both positive (5, 7–12) and negative (5,
13–21) regulators of transcription.
The murine Ikaros gene was initially reported to contain seven
exons (7) that were alternately spliced to generate transcripts for
multiple DNA-binding and nonbinding Ikaros isoforms (Fig. 1A)
(3, 7, 22, 23). Human and murine Ikaros were shown to be highly
homologous, and isoforms corresponding to murine Ik1 through
Ik6 (Fig. 1A) were detected in human cells (23). This suggested
that Ikaros mRNA splicing patterns might be identical in mice and
humans. However, subsequent studies of human Ikaros expression
in leukemia samples (24 –26) and in normal hematopoietic cells
*Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, Los Angeles, CA 90027; and †Department of Chemistry and Biochemistry, La Sierra University, Riverside CA 92515
Received for publication October 9, 2002. Accepted for publication January 9, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work is supported by National Institutes of Health Grants R01DK54567,
2P50HL54850, and P01CA59318 (to G.M.C.), National Research Service Award
1F32DK10101 (to K.J.P.), a fellowship from Childrens Hospital Los Angeles Research Institute (to K.J.P.), and a grant from the College of Arts and Sciences, La
Sierra University (to M.A.P.). G.M.C. is a Scholar of the Leukemia and Lymphoma
Society.
2
Address correspondence and reprint requests to Dr. Kimberly J. Payne, Childrens
Hospital Los Angeles, Division of Research Immunology/Bone Marrow Transplantation, Mail Stop #62, 4650 Sunset Boulevard, Los Angeles, CA 90027. E-mail address: [email protected]
3
Abbreviations used in this paper: Ik, Ikaros isoform; BM, bone marrow; CB, umbilical cord blood; HSC, hematopoietic stem cell; CTS, C-terminal sequence; NTS,
N-terminal sequence.
Copyright © 2003 by The American Association of Immunologists, Inc.
(27–29) identified transcripts for multiple Ikaros splice variants
that had not been reported in murine studies. Our studies showed
that Ikaros splice variants with a 60-base insertion following exon
2 (Fig. 1B) were expressed at the protein level in normal human
hematopoietic cells (29). We also detected transcripts for Ikaros-x
(Ikx; Fig. 1C), a new Ikaros isoform not previously described in
mice or humans. We found Ikx to be the predominant Ikaros protein in normal human umbilical cord blood (CB) and bone marrow
(BM) cells, although we observed little Ikx in T and B lymphoid
cell lines (29). Murine hematopoietic cells have not been examined
for expression of Ikx and the other novel Ikaros isoforms identified
in human studies.
Studies of mutant mice implicated Ikaros proteins as regulators
of critical events in hematopoietic differentiation and proliferation,
particularly in the lymphoid lineages. Ikaros-null mice fail to produce B cells and generate T cells only postnatally (30). Mice with
a defect in the Ikaros DNA-binding domain show more severe
lymphoid defects, including a complete loss of T, B, and NK cells
(31). In addition, mice with reduced Ikaros DNA-binding activity
show abnormal T cell proliferation and differentiation (32–34).
Although the initial reports of Ikaros mutant mice focused on
lymphoid defects, (30, 31), these mice display multiple hematopoietic abnormalities (4, 35, 36), including myeloid and hematopoietic stem cell (HSC) defects (4, 30, 31, 36) and aberrant erythropoiesis (6, 30, 31). Ikaros proteins have now been shown to act
as the DNA binding component of the PYR chromatin remodeling
complex that functions in globin switching (5, 6). A recent study
shows that Ikaros is important at early stages in granulocyte differentiation (37). These findings are consistent with erythroid and
myeloid defects exhibited by Ikaros mutant mice (4, 30, 31).
Reports of Ikaros isoform expression in primary cells outside
the lymphoid lineages are limited to a few studies that examined
Ikaros mRNA expression by RT-PCR (3, 8, 27). However, our
studies have shown a poor correlation between the expression of
Ikaros proteins and Ikaros mRNA, as assessed by RT-PCR (29).
The expression of Ikaros isoforms, at the protein level, has not
0022-1767/03/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
The Ikaros gene is alternately spliced to generate multiple DNA-binding and nonbinding isoforms that have been implicated as
regulators of hematopoiesis, particularly in the lymphoid lineages. Although early reports of Ikaros mutant mice focused on
lymphoid defects, these mice also show significant myeloid, erythroid, and stem cell defects. However, the specific Ikaros proteins
expressed in these cells have not been determined. We recently described Ikaros-x (Ikx), a new Ikaros isoform that is the predominant Ikaros protein in normal human hematopoietic cells. In this study, we report that the Ikx protein is selectively expressed
in human myeloid lineage cells, while Ik1 predominates in the lymphoid and erythroid lineages. Both Ik1 and Ikx proteins are
expressed in early human hematopoietic cells (LinⴚCD34ⴙ). Under culture conditions that promote specific lineage differentiation,
Ikx is up-regulated during myeloid differentiation but down-regulated during lymphoid differentiation from human LinⴚCD34ⴙ
cells. We show that Ikx and other novel Ikaros splice variants identified in human studies are also expressed in murine bone
marrow. In mice, as in humans, the Ikx protein is selectively expressed in the myeloid lineage. Our studies suggest that Ikaros
proteins function in myeloid, as well as lymphoid, differentiation and that specific Ikaros isoforms may play a role in regulating
lineage commitment decisions in mice and humans. The Journal of Immunology, 2003, 170: 3091–3098.
3092
Ikx IS SELECTIVELY EXPRESSED IN MYELOID DIFFERENTIATION
been examined in primary cells outside the lymphoid lineages, and
little is known of the role of Ikaros proteins in the myeloid lineage.
The lack of Ikx in human lymphoid cell lines led us to ask what
cells might be responsible for the abundance of Ikx proteins that
we detected in human CB and BM (29). One possibility was that
Ikx was expressed by erythroid and/or myeloid lineage cells. The
predominance of Ikx that we observed in normal human hematopoietic cells also raised the question of whether Ikx and other
novel Ikaros splice variants identified in human studies might be
expressed in the mouse. This is particularly important because experiments to determine the mechanisms of Ikaros function have
relied almost exclusively on the murine model. Neither murine nor
human studies have examined differential expression of Ikaros proteins between normal primary cells of the various hematopoietic
lineages or at different points in differentiation. Consequently,
questions concerning potential roles for specific Ikaros proteins in
hematopoietic lineage commitment have not been addressed.
Materials and Methods
Human CB and BM were collected according to guidelines approved by
the Childrens Hospital Los Angeles Committee on Clinical Investigation
(Investigational Review Board) and mononuclear cells were isolated as
previously described (38).
Nucleated erythroid lineage cells were isolated from mononuclear cells
that had been treated with PharM Lyse ammonium chloride lysing solution
(BD PharMingen, San Diego, CA) to lyse any remaining erythrocytes.
CD66B⫹ granulocytes were isolated (within 6 h of delivery) from fresh CB
cells located below the buffy layer after Ficoll-Hypaque separation. The
remaining human hematopoietic cells were isolated from total CB or BM
mononuclear cells.
Murine BM, spleen, and thymus were obtained from C57BL/6 mice
(The Jackson Laboratory, Bar Harbor, ME), aged 6 –12 wk, and rested for
at least 5 days after arrival before organs were harvested.
The murine S17 stromal cell line was a generous gift from Dr. K. Dorshkind
(University of Southern California, Los Angeles, CA). The Jurkat E6-1
cell line was obtained from American Type Tissue Culture Collection
(Manassas, VA).
Isolation of hematopoietic populations
Murine and human hematopoietic cells were incubated with Abs (see below) for 20 –30 min on ice, washed, and then cell populations were isolated
by FACS sorting or magnetic separation based on surface Ag expression.
For RT-PCR, cells were isolated by FACS (FACSVantage; BD Biosciences, San Jose, CA) based on forward scatter and side scatter gates
characteristic of living mononuclear cells and surface Ag staining as indicated below (typical purity of 99% or greater).
All HSC and progenitor populations were FACS sorted from CD34⫹enriched mononuclear cells. CD34⫹ enrichment was performed using the
MiniMacs CD34 progenitor isolation kit (Miltenyi Biotec, Auburn, CA)
according to the manufacturer’s instructions.
For immunoblot analysis, mature cells of the hematopoietic lineages
were isolated using the MiniMACS magnetic separation system (Miltenyi
Biotec). Murine and human B lineage cells were isolated using respective,
species-specific, CD19 MicroBeads (Miltenyi Biotec) to a purity of ⬃99%.
The remaining lineages were isolated by positive selection (typical purity
of 95% or greater) using anti-FITC or anti-PE MicroBeads (Miltenyi Biotec) following staining with FITC- or PE-conjugated Abs as described
below.
Abs used to isolate cells from human hematopoietic lineages were the
following: T cells, anti-CD3 FITC or anti-CD3 PE (clone Sk7); B cells,
anti-CD19 PE (clone 4G7); NK cells, anti-CD56 PE (clone NCAM16.2);
myeloid cells, anti-CD14 FITC (clone M0P9), anti-CD15 FITC (clone
MMA), anti-CD66B FITC (clone G10F5), and anti-CD11B PE (clone
D12) (all from BD Biosciences); and erythroid cells, anti-glycophorin A
FITC or anti-glycophorin A PE (clone KC16; Immunotech, Westbrook,
ME). Abs used to isolate cells from murine hematopoietic lineages were
the following: T cells, anti-CD3 FITC (clone 17A2); B cells, anti-CD19 PE
(1D3); and myeloid cells, anti-CD11B PE (M1/70) (all from BD Biosciences). Abs used to isolated CD34⫹ subsets were the following: antiCD34 allophycocyanin (clone 8G12; BD Biosciences) and biotinylated antiCD38 (clone HIT2; Caltag Laboratories, Burlingame, CA), visualized with
streptavidin red 613 (Invitrogen, Carlsbad, CA). Lin⫺CD34⫹ cells were
Culture conditions
Freshly sorted Lin⫺CD34⫹ cells were cultured in vitro under conditions
that promote lymphoid differentiation or myeloid differentiation (39). Lymphoid supporting conditions were as follows: coculture on the murine S17
stromal cell line (a generous gift from Dr. K. Dorshkind) in R10 medium
(RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) with 5% heatinactivated FCS, 0.5% penicillin/streptomycin, 1% L-glutamine, and 2-ME
(Sigma-Aldrich, St. Louis, MO)) supplemented with IL-3 at 5 ng/ml (R&D
Systems, Minneapolis, MN) and Flt-3 ligand at 50 ng/ml (Immunex, Seattle, WA) for the first 3 days, then with Flt-3 ligand at 50 ng/ml alone for
the remaining culture period. Myeloid supporting conditions were as follows: coculture on the murine S17 stromal cell line in BBMM medium
(IMDM (BioWhittaker, Walkersville, MD) with 20% heat-inactivated
FCS, 10% BSA, 0.5% penicillin/streptomycin, 1% L-glutamine, 10⫺4
mol/L 2-ME, and 10⫺6 mol/L hydrocortisone (to prevent lymphoid proliferation; Sigma-Aldrich)), supplemented with IL-3 at 5 ng/ml, IL-6 at 5
ng/ml, and stem cell factor at 25 ng/ml (R&D Systems).
RNA extraction and cDNA preparation
Total RNA was obtained using STAT 60 (Tel-Test, Friendswood, TX) as
per manufacturer’s directions. cDNA was prepared using oligo(dT) primers
(Pharmacia Biotech, Uppsala, Sweden) and the Omniscript kit (Qiagen,
Valencia, CA) as per manufacturer’s directions. cDNA from equivalent
cell numbers was used for RT-PCR analysis of human Ikaros expression in
hematopoietic lineages and in progenitor populations. Using primers and
conditions described previously (40), integrity of cDNA was assessed by
RT-PCR amplification of ␤2-microglobulin from the same cDNA master
mix used to assess Ikaros expression.
RT-PCR analysis
cDNA was subjected to PCR using HotStarTaq (Qiagen) as per manufacturer’s directions. Homologous primer pairs were designed to amplify selected murine and human Ikaros transcripts (29), including splice forms
with the 60-base insertion following exon 2.
Primers and conditions for amplification of human Ikaros cDNAs were
as described previously (29). Primers for amplification of mouse Ikaros
cDNAs were the following: forward, 5⬘-TGAGCCCATGCCTGTCCCT
GAG-3⬘, and reverse, 5⬘-GGTCTTCTGCCATCTCGTTGTGGTTA-3⬘.
PCR conditions for mouse primers were as follows: 15-min, 95° hot start
followed by 35 cycles of 1 min and 30 s at 95°, 30 s at 68°, and 3 min at
72°, followed by a final 10-min, 72° extension and cooling to 4°. To prevent heteroduplex formation between PCR products generated from different Ikaros isoforms as primer concentrations decreased, 20 additional
picomoles each of forward and reverse primers were added two cycles
before completion of the PCR.
Sequencing of RT-PCR products
Individual PCR products for sequencing were obtained by cutting individual bands from gel and purifying using UltraClean GelSpin DNA purification kit (MO BIO Laboratories, Solana Beach, CA) or by generating
single PCR products in a second PCR (identical with above, except run for
30 cycles) performed on samples aspirated by syringe from individual
bands. Sequencing was performed by the University of Southern California/Norris Comprehensive Cancer Center Microchemical Core Facility at
the Keck School of Medicine (Los Angeles, CA) or the Core Facility at the
Center for Molecular Biology and Gene Therapy (Loma Linda University,
Loma Linda, CA).
Immunoblots
For preparation of cell lysates, washed cells were frozen at ⫺80° and
thawed, on ice, in cold universal immunoprecipitation buffer (50 mM Tris,
150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM ␤-glycerol phosphate, and 0.3% IGEPAL in water), brought up to 1 mM PMSF,
1 ␮g/ml leupeptin, and 1 ␮g/ml aprotinin immediately before use. Suspended cells were sonicated on ice for ⬃10 s, checked for complete lysis
by microscope and then centrifuged at 14,000 rpm for 10 min at 4° to pellet
membranes. Supernatants were diluted in NuPage sample buffer (Invitrogen) and immediately run on gel or stored at ⫺80°. Lysates were run along
with Seeblue m.w. markers on a NuPage 10% Bis-Tris gel with MOPs
buffer using the XCell SureLock system (Invitrogen) and transferred to
Immobilon-P membrane (Millipore, Bedford, MA) using the XCell II Blot
Module (Invitrogen), all performed as per manufacturer’s instructions.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Cell sources and preparation
isolated using a mixture of Abs directed against glycophorin A, CD3,
CD19, CD56, CD14, CD15, and CD66B (Ab clones and sources listed
above).
The Journal of Immunology
Ikaros was detected using Ikaros M-20 and Ikaros E-20 (polyclonal goat
Abs specific to the N and C termini, respectively, common to all Ikaros
isoforms (Santa Cruz Biotechnology, Santa Cruz, CA)), anti-IkH (polyclonal rabbit Ab specific to residues encoded by the 60-base insert following Ikaros exon 2), or the mouse mAb 2A9 (specific to exon 3 of the Ikaros
protein) (generous gifts kindly provided by the laboratory of S. Smale
(Howard Hughes Medical Institute, University of California, Los Angeles,
School of Medicine, Los Angeles CA)). Blots were developed using the
ECL⫹Plus Western blotting detection system and accompanying Abs
(Amersham, Arlington Heights, IL) or anti-goat-HRP (Santa Cruz Biotechnology) as per manufacturer’s protocol, with the exception that blocking
reagent was present continually during Ab incubation.
Results
Ikx proteins, but not transcripts, are selectively expressed in
human myeloid lineage cells
throid lineage cells (Fig. 2B). Human T cells isolated from CB
showed only very faint bands representing multiple DNA-binding
and nonbinding Ikaros proteins (Fig. 2B). Ikx proteins are selectively expressed in myeloid lineage cells (Fig. 2B). The CD15⫹
cells isolated from CB include monocytic and granulocytic subsets
(41); however, the CD15 Ag is a differentiation and tumor-associated Ag that has been detected on nonhematopoietic cells (42,
43). To more precisely define Ikaros protein expression within the
myeloid lineages, we isolated CD14⫹ (primarily monocytes and
macrophages (44)) and CD66B⫹ (granulocytes (45, 46)) cells from
CB. Ikx predominates in both the CD14⫹ and CD66B⫹ myeloid
populations (Fig. 2B).
Ik1 predominated in the B, NK, and nucleated erythroid lineage
cells we examined. However, cells from these three lineages combined generally comprised ⬍20% (and in some cases ⬍10%) of
mononuclear cells in CB and only about a third of those in BM
(data not shown). This explains the predominance of Ikx that we
observed in our previous studies of total human CB and BM mononuclear cells (29).
Immunoblots specific to the amino acids encoded by the 60-base
insertion show that Ikaros insertion forms follow a pattern of lineage-specific expression that mirrors that observed for parent
Ikaros isoforms: Ikx⫹ is selectively expressed in myeloid cells
while Ik1⫹ predominates in B, NK, and erythroid lineage cells
(Fig. 2C).
These data show that the Ikx and Ikx⫹ proteins, previously reported to predominate in human BM and CB cells (29), are selectively expressed in myeloid lineage cells. This pattern of protein
expression was not predicted based on RT-PCR assessment of
Ikaros mRNA.
Murine hematopoietic cells express transcripts for Ikx
Given the predominance of Ikx in human hematopoietic cells, we
wanted to know whether murine hematopoietic cells express Ikx
and the novel Ikaros insertion forms identified in human studies.
Murine and human primer pairs were designed to amplify selected
Ikaros transcripts with the 60-base insertion following exon 2 (Fig.
1B). DNA sequencing confirmed the identity of murine RT-PCR
products consistent in size with Ik1, Ik2, Ik4, Ik8, and the new
Ikaros isoform, Ikx. (Fig. 3A). RT-PCR products consistent in size
with Ikx⫹ and, in some cases, Ik1⫹ were also detected (Fig. 3A).
However, we were unable to isolate RT-PCR products from murine insertion forms for DNA sequencing.
Ikx proteins predominate in mouse BM
FIGURE 1. Diagram of Ikaros isoforms and placement of RT-PCR
primers. A, Alternate splicing of exons 1–7 of the Ikaros gene gives rise to
isoforms Ik1 through Ik8. Zinc fingers are represented as vertical bars. B,
Placement of mouse and human primers designed to detect Ik1, Ikx, Ik2,
Ik4, Ik7, Ik8, and variants of these isoforms (designated Ik1⫹, Ikx⫹, Ik2⫹,
etc.) with the 60-base insertion following exon 2. C, Diagram of the new
Ikaros isoform, Ikx.
Immunoblots specific to both murine and human Ikaros proteins
show a similar pattern of Ikaros expression in mouse and human
BM (Fig. 3B). The predominant Ikaros protein is detectable as a
broad dense band consistent in size with Ikx forms (Fig. 3B). A
fainter band representing Ik1 forms is also detectable in both murine and human cells (Fig. 3B). Although we were unable to identify RT-PCR products for murine Ikaros insertion forms by DNA
sequencing, Abs specific to the insert show that the Ikaros insertion
forms are expressed at the protein level in mouse BM cells (Fig.
3C). These data show that mice, like humans, express proteins
representing the new Ikaros isoform, Ikx, and Ikaros splice forms
with the 60-base insertion. Furthermore, Ikx forms are the predominant Ikaros proteins in mouse, as well as human, BM.
Murine Ikx proteins are differentially expressed in lymphoid and
myeloid lineage cells
Previous studies of Ikaros protein expression in the mouse have
focused on the lymphoid lineage and failed to identify Ikx. To
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Our previous studies showed that Ikx (Fig. 1C) is the predominant
Ikaros protein in human CB and BM, but not in human lymphoid
cell lines (29). One explanation for these observations is that Ikx
is selectively expressed in nonlymphoid lineages. To determine
whether this explanation is correct, we examined expression of
Ikaros mRNA and protein in human lymphoid, myeloid, and nucleated erythroid lineage cells.
Multilineage RT-PCR analysis of Ikaros mRNA expression in
human CB and BM showed a similar pattern of products for all of
the hematopoietic lineages (Fig. 2A). PCR primers (Fig. 1B) generated products representing multiple DNA binding and nonbinding Ikaros isoforms, including many with the 60-base insertion, in
FACS-sorted cells from the T, B, NK, erythroid, and myeloid lineages (Fig. 2A). Isoforms with the 60-base insertion (insertion
forms) were designated as Ik1⫹, Ikx⫹, Ik2⫹, etc. The pattern of
RT-PCR products generated in the individual lineages matched the
pattern that we had previously observed in total human BM and
CB (29).
Contrary to what was observed in RT-PCR analysis of mRNA
expression, immunoblots showed that Ikaros isoforms are differentially expressed at the protein level in the hematopoietic lineages. Ik1 proteins are predominant in B, NK, and nucleated ery-
3093
3094
Ikx IS SELECTIVELY EXPRESSED IN MYELOID DIFFERENTIATION
determine whether murine Ikx is differentially expressed in lymphoid and myeloid cells, we examined expression of murine Ikaros
proteins in BM myeloid cells and in T and B lymphocytes from
multiple organs.
A dense band consistent in size with Ikx forms was expressed in
murine myeloid cells. In contrast, Ikx was only faintly detectable
in T and B lymphocytes in some immunoblots (Fig. 4). Ik1 forms
were the predominant Ikaros proteins in murine B and T cells but
represented a smaller fraction of the Ikaros proteins in myeloid
lineage cells. (Fig. 4). Surprisingly, total Ikaros proteins in general
appeared more abundant in myeloid than lymphoid lineage cells.
A comparison of immunoblots that detected all Ikaros isoforms
(Fig. 4A) with immunoblots that were visualized with Abs specific
to the insertion sequence (Fig. 4B) shows that both parent and
insertion forms of Ik1 and Ikx are expressed in murine cells.
Immunoblots specific to exon 3 (present in Ik1, Ikx, Ik3, and
Ik5) provide additional information on the Ikaros proteins expressed in the mouse. Ikaros proteins are more readily detected by
anti-Ik exon 3 (Fig. 4C) than by anti-Ik N-terminal sequence
(NTS) and C-terminal sequence (CTS) Abs (Fig. 4A) or anti-IkH
(Fig. 4B). This is likely due to differences in primary or secondary
Ab affinity as the same protein preparations were used for all blots
shown and identical results were obtained in several independent
experiments. The faint band detected by anti-NTS and anti-CTS
Abs just below Ikx forms in total BM and myeloid cells (Fig. 4A,
Ik2/3 forms) likely represents Ik2, as it was not detected (Fig. 4C)
by Abs specific to exon 3 (present in Ik1, Ikx, Ik3, and Ik5). Antiexon 3 Abs also identify a DNA-nonbinding isoform in both lymphoid and myeloid cells that contains exon 3.
Transcripts for multiple DNA-binding and nonbinding Ikaros
isoforms are expressed at early points in human hematopoietic
differentiation
The differential expression of Ikaros proteins that we observed in
mature murine and human hematopoietic cells of the various lineages raised the question of Ikaros expression at early points in
differentiation. To address this question, we first used RT-PCR to
determine whether human HSC and progenitor populations express Ikaros mRNA. Total human CD34⫹ CB and BM cells, and
subsets enriched for primitive HSCs (CD34⫹CD38⫺) or for multipotential and committed progenitors (CD34⫹CD38⫹) were
FACS sorted as shown in Fig. 5A. RT-PCR analysis of CB and BM
subsets showed that transcripts for a range of DNA-binding and
nonbinding Ikaros isoforms, including those with the 60-base insert, are expressed in progenitor and in primitive HSC populations
in human CB and BM (Fig. 5B). The pattern of RT-PCR products
generated by primitive hematopoietic cells (Fig. 5B) was similar to
that observed in mature human hematopoietic cells of the various
lineages (Fig. 2A).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. Ikx proteins, but not transcripts, are selectively expressed in human myeloid lineage cells. Analysis of Ikaros mRNA and protein expression in
human CB or BM isolated from the following hematopoietic lineages: T (CD3⫹), B (CD19⫹), erythroid (E; glycophorin A⫹ nucleated cells), NK (CD56⫹), and
myeloid (M, CD15⫹; Mon, CD14⫹; Gran, CD66B⫹). A, RT-PCR products were generated using primers shown in Fig. 1B. The human Jurkat T cell line was used
as a positive control and the murine S17 stromal cell line served as a negative control. Individual immunoblots were performed using equal numbers of cells and
detected with Abs specific to the NTS and the CTS common to all Ikaros isoforms (B) or the amino acids encoded by the 60-base insertion (C).
The Journal of Immunology
Ik1 and Ikx protein levels are differentially modulated during
myeloid and lymphoid differentiation
Our data demonstrate that the ability to generate transcripts for a
range of DNA-binding and nonbinding Ikaros isoforms is present
FIGURE 5. Transcripts for multiple DNA-binding and nonbinding
Ikaros isoforms are expressed at early points in human hematopoietic differentiation. A, Gates used for FACS sorting human progenitor and HSCenriched populations. B, RT-PCR analysis of FACS-sorted populations
from CB or BM (see Fig. 1B for primer placement).
very early in hematopoietic differentiation. However, we have observed a poor correlation between the expression of Ikaros transcripts and proteins. Therefore, to determine which of these transcripts are expressed as proteins, we examined Ikaros protein
expression in early hematopoietic cells and the progeny they generate during lymphoid and myeloid differentiation.
FACS-sorted CB cells that lack markers of lineage-committed
cells (CD3, CD19, CD56, glycophorin A, CD15, CD14, and
CD66B) but express CD34 (Lin⫺CD34⫹; Fig. 6A) were used to
prepare lysates for immunoblot analysis or placed into culture conditions that selectively support either lymphoid or myeloid lineage
FIGURE 4. Murine Ikx and Ikx⫹ are differentially expressed in lymphoid and myeloid lineage cells. Immunoblots performed using equal numbers of
cells from mouse BM, spleen, or thymus (Thy). B, T, and myeloid (M) lineage cells were isolated from indicated tissues based on expression of CD19,
CD3, or CD11B, respectively. Ikaros proteins were detected with Abs specific to the NTS and the CTS common to all Ikaros isoforms (A), the amino acids
encoded by the 60-base insertion (B), or exon 3 common to Ik1, Ikx, Ik3, and Ik5 (C).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 3. Transcripts and proteins for novel Ikaros isoforms, including Ikx, are expressed in mouse BM. A, RT-PCR analysis of Ikaros mRNA
expression in human (Hu) and mouse (M) BM (see Fig. 1B for primer
location). Immunoblots performed using equal numbers of murine or human BM cells detected with Abs specific to the NTS and the CTS common
to all Ikaros isoforms (B) or the amino acids encoded by the 60-base insertion (C).
3095
3096
Ikx IS SELECTIVELY EXPRESSED IN MYELOID DIFFERENTIATION
Discussion
differentiation (Fig. 6B). After 18 days in culture, lymphoid and
myeloid progeny were harvested for immunoblot analysis.
Unlike total CB cells which predominantly express Ikx, freshly
isolated Lin⫺CD34⫹ cells express both Ik1 and Ikx (Fig. 6C).
When Lin⫺CD34⫹ cells were placed in culture conditions that
selectively support myeloid differentiation, Ikx expression was upregulated in their progeny (Fig. 6C). In contrast, progeny generated
in culture conditions that promote lymphoid differentiation showed
down-regulation of Ikx (Fig. 6C). Thus, both Ik1 and Ikx are expressed in early hematopoietic cells and the expression of Ikx is modulated as cells undergo lineage commitment and differentiation.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Ik1 and Ikx proteins are differentially modulated during
myeloid and lymphoid differentiation. A, CD34⫹-enriched CB mononuclear cells that fall within a forward scatter and side scatter gate (R1)
characteristic of lymphoid cells are shown. Lin⫺CD34⫹ cells were FACS
sorted based on R1, R2 and R3 gates. B, Sorted Lin⫺CD34⫹ cells were
frozen for later immunoblot analysis or cultured under conditions that selectively support lymphoid or myeloid differentiation (see Materials and
Methods). C, Total CB mononuclear cells, FACS-sorted Lin⫺CD34⫹ cells,
and their progeny generated under lymphoid (L) or myeloid (M) culture
conditions were assessed by immunoblot analysis for total Ikaros protein
expression using Abs specific to the NTS and the CTS common to all
Ikaros isoforms.
In this report, we describe a comprehensive analysis of Ikaros
mRNA and protein expression during normal human hematopoiesis. Our studies show that Ikaros protein expression follows a
lineage- and stage-specific pattern of expression that correlates
poorly with Ikaros mRNA expression as assessed by RT-PCR.
Both Ik1 and Ikx proteins are expressed early in hematopoiesis.
Thereafter, Ikx is selectively expressed in the myeloid lineage,
while Ik1 predominates in erythroid and most lymphoid lineage
cells. The expression pattern of murine Ikaros proteins, including
Ikx, in lymphoid and myeloid cells is similar to that observed for
human Ikaros.
In contrast to expression at the protein level, RT-PCR analysis
of human Ikaros mRNA shows a similar pattern of expression for
DNA-binding and nonbinding Ikaros isoforms in all of the hematopoietic lineages and in HSC and progenitor populations. One
possible explanation for the poor correlation between RT-PCR
products and Ikaros proteins is that the expression of Ikaros is
regulated after transcripts for the various isoforms have been generated by RNA processing. A poor correlation between mRNA and
protein expression has been reported in a number of other cases.
These include genes expressed in hematopoietic cells (47) and
genes that, like Ikaros, are alternately spliced to generate multiple
isoforms (48).
Alternatively, simultaneous RT-PCR amplification of transcripts for multiple isoforms may not provide an accurate indicator
of the relative levels of each transcript i.e., amplification of some
transcripts may be more efficient than others. A difference in ability to compete for primers may seem unlikely given that primers
bind an identical sequence in all transcripts. However, particular
transcripts may be at a selective disadvantage for primer binding or
primer extension due to secondary structures conferred by specific
exons. For example, Ikaros isoforms that lack exon 3 (Ik2 and Ik4)
are disproportionately overrepresented among RT-PCR products
as compared with their representation at the protein level. In contrast, isoforms that contain exon 3 (Ik1 and particularly Ikx) are
underrepresented among RT-PCR products as compared with protein expression (Figs. 2, 3, 5, and 6).
Given the predominance of Ikx that we observe in normal human and murine hematopoietic cells, it might seem surprising that
Ikx was not identified in early studies. However, the Ikaros gene
gives rise to a number of splice forms, many of which were not
identified in the initial reports (7, 22). Ikaros expression has most
frequently been examined by RT-PCR and products consistent in
size with Ikx were detected in a number of murine (3, 23) and
human studies (23, 27, 49, 50). However, these products were not
identified and, in some cases, were thought to be artifacts. The
comparatively low frequency of Ikx among RT-PCR products did
not suggest that Ikx proteins might be abundantly expressed. In
addition, there was no indication that the unidentified RT-PCR
product (consistent in size with Ikx) might correspond to a myeloid-specific Ikaros protein, because the lineage-specific expression of Ikx proteins is not observed for Ikx transcripts (Fig. 2).
Due to the initial link between Ikaros and the lymphoid lineages,
most studies of Ikaros protein expression have been performed on
lymphoid cells which express little, if any, Ikx. Ikx was not identified in these studies, although a protein product consistent in size
with Ikx was detectable in some cases in both murine (22) and
human (25–27, 49, 50) cells. Thus, the identity of Ikx and its
abundance and almost exclusive expression in the myeloid lineage
were not known before our studies.
Murine HSC and progenitor populations have been shown to
express Ikaros mRNA (3, 8). As with mature human cells of the
The Journal of Immunology
have specific functions in hematopoiesis. Due to the relatively
large numbers of cells required for immunoblot analyses, there
have been few reports of Ikaros isoform expression, at the protein
level, in primary murine or human hematopoietic cells of any lineage. The poor correlation between Ikaros RT-PCR products and
Ikaros protein expression underscores the importance of examining Ikaros expression at the protein level. Given the tumor suppressor activity ascribed to Ikaros (30, 49 –53), aberrant Ikaros
expression in leukemia and other cell lines seems likely. Therefore, an understanding of the role of Ikaros in normal hematopoiesis may depend on knowledge of the specific Ikaros proteins expressed in primary hematopoietic cells.
The studies described in this report suggest that the expression of
Ik1 and Ikx could play a role in myeloid vs lymphoid and erythroid
lineage commitment. Current studies in our laboratory are aimed at
examining the effects of overexpression of Ik1 and Ikx on lineage
commitment and hematopoietic differentiation in human HSCs.
Acknowledgments
We thank the staff of Kaiser Permanente Hospital Sunset (Los Angeles,
CA) for collecting CB samples.
References
1. Lo, K., N. R. Landau, and S. T. Smale. 1991. LyF-1, a transcriptional regulator
that interacts with a novel class of promoters for lymphocyte-specific genes. Mol.
Cell. Biol. 11:5229.
2. Georgopoulos, K., D. D. Moore, and B. Derfler. 1992. Ikaros, an early lymphoidspecific transcription factor and a putative mediator for T cell commitment. Science 258:808.
3. Klug, C. A., S. J. Morrison, M. Masek, K. Hahm, S. T. Smale, and
I. L. Weissman. 1998. Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in
immature lymphocytes. Proc. Natl. Acad. Sci. USA 95:657.
4. Nichogiannopoulou, A., M. Trevisan, S. Neben, C. Friedrich, and
K. Georgopoulos. 1999. Defects in hematopoietic stem cell activity in Ikaros
mutant mice. J. Exp. Med. 190:1201.
5. O’Neill, D. W., S. S. Schoetz, R. A. Lopez, M. Castle, L. Rabinowitz, E. Shor,
D. Krawchuk, M. G. Goll, M. Renz, H. P. Seelig, et al. 2000. An Ikaros-containing chromatin-remodeling complex in adult-type erythroid cells. Mol. Cell.
Biol. 20:7572.
6. Lopez, R. A., S. Schoetz, K. DeAngelis, D. O’Neill, and A. Bank. 2002. Multiple
hematopoietic defects and delayed globin switching in Ikaros null mice. Proc.
Natl. Acad. Sci. USA 99:602.
7. Molnar, A., and K. Georgopoulos. 1994. The Ikaros gene encodes a family of
functionally diverse zinc finger DNA-binding proteins. Mol. Cell. Biol. 14:8292.
8. Morgan, B., L. Sun, N. Avitahl, K. Andrikopoulos, T. Ikeda, E. Gonzales, P. Wu,
S. Neben, and K. Georgopoulos. 1997. Aiolos, a lymphoid restricted transcription
factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J.
16:2004.
9. Kim, J., S. Sif, B. Jones, A. Jackson, J. Koipally, E. Heller, S. Winandy, A. Viel,
A. Sawyer, T. Ikeda, et al. 1999. Ikaros DNA-binding proteins direct formation
of chromatin remodeling complexes in lymphocytes. Immunity 10:345.
10. Ito, T., S. Nomura, M. Okada, Y. Katsumata, F. Kikkawa, T. Rogi, M. Tsujimoto,
and S. Mizutani. 2002. Ap-2 and Ikaros regulate transcription of human placental
leucine aminopeptidase/oxytocinase gene. Biochim. Biophys. Acta 290:1048.
11. Sun, P., and H. H. Loh. 2002. Transcriptional regulation of mouse ␦-opioid receptor gene: role of Ikaros in the stimulated transcription of mouse ␦-opioid
receptor gene in activated T cells. J. Biol. Chem. 277:12854.
12. Koipally, J., E. J. Heller, J. R. Seavitt, and K. Georgopoulos. 2002. Unconventional potentiation of gene expression by Ikaros. J. Biol. Chem. 277:13007.
13. Ernst, P., K. Hahm, and S. T. Smale. 1993. Both LyF-1 and an Ets protein interact
with a critical promoter element in the murine terminal transferase gene. Mol.
Cell. Biol. 13:2982.
14. Brown, K. E., S. S. Guest, S. T. Smale, K. Hahm, M. Merkenschlager, and
A. G. Fisher. 1997. Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell 91:845.
15. Koipally, J., J. Kim, B. Jones, A. Jackson, N. Avitahl, S. Winandy, M. Trevisan,
A. Nichogiannopoulou, C. Kelley, and K. Georgopoulos. 1999. Ikaros chromatin
remodeling complexes in the control of differentiation of the hemo-lymphoid
system. Cold Spring Harbor Symp. Quant. Biol. 64:79.
16. Brown, K. E., J. Baxter, D. Graf, M. Merkenschlager, and A. G. Fisher. 1999.
Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell
division. Mol. Cell 3:207.
17. Koipally, J., and K. Georgopoulos. 2000. Ikaros interactions with CtBP reveal a
repression mechanism that is independent of histone deacetylase activity. J. Biol.
Chem. 275:19594.
18. Cobb, B. S., S. Morales-Alcelay, G. Kleiger, K. E. Brown, A. G. Fisher, and
S. T. Smale. 2000. Targeting of Ikaros to pericentromeric heterochromatin by
direct DNA binding. Genes Dev. 14:2146.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
various lineages (Fig. 2), we found that highly purified, FACSsorted human HSC (CD34⫹CD38⫺) and progenitor (CD34⫹
CD38⫹) populations generate transcripts for multiple DNA-binding and nonbinding Ikaros isoforms (Fig. 5). Our data are consistent with a study by Nakayama et al. (27) that detected transcripts
for DNA-binding and nonbinding Ikaros isoforms in CD34⫹-enriched human CB cells and in culture-generated myeloid and erythroid cells.
The rarity of CD34⫹CD38⫺ cells (⬃0.05% of CB and BM
mononuclear cells (38)) precluded immunoblot analysis of protein
expression in this population. Therefore, for protein analysis, we
isolated CD34⫹ CB cells that lacked markers of lineage commitment (⬃0.75% of mononuclear cells). Both Ik1 and Ikx proteins
are detectable in the Lin⫺CD34⫹ population. However, this population is heterogeneous, including primitive HSCs, the CB multilymphoid progenitors (40), and potentially a common myeloid
progenitor. It is possible that Ik1 and Ikx are expressed in different
subsets of the Lin⫺CD34⫹ population, rather than coexpressed in
the entire population. Thus, our studies do not provide an analysis
of differential Ikaros protein expression in subpopulations of HSCs
and early progenitors. However, they do provide the first report of
Ikaros protein expression in early hematopoietic cells and demonstrate that Ik1 and Ikx proteins are expressed very early in normal
human hematopoietic differentiation.
Among the human hematopoietic lineages, Ik1 proteins predominate in BM nucleated erythroid lineage cells and in mature NK
and B cells isolated from CB. Surprisingly, CB T cells express
only low levels of multiple DNA-binding and nonbinding Ikaros
proteins. However, it is possible that the pattern of Ikaros expression we observed is unique to naive human T cells, because CB T
cells have not yet encountered Ag. In the murine hematopoietic
lineages, as in human, Ikx proteins predominate in BM and are
selectively expressed in myeloid lineage cells. In contrast to human CB T cells, murine T cells, isolated from spleen and thymus,
express Ik1 similarly to B cells. Surprisingly, total Ikaros proteins
appear to be more abundant in myeloid than in lymphoid lineage
cells, particularly in the mouse.
Using a mouse erythroid leukemia cell line, O’Neill et al. (5)
identified two Ikaros proteins (⬃65 and 55 kDa in size) that copurified with the PYR complex that functions in globin switching.
Based on size, these proteins were identified as Ik1 and Ik2, respectively. Although the 65-kDa protein does correspond to forms
of murine Ik1 identified in our studies, the 55-kDa protein is consistent in size with murine Ikx proteins that we identified using
Abs specific to Ikaros exon 3 (Ik2 does not contain exon 3). We did
not examine Ikaros expression in murine erythroid lineage cells.
Our human studies show abundant Ik1 expression in nucleated
erythroid lineage cells; however, Ikx is only faintly detectable.
Although there may be variation in Ikx expression between species, it is also possible that Ikx is selectively incorporated into the
PYR complex.
In the first study to describe alternate splicing of murine Ikaros
transcripts, Hahm et al. (22) describe a murine Ikaros sequence
that is homologous to the 60-base insert identified in human studies. Using murine cell lines, their study identified transcripts for
Ikaros splice forms that represent Ik5⫹ and Ik6⫹ (designated
Ikaros isoform IV and isoform II, respectively) (22). Our studies
show that splice variants with the insert (primarily Ik1⫹ and Ikx⫹)
are expressed at the protein level in normal murine and human
hematopoietic cells of multiple lineages. The pattern of expression
for Ikaros proteins with the insert mirrors that seen for parent isoforms that do not include the insert.
The selective expression of Ikx proteins in early progenitors and
in the myeloid lineage suggests that particular Ikaros isoforms may
3097
3098
Ikx IS SELECTIVELY EXPRESSED IN MYELOID DIFFERENTIATION
37. Dumortier, A., P. Kirstetter, P. Kastner, and S. Chan. 2002. Ikaros regulates
neutrophil differentiation. Blood. 5:1336.
38. Hao, Q. L., A. J. Shah, F. T. Thiemann, E. M. Smogorzewska, and G. M. Crooks.
1995. A functional comparison of CD34⫹CD38⫺ cells in cord blood and bone
marrow. Blood 86:3745.
39. Hao, Q. L., E. M. Smogorzewska, L. W. Barsky, and G. M. Crooks. 1998. In vitro
identification of single CD34⫹CD38⫺ cells with both lymphoid and myeloid
potential. Blood 91:4145.
40. Hao, Q. L., J. Zhu, M. A. Price, K. J. Payne, L. W. Barsky, and G. M. Crooks.
2001. Identification of a novel, human multilymphoid progenitor in cord blood.
Blood 97:3683.
41. Kansas, G. S., M. J. Muirhead, and M. O. Dailey. 1990. Expression of the CD11/
CD18, leukocyte adhesion molecule 1, and CD44 adhesion molecules during
normal myeloid and erythroid differentiation in humans. Blood 76:2483.
42. Gooi, H. C., S. J. Thorpe, E. F. Hounsell, H. Rumpold, D. Kraft, O. Forster, and
T. Feizi. 1983. Marker of peripheral blood granulocytes and monocytes of man
recognized by two monoclonal antibodies VEP8 and VEP9 involves the trisaccharide 3-fucosyl-N-acetyllactosamine. Eur. J. Immunol. 13:306.
43. Davidson, S. E., J. L. McKenzie, M. E. Beard, and D. N. Hart. 1988. The tissue
distribution of the 3 ␣-fucosyl-N-acetyl lactosamine determinant recognized by
the CD15 monoclonal antibodies CMRF-7 and 27. Pathology 20:24.
44. Wright, S. D., P. S. Tobias, R. J. Ulevitch, and R. A. Ramos. 1989. Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles for recognition
by a novel receptor on macrophages. J. Exp. Med. 170:1231.
45. Skubitz, K., K. Micklem, and E. van der Schoot. 1995. CD66 and CD67 cluster
workshop report. In Leukocyte Typing V, Vol. 1. B. L. Schlossmann, W. Gilks,
J. Harlan, T. Kishimoto, C. Morimoto, J. Ritz, T. A. Springer, T. F. Tedder,
R. F. Todd, eds. Oxford Univ. Press, Oxford, p. 889.
46. Eades-Perner, A. M., J. Thompson, H. van der Putten, and W. Zimmermann.
1998. Mice transgenic for the human CGM6 gene express its product, the granulocyte marker CD66b, exclusively in granulocytes. Blood 91:663.
47. Lian, Z., L. Wang, S. Yamaga, W. Bonds, Y. Beazer-Barclay, Y. Kluger,
M. Gerstein, P. E. Newburger, N. Berliner, and S. M. Weissman. 2001. Genomic
and proteomic analysis of the myeloid differentiation program. Blood 98:513.
48. Chen, G., T. G. Gharib, C. C. Huang, J. M. Taylor, D. E. Misek, S. L. Kardia,
T. J. Giordano, M. D. Iannettoni, M. B. Orringer, S. M. Hanash, and D. G. Beer.
2002. Discordant protein and mRNA expression in lung adenocarcinomas. Mol.
Cell. Proteomics 1:304.
49. Nakayama, H., F. Ishimaru, N. Avitahl, N. Sezaki, N. Fujii, K. Nakase,
Y. Ninomiya, A. Harashima, J. Minowada, J. Tsuchiyama, et al. 1999. Decreases
in Ikaros activity correlate with blast crisis in patients with chronic myelogenous
leukemia. Cancer Res. 59:3931.
50. Nakase, K., F. Ishimaru, N. Avitahl, H. Dansako, K. Matsuo, K. Fujii, N. Sezaki,
H. Nakayama, T. Yano, S. Fukuda, et al. 2000. Dominant negative isoform of the
Ikaros gene in patients with adult B-cell acute lymphoblastic leukemia. Cancer
Res. 60:4062.
51. Georgopoulos, K., S. Winandy, and N. Avitahl. 1997. The role of the Ikaros gene
in lymphocyte development and homeostasis. Annu. Rev. Immunol. 15:155.
52. Okano, H., Y. Saito, T. Miyazawa, T. Shinbo, D. Chou, S. Kosugi, Y. Takahashi,
S. Odani, O. Niwa, and R. Kominami. 1999. Homozygous deletions and point
mutations of the Ikaros gene in gamma-ray-induced mouse thymic lymphomas.
Oncogene 18:6677.
53. Karlsson, A., P. Soderkvist, and S. M. Zhuang. 2002. Point mutations and deletions in the znfn1a1/ikaros gene in chemically induced murine lymphomas. Cancer Res. 62:2650.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
19. Sabbattini, P., M. Lundgren, A. Georgiou, C. Chow, G. Warnes, and N. Dillon.
2001. Binding of Ikaros to the ␭5 promoter silences transcription through a mechanism that does not require heterochromatin formation. EMBO J. 20:2812.
20. Trinh, L. A., R. Ferrini, B. S. Cobb, A. S. Weinmann, K. Hahm, P. Ernst,
I. P. Garraway, M. Merkenschlager, and S. T. Smale. 2001. Down-regulation of
TDT transcription in CD4⫹CD8⫹ thymocytes by Ikaros proteins in direct competition with an Ets activator. Genes Dev. 15:1817.
21. Dorsam, G., and E. J. Goetzl. 2002. Vasoactive intestinal peptide receptor-1
(VPAC-1) is a novel gene target of the hemolymphopoietic transcription factor
Ikaros. J. Biol. Chem. 277:13488.
22. Hahm, K., P. Ernst, K. Lo, G. S. Kim, C. Turck, and S. T. Smale. 1994. The
lymphoid transcription factor LyF-1 is encoded by specific, alternatively spliced
mRNAs derived from the Ikaros gene. Mol. Cell. Biol. 14:7111.
23. Molnar, A., P. Wu, D. A. Largespada, A. Vortkamp, S. Scherer, N. G. Copeland,
N. A. Jenkins, G. Bruns, and K. Georgopoulos. 1996. The Ikaros gene encodes
a family of lymphocyte-restricted zinc finger DNA binding proteins, highly conserved in human and mouse. J. Immunol. 156:585.
24. Sun, L., N. Heerema, L. Crotty, X. Wu, C. Navara, A. Vassilev, M. Sensel,
G. H. Reaman, and F. M. Uckun. 1999. Expression of dominant-negative and
mutant isoforms of the antileukemic transcription factor Ikaros in infant acute
lymphoblastic leukemia. Proc. Natl. Acad. Sci. USA 96:680.
25. Sun, L., M. L. Crotty, M. Sensel, H. Sather, C. Navara, J. Nachman,
P. G. Steinherz, P. S. Gaynon, N. Seibel, C. Mao, et al. 1999. Expression of
dominant-negative Ikaros isoforms in T-cell acute lymphoblastic leukemia. Clin.
Cancer Res. 5:2112.
26. Sun, L., P. A. Goodman, C. M. Wood, M. L. Crotty, M. Sensel, H. Sather,
C. Navara, J. Nachman, P. G. Steinherz, P. S. Gaynon, et al. 1999. Expression of
aberrantly spliced oncogenic Ikaros isoforms in childhood acute lymphoblastic
leukemia. J. Clin. Oncol. 17:3753.
27. Nakayama, H., F. Ishimaru, Y. Katayama, K. Nakase, N. Sezaki, K. Takenaka,
K. Shinagawa, K. Ikeda, K. Niiya, and M. Harada. 2000. Ikaros expression in
human hematopoietic lineages. Exp. Hematol. 28:1232.
28. Olivero, S., C. Maroc, E. Beillard, J. Gabert, W. Nietfeld, C. Chabannon, and
C. Tonnelle. 2000. Detection of different Ikaros isoforms in human leukaemias
using real-time quantitative polymerase chain reaction. Br. J. Haematol. 110:826.
29. Payne, K. J., J. H. Nicolas, J. Y. Zhu, L. W. Barsky, and G. M. Crooks. 2001.
Cutting edge: predominant expression of a novel Ikaros isoform in normal human
hematopoiesis. J. Immunol. 167:1867.
30. Wang, J. H., A. Nichogiannopoulou, L. Wu, L. Sun, A. H. Sharpe, M. Bigby, and
K. Georgopoulos. 1996. Selective defects in the development of the fetal and
adult lymphoid system in mice with an Ikaros null mutation. Immunity 5:537.
31. Georgopoulos, K., M. Bigby, J. H. Wang, A. Molnar, P. Wu, S. Winandy, and
A. Sharpe. 1994. The Ikaros gene is required for the development of all lymphoid
lineages. Cell 79:143.
32. Winandy, S., L. Wu, J. H. Wang, and K. Georgopoulos. 1999. Pre-T cell receptor
(TCR) and TCR-controlled checkpoints in T cell differentiation are set by Ikaros.
J. Exp. Med. 190:1039.
33. Avitahl, N., S. Winandy, C. Friedrich, B. Jones, Y. Ge, and K. Georgopoulos.
1999. Ikaros sets thresholds for T cell activation and regulates chromosome propagation. Immunity 10:333.
34. Winandy, S., P. Wu, and K. Georgopoulos. 1995. A dominant mutation in the
Ikaros gene leads to rapid development of leukemia and lymphoma. Cell 83:289.
35. Boggs, S. S., M. Trevisan, K. Patrene, and K. Georgopoulos. 1998. Lack of
natural killer cell precursors in fetal liver of Ikaros knockout mutant mice. Nat.
Immun. 16:137.
36. Wu, L., A. Nichogiannopoulou, K. Shortman, and K. Georgopoulos. 1997. Cellautonomous defects in dendritic cell populations of Ikaros mutant mice point to
a developmental relationship with the lymphoid lineage. Immunity 7:483.