The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Payne, K. J.
Right arrow Articles by Crooks, G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Payne, K. J.
Right arrow Articles by Crooks, G. M.
The Journal of Immunology, 2003, 170: 3091-3098.
Copyright © 2003 by The American Association of Immunologists

Ikaros Isoform X Is Selectively Expressed in Myeloid Differentiation1

Kimberly J. Payne2,*, Grace Huang*, Eva Sahakian{dagger}, Judy Y. Zhu*, Natasha S. Barteneva*, Lora W. Barsky*, Marvin A. Payne{dagger} and Gay M. Crooks*

* Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, Los Angeles, CA 90027; and {dagger} Department of Chemistry and Biochemistry, La Sierra University, Riverside CA 92515


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 (Lin-CD34+). Under culture conditions that promote specific lineage differentiation, Ikx is up-regulated during myeloid differentiation but down-regulated during lymphoid differentiation from human Lin-CD34+ 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The 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, 4, 5, 6). In addition, Ikaros proteins were shown to act as both positive (5, 7, 8, 9, 10, 11, 12) and negative (5, 13, 14, 15, 16, 17, 18, 19, 20, 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, 25, 26) and in normal hematopoietic cells (27, 28, 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.



View larger version (28K):
[in this window]
[in a new window]
  		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.

 
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, 33, 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 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell sources and preparation

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: anti-CD34 allophycocyanin (clone 8G12; BD Biosciences) and biotinylated anti-CD38 (clone HIT2; Caltag Laboratories, Burlingame, CA), visualized with streptavidin red 613 (Invitrogen, Carlsbad, CA). Lin-CD34+ cells were isolated using a mixture of Abs directed against glycophorin A, CD3, CD19, CD56, CD14, CD15, and CD66B (Ab clones and sources listed above).

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% heat-inactivated 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 {beta}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'-TGAGCCCATGCCTGTCCCTGAG-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 {beta}-glycerol phosphate, and 0.3% IGEPAL in water), brought up to 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/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.

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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ikx proteins, but not transcripts, are selectively expressed in human myeloid lineage cells

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).



View larger version (97K):
[in this window]
[in a new window]
  		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).

 
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 erythroid 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.



View larger version (74K):
[in this window]
[in a new window]
  		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).

 
Ikx proteins predominate in mouse BM

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 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.



View larger version (53K):
[in this window]
[in a new window]
  		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).

 
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). Anti-exon 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).



View larger version (66K):
[in this window]
[in a new window]
  		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 HSC-enriched populations. B, RT-PCR analysis of FACS-sorted populations from CB or BM (see Fig. 1B for primer placement).

 
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 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 differentiation (Fig. 6B). After 18 days in culture, lymphoid and myeloid progeny were harvested for immunoblot analysis.



View larger version (45K):
[in this window]
[in a new window]
  		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.

 
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 up-regulated 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.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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, 26, 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 various lineages (Fig. 2), we found that highly purified, FACS-sorted 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 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, 50, 51, 52, 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.


   Footnotes
 
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. Back

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: kpayne{at}chla.usc.edu Back

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. Back

Received for publication October 9, 2002. Accepted for publication January 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lo, K., N. R. Landau, 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.[Abstract/Free Full Text]
  2. Georgopoulos, K., D. D. Moore, B. Derfler. 1992. Ikaros, an early lymphoid-specific transcription factor and a putative mediator for T cell commitment. Science 258:808.[Abstract/Free Full Text]
  3. Klug, C. A., S. J. Morrison, M. Masek, K. Hahm, S. T. Smale, 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.[Abstract/Free Full Text]
  4. Nichogiannopoulou, A., M. Trevisan, S. Neben, C. Friedrich, K. Georgopoulos. 1999. Defects in hematopoietic stem cell activity in Ikaros mutant mice. J. Exp. Med. 190:1201.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  6. Lopez, R. A., S. Schoetz, K. DeAngelis, D. O’Neill, A. Bank. 2002. Multiple hematopoietic defects and delayed globin switching in Ikaros null mice. Proc. Natl. Acad. Sci. USA 99:602.[Abstract/Free Full Text]
  7. Molnar, A., K. Georgopoulos. 1994. The Ikaros gene encodes a family of functionally diverse zinc finger DNA-binding proteins. Mol. Cell. Biol. 14:8292.[Abstract/Free Full Text]
  8. Morgan, B., L. Sun, N. Avitahl, K. Andrikopoulos, T. Ikeda, E. Gonzales, P. Wu, S. Neben, K. Georgopoulos. 1997. Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J. 16:2004.[Medline]
  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.[Medline]
  10. Ito, T., S. Nomura, M. Okada, Y. Katsumata, F. Kikkawa, T. Rogi, M. Tsujimoto, S. Mizutani. 2002. Ap-2 and Ikaros regulate transcription of human placental leucine aminopeptidase/oxytocinase gene. Biochim. Biophys. Acta 290:1048.
  11. Sun, P., H. H. Loh. 2002. Transcriptional regulation of mouse {delta}-opioid receptor gene: role of Ikaros in the stimulated transcription of mouse {delta}-opioid receptor gene in activated T cells. J. Biol. Chem. 277:12854.[Abstract/Free Full Text]
  12. Koipally, J., E. J. Heller, J. R. Seavitt, K. Georgopoulos. 2002. Unconventional potentiation of gene expression by Ikaros. J. Biol. Chem. 277:13007.[Abstract/Free Full Text]
  13. Ernst, P., K. Hahm, 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.[Abstract/Free Full Text]
  14. Brown, K. E., S. S. Guest, S. T. Smale, K. Hahm, M. Merkenschlager, A. G. Fisher. 1997. Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell 91:845.[Medline]
  15. Koipally, J., J. Kim, B. Jones, A. Jackson, N. Avitahl, S. Winandy, M. Trevisan, A. Nichogiannopoulou, C. Kelley, 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.[Medline]
  16. Brown, K. E., J. Baxter, D. Graf, M. Merkenschlager, A. G. Fisher. 1999. Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division. Mol. Cell 3:207.[Medline]
  17. Koipally, J., K. Georgopoulos. 2000. Ikaros interactions with CtBP reveal a repression mechanism that is independent of histone deacetylase activity. J. Biol. Chem. 275:19594.[Abstract/Free Full Text]
  18. Cobb, B. S., S. Morales-Alcelay, G. Kleiger, K. E. Brown, A. G. Fisher, S. T. Smale. 2000. Targeting of Ikaros to pericentromeric heterochromatin by direct DNA binding. Genes Dev. 14:2146.[Abstract/Free Full Text]
  19. Sabbattini, P., M. Lundgren, A. Georgiou, C. Chow, G. Warnes, N. Dillon. 2001. Binding of Ikaros to the {lambda}5 promoter silences transcription through a mechanism that does not require heterochromatin formation. EMBO J. 20:2812.[Medline]
  20. Trinh, L. A., R. Ferrini, B. S. Cobb, A. S. Weinmann, K. Hahm, P. Ernst, I. P. Garraway, M. Merkenschlager, 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.[Abstract/Free Full Text]
  21. Dorsam, G., 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.[Abstract/Free Full Text]
  22. Hahm, K., P. Ernst, K. Lo, G. S. Kim, C. Turck, 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.[Abstract/Free Full Text]
  23. Molnar, A., P. Wu, D. A. Largespada, A. Vortkamp, S. Scherer, N. G. Copeland, N. A. Jenkins, G. Bruns, 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.[Abstract]
  24. Sun, L., N. Heerema, L. Crotty, X. Wu, C. Navara, A. Vassilev, M. Sensel, G. H. Reaman, 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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  27. Nakayama, H., F. Ishimaru, Y. Katayama, K. Nakase, N. Sezaki, K. Takenaka, K. Shinagawa, K. Ikeda, K. Niiya, M. Harada. 2000. Ikaros expression in human hematopoietic lineages. Exp. Hematol. 28:1232.[Medline]
  28. Olivero, S., C. Maroc, E. Beillard, J. Gabert, W. Nietfeld, C. Chabannon, C. Tonnelle. 2000. Detection of different Ikaros isoforms in human leukaemias using real-time quantitative polymerase chain reaction. Br. J. Haematol. 110:826.[Medline]
  29. Payne, K. J., J. H. Nicolas, J. Y. Zhu, L. W. Barsky, G. M. Crooks. 2001. Cutting edge: predominant expression of a novel Ikaros isoform in normal human hematopoiesis. J. Immunol. 167:1867.[Abstract/Free Full Text]
  30. Wang, J. H., A. Nichogiannopoulou, L. Wu, L. Sun, A. H. Sharpe, M. Bigby, 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.[Medline]
  31. Georgopoulos, K., M. Bigby, J. H. Wang, A. Molnar, P. Wu, S. Winandy, A. Sharpe. 1994. The Ikaros gene is required for the development of all lymphoid lineages. Cell 79:143.[Medline]
  32. Winandy, S., L. Wu, J. H. Wang, 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.[Abstract/Free Full Text]
  33. Avitahl, N., S. Winandy, C. Friedrich, B. Jones, Y. Ge, K. Georgopoulos. 1999. Ikaros sets thresholds for T cell activation and regulates chromosome propagation. Immunity 10:333.[Medline]
  34. Winandy, S., P. Wu, K. Georgopoulos. 1995. A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma. Cell 83:289.[Medline]
  35. Boggs, S. S., M. Trevisan, K. Patrene, K. Georgopoulos. 1998. Lack of natural killer cell precursors in fetal liver of Ikaros knockout mutant mice. Nat. Immun. 16:137.[Medline]
  36. Wu, L., A. Nichogiannopoulou, K. Shortman, K. Georgopoulos. 1997. Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity 7:483.[Medline]
  37. Dumortier, A., P. Kirstetter, P. Kastner, S. Chan. 2002. Ikaros regulates neutrophil differentiation. Blood 5:1336.
  38. Hao, Q. L., A. J. Shah, F. T. Thiemann, E. M. Smogorzewska, G. M. Crooks. 1995. A functional comparison of CD34+CD38- cells in cord blood and bone marrow. Blood 86:3745.[Abstract/Free Full Text]
  39. Hao, Q. L., E. M. Smogorzewska, L. W. Barsky, G. M. Crooks. 1998. In vitro identification of single CD34+CD38- cells with both lymphoid and myeloid potential. Blood 91:4145.[Abstract/Free Full Text]
  40. Hao, Q. L., J. Zhu, M. A. Price, K. J. Payne, L. W. Barsky, G. M. Crooks. 2001. Identification of a novel, human multilymphoid progenitor in cord blood. Blood 97:3683.[Abstract/Free Full Text]
  41. Kansas, G. S., M. J. Muirhead, 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.[Abstract/Free Full Text]
  42. Gooi, H. C., S. J. Thorpe, E. F. Hounsell, H. Rumpold, D. Kraft, O. Forster, 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.[Medline]
  43. Davidson, S. E., J. L. McKenzie, M. E. Beard, D. N. Hart. 1988. The tissue distribution of the 3 {alpha}-fucosyl-N-acetyl lactosamine determinant recognized by the CD15 monoclonal antibodies CMRF-7 and 27. Pathology 20:24.[Medline]
  44. Wright, S. D., P. S. Tobias, R. J. Ulevitch, 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.[Abstract/Free Full Text]
  45. Skubitz, K., K. Micklem, E. van der Schoot. 1995. CD66 and CD67 cluster workshop report. B. L. Schlossmann, and W. Gilks, and J. Harlan, and T. Kishimoto, and C. Morimoto, and J. Ritz, and T. A. Springer, and T. F. Tedder, and R. F. Todd, eds. In Leukocyte Typing V Vol. 1:889. Oxford Univ. Press, Oxford.
  46. Eades-Perner, A. M., J. Thompson, H. van der Putten, W. Zimmermann. 1998. Mice transgenic for the human CGM6 gene express its product, the granulocyte marker CD66b, exclusively in granulocytes. Blood 91:663.[Abstract/Free Full Text]
  47. Lian, Z., L. Wang, S. Yamaga, W. Bonds, Y. Beazer-Barclay, Y. Kluger, M. Gerstein, P. E. Newburger, N. Berliner, S. M. Weissman. 2001. Genomic and proteomic analysis of the myeloid differentiation program. Blood 98:513.[Abstract/Free Full Text]
  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, D. G. Beer. 2002. Discordant protein and mRNA expression in lung adenocarcinomas. Mol. Cell. Proteomics 1:304.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  51. Georgopoulos, K., S. Winandy, N. Avitahl. 1997. The role of the Ikaros gene in lymphocyte development and homeostasis. Annu. Rev. Immunol. 15:155.[Medline]
  52. Okano, H., Y. Saito, T. Miyazawa, T. Shinbo, D. Chou, S. Kosugi, Y. Takahashi, S. Odani, O. Niwa, R. Kominami. 1999. Homozygous deletions and point mutations of the Ikaros gene in gamma-ray-induced mouse thymic lymphomas. Oncogene 18:6677.[Medline]
  53. Karlsson, A., P. Soderkvist, S. M. Zhuang. 2002. Point mutations and deletions in the znfn1a1/ikaros gene in chemically induced murine lymphomas. Cancer Res. 62:2650.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
T. Ronni, K. J. Payne, S. Ho, M. N. Bradley, G. Dorsam, and S. Dovat
Human Ikaros Function in Activated T Cells Is Regulated by Coordinated Expression of Its Largest Isoforms
J. Biol. Chem., January 26, 2007; 282(4): 2538 - 2547.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
E. Yamamoto, T. Ito, A. Abe, F. Sido, K. Ino, A. Itakura, S. Mizutani, S. Dovat, S. Nomura, and F. Kikkawa
Ikaros is expressed in human extravillous trophoblasts and involved in their migration and invasion
Mol. Hum. Reprod., November 1, 2005; 11(11): 825 - 831.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. M. Cupit, J. D. Hansen, A. S. McCarty, G. White, M. Chioda, F. Spada, S. T. Smale, and C. Cunningham
Ikaros Family Members from the Agnathan Myxine glutinosa and the Urochordate Oikopleura dioica: Emergence of an Essential Transcription Factor for Adaptive Immunity
J. Immunol., December 1, 2003; 171(11): 6006 - 6013.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Payne, K. J.
Right arrow Articles by Crooks, G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Payne, K. J.
Right arrow Articles by Crooks, G. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS