Role of a non-canonical splice variant of the Helios gene in the differentiation of acute lymphoblastic leukemic T cells

  • Authors:
    • Yinghui Li
    • Yanhua Liu
    • Can Liu
    • Fengyong Liu
    • Daolei Dou
    • Wenjie Zheng
    • Wei Liu
    • Feifei Liu
  • View Affiliations

  • Published online on: March 8, 2018     https://doi.org/10.3892/ol.2018.8214
  • Pages: 6957-6966
  • Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

T-cell acute lymphoblastic leukemia is a hematopoietic malignant disease, which arises from a genetic defect in the T‑cell maturation signaling pathway. As a result, it is necessary to identify the molecules that impact T‑cell development and control lymphoid‑lineage malignancy. The present study utilized Jurkat T lymphoblastic cells as a well‑established approach for the investigation into the function of the non‑canonical alternative splice variant of Helios for the in vitro study of T‑cell differentiation and leukemogenesis. In the present study, the Jurkat T‑cell lines with stable overexpression of the wild‑type (Helios‑1) or the non‑canonical short isoform (Helios‑Δ326‑1431), were established. RNA microarray, reverse transcription‑quantitative polymerase chain reaction and flow cytometry were used to assess changes in the gene expression profiles and to monitor the cell surface markers during T‑cell differentiation. Multiple genes associated with T‑cell differentiation and leukemogenesis were identified as being either activated or suppressed. In addition, the results indicated that the stable overexpression of the Helios isoforms stimulated the differentiation pathway of the T‑lineage lymphoblastic cells. Therefore, these results suggest that full‑length Helios‑1 has a tumor suppressor‑like and immunomodulatory role, in contrast to the oncogenic function of the non‑canonical short isoform Helios-Δ326-1431.

Introduction

The expressions of the distinct transcription factors, target genes, and cell surface molecules dictate the lineage commitment and differentiation of T lymphocytes. The transcription factors, such as Notch1, E2A, Id proteins, Ikaros, and PU.1 perform definitive functions in the T-cell differentiation (1). Along the pathway of commitment and maturation, the early T-cell precursors upregulate the expressions of the transcription factors, such as Notch1, Ikaros, GATA3, and Runx1. However, they downregulate others, such as C/EBPa, Lmo2, GATA2, and PU.1 (2,3). The expression patterns of the transcription factors are fundamental to the lineage commitment, specification, differentiation, and survival of the T-cells. Significantly, these regulatory factors which control normal development are frequently disturbed, and also implicated in T-cell transformations and leukemia (2).

Helios (Ikzf2), which is a zinc-finger DNA binding transcription factor and a key regulator of T-lineage differentiation, is a prime example (4,5). Helios belongs to the Ikaros transcription factor family, and shares a common structure, which is characterized by two zinc finger domains, an N-terminal DNA-binding domain (core motif GGGAA), and a C-terminal dimerization domain (4,6). Helios is specifically expressed in the T-cell lineage from the early stages of development. Furthermore, it has been shown that Helios is selectively induced by >10-fold in thymus-derived Foxp3+ regulatory T (Treg) cells, and also regulates the differentiation of T helper cells and production of cytokines (7,8). In addition, Helios augments the activation of Foxp3 by directly binding to the Foxp3 promoter (9). Previous research studies have demonstrated that Helios interacts with the nucleosome remodeling and histone deacetylase (NuRD) complex, which suggests that Helios plays a pivotal role in chromatin remodeling, as well as the expression of target genes (10).

It is known that Helios controls lymphopoiesis and leukemogenesis (6,1114). Recently we and other researchers identified multiple novel short isoforms of Helios which were overexpressed in patients with T-cell acute lymphoblastic leukemia, and demonstrated their dominant-negative function (6,11). The peripheral blood mononuclear cells (PBMCs) of some of the T-cell leukemia patients expressed various short protein isoforms (<55 kDa), which were not detected in healthy PBMCs. We found that one of these isoforms (Helios-Δ326-1431, 475 bp) lacks part of exon 3, all of exons 4 to 6, and embodies a nonsense mutation in exon 7 (6). Also, it has been determined that this novel short isoform lacks four N-terminal zinc fingers, which suggests that it is a putative dominant-negative isoform for the Ikaros gene family members (6). Therefore, the alternative splicing of Helios variants is possibly activated during leukemogenesis, and supports the role of a non-canonical isoform as an acute T-cell leukemic-type gene (ATL). These speculations have led to increased interest in the potential role of Helios in acute T-cell leukemia. However, the essential functions of the ATL-type Helios isoform have yet to be fully elucidated.

In this study, the ectopic overexpressions of the wild-type Helios-1, non-canonical short isoform Helios-Δ326-1431, and control in the Jurkat cells were utilized as the routine in vitro model of the T-cell acute lymphoblastic leukemia (T-ALL) for the purpose of investigating the function of Helios isoforms in the expression of important genes which are changed during T-cell development. This study examined in detail the modulated gene expression pattern using microarray. The results showed that the Helios isoforms regulated the transcriptional output of the target genes, as well as the epigenetic remodelers involved in leukocyte proliferation, cell cycle arrest, and growth. In addition, the Jurkat cells consisted of undifferentiated T lymphoblasts (15). The impact of the overexpression of the Helios isoforms on the differentiation of the Jurkat cells was also examined.

Materials and methods

Cell culture

In this study, the Jurkat cell line was purchased from the American Type Culture Collection (http://www.ATCC.com), and cultured as suggested by the manufacturer. The Jurkat T-cell lines with stable overexpression of wild-type Helios-1, non-canonical short isoform Helios-Δ326-1431, and control were established with lentiviral pLV-EF1α-MCS-IRES-Bsd expression vector (6). After selection by blasticidin (2 µg/ml) and confirmation by westernblot (6), the Jurkat cell lines with stable expression of Helios variants were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) which contained the following: 10% fetal calf serum, NaHCO3 1.5 g/l, glucose 2.5 g/l, sodium pyruvate 0.11 g/l, and 50 U/ml penicillin and 50 g/ml streptomycin at 37°C in 5% CO2 (6). The 293T cells were cultured in DMEM medium containing 10% FCS.

mRNA extraction, reverse transcription and quantitative PCR assays

The total RNA was extracted from the harvested Jurkat cell lines by using 1 ml of TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) per 5×106 cells. The first-strand cDNA was generated from 2 mg of the total RNA with the cDNA synthesis kit (Promega, Madison WI). The amplification of the Helios target transcripts was carried out with GoTaq qPCR master mix (Promega Corporation, Madison, WI, USA) in an ABI Prism 7000 analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). The data were normalized using endogenous β-actin and GAPDH controls. The fold-changes of the expression pattern were calculated using a ΔΔCq method according to the instructions (Applied Biosystems). The nucleotide sequences for the primers were as follows: FOXN2 forward (F): CATCCAGGTCTAGCGTGTCT, reverse (R): TGCATAGCCACTGTCTCCAA; RUNX3 (F): CCCCTCCGTTCCTAACTGTT, (R) CCCTGCCAAGAGAACAGAGA; WNT3 (F): TCCATGCAGTTCCCAAGGAT, (R) TGAAATCCATGTGCCTCCCT; MLLT4 (F): CACATCGTGGACATGCTGAG, (R) CATCATCGTCCTCCTCCTCC; MAST3 (F): CACCTCCCGCTACTTCCTAG, (R) AGTCTTAATGCCTTGGCCCT; MSX2 (F): ATATGAGCCCTACCACCTGC, (R) GCTTTTCCAGTTCTGCCTCC; TP53 (F): TGGCCATCTACAAGCAGTCA, (R) GGTACAGTCAGAGCCAACCT; CD79A(F): CTTCCCTCTAAACTGCCCCA, (R) CACTAAGTGGCCCTGACAGA; NDFIP2 (F): GTTTTATCCCGTGCCACCTC, (R) GCTGGTCTGCATCACTGAAG; CD40LG (F): TCAAATTGCGGCACATGTCA, (R) TGACTCGAAGCTTCCCGATT; SUMO1 (F): CTTCAACTGAGGACTTGGGG, (R) TCAGCAATTCTCTGACCCTCA; NOTCH2NL (F): AACATCGAGACCCCTGTGAG, (R) ATTCAGGCAAGGTCGAGACA; TCEAL8 (F): TCGAGGTGAGGGAAGAGAGA, (R) CTTCTGCCTCCTGTGGTACA; ZNF593 (F): CTTGGATGAGATTCACCGCG, (R) AAGTGGGTCTTCAGGTTGGT; NUDC (F): TTTCAGCCACCACAATCAGC, (R) AGCCTCTCTGCCTCTTCATC; CBX8 (F): GAAATGGAAGGGATGGTCGC, (R) GAGGAAGGTTTTGGGCTTGG; HCST (F): TTGCTACTTCTCTGCTCCCC, (R) ATCCGGAACAAGAGCCTGAA; TAB1 (F): GCAGAGCCAGAAATCCATGG, (R) GCTTGGCAAACTCAGTGTCA; NKAP (F): ATGGCCATGCTCTGTTACCT, (R) AGGGCTCTCTTCTCATCAGC; TGFA (F): GAAGCCACAAAGCCGGTAAA, (R) ATACTTACCGAGGGCTCACG; TNFSF9 (F): GGCCCAAAATGTTCTGCTGA, (R) CAAGTGAAACGGAGCCTGAG; MSX2 (F): ATATGAGCCCTACCACCTGC, (R) GCTTTTCCAGTTCTGCCTCC; HOXD11 (F): GGCTACGCTCCCTACTACG, (R) GTAGAACTGGTCGAAGCCCT; HEYL (F): ATGCAAGCCAGGAAGAAACG, (R) AGAATCCTGTCCCACCAGTG; DGKQ (F): CACGTCTCCCTGTTTGTTGG, (R) CCATGTCCTTCAGCAGCATG.

Flow cytometry

In this study, FITC-conjugated antibodies against human CD3 (clone UCHT1), CD8a (RPA-T8), CD25 (M-A251), CD7 (M-T701), and the PE-conjugated antibody against human CD4 (RPA-T4) (BioLegend, Inc., San Diego, CA, USA) were used. Also, mouse IgG1 antibody was used as the isotype control. Following the labeling, the Jurkat cell lines were washed and suspended in an ice-cold staining media (phosphate buffer saline containing 5% FBS, and 100 U/ml penicillin/streptomycin). The samples were processed in a FACS LSRFortessa (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed using Flowjo software (Tree Star, Inc., Ashland, OR, USA).

Gene-expression microarray and pathway analysis

The RNA was isolated from the Jurkat cell lines with stable expressions of the Helios isoforms. Gene expression microarray was performed with GeneChip human gene 2.0 ST array (Affymetrix; Thermo Fisher Scientific, Inc.). The details of the methods of the microarray analysis were previously described (16). The gene chip data were deposited into the Gene Expression Omnibus (GEO) database with the accession number GSE92416.

Statistical analysis

Statistical differences were determined using the unpaired Student's t-test and Ordinary one-way ANOVA with PRISM software (version 6.0c; GraphPad Software, Inc., La Jolla, CA, USA) for P-values. Multiple comparisons between the groups were performed using Tukey's HSD test. P<0.05 was considered to indicate a statistically significant difference.

Results

Modulation of target gene expression by Helios-1

We established the Jurkat T-cell lines with stable overexpression of wild-type Helios-1, non-canonical short isoform Helios-Δ326-1431, or control by lentiviral transduction in our recent study (6). We then compared the microarray data for transcriptional analysis of the Jurkat cells with stable overexpression of full-length Helios-1 and mock transfected control (Fig. 1A). With a >1.5-fold cut-off, the analysis revealed that the genes encoding tumor-suppressors RUNX3 (1.639-fold) and TP53 (0.622) were deregulated in the Helios-1 cells. In addition, the expressions of the genes WNT3 (1.748-fold), FOXN2 (1.565), MLLT4 (1.715), TGFa (1.700), and TGFbR2 (0.607), which were involved in the regulation of human T-cell leukemia, were found to be deregulated in the Helios-1 Jurkat cells when compared to the control (Table I). The genes encoding lymphoid-lineage cell markers, such as CD7 (1.660-fold), CCL1 (1.599), CXCR3 (2.098), CD79a (0.517), and CD40 ligand (0.548), were determined to display altered expression patterns. The genes encoding innate immunity molecules, such as TLR3 (1.581) and LRRC37 (0.528), were also found to be modulated when compared to the control cell line.

Table I.

T-cell target genes of Helios-1.

Table I.

T-cell target genes of Helios-1.

Target geneLocationExpression during T-cell specificationFunction
RAB27B18q21.2UpregulatedSmall GTPase mediated signal transduction
PIK3R517p13.1UpregulatedCell growth, proliferation, differentiation, motility, survival and oncogenesis
WNT317q21.32UpregulatedOncogenesis, regulation of cell fate and patterning, morphogenesis
TGFA2p13.3UpregulatedGrowth factor activity, angiogenesis and cancer
CCL117q12UpregulatedMonocyte chemotaxis, immunoregulatory and inflammatory processes
IRF816q24.1UpregulatedRegulation of lineage commitment and in myeloid cell maturation
RUNX31p36.11UpregulatedTranscription factor, and tumor suppressor, hematopoiesis
RASIP119q13.33UpregulatedVascular-specific regulator of GTPase signaling, cell architecture, and adhesion
CXCR3Xq13.1UpregulatedG protein-coupled receptor for chemokine, involved in leukocyte traffic
RIPK421q22.3UpregulatedSerine/threonine protein kinase that interacts with PKC, activate NF-κB
CD717q25.3UpregulatedExpressed on on thymocytes and mature T cells, play a role in T-cell interactions
MLLT46q27UpregulatedInvolved in acute myeloid leukemias with t(6;11)(q27;q23) translocation
FOXN22p16.3UpregulatedTranscription factor activity, T-Cell leukemia
RALB2q14.2UpregulatedPathways in cancer and signaling by GPCR
IL36A2q14.1UpregulatedActivate NF-κB and MAPK signaling pathways in inflammatory response
EEF1A220q13.33UpregulatedProtein biosynthesis, enzymatic delivery of aminoacyl tRNAs to the ribosome
TP5317p13.1DownregulatedCell cycle arrest, apoptosis, senescence, DNA repair, and tumor formation
MSX25q35.2DownregulatedTranscription factor activity, development
TGFBR23p24.1DownregulatedTGF-β signaling pathway
CD40LGXq26.3DownregulatedT-cell immune responses, T-cell proliferation and cytokine production

[i] RAB27B, member RAS oncogene family; PIK3R5, phosphoinositide-3-kinase regulatory subunit 5; wnt3, wnt family member 3; TGFA, transforming growth factor α; CCL1, C-C motif chemokine ligand 1; IRF8, interferon regulatory factor 8; RUNX3, runt related transcription factor 3; RASIP1, ras interacting protein 1; CXCR3, C-X-C motif chemokine receptor 3; RIPK4, receptor interacting serine/threonine kinase 4; FOXN2, forkhead box N2; RALB, RAS like proto-oncogene B; IL36A, interleukin 36, α; EEF1A2, eukaryotic translation elongation factor 1 α 2; TP53, tumor protein P53; MSX2, Msh homeobox 2; TGFBR2, transforming growth factor β receptor 2; CD40LG, CD40 ligand.

The genome-wide transcriptional analysis of the Helios-1 and mock transfected Jurkat T-cell lines were used to compare and identify the potential pathways which may have the ability to support the aberrant growth properties of the mutant population (6). The group of genes which were deregulated in the Helios-1 T-cells indicated considerable enrichment for the genes which were involved in the pathways of the lymphocyte differentiation, lymphocyte proliferation, and regulation of cell growth (P<0.05) (Fig. 1B). The genes encoding lymphoid-lineage tyrosine kinases and costimulatory molecules (for example, TYRO3, SYK, CD79a and CD40 ligand), as well as those encoding molecules which were involved in the cell proliferation, hematopoiesis and leukemogenesis (for example, IRF8, TP53, WNT3, TGFbR2, BST2 and DDR3), were identified to share those pathways (Fig. 1B). In addition, the pathways of the immune effector process, immune response, and adaptive immune response, were determined to be enriched in the group of genes which had been deregulated in the Helios-1 overexpressed Jurkat cells (for example IL28b, APOBEC3C, TLR3, CCL1, CD7, and IL36). Furthermore, the overexpression of the zinc-finger transcription factor Helios-1 correlated with the pathways of the regulation of DNA binding and chromatin assembly [SUMO1, MSX2, histone cluster 1 (H1), H2, H3 and H4]. Also, this study detected and identified the differences between the Helios-1 and the control Jurkat cell lines in their expression of target genes, such as FOXN2 (1.363-fold), RUNX3 (1.142), MLLT4 (1.544), MAST3 (1.367), MSX2 (0.325), TP53 (0.513), CD79a (0.785), DNFIP2 (0.613), CD40LG (0.241), and SUMO1 (0.694), by utilizing quantitative PCR (Fig. 1C).

Modulation of the target gene expression by non-canonical short isoform Helios-Δ326-1431

The transcription profile regulated by the leukemic-type short isoform Helios-Δ326-1431 was evaluated through RNA analysis and microarray (Fig. 2A). Among the 940 genes (>1.5-fold cut-off) which were dependent on Helios-Δ326-1431 for expression in the Jurkat lymphoblast cells, the changes were clustered in T-cell leukemogenesis and cell fate decisions (Table II). Several members, such as JUN (1.667-fold), MGMT (1.514), HRAS (1.963), TFPT (1.589), TAB1 (1.613), and KLF10 (0.643), were identified to be strong candidates for regulating T-lineage differentiation and involved in the development of leukemia. Other members showed the capacity to promote cell-fate decisions and morphogenesis, and were noted to be potential regulators of T-lineage commitment and homeostasis. Examples of these were HOXB7 (1.721-fold), GSC (2.036), HOXD11 (0.639), HES6 (1.646), NOTCH2NL (0.625), NKAP (2.059), and MSX2 (0.568). In addition, the modulators of the RAS signaling RAB40B (1.867-fold), RASSF7 (1.739), RASIP1 (1.683), and RASL10B (0.616) were deregulated as Helios-Δ326-1431 target genes. In addition, RNA polymerase and translation machinery factors, such as TAF1D (1.611-fold), POLR2G (1.644), EEF1G (2.173), TCEAL8 (1.666) and EIF1AY (1.610), were determined to be potential targets of the Helios-Δ326-1431.

Table II.

T-cell target genes of Helios-Δ326-1431.

Table II.

T-cell target genes of Helios-Δ326-1431.

Target geneLocationExpression during T-cell specificationFunction
GSC14q32.13UpregulatedAct as a transcription factor in the development during embryogenesis
IL23A12q13.3UpregulatedStimulate the production of IFN-γ, act on memory CD4(+) T cells
CBX817q25.3UpregulatedTranscriptional repressor, chromatin remodeling
TFPT19q13.42UpregulatedAssociated with childhood leukemia
TGFA2p13.3UpregulatedActivate a signaling pathway for cell proliferation, differentiation and development
JUN1p32.1UpregulatedSequence-specific DNA binding to regulate gene expression, involved in malignancies
HCST19q13.12UpregulatedActivate PI3K dependent signaling pathways, cell survival and proliferation
HES62q37.3UpregulatedRegulate cell differentiation, participate in Notch-mediated HES/HEY network
NKAPXq24UpregulatedActivation of the ubiquitous transcription factor NF-κB
HRAS11p15.5UpregulatedSignal pathways by intrinsic GTPase activity, involved in development, cancer
TNFSF919p13.3UpregulatedInvolved in the antigen presentation process and in the generation of cytotoxic T cells
HOXB717q21.32UpregulatedTranscription factor that is involved in cell proliferation, differentiation and development
HEYL1p34.2DownregulatedAn effector of Notch signaling and a regulator of cell fate decisions
PPIP5K115q15.3DownregulatedIntracellular signaling pathways of Inositol phosphates
LTA6p21.33DownregulatedMediate a large variety of inflammatory, immunostimulatory, and antiviral responses
KLF108q22.3DownregulatedTranscriptional repressor, inhibitory activity of cancer growth

[i] GSC, goosecoid homeobox; IL23A, interleukin 23 subunit α; CBX8, chromobox 8; TFPT, TCF3 (E2A) fusion partner; TGFA, transforming growth factor α; JUN, jun proto-oncogene; HCST, hematopoietic cell signal transducer; HES6, hes family BHLH transcription factor 6; NKAP, NF-κB activating protein; HRAS, HRas proto-oncogene, GTPase; TNFSF9, tumor necrosis factor superfamily member 9; HOXB7, homeobox B7; HEYL, hairy/enhancer-of-split related with YRPW motif 3; PPIP5K1, diphosphoinositol pentakisphosphate kinase 1; LTA, lymphotoxin α; KLF10, Kruppel like factor 10.

Pathway analysis of the genes which were targeted by the non-canonical Helios-Δ326-1431 or by the wild-type Helios-1, provided insight into the functional consequences of the differences in the Helios isoforms which impact T-cell development and leukemogenesis (P<0.05). The genes which were associated with the Helios-Δ326-1431 showed considerable and specific enrichment of the pathways involved in cell growth, such as lymph node development (lymphotoxin), and the positive regulation of leukocyte proliferation (Vav3, SYK, IL23, CD24), as well as the cytokine-mediated signaling pathway (GBP1, IRF8, SUMO1, CCR8, EGR1, and CXCR3) (Fig. 2B). In addition, the lymphoid-specific genes which encoded the molecules involved in the positive regulation of leukocyte-mediated immunity, positive regulation of chronic inflammatory response, and Type-I interferon-mediated signaling pathway (ISG15, HLA-A, -C, and -E) were found to be specifically targeted by the Helios-Δ326-1431 gene. When compared with the full-length Helios-1, the non-canonical Helios-Δ326-1431 were associated with the translation, gene expression, and chromatin remodeling, such as the protein-DNA complex assembly (RPA2, RAD51 Recombinase, and H1 and H2), regulation of DNA binding (JUN, ID2, SUMO1, and HEY2), and chromatin assembly. These findings indicate that the non-canonical Helios-Δ326-1431 variant targeted the genetic networks that support lymphocyte differentiation and regulation of gene expressions.

In order to examine the changes in the expression of the T-lineage transcription factors and regulatory genes during the Jurkat differentiation, we measured the transcript levels of the Notch2NL, TCEAL8, ZNF593, NUDC, CBX8, HCST, TAB1, NKAP, TGFa, TNFSF9, MSX2, HOXD11, HEYL, and DGKQ in the Helios-Δ326-1431 overexpressed Jurkat cells, as well as the control cells, using real-time PCR (Fig. 2C). The results were consistent with microarray data, and showed significant upregulations of the TCEAL8 (1.462-fold), ZNF593 (1.699), CBX8 (1.425), TAB1 (1.761), NKAP (1.558), TGFa (1.309), and TNFSF9 (3.673). In contrast, downregulations were observed in the expressions of the HOXD11 (0.103), HEYL (0.111), and DGKQ (0.082).

Surface marker expression

In order to evaluate the role of the Helios isoforms in the differentiation of the T lymphoblasts (17), the expressions of the CD4, CD8, CD3, CD25, and CD7 on the cell surface of Jurkat cells were tested on the 30th day after the transduction by flow cytometry (Fig. 3). The results showed that the cells which expressed CD3 at day 30 were decreased in the Helios-Δ326-1431 transduced cells (47.6±3%) when compared with the groups of the Helios-1 transduced cells (59.0±4%), or the control (57.6±3%) (Fig. 3A). The CD25 expression was determined to be increased in the Helios-Δ326-1431 cells (3.59±0.5%) when compared to the Helios-1 (0.59±0.3%) or control cell groups (0.33±0.2%) (Fig. 3C). However, there was no difference between Helios-1 and control for CD3 and CD25 expression (Fig. 3). This is consistent with the report that Helios functions as a master transcription factor in CD4+ CD25+ regulatory T cells (9,18). The expression patterns of CD7, which plays a role in T-cell interactions (19), were not changed upon the stable overexpression of Helios isoforms (Fig. 3D). Therefore, the non-canonical Helios-Δ326-1431 isoform regulates the cell surface expression of the T-lineage markers.

Discussion

In this study, the effects of the alternative splicing variants of Helios on the differentiation of the Jurkat T lymphoblast cell line were evaluated. Also, the data of mRNA microarray were assessed, and the deregulated expression of the vital regulatory gene involved in the T-cell proliferation, differentiation, and leukemic transformation was confirmed. This led to the identification of the gene profiles which were controlled by the full-length Helios-1, as well as the non-canonical leukemic-type short isoform Helios-Δ326-1431.

The presence of the alternative splice Helios variants was first revealed in research studies utilizing the primary leukemic cells from patients with newly diagnosed ALL (1214). In this study, we observed that dominant-negative Helios-Δ326-1431 potentially induces the expression of several oncogenes. According to the results, the Helios-Δ326-1431 significantly induced the upregulation of the molecules, such as the JUN proto-oncogene, which may be involved in the leukemogenesis of Helios-deregulated T-ALL (20). In addition, we observed that ectopic expression of Helios-Δ326-1431 led to the upregulated expression of HRAS, which exhibited leukemogenic potential in myeloid-lineage leukemia (21). Furthermore, this study identified that other components of the RAS signaling pathway, such as RASSF7, RAB40B, and RASIP1, were upregulated in the Helios-Δ326-1431 overexpressed cells. Interestingly, TFPT [TCF3 (E2A) fusion partner], which has been implicated in childhood leukemia (22), was also found to be upregulated in the Helios-Δ326-1431 cells. These targeted genes consolidated the activation of the programs which promoted survival and cell proliferation, even though their deregulation led to leukemic transformation. The overexpression of non-canonical Helios may provide aberrant survival properties to the differentiating thymocytes, which are predisposed for further selection in the activations of oncogene mutations and malignant phenotypes. These analyses from the microarray data indicate that the non-canonical short Helios isoform functions as an oncogenic variant and initiates leukemogenesis.

This study also identified that the genes which encode the T-lineage cell markers and chemotaxis molecules, such as CD7, CCL1, and CXCR3, are upregulated in the wild-type Helios-1 overexpressed Jurkat cells, whereas the B-lineage cell marker CD79a are downregulated. It has been reported that regulatory T-cells initiate recruitment and suppressive function via a CCL1 dependent pathway (23,24). In addition, the CXCR3 signaling directly induces the mobilization and recruitment of Tregs (25). Therefore, the Helios-1 may function as the transcription factor for regulatory T-cells by targeting the CCL1 and CXCR3 molecules. The microarray data also confirmed that the tyrosine kinases, TAM family receptor tyrosine kinase TYRO3 and SYK, are upregulated in the Helios-1 overexpressed Jurkat T-cells. The TYRO3 functions as a negative regulator of type 2 immunity (26), whereas the SYK has the ability to modulate the CD4+ T cell response (27). The GO analysis indicates that the pathways of the immune effector process and immune response (for example IL28, IL36, and TLR3) are enriched in the Helios-1 T cells. The results of the research by Asanuma et al were in agreement with this finding, and confirmed that the Helios variants regulated the pathway of the TNF receptor binding (11). Therefore, consistent with previous data, these deregulated patterns of gene expression suggest that wild-type Helios-1 has an immunomodulatory role in T-cell development.

It is interesting to note that the results of the previous research demonstrated that the dominant-negative isoform of Ikaros, Ik-6 was overexpressed in patients with blast crisis of chronic myelogenous leukemia, and acute B-lymphoblastic leukemia (28). These findings suggest that Ikaros plays the role of a tumor suppressor gene in the myeloid and B-cell lineages (28). In support of this viewpoint, Mullighan et al defined the oncogenic lesions that cooperate with BCR-ABL1 to induce ALL by genome-wide analysis, and identified that Ikaros was deleted in 83.7% of BCR-ABL1 lymphoblastic leukemia (29). In contrast, the T-cell malignancies were only observed in the gene-targeted mice of Ikaros (30). The related gene Helios has been shown to frequently exhibit multiple dominant-negative isoforms in patients with T-cell acute leukemia (6,1114). In the mouse model, the overexpression of the artificial dominant-negative Helios isoform leads to increased T-cell proliferation, as well as the development of T-cell lymphomas (31). However, in Helios-deficient mouse line, Helios is not essential for the development, homeostasis, and function of the thymic-derived T lymphocytes, which suggests that other Ikaros family members possibly compensate for the Helios in the T-cells (32). Overall, these data indicate that the coordinated deregulation of the Ikaros gene family may lead to human hematologic malignancies.

In this study, the roles of Helios in target gene activations and repressions, as well as T-cell development, were investigated. The findings in this study suggest that the full-length Helios-1 may play a decisive role in shaping the Treg cell identity. In addition, Helios family members are thought to coordinate gene transcription through chromatin remodeling, and previous research showed that Ikaros and Helios interact in the nucleosome remodeling complex of DNA-dependent ATPase Mi-2 and histone deacetylases (6,33). Thus, it is possible that Helios-1 modulates the expression of CD25, which is the marker for distinguishing Treg cells (Fig. 3C). In contrast, the proportion of CD25+ cells was increased in the Jurkat T lymphoblast cells that were stably transduced with dominant-negative Helios-Δ326-1431. Therefore, the dominant-negative Helios-Δ326-1431 isoform promotes the leukemogenesis and T-cell differentiation by inhibiting the activity of the functional Ikaros proteins, and also defining the leukemic transcriptional program, such as HRAS, TGF, and JUN.

The events initiated by Helios overexpression consolidate activation of the gene expression programs, which promote cell growth and survival. When deregulated, this process gives rise to leukemic transformation. The alternative splicings of Helios isoforms are part of a regulatory mechanism, which is effective during T-cell development and leukemogenesis. Helios also controls a network of epigenetic and transcriptional regulators during the normal T-cell development and leukemogenesis. Therefore, targeting the transcriptional regulation of Helios may open new avenues for leukemia treatment.

Acknowledgements

The authors would like to thank Dr. Alan Holt and Dr. Phillip Bryant for their assistance in editing this manuscript.

Funding

This study was supported by grants from National Science Foundation (grant no. NSF 81202309; nsfc.gov.cn/), Tianjin City Foundation of Basic and Advanced Research (grant no. 13JCQNJC11200; tstc.gov.cn/) and the New Teacher Fund from the Ministry of Education (grant no. 20120031120057) to FL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

YIL, YAL and CL performed experiments and analyzed the data. FL designed research, analyzed data and wrote the paper. FL, JL and DL performed experiments and provided technical support. WZ and WL assisted with the experiments.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Yui MA and Rothenberg EV: Developmental gene networks: A triathlon on the course to T cell identity. Nat Rev Immunol. 14:529–545. 2014. View Article : Google Scholar : PubMed/NCBI

2 

Gómez-del Arco P, Kashiwagi M, Jackson AF, Naito T, Zhang J, Liu F, Kee B, Vooijs M, Radtke F, Redondo JM and Georgopoulos K: Alternative promoter usage at the Notch1 locus supports ligand-independent signaling in T cell development and leukemogenesis. Immunity. 33:685–698. 2010. View Article : Google Scholar : PubMed/NCBI

3 

McCormack MP, Shields BJ, Jackson JT, Nasa C, Shi W, Slater NJ, Tremblay CS, Rabbitts TH and Curtis DJ: Requirement for Lyl1 in a model of Lmo2-driven early T-cell precursor ALL. Blood. 122:2093–2103. 2013. View Article : Google Scholar : PubMed/NCBI

4 

Kelley CM, Ikeda T, Koipally J, Avitahl N, Wu L, Georgopoulos K and Morgan BA: Helios, a novel dimerization partner of Ikaros expressed in the earliest hematopoietic progenitors. Curr Biol. 8:508–515. 1998. View Article : Google Scholar : PubMed/NCBI

5 

Kim HJ, Barnitz RA, Kreslavsky T, Brown FD, Moffett H, Lemieux ME, Kaygusuz Y, Meissner T, Holderried TA, Chan S, et al: Stable inhibitory activity of regulatory T cells requires the transcription factor Helios. Science. 350:334–339. 2015. View Article : Google Scholar : PubMed/NCBI

6 

Zhao S, Liu W, Li Y, Liu P, Li S, Dou D, Wang Y, Yang R, Xiang R and Liu F: Alternative splice variants modulates dominant-negative function of Helios in T-Cell leukemia. PLoS One. 11:e01633282016. View Article : Google Scholar : PubMed/NCBI

7 

Fontenot JD, Rasmussen JP, Gavin MA and Rudensky AY: A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol. 6:1142–1151. 2005. View Article : Google Scholar : PubMed/NCBI

8 

Hill JA, Feuerer M, Tash K, Haxhinasto S, Perez J, Melamed R, Mathis D and Benoist C: Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature. Immunity. 27:786–800. 2007. View Article : Google Scholar : PubMed/NCBI

9 

Getnet D, Grosso JF, Goldberg MV, Harris TJ, Yen HR, Bruno TC, Durham NM, Hipkiss EL, Pyle KJ, Wada S, et al: A role for the transcription factor Helios in human CD4(+)CD25(+) regulatory T cells. Mol Immunol. 47:1595–1600. 2010. View Article : Google Scholar : PubMed/NCBI

10 

Sridharan R and Smale ST: Predominant interaction of both Ikaros and Helios with the NuRD complex in immature thymocytes. J Biol Chem. 282:30227–30238. 2007. View Article : Google Scholar : PubMed/NCBI

11 

Asanuma S, Yamagishi M, Kawanami K, Nakano K, Sato-Otsubo A, Muto S, Sanada M, Yamochi T, Kobayashi S, Utsunomiya A, et al: Adult T-cell leukemia cells are characterized by abnormalities of Helios expression that promote T cell growth. Cancer Sci. 104:1097–1106. 2013. View Article : Google Scholar : PubMed/NCBI

12 

Nakase K, Ishimaru F, Fujii K, Tabayashi T, Kozuka T, Sezaki N, Matsuo Y and Harada M: Overexpression of novel short isoforms of Helios in a patient with T-cell acute lymphoblastic leukemia. Exp Hematol. 30:313–317. 2002. View Article : Google Scholar : PubMed/NCBI

13 

Fujii K, Ishimaru F, Nakase K, Tabayashi T, Kozuka T, Naoki K, Miyahara M, Toki H, Kitajima K, Harada M and Tanimoto M: Over-expression of short isoforms of Helios in patients with adult T-cell leukaemia/lymphoma. Br J Haematol. 120:986–989. 2003. View Article : Google Scholar : PubMed/NCBI

14 

Tabayashi T, Ishimaru F, Takata M, Kataoka I, Nakase K, Kozuka T and Tanimoto M: Characterization of the short isoform of Helios overexpressed in patients with T-cell malignancies. Cancer Sci. 98:182–188. 2007. View Article : Google Scholar : PubMed/NCBI

15 

Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, et al: A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 129:1401–1414. 2007. View Article : Google Scholar : PubMed/NCBI

16 

Joshi I, Yoshida T, Jena N, Qi X, Zhang J, Van Etten RA and Georgopoulos K: Loss of Ikaros DNA-binding function confers integrin-dependent survival on pre-B cells and progression to acute lymphoblastic leukemia. Nat Immunol. 15:294–304. 2014. View Article : Google Scholar : PubMed/NCBI

17 

Saki N, Abroun S, Soleimani M, Mortazavi Y, Kaviani S and Arefian E: The roles of miR-146a in the differentiation of Jurkat T-lymphoblasts. Hematology. 19:141–147. 2014. View Article : Google Scholar : PubMed/NCBI

18 

Thornton AM, Korty PE, Tran DQ, Wohlfert EA, Murray PE, Belkaid Y and Shevach EM: Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol. 184:3433–3441. 2010. View Article : Google Scholar : PubMed/NCBI

19 

Mitchell JL, Seng A and Yankee TM: Ikaros, Helios, and Aiolos protein levels increase in human thymocytes after β selection. Immunol Res. 64:565–575. 2016. View Article : Google Scholar : PubMed/NCBI

20 

Kollmann K, Heller G, Ott RG, Scheicher R, Zebedin-Brandl E, Schneckenleithner C, Simma O, Warsch W, Eckelhart E, Hoelbl A, et al: c-JUN promotes BCR-ABL-induced lymphoid leukemia by inhibiting methylation of the 5′ region of Cdk6. Blood. 117:4065–4075. 2011. View Article : Google Scholar : PubMed/NCBI

21 

Parikh C, Subrahmanyam R and Ren R: Oncogenic NRAS, KRAS, and HRAS exhibit different leukemogenic potentials in mice. Cancer Res. 67:7139–7146. 2007. View Article : Google Scholar : PubMed/NCBI

22 

Brambillasca F, Mosna G, Ballabio E, Biondi A, Boulukos KE and Privitera E: Promoter analysis of TFPT (FB1), a molecular partner of TCF3 (E2A) in childhood acute lymphoblastic leukemia. Biochem Biophys Res Commun. 288:1250–1257. 2001. View Article : Google Scholar : PubMed/NCBI

23 

Mira E, León B, Barber DF, Jiménez-Baranda S, Goya I, Almonacid L, Márquez G, Zaballos A, Martínez-A C, Stein JV, et al: Statins induce regulatory T cell recruitment via a CCL1 dependent pathway. J Immunol. 181:3524–3534. 2008. View Article : Google Scholar : PubMed/NCBI

24 

Hoelzinger DB, Smith SE, Mirza N, Dominguez AL, Manrique SZ and Lustgarten J: Blockade of CCL1 inhibits T regulatory cell suppressive function enhancing tumor immunity without affecting T effector responses. J Immunol. 184:6833–6842. 2010. View Article : Google Scholar : PubMed/NCBI

25 

Li CX, Ling CC, Shao Y, Xu A, Li XC, Ng KT, Liu XB, Ma YY, Qi X, Liu H, et al: CXCL10/CXCR3 signaling mobilized-regulatory T cells promote liver tumor recurrence after transplantation. J Hepatol. 65:944–952. 2016. View Article : Google Scholar : PubMed/NCBI

26 

Chan PY, Silva Carrera EA, De Kouchkovsky D, Joannas LD, Hao L, Hu D, Huntsman S, Eng C, Licona-Limón P, Weinstein JS, et al: The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity. Science. 352:99–103. 2016. View Article : Google Scholar : PubMed/NCBI

27 

Chauhan AK, Moore TL, Bi Y and Chen C: FcγRIIIa-Syk Co-signal modulates CD4+ T-cell response and Up-regulates Toll-like Receptor (TLR) expression. J Biol Chem. 291:1368–1386. 2016. View Article : Google Scholar : PubMed/NCBI

28 

Beer PA, Knapp DJ, Miller PH, Kannan N, Sloma I, Heel K, Babovic S, Bulaeva E, Rabu G, Terry J, et al: Disruption of IKAROS activity in primitive chronic-phase CML cells mimics myeloid disease progression. Blood. 125:504–515. 2015. View Article : Google Scholar : PubMed/NCBI

29 

Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J, White D, Hughes TP, Le Beau MM, Pui CH, et al: BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature. 453:110–114. 2008. View Article : Google Scholar : PubMed/NCBI

30 

Winandy S, Wu P and Georgopoulos K: A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma. Cell. 83:289–299. 1995. View Article : Google Scholar : PubMed/NCBI

31 

Zhang Z, Swindle CS, Bates JT, Ko R, Cotta CV and Klug CA: Expression of a non-DNA-binding isoform of Helios induces T-cell lymphoma in mice. Blood. 109:2190–2197. 2007. View Article : Google Scholar : PubMed/NCBI

32 

Cai Q, Dierich A, Oulad-Abdelghani M, Chan S and Kastner P: Helios deficiency has minimal impact on T cell development and function. J Immunol. 183:2303–2311. 2009. View Article : Google Scholar : PubMed/NCBI

33 

Kim J, Sif S, Jones B, Jackson A, Koipally J, Heller E, Winandy S, Viel A, Sawyer A, Ikeda T, et al: Ikaros DNA-binding proteins direct formation of chromatin remodeling complexes in lymphocytes. Immunity. 10:345–355. 1999. View Article : Google Scholar : PubMed/NCBI

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May-2018
Volume 15 Issue 5

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Spandidos Publications style
Li Y, Liu Y, Liu C, Liu F, Dou D, Zheng W, Liu W and Liu F: Role of a non-canonical splice variant of the Helios gene in the differentiation of acute lymphoblastic leukemic T cells. Oncol Lett 15: 6957-6966, 2018
APA
Li, Y., Liu, Y., Liu, C., Liu, F., Dou, D., Zheng, W. ... Liu, F. (2018). Role of a non-canonical splice variant of the Helios gene in the differentiation of acute lymphoblastic leukemic T cells. Oncology Letters, 15, 6957-6966. https://doi.org/10.3892/ol.2018.8214
MLA
Li, Y., Liu, Y., Liu, C., Liu, F., Dou, D., Zheng, W., Liu, W., Liu, F."Role of a non-canonical splice variant of the Helios gene in the differentiation of acute lymphoblastic leukemic T cells". Oncology Letters 15.5 (2018): 6957-6966.
Chicago
Li, Y., Liu, Y., Liu, C., Liu, F., Dou, D., Zheng, W., Liu, W., Liu, F."Role of a non-canonical splice variant of the Helios gene in the differentiation of acute lymphoblastic leukemic T cells". Oncology Letters 15, no. 5 (2018): 6957-6966. https://doi.org/10.3892/ol.2018.8214