Table 1.
Clinical characteristics of patients with T-cell leukemia and myeloid leukemia.
Fig 1.
Aberrant expression of Helios transcripts in cell lines and primary T-cell or myeloid leukemic cells.
(A) RT-PCR to detect Helios expression in hematopoietic cells derived from human T leukemic (Jurkat, CCRF-CEM, MOLT-4), B leukemic (Val, SUDHL4, HMy2.CIR) and myeloid cell lines (HL-60, U937). Samples of non-hematopoietic lineage cells lines derived from breast cancer (MCF-7, MDA-MB 231), hepatic cancer (HepG2, SK-Hep1), renal cancer (HEK 293T), and mesenchymal stem cells (MSC) were included for the test. To identify alternative splicing isoforms, RT-PCR analyses were performed with the primers designed in exons 1 and 7 of canonical full-length Helios transcript. Amplified cDNAs were cloned and sequencing analyses were performed. (B) Westernblot analysis of Helios in hematopoietic and non-hematopoietic lineage cell lines to show the presence of multiple Helios isoforms. The molecular weights of Helios short variants were 53 kDa (Helios V1), 10 kDa (Helios-V2) and 16 kDa (Helios-V3). (C) Increased expression of Helios short variants in hematological malignancies. RT-PCR analysis of Helios mRNA was performed in peripheral mononuclear blood cells from individuals with acute T-cell-derived leukemia (n = 9) or myeloid leukemia (n = 3), as well as samples from healthy subjects. (D) Diagram of Helios-1, Helios-2, and short Helios splice variants identified in this study. Helios variant 1 (V1), variant 2 (V2) and variant 3 (V3) are novel alternative splice isoforms identified in leukemic cell lineages. The brown rectangles in exons 3–5 represent the four N-terminal zinc finger motifs utilized in DNA consensus-sequence binding. The two purple rectangles in exon 7 show the C-terminal zinc finger motifs. Three novel Helios isoforms are nominated according to the exons or nucleotides deleted from the wild-type Helios transcript. The canonical full-length Ikaros 1 is included for comparison.
Fig 2.
Subcellular distribution of leukemia-type Helios isoforms.
(A, B) Cos-7 cells were transfected with the indicated five Helios isoforms and subjected to RT-PCR (A) and Western blot analyses (B). The COS-7 cell extracts were developed with the mouse monoclonal anti-Flag antibody (left) and an anti-Helios antibody (right). (C) Immunofluorescence localization of Helios proteins. The COS-7 cells were transfected with different Helios isoforms. The cellular localization of each protein was visualized with the anti-Flag antibody and then stained with a secondary anti-mouse antibody conjugated with Cy2 (green). The nuclei were counterstained by DAPI staining (blue). Helios-V1, Helios variant 1; Helios-V2, Helios variant 2; Helios-V3, Helios variant 3. (D) Graphic representation of the subcellular localization of Helios alternative-splicing variants. The analysis was conducted by counting nuclear or cytoplasmic staining, or both (nuclear and cytoplasmic), of the five isoforms as percent point. Eight fields were counted for each transfection.
Fig 3.
In vivo interaction between Helios isoforms and histone deacetylase (A) Association of Helios splice variants and constituents of the Mi-2β/NuRD and SIN3 histone deacetylase complexes. The Helios complexes were immunoprecipitated with an anti-FLAG antibody and the eluates were subjected to Westernblot with anti-FLAG, anti-SIN3A, anti-MTA2, and anti-HDAC1 antibodies which identify the sub-fractions of the Mi-2β/NuRD complex. (B) Confocal microscopy of untransfected COS-7 cells to show the nuclear localization of HDAC1, SIN3A and MTA2. (C-E) Visualization of COS-7 cells transfected with each Helios isoform by confocal microscopy. Helios splice variants were visualized with an anti-FLAG and a secondary Ab conjugated with Cy2 (green). The proteins HDAC1 (C), MTA2 (D) and SIN3A (E) were stained by antibodies conjugated with Cy3 (red). The nuclei were detected by DAPI staining (blue).
Fig 4.
Enrichment of Helios isoforms at the Foxp3 promoter.
(A) Clustal alignment of human, monkey (COS-7 cells) and mouse genomic sequences showing the conservation of Foxp3 core promoter. The black arrow indicates the location of transcription start site. The locations of the CAAT box, GC box and TATA box are shown by black box. Green boxes illustrate the CpG dinucleotides. The binding sites of transcription factor NF-AT, AP-1 and Helios are marked with red, blue and yellow boxes, respectively. (B) ChIP-coupled PCR assay of the enrichment of Helios isoforms at binding motifs at distinct positions on the Foxp3 promoter. ChIP was performed using anti-Flag and anti-RNA polymerase II (positive control) antibodies in COS-7 cells transfected with different Helios isoforms. Negative controls (Neg) are also shown, as are the Input and bound fractions.
Fig 5.
Helios short splice variants exhibit dominant-negative function in the suppressive activities of full-length Ikaros or Helios.
(A) Co-localization of wild-type Ikaros and leukemia-type short Helios isoforms. COS-7 cells were co-transfected with pLV expression plasmids encoding Ikaros and FLAG-tagged leukemia-type Helios variants. Staining for expressed factors was performed with anti-FLAG or anti-Ikaros antibodies, and visualized with Cy2 (green) and Cy3 (red) respectively. The nucleus was counterstained with DAPI (blue). The samples were analyzed by confocal laser microscopy. (B) Suppressive transcriptional activities of individual Ikaros or Helios isoforms demonstrated by Hes1 promoter dual-luciferase reporter systems. The diagram shows the relative changes in luciferase activity in COS-7 cells co-transfected with promoter reporter construct of Hes1 and with the pLV expression constructs encoding various Helios or Ikaros isoforms. Basal activity of Hes1 promoter was defined as firefly ⁄ renilla ratio. Data are Mean ± SD, n = 3. (*p < 0.05; **p < 0.01 vs. the control group). (C) Functional dominant-negative activity of Helios-V2 and Helios-V3 against Ikaros-1 and Helios-1 determined by Hes1 promoter assay. The molar ratios of Ikaros-1 or Helios-1 to Helios-V2 and-V3 plasmids are 1:1, 1:2, and 1:3 respectively. Relative luciferase activities were measured as the firefly ⁄ renilla ratio. Each bar indicates the Mean ± SD of three replicates.
Fig 6.
Short Helios variants function in T cell proliferation and survival.
(A) Cell proliferation analysis of Helios-expressing Jurkat cells was examined by BrdU incorporation. The Jurkat cells stably expressing Helios variants were starved with no-serum media for 40 hr, and pulse-labeled with BrdU for 3 hr. Proliferation was determined by BrdU incorporation. Mean ± SD is shown (*** p< 0.05). (B) EdU assay of relative Hoechst33342 stained cells and EdU add-in cells. The proliferating capability of Helios-expressing Jurkat cells was evaluated using EdU incorporation. (*p<0.05) (C, D) The overexpression of Helios-V2 and–V3 protects against apoptosis in the presence of Cisplatin. Jurkat cells stably expressing Helios variants were evaluated for apoptosis by measurement of Annexin V staining (C) (*p<0.05) or evaluated with the treatment of Cisplatin (D). The data shown are representative of three different experiments. (***p<0.05)
Fig 7.
In-depth search of Helios targets by microarray analysis.
(A) Hierarchical clustering of deregulated genes between Jurkat stable cells. Jurkat cells stably expressing Helios-1, and short variants Helios-V2, and Helios-V3 were used to analyze their gene expression patterns by the microarray. The 2D hierarchical clusters between the cells expressing Helios variants are indicated. (B) Venn diagram showing numbers of genes differentially expressed in the Jurkat stable cell lines versus control. The differential expression gene set (1.5-fold changes) was compared. (C) Pathway analysis of genes deregulated in Helio-V3 Jurkat cells relative to their expression in control cells. Number along vertical line indicates P value (-log) for pathway discovery. (D) Analysis of relative gene expression by quantitative RT-PCR in Jurkat cells stably expressing Helios splice variants compared with mock transfected Jurkat cells.