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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Journal of Hematology & Oncology 1/2017

The antiproliferative ELF2 isoform, ELF2B, induces apoptosis in vitro and perturbs early lymphocytic development in vivo

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2017
Autoren:
Fiona H. X. Guan, Charles G. Bailey, Cynthia Metierre, Patrick O’Young, Dadi Gao, Teh Liane Khoo, Jeff Holst, John E. J. Rasko
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13045-017-0446-7) contains supplementary material, which is available to authorized users.

Abstract

Background

ELF2 (E74-like factor 2) also known as NERF (new Ets-related factor), a member of the Ets family of transcription factors, regulates genes important in B and T cell development, cell cycle progression, and angiogenesis. Conserved ELF2 isoforms, ELF2A, and ELF2B, arising from alternative promoter usage can exert opposing effects on target gene expression. ELF2A activates, whilst ELF2B represses, gene expression, and the balance of expression between these isoforms may be important in maintaining normal cellular function.

Methods

We compared the function of ELF2 isoforms ELF2A and ELF2B with other ELF subfamily proteins ELF1 and ELF4 in primary and cancer cell lines using proliferation, colony-forming, cell cycle, and apoptosis assays. We further examined the role of ELF2 isoforms in haemopoietic development using a Rag1 -/-murine bone marrow reconstitution model.

Results

ELF2B overexpression significantly reduced cell proliferation and clonogenic capacity, minimally disrupted cell cycle kinetics, and induced apoptosis. In contrast, ELF2A overexpression only marginally reduced clonogenic capacity with little effect on proliferation, cell cycle progression, or apoptosis. Deletion of the N-terminal 19 amino acids unique to ELF2B abrogated the antiproliferative and proapoptotic functions of ELF2B thereby confirming its crucial role. Mice expressing Elf2a or Elf2b in haemopoietic cells variously displayed perturbations in the pre-B cell stage and multiple stages of T cell development. Mature B cells, T cells, and myeloid cells in steady state were unaffected, suggesting that the main role of ELF2 is restricted to the early development of B and T cells and that compensatory mechanisms exist. No differences in B and T cell development were observed between ELF2 isoforms.

Conclusions

We conclude that ELF2 isoforms are important regulators of cellular proliferation, cell cycle progression, and apoptosis. In respect to this, ELF2B acts in a dominant negative fashion compared to ELF2A and as a putative tumour suppressor gene. Given that these cellular processes are critical during haemopoiesis, we propose that the regulatory interplay between ELF2 isoforms contributes substantially to early B and T cell development.

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Zusatzmaterial
Additional file 1: Table S1. PCR primers used in this study (DOC 69 kb)
13045_2017_446_MOESM1_ESM.doc
Additional file 2: Table S2. List of primary and secondary antibodies used in immunofluorescence (IF) and western blot (WB) analysis. All antibodies were diluted to their working concentrations in the appropriate blocking solution (DOC 43 kb)
13045_2017_446_MOESM2_ESM.doc
Additional file 3: Table S3. Antibody-fluorophore conjugates and filter combinations used to distinguish haemopoietic-specific cell surface markers (DOC 40 kb)
13045_2017_446_MOESM3_ESM.doc
Additional file 4: Figure S1. Confirmation of DNA binding of ELF2 isoforms by ChIP. A) The doxycycline-regulatable ‘dox-off’ lentiviral vector used to co-express eGFP and ELF2 isoforms. B) Flow cytometric analysis of HEK293T cells transfected with eGFP only (control)-, HA-ELF2A- and HA-ELF2B-containing vectors. C) ChIP PCR of known ELF2 targets (VCP, PYGO2, LMO2, and LYN promoters), novel ELF2-binding sites in ELF2 promoter regions (P1, P2, and P3) as well as a negative control region spanning H19 exons 4 and 5. (PDF 1124 kb)
13045_2017_446_MOESM4_ESM.pdf
Additional file 5: Figure S2. Validation of reagents used to detect ELF2 isoform expression. Design A) and validation B) of RT-qPCR primers used to detect Elf2a and Elf2b major and minor isoforms with expected amplicon sizes (bp). C) RT-qPCR detection of Elf2 isoform expression in murine haemopoietic cell lines. D) Specific N-terminal sequences used as immunising peptides to produce isoform-specific antibodies. The amino acid identity between mouse and human sequences is shown. E) Validation of specificity and species cross-reactivity of ELF2A and ELF2B antibodies in control-transduced (GFP vector only; Con) HEK293T cells and cells transduced with mouse Elf2A (mA), mouse Elf2b (mB), human ELF2A (hA), or human ELF2B (hB)-containing lentiviral vectors (PDF 1535 kb)
13045_2017_446_MOESM5_ESM.pdf
Additional file 6: Table S4. Somatic mutations in ELF2 in cancer. Mutations are compiled from the TCGA CBIO portal and COSMIC databases. Mutations for ELF2A are shown; no mutations in ELF2B’s 19 aa N-terminus have been recorded (DOC 99 kb)
13045_2017_446_MOESM6_ESM.doc
Additional file 7: Figure S3. Confirmation of ELF protein expression in vitro. A) Determination of endogenous ELF family protein levels in immortalised and primary cells; Con = HeLa cells overexpressing the respective HA-tagged ELF protein. Numbers indicate molecular weight markers (in kDa). B) Confirmation of subcellular localisation of ELF family members and ELF2∆ truncation mutant in HeLa cells: GFP expression confirms transduction efficiency; HA staining confirms ELF family protein overexpression; DAPI confirms DNA staining; scale bar = 50 μm. (PDF 3489 kb)
13045_2017_446_MOESM7_ESM.pdf
Additional file 8: Figure S4. ELF subfamily protein expression. A) Gating strategy for FACS enrichment of ELF protein-expressing HeLa cells indicating total GFP+ population or low, medium or high GFP-expressing cells. Total CFSE-labelled GFP+ HeLa cells B) and low and medium GFP subpopulations C) were incubated ± dox for 3 d. D) Gating strategy of BrdU and 7-AAD staining of ELF overexpressing HeLa cells for cell cycle analysis. E) Representative differential interference microscopy (DIC) and fluorescence images of cells overexpressing ELF subfamily members. Morphologically dead or dying cells are indicated with red arrows; scale bar = 50 μm. B). (PDF 17858 kb)
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Additional file 9: Table S5. Summary of validated ELF2 targets involved in B and T cell development. All targets have been validated by reporter gene assay or by EMSA. (DOC 52 kb)
13045_2017_446_MOESM9_ESM.doc
Additional file 10: Figure S5. Reconstitution efficiency in ELF2+ retrogenic mice. A) Murine stem cell virus-based (MSCV) retroviral vector (pMIG) used for expressing HA-tagged Elf2 isoforms; primer sequences used for detecting specific isoform expression are indicated (arrowheads); a common 5’ primer within the HA-tag and 3’ primer able to detect all Elf2 isoforms were used. B) RT-qPCR of ectopic Elf2a isoform expression in the spleens of retrogenic mice after 3 months reconstitution. Analysis of GFP expression after 4 weeks in peripheral blood mononuclear cells: total C); T cell population D); B cell population E); and granulocytes F). Reconstitution efficiency in the haemopoietic compartment after 3 months. Data represents the mean ± SEM of 3 experiments each performed with 4–5 mice per experimental arm. Statistical analysis performed using Student’s t test (ns, not significant; *, p < 0.05; **, p < 0.01) (PDF 1218 kb)
13045_2017_446_MOESM10_ESM.pdf
Additional file 11: Figure S6. Analysis of lymphocytic subsets in ELF2 retrogenic mice. A) Detection of TCRβ surface expression in thymocytes. Analysis of splenic T cells for TCRβ B) and CD4 and CD8 expression C). Analysis of mature T subsets in the spleen: CD4+ D) or CD8+ E) and CD4+ Tregs. Data represents the mean ± SEM. of 3 experiments each performed with 4–5 mice per experimental arm. Statistical analysis performed using Student’s t test (ns, not significant; *, p < 0.05; **, p < 0.01). (PDF 287 kb)
13045_2017_446_MOESM11_ESM.pdf
Additional file 12: Figure S7. ELF2 isoform expression decreases during ATRA-induced myeloid differentiation. A) MPRO cells were induced to differentiate with 10 μM all-trans retinoic acid (ATRA) and were co-stained with FITC-conjugated anti-Gr-1 antibodies and propidium iodide (PI) DNA dye. Stained MPRO cells were FACS-enriched for different Gr-1 populations: Gr-1Neg, Gr-1Low, Gr-1Mid, and Gr-1High. B) May-Grünwald-Giemsa staining of treated MPRO cells: Gr-1Neg cells showing predominantly promyelocytes (line-arrows); Gr-1Low cells showing promyelocytes and myelocytes (closed arrows); Gr-1Mid cells showing myelocytes and granulocytes (open arrows); and Gr-1High showing mature granulocytes. Scale bars represent 25 μm. C) Each population was examined by RT-qPCR to measure marker genes differentially expressed during granulopoiesis, including cathepsin G (Ctsg), lactoferrin (Ltf) and metalloproteinase 9 (Mmp9). Gene expression was normalised to β-actin and expressed relative to the Gr-1Neg population (set as 1.0). Error bars represent SEM from 4 independent replicates, each performed in duplicate. D) Expression of Elf2 isoforms was examined as in C). Two-sided t test was performed to compare Gr-1High to Gr-1Neg for each Elf2 isoform (p < 0.01**, p < 0.001 ***) (PDF 151 kb)
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