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First published online July 23, 2007
doi: 10.1242/10.1242/jcs.007344

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Combinatorial effects of splice variants modulate function of Aiolos

¹ Cancer Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain
² Molecular Medicine Unit-Department of Medicine, Center for Cancer Research (CIC), University of Salamanca, Salamanca, Spain
³ Leukaemia Research Fund Centre at the Institute of Cancer Research, London, UK
⁴ Confocal Microscopy and Cytometry Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
⁵ Department of Histology (University of Granada) and Fundación Hospital Clínico, Granada, Spain

View larger version (32K): [in this window] [in a new window]   Fig. 1. The Aiolos gene codes multiple isoforms. (A) A representative RT-PCR with primers corresponding to exon 1 and 7 in hematopoietic cell lines derived from human B leukemic (697, RAMOS, RS11;846, REH) and T leukemic (JURKAT, SUPT11) cells and the NKL cell line, derived from natural killer (NK) cells. (B) Schematic representation of Aiolos isoforms. Green boxes in exons 3-5 indicate the four N-terminal zinc finger motifs involved in DNA binding and the two violet boxes in exon 7 indicate the C-terminal motifs required for dimer formation. The two Aiolos isoforms (at the bottom) contain an alternative exon, which we have named exon 5a (see Results). Aiolos isoforms have been named according to the exons missing from the full-length isoform. Ikaros full-length isoform Ik-1 is also shown at the bottom. Nine novel Aiolos isoforms are indicated on the right by asterisks. (C) RT-PCR analysis of non-hematopoietic human cell lines (monkey SV40-transformed fibroblast cell line COS-7, breast cell line MCF 7, liver cell line HEPG2, a neuroblastoma-derived cell line SHSY-5Y and kidney cell line HEK) also show the presence of multiple Aiolos isoforms. Molecular weight (MW) markers are included. PCR products were transferred onto a nylon membrane and hybridized with a specific C-terminal Aiolos probe in order to confirm the identity of the PCR bands (bottom panel). (D) Representative RT-PCR analysis of B cells including B-cell-derived lymphoblastoid cells (B-PBL), and samples from healthy subjects (N1-N4) and individuals with systemic lupus erythematosus (SLE1-SLE4) and rheumatoid arthritis (RA1-RA4).

View larger version (33K): [in this window] [in a new window]   Fig. 2. Transfection of Aiolos isoforms in mammalian cells. (A) Schematic representation of the five selected isoforms. (B) Western blot of COS-7 cells extracts transfected with the five Aiolos isoforms and developed with the mouse monoclonal anti-FLAG antibody (left) and a polyclonal anti-Aiolos antiserum (right). (C) Subcellular localization of Aiolos isoforms. Confocal images of COS-7 cells transfected with different Aiolos isoforms and labeled with antibody to Aiolos (green). Nuclear DNA was counterstained with DAPI (blue). Images of different subpopulations are included (indicated in Results).

View larger version (73K): [in this window] [in a new window]   Fig. 3. Subcellular localization in COS-7 cells of Aiolos isoforms cotransfected with Ikaros-1. Aiolos isoforms were labeled with Cy2 (green) and Ikaros-1 was labeled with Cy3 (red). Nuclei were counterstained with DAPI (blue).

View larger version (37K): [in this window] [in a new window]   Fig. 4. Association of Aiolos isoforms with histone deacetylase. (A) In vivo interaction between Aiolos isoforms and components of the Mi-2/NurD and SIN3 histone deacetylase complexes. Immunoprecipitation of Aiolos was accomplished using an anti-FLAG antibody and eluates were tested by immunoblot analysis with anti-FLAG, anti-HDAC1, anti-SIN3A and three antibodies that each distinguish a subfraction of the Mi-2/NuRD complex, namely anti-MTA1, anti-MTA2 and anti-MTA3. Input COS-7 cell extracts were tested for all antibodies (not shown). (B) Confocal images of untransfected COS-7 cells showing distribution of HDAC1, SIN3A and MTA2. (C) Confocal images of COS-7 cells transfected with different Aiolos isoforms. Aiolos isoforms were stained with an anti-FLAG and a secondary anti-mouse labeled with Cy2 (green). HDAC1, MTA2 and SIN3A were labeled with Cy3 (red). Nuclei were counterstained with DAPI (blue).

View larger version (28K): [in this window] [in a new window]   Fig. 5. Effects on global histone acetylation patterns of COS-7 cells transfected with Aiolos isoforms. (A) Western blot analysis of histone modifications of cell extracts. Analysis with antibodies against acetyl-H4, acetyl K16 and K8 of H4, acetyl-H3 and acetyl K9 of histone H3. A Coomassie Blue staining is also shown as a loading control. (B) Analysis of histone H4 acetylation by high performance capillary electrophoresis (HPCE). An example of HPCE separation of acetylated species of histone H4 from pcDNA3 and Aiolos-transfected COS-7 cells. The non-acetylated (Ac0), monoacetylated (Ac1) and diacetylated (Ac2) forms of histone H4 are indicated. (C) A quantification of the HPCE analysis showing the acetylation of histone H4 corresponding to COS-7 cells transfected with all Aiolos isoforms.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Analysis of the interaction of Aiolos isoforms with the promoter of SIRT1. (A) ChIP assays were performed with anti-FLAG, anti-Aiolos and anti-RNA pol II antibodies in COS-7 cells transfected with all the Aiolos isoforms studied in this work. Negative controls (No antibody, NAB) are also shown. Input and bound fractions are shown. In addition to the SIRT1 promoter, actin B gene promoter was used as a negative control. (B) Association of Aiolos to the SIRT1 promoter in B cells. ChIP assays in untransfected RAJI and B-PBL cells. Anti-Aiolos and anti-RNA pol II antibodies were used. Actin B was used as a negative control.

View larger version (49K): [in this window] [in a new window]   Fig. 7. Transfection of Aiolos isoforms in B cells. (A) Subcellular localization of Aiolos isoforms. Confocal images of RAJI and B-PBL cells transfected with different Aiolos isoforms and labeled with Aiolos antibody (green). Nuclear DNA was counterstained with DAPI (blue). Images of different subpopulations are included (indicated in Results). (B) Association of Aiolos isoforms with histone deacetylase. Confocal images of RAJI and B-PBL cells transfected with different Aiolos isoforms. Aiolos isoforms were stained with anti-FLAG and a secondary anti-mouse antibody, labeled with Cy2 (green). HDAC1 was labeled with Cy3 (red). Nuclei were counterstained with DAPI (blue).