Aiolos collaborates with Blimp-1 to regulate the survival of multiple myeloma cells

Abstract

The transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) has crucial roles in the control of plasma cell differentiation and in maintaining survival of plasma cells. However, how Blimp-1 ensures the survival of plasma cell malignancy, multiple myeloma (MM), has remained elusive. Here we identified Aiolos, an anti-apoptotic transcription factor of MM cells, as a Blimp-1-interacting protein by mass spectrometry. ChIP coupled with DNA microarray was used to profile the global binding of Aiolos and Blimp-1 to endogenous targets in MM cells, which revealed their co-binding to a large number of genes, including apoptosis-related genes. Accordingly, Blimp-1 and Aiolos regulate similar transcriptomes in MM cells. Analysis of the binding motifs for Blimp-1 and Aiolos uncovered a partial motif that was similar across sites for both proteins. Aiolos promotes the binding of Blimp-1 to target genes and thereby enhances Blimp-1-dependent transcriptional repression. Furthermore, treatment with an anti-MM agent, lenalidomide, caused ubiquitination and proteasomal degradation of Blimp-1, leading to the de-repression of a new Blimp-1 direct target, CULLIN 4A (CUL4A), and reduced Aiolos levels. Accordingly, lenalidomide-induced cell death was partially rescued by reintroduction of Blimp-1 or knockdown of CUL4A. Thus, we demonstrated the functional impacts and underlying mechanisms of the interaction between Aiolos and Blimp-1 in maintaining MM cell survival. We also showed that interruption of Blimp-1/Aiolos regulatory pathways contributes to lenalidomide-mediated anti-MM activity.

Figure 1

Blimp-1 directly interacts with Aiolos. (a and b) H929 (a) and U266 (b) nuclear extracts were used for immunoprecipitation (IP) with anti-Blimp-1 (upper panels) or anti-Aiolos (lower panels). Immunoprecipitates and input lysates were analyzed with immunoblotting (IB) using the indicated antibodies. Rabbit IgG (rIgG) was used as the control IP antibody. (c) Co-IP using anti-Aiolos (left panel) or anti-Blimp-1 (right panel) demonstrated the interaction of Blimp-1 and Aiolos in bone marrow aspirates of MM patients. Results are one representative from two independent experiments. (d) Schematics of constructs for domain mapping of FLAG-tagged full-length Blimp-1 (a) and its various deletion mutants (b–g). Known motifs: acidic, acidic domain; PR, PR domain; proline-rich, proline-rich domain; Zn, the five zinc-finger motifs. (e) The HA-tagged Aiolos expression plasmid (HA-Aiolos) was co-transfected into 293T cells with the plasmids encoding various lengths of FLAG-tagged Blimp-1 or empty vector (−). Cell lysates were prepared for IP using anti-FLAG followed by IB. Arrows indicate FLAG-tagged Blimp-1 and its variants. (f) Schematics of constructs for domain mapping of HA-tagged full-length Aiolos (a) and its deletion mutants (b–f). (g) FLAG-tagged Blimp-1 was co-transfected with HA-Aiolos, its deletion constructs or empty vector (−) into 293T cells followed by co-IP and IB as described in e. (h) Schematics of constructs of GST-fused full-length Aiolos (a) and various deletion mutants of Aiolos (b–e). (i) A GST pull down assay was performed using recombinant His-tagged △1–526 Blimp-1 and GST or various forms of GST-Aiolos fusion proteins followed by IB. Arrows indicate GST or GST-fused Aiolos fragments

Figure 2

Identification of direct binding sites of Aiolos and Blimp-1 in the MM cell line H929. (a and b) Chromatin from H929 cells was subjected to a ChIP assay with anti-Aiolos or anti-Blimp-1. (a) The Venn diagram shows the number of overlapping Aiolos target genes and Blimp-1 target genes in H929 cells. (b) Pie charts show the results of GO analysis of the unique targets of Blimp-1 (upper left panel), the unique targets of Aiolos (upper right panel), their common targets (lower left panel) and genes that were not bound by Blimp-1 or Aiolos (lower right panel); (*P<0.05). (c–h) The graphs are the average values of the binding probability (pXbar) of Aiolos (red line) or Blimp-1 (blue line) in H929 cells from two ChIP–chip experiments. The x axis represents the probe regions in the target genes, and the y axis shows the pXbar value. The position of transcription start sites and the direction of transcription are indicated

Figure 3

Aiolos/Blimp-1 and Aiolos/Ikaros heterodimers bind to two distinct motifs. (a) Sequences shown are the Blimp-1- and Aiolos-binding consensus motifs analyzed with the motif discovery web server eTFBS. (b) Lysates from H929 cells were used to perform IP with the indicated antibodies, followed by IB. Arrow indicates a nonspecific band. (c and d) Chromatin prepared from H929 cells was subjected to a ChIP and re-ChIP assay using anti-Blimp-1 (c) or anti-Ikaros (d) in the first ChIP and anti-Aiolos or rabbit IgG in the re-ChIP. Results in (c) and (d) represent the mean±S.E.M. (n=3). **P<0.01; ***P<0.005

Figure 4

Aiolos enhances the binding of Blimp-1 to DNA. (a and b) H929 cells, transduced with shCtrl, shAiolos, or shBlimp-1 for 4 days, were subjected to the ChIP assay using anti-Blimp-1 (a) or anti-Aiolos (b). All data represent the mean±S.E.M. (n=3). (c) Immunoblot of Blimp-1 or Aiolos captured in DNA pull down assays with biotinylated CIITA (upper panel) or c-Myc (lower panel) probes containing the Blimp-1-binding site and recombinant His6-tBlimp-1 (506−825) and/or His6-Aiolos (1−509 or 120−509). (d) Luciferase reporter assay using lysates from 293T cells transfected with plasmids encoding Blimp-1 and/or Aiolos (1−509 or 120−509) or empty vector alone (mock) together with wild-type (WT) or Blimp-1 binding site-mutated (Mutant) CIITA-pIII-Luc and RL-tk. (e) Immunoblots show the expression of the indicated proteins from corresponding transfectants described in d. Results represent the mean±S.D. (n=3). *P<0.05; ***P<0.005

Figure 5

Aiolos and Blimp-1 collaboratively regulate gene expression and maintain the survival of MM cells. (a) Heatmap of microarray data from the H929 line transduced with shCtrl, shAiolos or shBlimp-1 for 4 days. The changes in expression for each gene were calculated as the ratio of expression in shAiolos- or shBlimp-1-transduced cells versus shCtrl-transduced cells. Microarray experiments were performed in duplicate. Each row represents a significantly induced (red) or repressed (green) gene following knockdown of Aiolos or Blimp-1. (b) Pie charts show the results of GO analysis of Blimp-1-dependent genes (left panel) or Aiolos-dependent genes (right panel) in H929 cells. (c) RT-QPCR analysis with samples from (a) validated the expression of apoptosis-related genes in shRNA-expressing cells. (d) Chromatin from H929 cells was subjected to the ChIP assay using anti-Blimp-1 or anti-Aiolos, followed by QPCR to quantify the binding of Blimp-1 or Aiolos to the indicated genes. (e) Immunoblots show the knockdown efficiency of shBlimp-1 and shAiolos in three MM cell lines expressing indicated shRNA for 3 days. (f) Flow cytometric analysis of annexin V staining shows the increased apoptosis in MM cells expressing shAiolos or shBlimp-1 for 3 days. Data in c and d represent the mean±S.E.M. (n=3). *P<0.05; **P<0.01; ***P<0.005

Figure 6

Blimp-1 directly suppresses CUL4A. (a) Schematic representation of two potential Blimp-1-binding sites on CUL4A. The transcriptional start site (+1) is indicated. (b) ChIP shows that Blimp-1 (upper panel), but not Aiolos (lower panel), binds to intron 3 of CUL4A in H929 cells. IgG was used as an isotype control antibody. CIITA promoter III region and its the 3′ untranslated regions (UTR) region were served as the positive and negative control loci for Blimp-1 binding. (c) RT-QPCR shows the increased CUL4A mRNA in shBlimp-1-expressing MM cells. (d) Immunoblots show the reduced expression of Aiolos and Ikaros but increased expression of CUL4A in three MM cell lines depleted of Blimp-1. Data in b and c represent the mean±S.D. (n=3). *P<0.05; **P<0.01

Figure 7

The cytotoxic effects of lenalidomide in MM cells require proteolysis of Blimp-1. (a) Immunoblots show the effects of lenalidomide (20 μM) on the expression of the indicated proteins in three MM cell lines at the indicated time points. (b) Co-IP shows the effect of lenalidomide on ubiquitination of Blimp-1. Pre-cleared nuclear extract from H929 cells treated with lenalidomide and MG132 (10 μM) at indicated time points was used for IP with anti-Ub, followed by immunoblot analysis with anti-Blimp-1. Arrow indicates a nonspecific protein. (c) Percentage of annexin V⁺ cells in MM cells that expressed Blimp-1–EGFP or EGFP and were treated with lenalidomide or DMSO for 24 h. (d) Percentage of annexin V⁺ cells in shCUL4A#2- or shCtrl-expressing cells treated with lenalidomide or DMSO for 24 h. (e) Working model of the Blimp-1/CUL4A/Aiolos regulatory axis in inducing apoptosis after lenalidomide treatment. Data in c and d represent the mean±S.D. (n=3). *P<0.05; **P<0.01