Recruitment of the SWI/SNF Chromatin Remodeling Complex by NFATc1 in the Transcriptional Regulation of the C-MYC Oncogene in Aggressive B-Cell Lymphomas

Abstract

The NFAT (nuclear factor of activated T-cells) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway for involvement in the pathogenesis of various malignancies, including large B-cell lymphoma (LBCL), a non-Hodgkin's lymphoma subgroup that is generally responsive to conventional cancer therapies (R-CHOP), but relapse is common that subsequently leads to therapeutic resistance. Although we have shown previously that NFAT family member NFATc1 is constitutively activated and has the ability to maintain cell growth and survival in LBCL cell lines and primary cells, the molecular mechanism(s) underlying how NFATc1 regulates cell growth and survival in LBCL is still unclear. In this study, we demonstrate that the well-known oncogene c-myc is transcriptionally regulated by the transcription factor NFATc1 in LBCL, through a chromatin remodeling mechanism that involves the recruitment of the SWI/SNF chromatin-remodeling complex. In aggressive B-cell lymphoma cell lines, c-myc oncogene protein expression was shown to correlate with NFATc1 protein expression. We further showed that NFATc1 binds to a specific DNA binding element within the proximal c-myc promoter and up-regulates c-myc transcription. The SWI/SNF proteins Brg-1 and Brm, chromatin-remodeling proteins that utilize ATP hydrolysis for energy to modify chromatin structure in order to regulate gene expression, also were shown to bind to the NFAT binding site on the c-myc promoter. Confocal microscopic analysis showed that NFATc1 colocalizes with Brg-1, and co-immunoprecipitation assays showed that Brg-1 interacts with NFATc1. Both proteins interact with the c-myc promoter within the NFAT binding site, as demonstrated by chromatin-immunoprecipitation (ChIP) analysis. Induction of a constitutively active mutant of NFATc1 (caNFATc1) in an NFATc1 negative lymphoma cell line induces c-myc protein expression. Constitutively active NFATc1 also enhances Brg-1 binding to the c-myc promoter when analyzed by ChIP-qPCR assays, suggesting that NFATc1 recruits Brg-1 to the NFAT binding site in the c-myc promoter. Down-regulation of NFATc1 by chemical inhibitors (FK-506) or by validated shRNA of NFATc1, inhibited c-myc protein expression and in-vitro lymphoma cell growth. Our data indicates a novel control mechanism for the transcriptional regulation of c-myc in the pathophysiology of aggressive lymphoma B cells and suggests that targeting NFATc1 could have therapeutic value.