The Histone Demethylase LSD1 Acts As a BCL6 Corepressor In Germinal Center B Cells

Abstract

The BCL6 transcription repressor (B-Cell Lymphoma 6) is required for B cells to form germinal centers (GC) and undergo antibody affinity maturation. BCL6 mediates its functions mainly through the recruitment of corepressor complexes with distinct enzymatic activities including the SMRT/NCOR, BCOR and MTA3 corepressors. Preliminary chromatin immunoprecipitation (ChIP) studies designed to dissect the biochemical actions of BCL6 on chromatin, showed that BCL6 inhibition results in considerable induction of H3K4me2 levels at selected BCL6 target loci. Consistent with these observations a BCL6 expressing lymphoma cell line had lower levels of H3K4me2 compared to BCL6 negative lymphoma cells. Hence, we hypothesized that in addition to HDAC and PRC1-like complexes BCL6 associates with distinct complexes bearing H3K4me2 demethylase activity. We observed that one of the few H3K4 demethylases known to target H3K4me1/2 residues, Lysine Specific Demethylase-1 (LSD1) is highly expressed in GC B cells. More specifically both LSD1 transcript and protein levels were induced in the transition from naïve to GC B cell state, similar to BCL6 levels. Collectively these results indicate that BCL6 might functionally interact with LSD1 in GC B cells to mediate transcriptional repression of key gene networks. We initially asked if BCL6 and LSD1 proteins interact in GC-derived B cells using coimmunoprecipitation and GST pull down experiments. We found that BCL6 and LSD1 proteins endogenously interact in these cells. Additional GST pull-down experiments using full length GST-LSD1 and in-vitro translated S35 BCL6 confirmed this interaction suggesting it is possibly direct. Moreover, immunoprecitated BCL6 complexes could demethylate H3K4me1 but not H3K4me3 in in vitro assays using histone substrates. To further evaluate the role of LSD1 in the physiological GC response we challenged C57BL/6 mice with T-cell dependent antigen (sheep red blood cells) and we examined GC formation in mice treated with a specific LSD1 inhibitor compared to untreated control mice. LSD1 inhibition resulted in GC formation deficiency indicating that LSD1 enzymatic activity plays key roles in normal immune response. To study the functional role of BCL6-LSD1 complexes we performed Q-ChIP assays for LSD1 at BCL6 target loci and showed that several BCL6 target loci, including MLL2, CDKN1A, BRAF and CCR6 are also bound by LSD1. To extend these findings we performed LSD1 ChIP-seq in a GC-derived lymphoma cell line. These genomic studies revealed an extensive overlap of BCL6 and LSD1 binding sites (1509 common loci). Roughly 560 gene promoters were bound by both BCL6 and LSD1 at the same site. Moreover, about 1,000 BCL6-LSD1 binding sites were outside promoters and might play a role in the regulation of inter/intragenic elements similar to the role of LSD1 in enhancer regulation in embryonic stem cells. Loss of BCL6 at several targeted loci resulted in LSD1 release from the chromatin suggesting that BCL6 mediates tethering of LSD1 at these loci. Genes bound or associated with BCL6-LSD1 complexes were significantly enriched in pathways such as B cell activation, chromatin organization and cell cycle control. Taken together these results suggest that LSD1 acts as a BCL6 corepressor in GC B cells and is an essential transcriptional regulator for the formation of normal GC response. BCL6 and LSD1 possibly cooperate to establish a transcriptional program crucial for the GC stage of B cell differentiation. Because LSD1 is expressed at high levels in lymphoma cells, targeting the LSD1 enzymatic activity could potentially be exploited as a lymphoma targeted therapy. Disclosures:No relevant conflicts of interest to declare.