Confirming the tamoxifen-induced deletion in ERT2-Ezh2KO ASC, an 18-fold reduction in the transcripts across deleted exons was observed compared to wild type (Supplemental Figure 4A). enzymes using genetic approaches (5). Deletion of the histone acetyltransferase, MOZ, reduces GC B cells and skews responding B cells towards low affinity IgM+ memory B cells (17). Additionally, treatment of mice with histone deacetylase inhibitors reduces B cell responses (18), indicating that both erasing and writing epigenetic modifications is an essential process in B cell differentiation. Importantly, epigenetic modifiers are frequent targets of both activating and inactivating mutations in lymphomas (19, 20). Therefore, a full understanding of epigenetic mechanisms and targets for distinct enzymes is important to manipulate B cell differentiation and understand the effects of therapeutics targeting these enzymes. One of the best characterized repressive epigenetic histone modifications is the trimethylation of histone H3 at lysine 27 (H3K27me3), which is mediated by the polycomb repressive complex 2 (PRC2) (21, 22). Enhancer of zest 2 (EZH2) is the catalytic subunit of the PRC2 complex and functions as an essential transcriptional silencer (23C25). EZH2 is upregulated in pre-B cells, in which it is necessary for VDJ recombination during B cell development (26) and to repress germline Ig transcription (27). EZH2 is expressed at low levels in quiescent, na?ve B cells, but is highly upregulated in GC B cells where it facilitates cellular proliferation, protects from activation-induced cytidine deaminase (AID) off target activity, and represses the differentiation of GC B cells into ASC (15, 16, 28, 29). EZH2 interacts with distinct sets of transcription factors, such as BCL6 in GC B cells (29) and Blimp-1 in ASC (30), to direct cell-type specific gene repression programs. B cell specific-deletion of EZH2 leads to a loss of GC formation, thereby leading to defects in the formation of ASC (15, 16). However, no studies have directly assessed whether or how EZH2 functions in ASC. Here we tested the role of EZH2 in two T-independent models of ASC differentiation, one initiated by the T-independent antigen, LPS, and the other initiated by influenza infection in the absence of CD4 T cells. We found that EZH2 was progressively upregulated in stimulated B cells, with expression peaking in ASC. In addition, B cell specific genes gained H3K27me3 in their promoters as ASC differentiation progressed, indicating that EZH2 may repress these genes. Following immunization Rabbit polyclonal to ALDH1L2 with the T-independent antigen, LPS, or infection with influenza virus in the absence of T cells, mice with a tamoxifen-inducible deletion generated fewer ASC. The EZH2-dependent defect was cell intrinsic to B cells and resulted in the enhanced expression and increased chromatin accessibility of B cell genes that gain H3K27me3 and are normally repressed in ASC, including Blimp-1 target genes and inflammatory genes. mRNA levels (right) quantitated by RT-qPCR and expressed relative to 18s rRNA as mean SD. *p 0.05 by Students two-tailed T-test. These data are representative of three independent experiments. (B) Protein levels of EZH2 in naive B cells (nB) and LPS induced activated B cells (actB) and CD138+ antibody secreting cells (ASC). FMO, fluorescence minus one. Data is representative of two experiments. (C) Scatter plot of the average promoter H3K27me3 in nB and ASC versus the log2 fold change of H3K27me3 as determined by ChIP-seq. ChIP-seq data is summarized from 2 biological replicates of nB and ASC. (D) REVIGO (50) plot summarizing Gene Ontology terms for the 1,623 genes that gain promoter H3K27me3 in ASC from C. Flow cytometry and cell sorting For staining, cells were resuspended at 1 106 cells/100 l in FACS buffer (1 PBS, 1% BSA, 2 mM EDTA) and blocked with anti-Fc (Tonbo Biosciences, 2.4G2) at a concentration.GSEA analysis was performed using a set of transcription factors specifically expressed in Follicular B cells (FoB) compared to bone marrow ASC (57) and showed that this set of transcription factors were significantly upregulated in ERT2-Ezh2KO ASC (Figure 5B). characterized by ChIP-seq, have cell-type specific patterns in na?ve B cells, differentiated ASC, and in GC B cells (12C16). However, few studies have examined the role of histone modifying enzymes using genetic approaches (5). Deletion of the histone acetyltransferase, MOZ, reduces GC Ro 28-1675 B cells and skews responding B cells towards low affinity IgM+ memory B cells (17). Additionally, treatment of mice with histone deacetylase inhibitors reduces B cell responses (18), indicating that both erasing and writing epigenetic modifications is an essential process in B cell differentiation. Importantly, epigenetic modifiers are frequent targets of both activating and inactivating mutations in lymphomas (19, 20). Therefore, a full understanding of epigenetic mechanisms and targets for distinct enzymes is important to manipulate B cell differentiation and understand the effects of therapeutics targeting these enzymes. One of the best characterized repressive epigenetic histone modifications is the trimethylation of histone H3 at lysine 27 (H3K27me3), which is mediated by the polycomb repressive complex 2 (PRC2) (21, 22). Enhancer of zest 2 (EZH2) is the catalytic subunit of the PRC2 complex and functions as an essential transcriptional silencer (23C25). EZH2 is upregulated in pre-B cells, Ro 28-1675 in which it is necessary for VDJ recombination during B cell development (26) and to repress germline Ig transcription (27). EZH2 is expressed at low levels in quiescent, na?ve B cells, but is highly upregulated in GC B cells where it facilitates cellular proliferation, protects from activation-induced cytidine deaminase (AID) off target activity, and represses the differentiation of GC B cells into ASC (15, 16, 28, 29). EZH2 interacts with distinct sets of transcription factors, such as BCL6 in GC B cells (29) and Blimp-1 in ASC (30), to direct cell-type specific gene repression programs. B cell specific-deletion of EZH2 leads to a loss of GC formation, thereby leading to defects in the formation of ASC (15, 16). However, no studies have directly assessed whether or how EZH2 functions in ASC. Here we tested the role of EZH2 in two T-independent models of ASC differentiation, one initiated by the T-independent antigen, LPS, and the other initiated by influenza infection in the absence of CD4 T cells. We found that EZH2 was progressively upregulated in stimulated B cells, with expression peaking in ASC. In addition, B cell specific genes gained H3K27me3 in their promoters as ASC differentiation progressed, indicating that EZH2 may repress these genes. Following immunization with the T-independent antigen, LPS, or infection with influenza virus in the lack of T cells, mice having a tamoxifen-inducible deletion produced fewer ASC. The EZH2-reliant defect was cell intrinsic to B cells and led to the enhanced manifestation and improved chromatin availability of B cell genes that gain H3K27me3 and so are normally repressed in ASC, including Blimp-1 focus on genes and inflammatory genes. mRNA amounts (correct) quantitated by RT-qPCR and indicated in accordance with 18s rRNA as mean SD. *p 0.05 by Students two-tailed T-test. These data are representative of three 3rd party tests. (B) Protein degrees of EZH2 in naive B cells (nB) and LPS induced triggered B cells (actB) and Compact disc138+ antibody secreting cells (ASC). FMO, fluorescence minus one. Data can be representative of two tests. (C) Scatter storyline of the common promoter H3K27me3 in nB and ASC versus the log2 collapse modification of H3K27me3 as dependant on ChIP-seq. ChIP-seq data can be summarized from 2 natural replicates of nB and ASC. (D) REVIGO (50) Ro 28-1675 storyline summarizing Gene Ontology conditions for the 1,623 genes that gain promoter H3K27me3 in ASC from C. Movement cytometry and cell sorting For staining, cells had been resuspended at 1 106 cells/100 l in FACS buffer (1 PBS, 1% BSA, 2 mM EDTA) and clogged with anti-Fc (Tonbo Biosciences, 2.4G2) in a focus of 0.25 g per 1 106 cells for 15 min on ice. The next antibodies were useful for FACS evaluation: B220-PE-Cy7 (Tonbo Biosciences, #60-0452-U100,.
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