Therefore hypomorphic GC B cells show evidence of increased levels of DNA damage in vivo, suggesting that DNMT1 plays a role in genomic stability during affinity maturation. Open in a separate window Figure 7 Increased H2AX phosphorylation in the GCs of hypomorphic mice. methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair. Introduction On T-cell dependent activation, resting/naive B cells (NBCs) can be induced to migrate into lymphoid follicles and form germinal centers (GCs).1,2 GC B cells subsequently undergo massive clonal expansion and mutagenesis mediated by activation-induced cytosine deaminase (AICDA).2 Tolerance of simultaneous proliferation and genomic instability is a hallmark of the GC B-cell phenotype and is required for development of B-cell clones able to generate high-affinity antibodies.1,2 AICDA not only induces mutations within the immunoglobulin loci but also localizes to many other sites of the genome including promoters and coding sequences of actively transcribed genes enriched in RGYW DNA motifs.3C6 AICDA-induced mutations can thus occur at many sites throughout the genome in normal GCs.3,6 In accordance with these observations, AICDA has been demonstrated to play a critical role in lymphomagenesis.7 While genetic diversity of B-cell clones within GCs is important for the emergence of cells encoding high-affinity immunoglobulins, it also provides opportunities for the emergence of malignant clones. In fact a majority of B-cell neoplasms originate from cells that have transited the GC reaction.1 Induction of the GC phenotype requires that NBCs undergo major changes in gene expression patterning, the basis of which are not fully understood. These shifts are mediated in part by transcription factors such as BCL6 and BACH28C10 and histone modifying enzymes Vilazodone D8 such as EZH2.11 However, differential methylation of CpG dinucleotides is also known to control tissue specific gene expression.12,13 CpG methylation is mediated by a family of DNA methyltransferase enzymes (DNMTs).14 Of these, DNMT1 primarily mediates maintenance methylation, because of its preference for hemimethylated DNA15; while DNMT3A and 3B primarily mediate de novo DNA methylation. Differential methylation occurs at the earliest stages of lymphopoiesis16 and hypomorphic mice accordingly display skewed hematopoietic differentiation toward the myeloid lineage,17 but the role of DNMT1 in Vilazodone D8 mature B cells has not been studied in a detailed manner. Both aberrant DNA hypermethylation and hypomethylation have been shown to occur in lymphomas derived from GC B-cells such as diffuse large B-cell lymphomas (DLBCL).18,19 Furthermore, DLBCLs with GCB (Germinal Center B-cell like) versus ABC (Activated B cell-like) gene expression signatures display distinct DNA methylation profiles,18 suggesting that cytosine methylation may contribute to the distinct phenotypes of these tumors. Very little is known regarding mechanisms of DNA demethylation, but reports have suggested that cytosine deamination mediated by AICDA followed by base excision repair might contribute to this process by replacing methylated cytosines with new, unmethylated nucleotides.20C23 To determine whether differential DNA methylation patterning occurs naturally in GC B-cells, we examined DNA methylation profiles and the potential role of DNMTs in mediating the GC B cell phenotype. The data suggest a function Vilazodone D8 for cytosine methylation in mature B-cell gene expression patterning with implications for the contribution of AICDA and DNMT1 to genetic and epigenetic instability during lymphomagenesis. Methods B-cell fractionation Leftover human tonsils were obtained after routine tonsillectomies, performed at New York Presbyterian Hospital. All tissue collection was approved by the Weill Cornell Medical College Institutional Review Board. Tonsils were minced on ice and mononuclear cells were isolated using Histopaque density centrifugation. All washes were performed in PBS/2% BSA/2% EDTA. All antibodies were used at 1:100 dilution in cold PBS and staining was done for 10 minutes on Rabbit Polyclonal to DNL3 ice, followed by 3 washes. The B-cell populations were separated using AutoMACS system (Milteny Biotec) using posselD program. Naive B cells (NBCs) were separated using depletion of GC cells, T cells and plasma and memory cells (CD10, CD3, and CD27), followed by enrichment for IgD+ B cells; Germinal Center B (GCB) cells were separated by positive selection with CD77 (anti-CD10: BD Biosciences; anti-CD3: BD.