| The mechanism(s) by which germinal center (GC) B lymphocytes differentiate into antibody-secreting plasma cells or long-lived memory B cells is not well elucidated. We hypothesized that regulation of the expression of multiple genes during B cell differentiation may, in-part, be mediated by alteration in chromatin accessibility to transcription factors. The murine B lymphoma cell line L10A and murine splenic B cells were treated with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor. TSA upregulated B lymphocyte-induced maturation protein-1 (Blimp-1), a master gene that regulates late B cell differentiation to plasma cells, Mad, and J chain, down-regulated c-Myc and BSAP/ Pax-5 gene expression, increased cell surface expression of Syndecan-1 (CD138), and decreased surface IgM expression. However, TSA failed to induce L10A to secrete IgM. Incubation of murine splenic B cells treated with dextran conjugated anti-IgD (anti-δ-dex) Ab with TSA, increased Blimp-1 gene and Syndecan-1 surface expression, consistent with induction of the onset of terminal B cell differentiation. Co-incubation of L10A cells with TSA and cycloheximide (CHX), a protein synthesis inhibitor abrogated the up-regulation of Blimp-1 expression, indicating that TSA-activated Blimp-1 expression required synthesis of a transcriptional activator. In contrast, Mad expression was increased in L10A cells cultured with TSA and CHX or CHX alone, suggesting Mad expression may occur independent of Blimp-1 expression and is regulated by a labile, HDAC associated transcriptional repressor. Because Mad consensus binding sites were present in the upstream promoter region of Bcl-6, Mad regulation of Bcl-6 was analyzed. Co-transfection of Mad with the upstream promoter region of Bcl-6 down-regulated Bcl-6 expression. Anti-sense Mad1 oligonucleotides abrogated the downregulation of Bcl-6 expression that occurred with differentiation of mouse splenic B induced by activation with anti-6-dex and IL-5. Bcl-6 expression was down-regulated in WEHI 231 B cell lines that were transduced with retroviruses expressing Mad1. These results suggest that late B cell differentiation may be regulated by chromatin remodeling and Mad1 regulates Bcl-6 expression but alone is not sufficient to alter downstream gene expression for late B cell differentiation to plasma cells. These studies provide novel insights into the regulation of late B cell differentiation. |
