Results support our hypothesis that nuclear hormone receptors directly influence machinery pertinent to antibody expression. in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ER antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to C, with peaks in E and S sites. Additional peaks of ER binding were coincident with hs1,2 and hs4 sites in the 3 regulatory region (3RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and CSR. Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease. or em in vitro /em , changes in cytokine levels, B cell growth, and isotype expression patterns are readily apparent [2C17] The isotype profile of a B cell population is the outcome of sophisticated mechanisms of B cell activation by foreign- or self-antigens. Depending on antigen triggers and the environment of activation, B cells will experience proliferation, cell maturation, somatic hyper-mutation (SHM), and/or class switch recombination (CSR). In a na?ve B cell, the V-D-J gene sequence of an expressed antibody heavy chain is positioned upstream of C, and C, and both IgM and IgD isotypes can be transcribed/translated as a consequence of differential RNA splicing. When CSR is triggered, double strand DNA breaks occur in S regions upstream of donor and acceptor constant (C) region genes. Excision BD-1047 2HBr of a large DNA loop and non-homologous end joining between S region segments re-positions V-D-J genes upstream of a new C region gene to support IgG, IgE, or IgA production [19C21]. Enhancer regions upstream of C (the intronic enhancer E) and downstream of C (the 3regulatory region [3RR]) each influence the quality and magnitude of antibody expression [22,23]. How might estrogen alter the expression of antibody isotypes? Estrogen is classically known for its binding to estrogen receptor (ER), a type I member of the nuclear hormone receptor BD-1047 2HBr superfamily. ER modifies DNA accessibility to transcription machinery [24C26], a function that is altered by ligand binding [24C26]. Importantly, estrogen and ER regulate the expression of cytokines, the anti-apoptotic Bcl-2 protein, and AID, an enzyme required for CSR [27C29]. Although these influences may be sufficient to explain estrogens impact on antibody isotype expression patterns, we asked if ER might also bind the immunoglobulin heavy chain locus directly. We performed chromatin immunoprecipitation (ChIP) analyses with DNA from activated B cells BD-1047 2HBr and showed that ER was bound to several DNA elements pertinent to CSR. Based on these data, we present our hypothesis that estrogen and ER instruct the magnitude and quality of CSR and isotype expression in B cells by binding the immunoglobulin heavy chain locus. Such events may, in turn, define B cell responses to foreign- and self-antigens, explaining, at least in part, differences in disease outcomes between males and females. 2. MATERIALS AND METHODS 2.1 B cell purification Single cell suspensions from C57Bl/6J mouse spleens were made by mechanically disrupting the tissues and passing through a 70 m cell strainer. Lymphocytes were separated from erythrocytes by centrifugation on a cushion of Lymphocyte Separation Medium (MP Biomedicals). B cells were purified from the lymphocytes by negative selection with anti-CD43 and anti-CD11b microbeads (Miltenyi Biotec) and passing through a MACS LD Column (Miltenyi Biotec) BD-1047 2HBr followed by the elution of the unbound B cells. 2.2 Culture of B cells for ChIP analyses Purified B cells were plated in a 96-well flat-bottomed tissue culture plate at a final concentration of 4 106 cells/ml in a volume Rabbit polyclonal to AADACL3 of 200 l/well in freshly prepared RPMI medium (Life Technologies) containing 10% fetal bovine serum (Atlanta Biologicals), 2mM L-glutamine (Life Technologies), 50 g/ml gentamicin (Lonza), and 55 M 2-mercaptoethanol (Life Technologies). LPS (Sigma) was added to a final concentration of 5 g/ml and cultures were incubated BD-1047 2HBr overnight at 37C in 5% CO2. 2.3.