This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Plasma cells, the final effectors of humoral immunity, are terminally differentiated non-dividing cells dedicated solely to the synthesis and secretion of immunoglobulin (Ig). A subset of plasma cells are long lived and can reside in the bone marrow for years. Therefore, plasma cells have developed mechanisms to ensure the long-term maintenance of their differentiated state. Blimp-1 is the master regulator of plasma cell differentiation. Forced expression of Blimp-1 in primary B cells is sufficient to drive mature B cells to become antibody secreting plasma cells and B cells lacking Blimp-1 are unable to terminally differentiate. Blimp-1 induces terminal B cell differentiation by extinguishing the mature B cell gene expression program and by promoting exit from the cell cycle by directly repressing c-myc. The Blimp-1 protein contains several functional domains. Five zinc fingers are located at the c-terminal of the protein mediate DNA binding of Blimp-1 to its cognate sequence motif. Blimp-1 interacts directly with histone deacetylases (HDACs) through the Proline domain located in the center of protein. Blimp-1 mediated repression is dependent upon HDACs in transient repression assays. Blimp-1 also contains a SET domain at the N-terminus. The SET domain is found in many chromatin regulators and several SET domains from diverse proteins have recently been shown to harbour an intrinsic histone methyltransferase (HMTase) activity. Histone acetylation and phosphorylation are dynamic and reversible, whereas histone methylation appears stable on the basis on thermodynamic calculations and the fact that histone de-methylases have yet to be identified. The current data suggest that lysine methylation of histones may function as long term epigenetic imprint for maintaining chromatin states. We hypothesize that SET domain of Blimp-1 is an HMTase and that this activity is required for the stable maintenance of terminally differentiated long lived plasma cell state. To test this hypothesis, we constructed Flag tagged Blimp-1 inducible RAJI and BJAB cell lines. Blimp-1 was immunoprecipitated from both cells line and subjected to an HMTase assay against histones in the form of recombinant octamer and native polynucleosomes. Blimp-1 immunoprecipitated from RAJI, but not BJAB cells, methylated histone H3. The target lysine residue was mapped by performing HMTase assays with H3 peptides. In summary, Blimp-1 immunoprecipitate from RAJI cells possessed H3 lysine 9 MTase activity that can methylate naked or mono-methyl lysine 9 to di-methylated but not tri-methylated lysine. Two scenarios were envisaged whereby Blimp-1 immunoprecipitated from one cell line contains activity but not form the other. The first is that Blimp-1 requires accessory factors for activity, as is the case for the HMTase Ezh2, that are only expressed in RAJI cells. The second is that Blimp-1 is not an HMTase itself but associates with an HMTase expressed in the RAJI cell line. To discriminate between these possibilities, we performed immunoprecipitations of Blimp-1 from both cell lines and eluted the protein from the beads using Flag peptide and compared the immunoprecipitates by Silver staining. This revealed a difference between the cell lines, with additional proteins co-precipitating with Blimp-1 from the RAJI cells. These proteins were then subjected to mass spectrometry for identification. The co-precipitated proteins were identified as mainly HSPs.
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