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Department of Immunology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261
B cells produce Ig H chain (IgH) mRNA and protein, primarily of the membrane-bound specific form. Plasma cells produce 20- to 50-fold higher amounts of IgH mRNA, most processed to the secretory specific form; this shift is mediated by substantial changes in RNA processing but only a small increase in IgH transcription rate. We investigated RNA polymerase II (RNAP-II) loading and phosphorylation of its C-terminal domain (CTD) on the IgG2a H chain gene, comparing two mouse cell lines representing B (A20) and plasma cells (AxJ) that express the identical H chain gene whose RNA is processed in different ways. Using chromatin immunoprecipitation and real-time PCR, we detected increased RNAP-II and Ser-2 and Ser-5 phosphorylation of RNAP-II CTD close to the IgH promoter in plasma cells. We detected increased association of several 3' end-processing factors, ELL2 and PC4, at the 5' end of the IgH gene in AxJ as compared with A20 cells. Polymerase progress and factor associations were inhibited by 5,6-dichlorobenzimidazole riboside, a drug that interferes with the addition of the Ser-2 to the CTD of RNAP-II. Taken together, these data indicate a role for CTD phosphorylation and polyadenylation/ELL2/PC4 factor loading on the polymerase in the choice of the secretory poly(A) site for the IgH gene.
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1 This work was supported by National Institutes of Health Grant CA86433 (to C.M.) and Training Grant T32 CA82084 (to S.A.S.).
2 Current address: Targeted Molecular Diagnostics, 610 Oakmont Lane, Westmont, IL 60559.
3 Address correspondence and reprint requests to Dr. Christine Milcarek, Department of Immunology, University of Pittsburgh, E1054 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail address: milcarek{at}pitt.edu
4 Abbreviations used in this paper: blimp-1, B lymphocyte induction of maturation protein 1; IgH, Ig H chain; sec, secretory specific; mb, membrane bound; hnRNP, heterogeneous nuclear ribonucleoprotein; CTD, C-terminal domain; RNAP-II, RNA polymerase II; IP, immunoprecipitation; ChIP, chromatin IP; GSP, gene-specific antisense primer; QPCR, quantitative PCR; Ct, cycle threshold; RT, reverse transcription; EH, enhancer region; DRB, 5,6-dichlorobenzimidazole riboside; Tm, melting temperature; IVS, intervening sequence.
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