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Increased Phosphorylation of the Carboxyl-Terminal Domain of RNA Polymerase II and Loading of Polyadenylation and Cotranscriptional Factors Contribute to Regulation of the Ig Heavy Chain mRNA in Plasma Cells¹

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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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The JI 2007 179: 7191-7192. [Full Text]  

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