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The B cell-specific activity of immunoglobulin (Ig) gene promoters is to a large extent mediated by the conserved octamer motif ATTTGCAT. This requires the DNA binding octamer factors Oct-1 and/or Oct-2, as well as an additional B cell-restricted non-DNA binding cofactor. We recently cloned such a coactivator specific for Oct-1 or Oct-2 from human B cells and called it OBF-1. Here we report the isolation and characterization of the murine homologue. Full-length cDNA clones as well as genomic clones were isolated and the gene structure was determined. The deduced protein sequence shows that the mouse protein has an identical length, is likewise proline rich and shows 89% overall identity to the human protein. The OBF-1 gene is expressed in a very highly B cell-specific manner and is transcribed in cells representative of all stages of B cell differentiation, including the earliest ones. We show that OBF-1 interacts in the absence of DNA with the POU domain of Oct-1 or Oct-2 and also with the general transcription factors TBP and TFIIB. Furthermore, we demonstrate that although OBF-1 efficiently activates promoter octamer sites, it does not activate enhancer octamer sites.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is heavily phosphorylated during the transition from transcription initiation to the establishment of an elongationcompetent transcription complex. FCP1 is the only phosphatase known to be specific for the CTD of the largest subunit of RNAPII, and its activity is believed to be required to reactivate RNAPII, so that RNAPII can enter another round of transcription. We demonstrate that FCP1 is a phosphoprotein, and that phosphorylation regulates FCP1 activities. FCP1 is phosphorylated at multiple sites in vivo. The CTD phosphatase activity of phosphorylated FCP1 is stimulated by TFIIF, whereas dephosphorylated FCP1 is not. In addition to its role in the recycling of RNAPII, FCP1 also affects transcription elongation. Phosphorylated FCP1 is more active in stimulating transcription elongation than the dephosphorylated form of FCP1. We found that only phosphorylated FCP1 can physically interact with TFIIF. We set out to purify an FCP1 kinase from HeLa cells and identified casein kinase 2, which, surprisingly, displayed a negative effect on FCP1-associated activities. N umerous studies have extensively documented the effects of phosphorylation on the transcriptional properties of RNA polymerase II (RNAPII) (1-5). The target of these regulatory phosphorylation events is the C-terminal domain (CTD) of the largest subunit of RNAPII. The hypophosphorylated form of RNAPII, designated RNAPIIA, was demonstrated to be essential for preinitiation complex formation and transcription initiation, whereas hyperphosphorylated RNAPII, designated RNAPIIO, is involved in transcription elongation (6, 7). During the transition from transcription initiation to elongation, the CTD of the largest subunit of the RNAPII is heavily phosphorylated at Ser-2 and Ser-5 within the multiply repeated consensus sequence YS 2 PTS 5 PS (2,4,8). The CTD of the mammalian protein contains 52 repeats, which are phosphorylated by a number of different protein kinases, including CDK7 (5, 9), CDK9 (10, 11), and CDK8 (3, 12). The CDK7 and CDK9 kinases are tightly linked to the transcription cycle. In contrast, the kinase activity of CDK8, a component of the Mediator complex, was shown to negatively regulate transcription initiation.Although multiple kinases target the CTD of RNAPII, the only protein phosphatase known to dephosphorylate the CTD is FCP1. FCP1 was identified in both mammalian cells (13-15) and Saccharomyces cerevisiae (16,17). Dephosphorylation of the CTD of the largest subunit of RNAPII by FCP1 can facilitate recycling of the hyperphosphorylated form of RNAPII, allowing RNAPII to enter another round of transcription (15). FCP1 dephosphorylates the CTD of the largest subunit of RNAPII in solution (14)(15)(16)(18)(19)(20) and when associated with transcription elongation complexes (15,21,22). The phosphatase activity of FCP1 is stimulated by the general transcription factor TFIIF; however, TFIIB inhibits this stimulation (14). Mapping of the interaction domains betwee...

The lymphoid-specific transcription factor Oct-2a contains two transcriptional activation domains which are located within the N-terminal and C-terminal regions. To study their differential activation properties, we linked the isolated effector domains to the GAL4 DNA-binding domain. We have shown that both activating regions of Oct-2a, isolated from their natural context, can activate transcription as promoter factors. In contrast to the C-terminus, activation by the N-terminal domain is dependent on a yet unidentified factor(s) binding to the simian virus 40 enhancer. The results obtained by duplication of activation domains or their mixed combination suggest that the domains are functionally independent. However, activation from a remote position could only be achieved with the C-terminus of Oct-2a in B cells. In lymphoid cells, higher activation levels were observed, suggesting that distinct B-cell-specific cofactors in concert with the effector domains of Oct-2a might be involved in mediating transcription from proximal and remote positions. Furthermore, we identified a repression domain at the N-terminus of Oct-2a. When transferred to a potent activator, transcriptional stimulation was inhibited efficiently. These results underscore the modular structure of Oct-2a with separable domains for activation and repression and suggest that Oct-2a might have complex regulatory functions in vivo.

The ILRE (interleukin response element) contained within the promoter of the interleukin-6 (IL-6) gene is defined as the site recognized by the p65 NF-B transcriptional activator and is crucial for activation of the IL-6 gene. The region of the promoter containing the ILRE is complex containing a CCAAT enhancer-binding protein (C/EBP) site immediately upstream of the ILRE, which is required for optimal activation of the IL-6 gene. Additionally, the ILRE overlaps a site that is recognized by the mammalian transcriptional repressor RBP (CBF-1), and RBP binding within the ILRE region represses activated IL-6 expression. In this study, the complexity of this region is further revealed by the identification of a second nested C/EBP site, which overlaps that of RBP and therefore also the ILRE. Optimal activation requires both the upstream and newly identified C/EBP sites in conjunction with the p65 NF-B binding site. We previously reported that RBP represses IL-6 activation but does not target p65. We extend these analyses here to show that RBP binding does not occlude p65 from binding but instead directly overlaps the newly identified downstream C/EBP site, thereby impeding p65-C/ EBP-mediated co-activation. This result suggests a role for RBP in the repression of other genes containing a C/EBP site that exhibits sequence overlap with the RBP site.

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