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Regulation of the Murine D2 Promoter by Upstream Stimulatory Factor 1, Runx1, and c-Myb¹

Department of Immunology, Duke University Medical Center, Durham, NC 27710

Accessibility control of V(D)J recombination at Ag receptor loci depends on the coordinate activities of transcriptional enhancers and germline promoters. Recombination of murine Tcrd gene segments is known to be regulated, at least in part, by the Tcrd enhancer (E) situated in the J2-C intron. However, there has been little characterization of promoters and other cis-acting elements that are activated by or collaborate with E and that might function to regulate Tcrd gene recombination events. We now describe a strong promoter that is tightly associated with the murine D2 gene segment. EMSAs reveal that upstream stimulatory factor 1, Runx1, c-Myb, lymphoid enhancer binding factor 1, NF1, and E47 all interact with this promoter in vitro. Of these, upstream stimulatory factor 1, Runx1, and c-Myb appear necessary for full promoter activity in transiently transfected cells. Moreover, the same three factors were found to interact with the promoter in vivo by chromatin immunoprecipitation. We suggest that these factors play important roles as E-dependent regulators of D2 accessibility in vivo. Consistent with the established roles of c-Myb and Runx factors in E function, we detected low level, enhancer-independent activity of the D2 promoter in transient transfection experiments. We speculate that the D2 promoter may play a role as a weak, enhancer-independent regulator in vivo, and might contribute to residual Tcrd rearrangement in E^–/– mice.

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