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Differential Activation of Dual Promoters Alters Dβ2 Germline Transcription during Thymocyte Development¹

Department of Microbiology, North Carolina State University, Raleigh, NC 27695

Ag receptor genes are assembled through somatic rearrangements of V, D, and J gene segments. This process is directed in part by transcriptional enhancers and promoters positioned within each gene locus. Whereas enhancers coordinate reorganization of large chromatin stretches, promoters are predicted to facilitate the accessibility of proximal downstream gene segments. In TCR β locus, rearrangement initiates at two D-J cassettes, each of which exhibits transcriptional activity coincident with DJ rearrangement in CD4/CD8 double-negative pro-T cells. Consistent with a model of promoter-facilitated recombination, assembly of the DJβ1 cassette is dependent on a Dβ1 promoter (PDβ1) positioned immediately 5' of the D. Assembly of DJβ2 proceeds independent from that of DJβ1, albeit with less efficiency. To gain insight into the mechanisms that selectively alter D usage, we have defined transcriptional regulation at Dβ2. We find that both DJβ cassettes generate germline messages in murine CD44⁺CD25^– double-negative 1 cells. However, transcription of unrearranged DJβ2 initiates at multiple sites 400–550 bp downstream of the Dβ2. Unexpectedly, loci from which germline promoter activity has been deleted by DJ rearrangement redirect transcription to sites immediately 5' of the new DJβ2 joint. Our analyses suggest that 3'-PDβ2 activity is largely controlled by NF-B RelA, whereas 5'-PDβ2 activity directs germline transcription of DJβ2 joints from initiator elements 76 bp upstream of the Dβ2 5' recombination signal sequence. The unique organization and timing of Dβ2 promoter activity are consistent with a model in which promoter placement selectively regulates the rearrangement potential of Dβ2 during TCR β locus assembly.

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 grants from the Elsa U. Pardee Cancer Foundation (2005-0069) and the North Carolina Agriculture Research Service (NC06711).

² Address correspondence and reprint requests to Dr. Michael L. Sikes, Department of Microbiology, North Carolina State University, P.O. Box 7615, Raleigh, NC 27695. E-mail address: mlsikes{at}ncsu.edu

³ Abbreviations used in this paper: RSS, recombination signal sequence; Ct, cycle threshold; DN, double negative; DP, double positive; Eβ, Tcrb enhancer; inr, transcription initiator; IP, immunoprecipitation; PDβ, Dβ promoter; SP, single positive; Tcra, TCR locus; Tcrb, TCR β locus; USF1, upstream stimulatory factor 1.

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