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Regulatory Elements in the Promoter of a Murine TCRD V Gene Segment^{1 ,2}

Laura J. Kienker^*, Maya R. Ghosh and Philip W. Tucker^3,

^* Harold C. Simmons Arthritis Research Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75235; and Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712

TCRD V segments rearrange in an ordered fashion during human and murine thymic development. Recombination requires the accessibility of substrate gene segments, and transcriptional enhancers and promoters have been shown to regulate the accessible chromatin configuration. We therefore investigated the regulation of TCRD V rearrangements by characterizing the promoter of the first TCRD V segment to be rearranged, DV101S1, under the influence of its own enhancer. Sequences required for full promoter activity were identified by transient transfections of normal and mutated promoters into a human lymphoma, and necessary elements fall between -86 and +66 nt, relative to the major transcription start site. They include a cAMP responsive element (CRE) at -62, an Ets site at -39, a TATA box at -26, the major transcriptional start site sequence (-8 to -5 and -2 to +11), and a downstream sequence (+12 to +33). Gel shift analyses and in vitro DNase I footprinting showed that nuclear proteins bind to the functionally relevant CRE, Ets, +1 to +10 sequence, and the +17 to +21 sequence. Nuclear proteins also bind to an E box at -52, and GATA-3 binds to a GATA motif at -5, as shown by Ab ablation-supershift experiments, but mutations that abrogated protein binding to these sites failed to affect DV101S1 promoter activity. We conclude that not all protein-binding sites within the DV101S1 minimal promoter are important for enhancer driven TCRD gene transcription. Further, the possibility remains that the GATA and E box sites function in enhancer independent DV101S1 germline transcription.