Functional characterization of the murine gamma/delta TCR V-gamma-3 promoter region
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The first functional T cells in the murine thymus express γδ T cell receptors (TCR), utilizing a Vγ3/Jγ1/Cγ1 rearrangement. Distinctive rearrangement of these segments has been linked to an accessible chromatin configuration. Elements within transcriptional promoters and enhancers have been demonstrated to regulate accessibility of the recombinase machinery to these gene segments. Therefore, an analysis of the Vγ3 promoter region was performed to determine possible regulatory regions responsible for this particular expression pattern. Within the Vγ3 minimal promoter region (-147 to +208 bp with respect to the start site, as determined by reporter gene studies), DNase I footprinting identified three regions of partial protection and hypersensitivity within +37/+208. These include an AT-rich tract, which maps to the first intron (+65/+181) and resembles matrix-associated regions (MARs) such as those located within immunoglobulin heavy chain (IgH) enhancer and promoter elements. Transcription factors, such as Bright (B-cell regulator of immunoglobulin heavy chain transcription), CDP/Cux (CCAAT displacement protein) and SATB1 (Special AT-rich sequence binding protein), have been shown to act as positive and negative regulators of IgH genes by binding to these MARs. EMSA experiments established that Bright, CDP/Cux and SATB1 bound to regions within +65/+181. Bright and SATB1 activated Vγ3-mediated transcription, while Cux was shown to repress transcription. Additional reporter data established a putative repressor region upstream of the start site between -897 and -586. Although -897/-586 contained six consensus GATA binding sites, unexpectedly, no functional role for this category of transcription factors was found. Instead, this region enhanced Cuxmediated repression of -147/+208 whereas Bright and SATB1 were able to abrogate its repressive effect. CDP/Cux and SATB1 have been shown to negatively and positively regulate transcription of T cell specific genes. Although Bright protein expression previously had been observed only in B lymphocytes, Bright protein does accumulate in γδ tumors and clones, and Bright mRNA can be detected in fetal thymus at a time concomitant with appearance of Vγ3-expressing T cells. Together these results provide a new role for Bright and SATB1 as positive transcriptional regulators, and extend the role for CDP/Cux, as a negative transcriptional regulator, of the murine TCR Vγ3 gene locus.