Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure

Abstract

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4- CD8- (double negative [DN]) and CD4+ CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of approximately 100 variable gene segments contributes substantially to the adult TCRdelta repertoire. Moreover, although most contribute to the TCRalpha repertoire, variable gene segments that are Jalpha proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRdelta repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

Figure 1.

Schematic map of the TcraTcrd locus. The map shows the locations of the V gene segments analyzed in this study (approximately one third of the total), as well as Dad1. Eδ and Eα are identified by black circles. The gray bars classify the V gene segments according to location: proximal, central, central duplication, and distal. V gene segment nomenclature is according to IMGT (reference 8), with conversion to the prior nomenclature of Arden (reference 7) provided. The map is drawn to scale with distances indicated on the upper line.

Figure 2.

V gene segment histone modifications. Histone H3 and H4 acetylation was measured in chromatin prepared from DN thymocytes (Rag2^−/−), DP thymocytes (Rxβ), Eα^−/− DP thymocytes (Eα^−/−Rxβ), and non–T cells (T-deficient splenocytes of Tcrb^−/−Tcrd^−/− mice). Data for all individual gene segments are displayed according to the order in which the gene segments appear in the TcraTcrd locus. In cases where multiple family members are analyzed in a single PCR, the data is displayed according to the locus position of the most distal family member. Arrows identify the dominant adult Vδ gene segments. The data represent the mean ± SEM of triplicate PCR reactions. Analysis of two independent chromatin preparations gave similar results.

Figure 3.

V gene segment germline transcription. Germline transcription was measured by RT-PCR using serial threefold dilutions of cDNA prepared from DN thymocytes (Rag2^−/−), DP thymocytes (Rxβ), and Eα^−/− thymocytes (Eα^−/−). − identifies control PCR reactions using templates prepared without the addition of reverse transcriptase. Wedges indicate graded amounts of input cDNA. The TRAV15 primers detect all four members of the TRAV15 family. Analysis of two independent cDNA preparations gave similar results. White line indicates that intervening lanes have been spliced out.