Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination

Abstract

During the recombination of variable (V) and joining (J) gene segments at the T cell receptor alpha locus, a ValphaJalpha joint resulting from primary rearrangement can be replaced by subsequent rounds of secondary rearrangement that use progressively more 5' Valpha segments and progressively more 3' Jalpha segments. To understand the mechanisms that target secondary T cell receptor alpha recombination, we studied the behavior of a T cell receptor alpha allele (HYalpha) engineered to mimic a natural primary rearrangement of TRAV17 to Jalpha57. The introduced ValphaJalpha segment was shown to provide chromatin accessibility to Jalpha segments situated within several kilobases downstream and to suppress germ-line Jalpha promoter activity and accessibility at greater distances. As a consequence, the ValphaJalpha segment directed secondary recombination events to a subset of Jalpha segments immediately downstream from the primary rearrangement. The data provide the mechanistic basis for a model of primary and secondary T cell receptor alpha recombination in which recombination events progress in multiple small steps down the Jalpha array.

Fig. 1.

Tcra allele structure and germ-line transcription. (A) Schematic maps of the wild-type, ΔTEA, and HYα Tcra alleles are shown. The gray rectangles identify Vα segments, and black vertical lines represent Jα segments (numbered). Bent arrows identify the locations of active promoters. The diagram depicts changes in promoter activity on the ΔTEA and HYα alleles. (B) RT-PCR analysis of Tcra transcripts. The structure of spliced Jα transcripts and the PCR strategies used to detect individual Jα transcripts and total Cα-containing transcripts are presented. PCR primers are indicated by arrowheads. Serial 3-fold dilutions of cDNA prepared from R×β, ΔTEA R×β, and HYα R×β thymocytes were analyzed by RT-PCR to detect Jα transcripts, total Cα-containing transcripts, or β-actin (Actb) transcripts. (−) indicates a control PCR lacking cDNA. PCRs were analyzed by Southern blotting with Cα or Actb probes. Because of variations in PCR efficiency, PCR cycle number, and exposure time, the figure should not be taken to indicate quantitative relationships among the different transcripts; however, for any individual transcript, quantitative comparisons are valid among the different thymus RNA samples.

Fig. 2.

Tcra allele histone modifications. Histone H3 and H4 acetylation was analyzed by immunoprecipitation of chromatin prepared from thymocytes of R×β, ΔTEA R×β, HYα R×β, and Rag2^−/− mice. Sites associated with Jα segments (numbered) were analyzed. Bound and input fractions were quantified using real-time PCR, and ratios of bound/input were normalized to that for β₂-microglobulin in each sample. The data shown are representative of two experiments and are expressed as the mean ± SEM of triplicate PCRs.

Fig. 3.

Targeting of secondary Vα to Jα recombination on the HYα allele. TRAV12 (Vα8) to Cα RT-PCR products obtained from thymocytes of 3- to 6-week-old ΔTEA Rorc^−/− and HYα Rorc^−/− mice were cloned and sequenced to evaluate Jα usage. The fraction of clones using particular Jα segments (numbered) is plotted. Numbers of clones analyzed for ΔTEA Rorc^−/− and HYα Rorc^−/− were 96 and 99, respectively. Because the TRAV12 primer detects multiple dispersed TRAV12 family members, TRAV12 rearrangements are relatively unbiased with respect to Jα usage.

Fig. 4.

Thymic Jα repertoires of mice carrying the HYα allele. TRAV12 (Vα8) to Cα and glucose-6-phosphate dehydrogenase (G6PD) RT-PCR products obtained from thymocytes of 4- to 5-week-old wild-type, ΔTEA, and HYα mice were fractionated on agarose gels and immobilized to nylon filters. Jα usage was evaluated by hybridization with ³²P-labeled Jα and G6PD oligonucleotide probes and was expressed as (variant allele Jα/G6PD)/(wild-type allele Jα/G6PD). Results represent the mean ± SEM of three or four determinations.

Fig. 5.

Model for primary and secondary Vα to Jα recombination. The diagram depicts Vα segments as gray rectangles, accessible Jα segments as long black vertical lines, and inaccessible Jα segments as short black vertical lines. Promoters are depicted as bent arrows. In the model, primary Vα to Jα rearrangement is directed to a set of Jα segments made accessible by the germ-line TEA and Jα49 promoters. Secondary Vα to Jα rearrangements are directed to discrete sets of Jα segments made accessible by successively introduced Vα promoters. Rearrangements would progress down the Jα array in multiple small steps.