Interleukin 7 receptor control of T cell receptor gamma gene rearrangement: role of receptor-associated chains and locus accessibility

Abstract

VDJ recombination of T cell receptor and immunoglobulin loci occurs in immature lymphoid cells. Although the molecular mechanisms of DNA cleavage and ligation have become more clear, it is not understood what controls which target loci undergo rearrangement. In interleukin 7 receptor (IL-7R)alpha-/- murine thymocytes, it has been shown that rearrangement of the T cell receptor (TCR)-gamma locus is virtually abrogated, whereas other rearranging loci are less severely affected. By examining different strains of mice with targeted mutations, we now observe that the signaling pathway leading from IL-7Ralpha to rearrangement of the TCR-gamma locus requires the gammac receptor chain and the gammac-associated Janus kinase Jak3. Production of sterile transcripts from the TCR-gamma locus, a process that generally precedes rearrangement of a locus, was greatly repressed in IL-7Ralpha-/- thymocytes. The repressed transcription was not due to a lack in transcription factors since the three transcription factors known to regulate this locus were readily detected in IL-7Ralpha-/- thymocytes. Instead, the TCR-gamma locus was shown to be methylated in IL-7Ralpha-/- thymocytes. Treatment of IL-7Ralpha-/- precursor T cells with the specific histone deacetylase inhibitor trichostatin A released the block of TCR-gamma gene rearrangement. This data supports the model that IL-7R promotes TCR-gamma gene rearrangement by regulating accessibility of the locus via demethylation and histone acetylation of the locus.

Figure 3

Defective rearrangement of the TCR-γ locus in Jak3^−/− thymocytes. Thymocytes from Jak3^−/− mice were compared with normal adult C57BL/6, fetal (day 15), and RAG2^−/− mice for rearrangements of the TCR-γ locus as in Fig. 2. Control lanes represent a pool of five individuals, whereas individual mice are shown from the Jak3^−/− and Jak3^+/− strains.

Figure 1

Location of primers and probes within the TCR-γ1 locus.

Figure 2

Defective rearrangement of the TCR-γ locus in IL-7R^−/− and γ_c ^−/− thymocytes. Thymocyte suspensions were prepared from normal adult C57BL/6 or fetal (day 15) mice or from mice deficient in IL-7Rα, Rag2, or γ_c. DNA was extracted and PCR reactions were performed to detect the indicated rearrangements of the TCR-γ locus. The control PCR reaction uses internal primers for Vγ2 (which detects both germ line and rearranged DNA). Each lane represents a pool of five individuals except for γ_c ^−/− and ^+/−, for which three individuals are shown.

Figure 6

Methylation of the TCR-γ locus in IL-7Rα^−/− and γ_c ^−/− thymocytes. Thymocyte cell suspensions were prepared from IL-7Rα^−/−, γ_c ^−/−, RAG2^−/−, or SCID mice. DNA was extracted and digested with MspI or HpaII. Southern blot analysis was performed using a probe for the TCR-γ constant region. Each lane represents a pool of two to five individuals except γ_c ^−/− representing one individual per lane. Position of MspI/HpaII cleavage sites within the TCR-γ locus are indicated with an arrow. Position of the probe used for hybridization is indicated with a closed bar.

Figure 4

Defective expression of sterile TCR-γ transcripts and constant region transcripts in IL-7Rα^−/− thymocytes. Thymocyte suspensions were prepared from normal C57BL/6 mice (embryonic day 15), IL-7R^−/−, or RAG2^−/− mice. RNA was extracted and cDNA was synthesized. PCR reactions were performed for unrearranged transcripts from the TCR-γ locus, including the constant region and the indicated V regions. GAPDH transcripts were analyzed as a positive control for RNA loading. Three dilutions of each cDNA were tested to reflect quantitation.

Figure 5

Nuclear proteins from IL-7Rα^−/− thymocytes binding to regions of the TCR-γ enhancer. Thymocyte cell suspensions were prepared from normal C57BL/6 embryos (day 15 of gestation), IL-7Rα^−/−, or Rag2^−/− mice. Nuclear extracts were prepared. Gel mobility shift assays were performed using labeled oligonucleotides from three sites in the enhancer region of the TCR-γ locus. To assess specificity of binding, unlabeled competitors were added in 10-fold excess to the labeled oligonucleotide (Specific denotes the same sequence used in the labeled probe; Non-specific is an unrelated sequence of the same size). Antibodies were added to determine whether the NFγ2-binding protein complex contained STAT5a or -b proteins.

Figure 7

TSA restores rearrangement of the TCR-γ locus in IL-7Rα^−/− thymocytes. Bone marrow from IL-7R^−/− mice was used to reconstitute irradiated embryonic thymic lobes (from C57BL/6 mice) in hanging drops. Reconstituted lobes were then cultured in the indicated concentrations of TSA for 96 h and analyzed for rearrangement of the TCR-γ locus.