Members of the Runx family of transcriptional regulators are required for the appropriate expression of CD4 and CD8 at discrete stages of T cell development. The roles of these factors in other aspects of T cell development are unknown. We used a strategy to conditionally inactivate the genes encoding Runx1 or Runx3 at different stages of thymocyte development, demonstrating that Runx1 regulates the transitions of developing thymocytes from the CD4−CD8− double-negative stage to the CD4+CD8+ double-positive (DP) stage and from the DP stage to the mature single-positive stage. Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages, respectively. Runx1-deficient CD4+ T cells had markedly reduced expression of the interleukin 7 receptor and exhibited shorter survival. In addition, inactivation of both Runx1 and Runx3 at the DP stages resulted in a severe block in development of CD8+ mature thymocytes. These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells.

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T cell receptor (TCR) ␦ and ␣ variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCR␦ segments are situated between TCR␣ segments. The TCR␣ enhancer (E␣) located at the 3 end of the TCR␣͞␦ locus functions over a long chromosomal distance to promote TCR␣ rearrangement and maximal TCR␦ expression; whereas the TCR␦ enhancer (E␦) is located among the TCR␦ segments and functions with additional element(s) to mediate TCR␦ rearrangement. We used gene-targeted mutation to evaluate whether the identity of E␣ and the position of E␦ are critical for the developmental stage-specific assembly of TCR ␦ and ␣ variable region genes. Specific replacement of E␣ with E␦, the core E␣ element (E␣C), or the Ig heavy chain intronic enhancer (iE ), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCR␣ rearrangement beyond that observed in the absence of E␣. Therefore, the identity and full complement of E␣-binding sites are critical for promoting accessibility within the TCR␣ locus. In the absence of the endogenous E␦ element, specific replacement of E␣ with E␦ also did not promote TCR␦ rearrangement. However, deletion of intervening TCR␣͞␦ locus sequences to restore the inserted E␦ to its normal chromosomal position relative to 5 sequences rescued TCR␦ rearrangement. Therefore, unlike E␣, E␦ lacks ability to function over the large intervening TCR␣ locus and͞or E␦ function requires proximity to additional upstream element(s) to promote TCR␦ accessibility.T he exons that encode T cell receptor (TCR) and Ig variable regions are assembled during lymphocyte development from component variable (V), diversity (D), and joining (J) gene segments (1, 2). Both ␣␤ and ␥␦ T cells develop from CD4 Ϫ ͞ CD8 Ϫ (double negative, DN) thymocytes in which TCR␤, -␥, and -␦ genes are all assembled (3). Productive VDJ␤ rearrangements generate TCR␤ chains that associate with surrogate TCR␣ chains to form preTCRs that signal expansion and differentiation to the CD4 ϩ ͞CD8 ϩ (double positive, DP) thymocyte stage (4, 5). Functional VJ␣ rearrangements in DP cells generate TCR␣ chains that, when expressed as surface ␣␤ TCR, direct cellular selection and development to the CD4 ϩ or CD8 ϩ (single positive, SP) thymocytes that exit the thymus as mature ␣␤ T cells (6). Alternatively, productive VDJ␦ and VJ␥ rearrangements in DN progenitor (pro-) T cells generate TCR␦ and -␥ chains that form cell surface ␥␦ TCR and direct ␥␦ T cell development (4, 5).The TCR␣͞␦ locus spans 1 Mb and contains variable region gene segments for two distinct antigen receptors (7). The 5Ј end of the locus consists of a cluster of Ϸ90 V␣͞V␦ gene segments with V␦s concentrated near the 3Ј end of the cluster, and the 3Ј end of the locus consists of a cluster of 50 J␣ segments that spans Ϸ60 kb followed by the TCR␣ constant region (C␣). The D␦ (D␦1 and D␦2) and J␦ (J␦1 and J␦2) segments, the TCR␦ constant region (C␦) and V␦5 lie between the V␣͞V␦ and...

Assembly of TCRβ variable region genes is ordered during thymocyte development with Dβ to Jβ rearrangement preceding Vβ to DJβ rearrangement. The 5′Dβ 12-RSS is required to precisely and efficiently target Vβ rearrangement beyond simply enforcing the 12/23 rule. By prohibiting direct Vβ to Jβ rearrangement, this restriction ensures Dβ gene segment use in the assembly of essentially all TCRβ variable region genes. In this study, we show that rearrangement of Vβ 23-RSSs is significantly biased to the Dβ 12-RSS over Jβ 12-RSSs on extrachromosomal recombination substrates in nonlymphoid cells that express the recombinase-activating gene-1/2 proteins. These findings demonstrate that targeting of Vβ to Dβ rearrangement can be enforced by the V(D)J recombinase in the absence of lymphoid-specific factors other than the recombinase-activating gene-1/2 proteins.