Chromatin remodeling by the T cell receptor (TCR)-beta gene enhancer during early T cell development: Implications for the control of TCR-beta locus recombination.

Gene targeting studies have shown that T cell receptor (TCR)-beta gene expression and recombination are inhibited after deletion of an enhancer (Ebeta) located at the 3' end of the approximately 500-kb TCR-beta locus. Using knockout mouse models, we have measured, at different regions throughout the TCR-beta locus, the effects of Ebeta deletion on molecular parameters believed to reflect epigenetic changes associated with the control of gene activation, including restriction endonuclease access to chromosomal DNA, germline transcription, DNA methylation, and histone H3 acetylation. Our results demonstrate that, in early developing thymocytes, Ebeta contributes to major chromatin remodeling directed to an approximately 25-kb upstream domain comprised of the Dbeta-Jbeta locus regions. Accordingly, treatment of Ebeta-deleted thymocytes with the histone deacetylase inhibitor trichostatin A relieved the block in TCR-beta gene expression and promoted recombination within the Dbeta-Jbeta loci. Unexpectedly, however, epigenetic processes at distal Vbeta genes on the 5' side of the locus and at the 3' proximal Vbeta14 gene appear to be less dependent on Ebeta, suggesting that Ebeta activity is confined to a discrete region of the TCR-beta locus. These findings have implications with respect to the developmental control of TCR-beta gene recombination, and the process of allelic exclusion at this locus.

Definition of a T-cell receptor beta gene core enhancer of V(D)J recombination by transgenic mapping.

V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activating cis-recombination events. A striking example is the T-cell receptor beta-chain (TCRbeta) gene enhancer (Ebeta), which in the mouse consists of at least seven nuclear factor binding motifs (betaE1 to betaE7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within Ebeta required for recombination enhancer activity. The Ebeta core is comprised of a limited set of motifs (betaE3 and betaE4) and an additional previously uncharacterized 20-bp sequence 3' of the betaE4 motif. This core element confers cell lineage- and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the betaE1 through betaE4 motifs but not the 3' sequences, although active in inducing germ line transcription within the transgenic array, did not retain the Ebeta recombinational activity. Our results emphasize the multifunctionality of the TCRbeta enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.

The Pim-1 kinase stimulates maturation of TCRbeta-deficient T cell progenitors: implications for the mechanism of Pim-1 action.

We demonstrate that overexpression of Pim-1, a cytoplasmic serine/threonine kinase of poorly defined function, results in the development of substantial numbers of CD4(+)CD8(+) double-positive thymocytes in two independent knock-out mouse models (i.e. the RAG-1-deficient and TCRbeta gene enhancer-deleted mice) in which production of a functionally rearranged TCRbeta gene (hence the pre-TCR) is impaired. This activity of Pim-1, however, does not affect signaling through the Ras/Raf/MAP kinase cascade nor signaling which mediates suppression of TCRbeta gene recombination (i.e. allelic exclusion). While overexpression of Pim-1 positively affects cell cycle progression in selected CD4(-)CD8(-) double-negative precursors, it did not affect expression of components of the cell cycle machinery, with the exception of the G(1)-specific phosphatase Cdc25A upon antigen receptor stimulation. We propose that Pim-1 acts downstream, or in parallel, to pre-TCR-mediated selection as one factor involved in the proliferative expansion of beta-selected pre-T cells.

Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development.

Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.

TCRalpha enhancer activation occurs via a conformational change of a pre-assembled nucleo-protein complex.

The TCR alpha enhancer (Ealpha) has served as a paradigm for studying how enhancers organize trans-activators into nucleo-protein complexes thought to recruit and synergistically stimulate the transcriptional machinery. Little is known, however, of either the extent or dynamics of Ealpha occupancy by nuclear factors during T cell development. Using dimethyl sulfate (DMS) in vivo footprinting, we demonstrate extensive Ealpha occupancy, encompassing both previously identified and novel sites, not only in T cells representing a developmental stage where Ealpha is known to be active (CD4(+)CD8(+)-DP cells), but surprisingly, also in cells at an earlier developmental stage where Ealpha is not active (CD4(-)CD8(-)-DN cells). Partial occupancy was also established in B-lymphoid but not non-lymphoid cells. In vivo DNase I footprinting, however, implied developmentally induced changes in nucleo-protein complex topography. Stage-specific differences in factor composition at Ealpha sequences were also suggested by EMSA analysis. These results, which indicate that alterations in the structure of a pre-assembled nucleo-protein complex correlate with the onset of Ealpha activity, may exemplify one mechanism by which enhancers can rapidly respond to incoming stimuli.

Natural killer cell acceptance of H-2 mismatch bone marrow grafts in transgenic mice expressing HLA-Cw3 specific killer cell inhibitory receptor.

Natural killer (NK) cells express killer cell inhibitory receptors (KIRs) for major histocompatibility complex class I molecules. Engagement of these surface receptors inhibits NK cell cytotoxic programs. KIR can also be expressed on T cell subsets, and their engagement similarly results in inhibition of effector functions initiated by the CD3/T cell receptor complex. KIR genes belong to two distinct families: the immunoglobulin superfamily (IgSF KIRs) and dimeric C2 lectins (lectin-like KIRs). Whereas both IgSF (p58: CD158, p70, and p140) and lectin-like KIRs (CD94/NKG2A heterodimers) have been found in human, only lectin-like KIRs (all members of the Ly-49 family) have been described in the mouse. We have generated transgenic mice expressing an IgSF KIR, CD158b (p58.2), which recognizes HLA-Cw3. Our data show that CD158b is necessary and sufficient to confer specificity to NK cells, as well as to modulate T cell activation programs in vitro. In addition, we did not detect any adaptation of CD158b cell surface expression to that of HLA class I ligands in the CD158b x HLA-Cw3 double transgenic mice, in contrast to observations with Ly-49 in the mouse. Therefore, distinct strategies of selection/calibration appear to be used by IgSF and lectin-like KIRs. Finally, the transgenic expression of CD158b KIR prevents the in vivo rejection of H-2 mismatch bone marrow grafts, which express the cognate major histocompatibility class I HLA-Cw3 allele, demonstrating for the first time the in vivo implication of human IgSF KIRs in the negative regulation of NK cell function.

T cell development in TCR beta enhancer-deleted mice: implications for alpha beta T cell lineage commitment and differentiation.

T cell differentiation in the mouse thymus is an intricate, highly coordinated process that requires the assembly of TCR complexes from individual components, including those produced by the precisely timed V(D)J recombination of TCR genes. Mice carrying a homozygous deletion of the TCR beta transcriptional enhancer (E beta) demonstrate an inhibition of V(D)J recombination at the targeted TCR beta locus and a block in alpha beta T cell differentiation. In this study, we have characterized the T cell developmental defects resulting from the E beta-/- mutation, in light of previously reported results of the analyses of TCR beta-deficient (TCR beta-/-) mice. Similar to the latter mice, production of TCR beta-chains is abolished in the E beta-/- animals, and under these conditions differentiation into cell-surface TCR-, CD4+CD8+ double positive (DP) thymocytes depends essentially on the cell-autonomous expression of TCR delta-chains and, most likely, TCR gamma-chains. However, contrary to previous reports using TCR beta-/- mice, a minor population of TCR gamma delta+ DP thymocytes was found within the E beta-/- thymi, which differ in terms of T cell-specific gene expression and V(D)J recombinase activity, from the majority of TCR-, alpha beta lineage-committed DP thymocytes. We discuss these data with respect to the functional role of E beta in driving alpha beta T cell differentiation and the mechanism of alpha beta T lineage commitment.