Specific projectsThe role of IL-7 in regulation of T cell receptor gene rearrangementFor an ordered recombination process to occur, alteration of chromatin structure is required allowing access of the specific V(D)J recombinase to the T cell receptor locus. We have previously identified interleukin-7, a stromal cell derived cytokine, that promotes rearrangement in vitro. Mice that are deficient in part of the IL-7R signal transduction pathway (IL-7R-/-, c--/- or Jak3-/-) are unable to rearrange their T cell receptor chain in vivo. By searching for recombination intermediates we found that IL-7R-/- thymocytes are unable to initiate the cleavage at the appropriate site at the TCR locus. Thymocytes from these mice have no sterile transcripts of the TCR locus suggesting a repressed form of chromatin. Nuclei prepared from IL-7R -/- thymocytes were not accessible for cleavage by recombinant rag proteins in vitro indicating that the chromatin is not accessible to V(D)J recombinase in the absence of the IL-7 signal. The TCR locus is heavily methylated in thymocytes of IL-7R-/- mice suggesting a possible mechanism for suppression of chromatin. Modification of histone acetylation with the specific histone deacetylase inhibitor, trichostatin A, could overcome the inhibition of rearrangement in the absenec of the IL-7 signal. Thus interleukin-7 may specifically control chromatin accesibiltiy of the TCR locus by alteration of methylation and histone acetylation. We are currently identifying sites within the TCR locus that are responsible for specific methylation and chromatin accessibilty under the control of IL-7.Role of a LSH, an SNF2/helicase family member in lymphocyte activation and chromatin alterationWe have recently cloned a novel member of the SNF2/helicase family (LSH) that is marked by the presence of seven conserved helicase domains. Members of the SNF2/helicase family are known to modulate chromatin accessibilty and thus regulate transcription, recombination and DNA repair. LSH (lymphoid specific helicase), is preferentially expressed in lymphoid tissue and is highly inducible in T cells by T cell receptor triggering and CD28 co- stimulation. Its time course of expression correlates closely with the onset of the S-phase in activated T cells. In order to test whether LSH and its presumed chromatin remodeling activity plays a distinct role in lymphocyte development we deleted the LSH gene in embryonal stem cells by homologous recombination. Fetal liver of LSH-/- embryos were used as a source for lymphoid precursor cells in order to reconstitute Rag2-/- mice that are devoid of a lymphoid system. Peripheral T lymphocytes are reduced by 50% and B lymphocytes are reduced to one third of their wild type controls in the rag-chimeric animals. Analysis of thymic subpopulations reveals a normal number of CD4-CD8- thymic precursors but reduced numbers of more mature CD4+CD8+ cells or single positive cells suggesting a moderate arrest at the transition to the double positive stage. Mature T cells in the periphery can be stimulated to efficiently secrete cytokines (such as IL-2 or IFN ), however, they fail to proliferate in response to polyclonal T cell stimuli. Instead T cells undergo apoptosis. Thus LSH is required for normal development of the lymphoid system and in addition is required for normal growth of mature T cells. We are currently examining the peptide complex that associates with LSH and studying the molecular mechanism by which LSH protects the cells against apoptosis and leads them into normal cell cycle.AIDS TITLE: N/A - T cell Development, Gene Rearrangement, SNF2/Helicase, Chromatin,
Showing the most recent 10 out of 11 publications
