The role of IL-7 in regulation of T cell receptor gene rearrangement For 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 alpha-/-, c gamma -/- 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 gamma locus. Thymocytes from these mice have no sterile transcripts of the TCR gamma 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 gamma 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 absence of the IL-7 signal. Chromatin immuneprecipitation with specific antibodies raised against acetylated histones reveal a profound difference at the histone 3 acteylation levels at the TCR gamma locus in dependence of the IL-7 signal. Thus interleukin-7 may specifically control chromatin accessibility of the TCR gamma locus by alteration of methylation and histone acetylation. We are currently identifying sites within the TCR gamma 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 alteration We 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 methylation. Deletion of Lsh in embryonal stem cells by homologous recombination results in early death of newborns indicating a crucial role for Lsh in murine development. Lsh deleted mice are slightly smaller in size at birth and show renal defects with signs of tissue necrosis/apoptosis but are otherwise indistinguishable form control littermates. Since Lsh is preferentially expressed in lymphoid tissue in the adult mice we also tested its role in lymphocyte development. 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 gamma ), 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 effect of Lsh on chromatin structure.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010014-07
Application #
6762325
Study Section
(LMI)
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Xi, Sichuan; Geiman, Theresa M; Briones, Victorino et al. (2009) Lsh participates in DNA methylation and silencing of stem cell genes. Stem Cells 27:2691-702
Zhu, Heming; Geiman, Theresa M; Xi, Sichuan et al. (2006) Lsh is involved in de novo methylation of DNA. EMBO J 25:335-45
Fan, Tao; Hagan, John P; Kozlov, Serguei V et al. (2005) Lsh controls silencing of the imprinted Cdkn1c gene. Development 132:635-44
Huang, Jiaqiang; Fan, Tao; Yan, Qingsheng et al. (2004) Lsh, an epigenetic guardian of repetitive elements. Nucleic Acids Res 32:5019-28
Yan, Qingsheng; Huang, Jiaqiang; Fan, Tao et al. (2003) Lsh, a modulator of CpG methylation, is crucial for normal histone methylation. EMBO J 22:5154-62
Muegge, Kathrin (2003) Modifications of histone cores and tails in V(D)J recombination. Genome Biol 4:211
Muegge, Kathrin; Young, Howard; Ruscetti, Francis et al. (2003) Epigenetic control during lymphoid development and immune responses: aberrant regulation, viruses, and cancer. Ann N Y Acad Sci 983:55-70
Fan, Tao; Yan, Qingsheng; Huang, Jiaqiang et al. (2003) Lsh-deficient murine embryonal fibroblasts show reduced proliferation with signs of abnormal mitosis. Cancer Res 63:4677-83
Geiman, T M; Muegge, K (2000) Lsh, an SNF2/helicase family member, is required for proliferation of mature T lymphocytes. Proc Natl Acad Sci U S A 97:4772-7
Lee, C K; Kim, K; Geiman, T M et al. (1999) Cloning thymic precursor cells: demonstration that individual pro-T1 cells have dual T-NK potential and individual pro-T2 cells have dual alphabeta-gammadelta T cell potential. Cell Immunol 191:139-44

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