| T cell receptors (TCR) and immunoglobulin molecules (Ig) are essential for activating a targeted immune response. Vertebrates use a highly regulated, site-specific recombination reaction termed V(D)J recombination to produce an adequately diverse array of TCR and Ig. One poorly characterized means of regulating V(D)J recombination is through developmentally regulated changes in chromatin structure. The three sections of this thesis aim to expand our understanding of how chromatin structure and chromatin-associated proteins are involved in the multi-level regulation of V(D)J recombination.;First, we demonstrate that the inability of the V(D)J recombinase to cleave mononucleosomes in vitro is due to the interfering effects of the histone tails. Binding and cleavage are restored upon removal or acetylation of the histone tails and when an ATP dependent remodeling complex alters the nucleosome structure.;Second, using chromatin immunoprecipitation analysis we have defined distinct combinations of modified histones at recombinationally accessible and inaccessible domains within the antigen receptor loci. Unexpectedly, di-methylated histone H3-lysine 4 is enriched at the transition points between active and inactive domains. We speculate that this modification may mark DNA regulatory elements within antigen receptor loci.;Third, we have identified an extensive series of putative CTCF (a factor that binds chromatin insulator elements) binding sites within the murine and human Ig heavy chain loci. We demonstrate that one of these sites is actively bound by CTCF in both lymphoid and non lymphoid cells and confers enhancer-blocking activity in vitro, when the CTCF binding site is intact. A series of potential roles for CTCF in the regulation of the Ig heavy chain locus are discussed. |
