The vertebrate immune system has developed an elegant process to generate a vast array of cell-surface and secreted receptors designed to recognize virtually any non-self molecule. This is achieved through the splicing together of two or three gene segments from a large pool of potential segments encoded by the T-cell receptor (TCR) and immunoglobulin (Ig) gene loci through a process known as V(D)J recombination. In recent years much has been learned about the mechanism of the basic V(D)J recombination reaction while little progress was made towards the understanding of how this process is regulated. The evidence that exists suggests that regulation is achieved at the level of chromatin. This is based on the observation that expression of the recombinase in virtually any cell type is sufficient for the recombination of heterologous substrates but not the wild type TCR or Ig loci indicating that the chromatin is in a state that is refractory to rearrangement at these loci in inappropriate cells. Specificity is also imparted to the rearrangement of the TCR and Ig gene loci within actively recombining cell types. Complete rearrangement of the TCR gene occurs only in T-cells while the Ig locus is only rearranged in antibody producing B-cells.
The aim of this proposal is to address specific unanswered questions in the field of V(D)J recombination. The role that cell-type-specific factors play in modulating chromatin structure such that the V(D)J sites are rendered accessible to recombinase action will be addressed through the use of TCR V(D)J recombination templates that have been reconstituted into chromatin in vitro. Additionally, the specific nature of the biochemical activity of any identified factor will be determined. Finally, the role that transcription of the TCR locus plays in the regulation of V(D)J recombination will be addressed.
