| Lineage specificity and temporal ordering of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangement are reflected in the accessibility of recombination signal sequences (RSSs) within chromatin to in vitro cleavage by the V(D)J recombinase. In the present work, I investigated the basis of this regulation by testing the ability of purified RAG1 and RAG2 proteins to initiate cleavage on positioned nucleosomes containing RSS substrates. I found that nicking and double-strand DNA cleavage of RSSs positioned on the face of an unmodified nucleosome are entirely inhibited. This inhibition was independent of translational position or rotational phase and could not be overcome either by addition of the DNA-bending protein HMG-1 or by the use of hyperacetylated histones. I suggest that the nucleosome could act as the stable unit of chromatin that limits recombinase accessibility to potential RSS targets, and that actively rearranging gene segments might be packaged in a modified or disrupted nucleosome structure. Using techniques based on MNase and restriction enzyme digestion of intact nuclei, I examined the in vivo chromatin structure of the immunoglobulin kappa locus J genes in non-lymphoid cells as compared to lymphoid cells poised at different stages of development. I present preliminary data from general MNase sensitivity, indirect end-labeling, high resolution mapping of MNase breaks by LMPCR, and restriction enzyme accessibility. |
