Distinct Modifying Activities Are Required to Render Chromatin Accessible to Transcription versus V(D)J Cleavage

다국어 초록 (Multilingual Abstract)

Assembly of antigen receptor genes is a highly ordered process that depends on regulating access of the recombination machinery to the recombining loci. We previously have shown that V(D)J recombination can be regulated by alterations of chromatin structure at the level of a single nucleosome. V(D)J cleavage is greatly impeded when the recombination signal sequences(RSSs) are assembled into nucleosome. However, cleavage is enhanced when the histones are acetylated. Moreover, hSWI/SNF, a chromatin remodeling factor, was found to function alone and in combination with acetylation to greatly stimulate V(D)J cleavage on reconstituted nucleosome.
To understand more about the regulation of V(D)J recombination within chromatin, we compared the requirements for generating chromatin that is accessible for either cleavage or transcription. For these studies, we established an in vitro system in which promoter-reporter gene plasmids containing inserted RSSs were assembled into chromatin. These templates were then used to examine the effect of specific chromatin remodeling or histone modifying complexes on RAG-mediated cleavage and transcription. Although addition of the p300 histone acetyltransferase in combination with Sp1 activates transcription, these factors do not permit V(D)J cleavage. Conversely, modification by hSWI/SNF alone facilitates V(D)J cleavage, but hSWI/SNF in the absence of Sp1 does not generate a transcriptionally active template. Thus the requirements for transcriptional and recombinational access to chromatin are distinct and V(D)J cleavage through a nucleosomal array is uncoupled from transcription.