RIGS (REPEAT-INDUCED GENE SILENCING) IN ARABIDOPSIS IS TRANSCRIPTIONAL AND ALTERS CHROMATIN CONFIGURATION

We have previously reported repeat-induced gene silencing (RIGS) in Ar abinopsis, in which transgene expression may be silenced epigeneticall y when repeated sequences are present, Among an allelic series of line s comprising a primary transformant and various recombinant progeny ca rrying different numbers of drug resistance gene copies at the same lo cus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA, We now report chara cterization, in nuclei isolated from the same transgenic lines, of gen e expression by nuclear run-on assay and of chromatin structure by nuc lease protection assay. We find that silencing is correlated with abse nce of run-on transcripts, indicating that expression is silenced at t he level of transcription, We find further that silencing is also corr elated with increased resistance to both DNase I and micrococcal nucle ase, indicating that the silenced state reflects a change in chromatin configuration. We propose that silencing results when a locally paire d region of homologous repeated nucleotide sequences is flanked by unp aired heterologous DNA, which leads chromatin to adopt a local configu ration that is difficult to transcribe, and possibly akin to heterochr omatin.