NUCLEAR-ORGANIZATION AND TRANSCRIPTIONAL SILENCING IN YEAST

Transcriptional repression at the yeast silent mating type loci requir es the formation of a nucleoprotein complex at specific cis-acting ele ments called silencers, which in turn promotes the binding of a histon e-associated Sir-protein complex to adjacent chromatin. A similar mech anism of long-range transcriptional repression appears to function nea r telomeric repeat sequences, where it has been demonstrated that Sir3 p is a limiting factor for the propagation of silencing. A combined im munofluorescence/in situ hybridization method for budding yeast was de veloped that maintains the three-dimensional structure of the nucleus. In wild-type cells the immunostaining of Sir3p. Sir4p and Rap1 coloca lizes with Y' subtelomeric sequences detected by in situ hybridization . All three antigens and the subtelomeric in situ hybridization signal s are clustered in foci, which are often adjacent to, but not coincide nt with, nuclear pores. This colocalization of Rap1, Sir3p and Sir4p w ith telomeres is lost in sir mutants, and also when Sir4p is overexpre ssed. To test whether the natural positioning of the two HM loci, loca ted roughly 10 and 25 kb from the ends of chromosome III, is important for silencer function, a reporter gene flanked by wild-type silencer elements was integrated at various internal sites on other yeast chrom osomes. We find that integration at internal loci situated far from te lomeres abrogates the ability of silencers to repress the reporter gen e. Silencing can be restored by creation of a telomere at 13 kb from t he reporter construct, or by insertion of 340 bp of yeast telomeric re peat sequence at this site without chromosomal truncation. Elevation o f the internal nuclear pools of Sir1p, Sir3p and Sir4p can relieve the lack of repression at the LYS2 locus in an additive manner, suggestin g that in wild-type cells silencer function is facilitated by its juxt aposition to a pool of highly concentrated Sir proteins, such as those created by telomere clustering.