COOPERATION AT A DISTANCE BETWEEN SILENCERS AND PROTO-SILENCERS AT THE YEAST HML LOCUS

Transcriptional repression at the silent yeast mating type loci is ach ieved through the formation of a particular nucleoprotein complex at s pecific cis-acting elements called silencers. This complex in turn app ears to initiate the spreading of a histone binding protein complex in to the surrounding chromatin, which restricts accessibility of the reg ion to the transcription machinery, We have investigated long-range, c ooperative effects between silencers by studying the repression of a r eporter gene integrated at the HML locus flanked by various combinatio ns of wild-type and mutated silencer sequences, Two silencers can coop erate over >4000 bp to repress transcription efficiently. More importa ntly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functio nal cooperativity is demonstrated using a quantitative assay for repre ssion, and varies with the affinity of the binding sites used, Since t he repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 pr oteins bound to distant I)NB elements may interact to create an interf ace of sufficiently high affinity such that Sir-containing complexes b ind, nucleating the silent chromatin state.