To determine whetherHMR-Ealso promotes the association of Sir proteins with neighboring nucleosomes in the context of telomere VI-R, we examined a strain in which a 430-bp fragment containing theHMR-Esilencer was integrated adjacent to telomere VI-R (Determine 2A). secondary silencer,HMR-I, boosted the level of Sir proteins atHMR, apparently beyond the level necessary to repress transcription. Finally, we discovered that a tRNAThrgene nearHMR-Ihelped maintain silenced chromatin and transcriptional repression under conditions of reduced deacetylase activity. This study highlights the importance of NMDA auxiliary elements, such asHMR-Iand the tRNAThrgene, in enhancing the association of Sir silencing proteins with appropriate genomic locations, thereby buffering the capacity of silenced chromatin to assemble under suboptimal conditions. SILENCED chromatin and some other specialized chromatin says have the capacity to propagate along a chromosome. This ability is usually important for assembling extended chromatin domains but must be controlled to prevent one chromatin domain name from encroaching into another. The prevailing model of the spreading process postulates that silencing proteins are first recruited to specific sequences, termed silencers, and then spread from these sites through interactions with nucleosomes (Hoppeet al.2002;Ruscheet al.2002;Grewaland Elgin2007). Thus, the primary role of silencers is usually thought to be the nucleation of silenced chromatin, and the spreading process is usually thought to occur independently of the silencer. However, we recently discovered that at least one silencer inSaccharomyces cerevisiaecan promote the assembly of silenced NMDA chromatin at a step after recruitment (Lynchand Rusche2009). We have now compared the abilities of various silencers and combinations of silencers to shape the steady-state distributions of silencing proteins in the yeastS. cerevisiaeand find PRKCB that silencers differ in their abilities to promote the steady-state association of silencing proteins with neighboring nucleosomes. InS. cerevisiae, domains of silenced chromatin are found at the silent mating-type, loci (HMRandHML) and at most telomeres (reviewed inRuscheet al.2003). At the silent mating-type loci, silenced chromatin maintains haploid cell identity by preventing the expression of extra copies of the mating-type genes. This silenced chromatin also protectsHMRandHMLfrom cutting by the HO endonuclease that triggers mating-type switching (reviewed inHaber1998). The role of silenced chromatin at subtelomeric domains is usually less well comprehended, although it is usually speculated to contribute to the stability of the ends of the chromosomes. The structural components of silenced chromatin are the silent information regulator (SIR) proteins, Sir2p, Sir3p, and Sir4p, which are recruited to the chromosome by silencers. At theHMRlocus, theHMR-EandHMR-Isilencers flank auxiliary copies of theamating-type genes, and at theHMLlocus theHML-EandHML-Isilencers flank copies of the mating-type genes. Each of these four silencers is composed of binding sites for the origin recognition complex (ORC) and for Rap1p, Abf1p, or both. In contrast, multiple Rap1p binding sites embedded within the terminal telomeric repeats facilitate the recruitment of Sir proteins to the chromosome ends. In addition, binding sites for ORC and Abf1p occur in the core X subtelomeric element associated with all telomeres, and these binding sites contribute to silencing at some (Fourelet al.1999;Prydeand Louis1999) but not all telomeres (Mondouxand Zakian2007). Following recruitment to silencers, the Sir protein complex is usually thought to spread along the chromosome by repetitive cycles of histone deacetylation and binding in a process referred to as sequential deacetylation (Hoppeet al.2002;Ruscheet al.2002). Sir2p, a histone deacetylase, generates hypoacetylated nucleosomes that are preferentially bound by Sir3p and Sir4p, which in turn recruit an additional molecule of Sir2p to the chromatin (Hoppeet al.2002;Ruscheet al.2002). Thus, the Sir protein complex is usually predicted to spread away from the silencers in a linear step-wise manner. Our discovery that theHMR-Esilencer promotes the establishment of silenced chromatin over NMDA several kilobases more efficiently than does the telomeric repeat at chromosome VI-R (Lynchand Rusche2009) suggests that spreading may not be strictly linear. We hypothesize that proteins associated with theHMR-Esilencer promote a looped or compact arrangement of the chromatin fiber that brings the silencer-associated Sir complex into close proximity with multiple nucleosomes at once, such that multiple nucleosomes can be deacetylated independently of one another, enabling assembly to occur in a nonlinear fashion. Consistent with this model, chromosome conformation capture (3C) experiments have indicated that silenced chromatin adopts either a looped or compact chromatin structure at theHMRlocus (Valenzuelaet al.2008;Mieleet al.2009). To explore how silencers shape the steady-state distribution of Sir proteins atHMR, we have now examined the association of silencing proteins withHMRin the presence and absence of three known regulatory sequencestwo silencers and a boundary element. TheHMR-Esilencer is composed of binding sites for ORC, Rap1p, and Abf1p and is required for silencing atHMR. A second silencer,HMR-I, is not required for the silencing ofHMRa1, although it does contribute to the silencing.