Functional studies of the C-terminal domain of Sir3 (CTD) and histone H2A in telomeric position effect (TPE)
Description
Heterochromatin is nucleated at a specific site and spreads into adjacent sequences through interaction between modified histories and non-histone proteins. In the yeast Saccharomyces cerevisiae, these non-histone proteins are Sir2, Sir3, and Sir4. We have previously used a tethered silencing assay to isolate the C-terminal domain 144 amino acids of Sir3 (CTD) that, when tethered adjacent to telomere, is able to restore the silencing defect conferred by the rap1-17 allele that encodes a C-terminal truncation of Rap1. Here, we explore the function and structure of CTD involving in the nucleation of silent chromatin. We demonstrate that CTD is the minimum Sir3 dimerization domain that is conserved in divergent yeasts. The CTD is able to recruit Sir2, Sir3, and Sir4 at tethering sites and to spread Sir proteins into adjacent sequences. However, the CTD-Sir3 interaction is not sufficient to restore tethered silencing because a specific CTD mutation is severely defective in tethered silencing despite its ability to recruit Sir3. In addition, CTD is able to facilitate deacetylated H4-K16. Our data suggest that cooperative interactions between CTD, Sir3p, and deacetylated H4-K16 are essential to nucleate silent chromatin. Furthermore, the sequence alignment predicts that the CTD has a Cdc6 domain III winged-helix structure found in many transcription and replication, proteins, including Orc1. Consistent with the prediction, insertion mutations in the junction of CTD abrogate silencing, suggesting that the projection of CTD toward its interaction partners is critical for Sir3 function. Interestingly, tethering of the corresponding C-terminus of Orc1 confers significant, albeit low levels, of silencing. Our results suggest that Sir3 and Orc1p may derive from the same ancestral gene Additionally, we have isolated histone H2A alleles, hta1tpe, which are defective in telomeric position effect (TPE) and the nonhomologous end joing (NHEJ) repair pathway, and confer Spt- phenotype, suggesting that hta1tpe alleles may change subtelomeric chromatin structure, resulting in impairment to recruit silencing or repair factors