UC San Diego

Transcriptional silencing is a crucial process that is mediated through chromatin structure. The histone deacetylase Sir2 silences genomic regions that include telomeres, ribosomal DNA, and the cryptic mating-type loci. The formation and regulation of silent chromatin has been much studied but remains incompletely understood. Additional factors controlling silent chromatin were suggested by a previously completed Sir2 two-hybrid screen. In this screen, Gas1 was identified as a Sir2- interacting protein. In the research reported here, an unsuspected role for the enzyme Gas1 in locus-specific transcriptional silencing is presented using genetic and biochemical methods. GAS1 encodes a \[Beta\]-1,3- glucanosyltransferase previously characterized for its role in cell wall biogenesis. In gas1 mutants, telomeric silencing is defective and rDNA silencing is enhanced. The catalytic activity of Gas1 is required for normal silencing, established through analysis of gas1catalytically inactive mutants. Gas1's role in silencing is distinct from its role in cell wall biogenesis. Established hallmarks of silent chromatin, such as Sir2 binding and H4K16 and H3K56 deacetylation, appear unaffected in gas1 mutants in chromatin immunoprecipitation analysis. Thus, another event required for telomeric silencing must be influenced by GAS1. Sir2 itself is present in immunoprecipitations of \[Beta\]-1,3- glucan, the substrate of Gas1 activity. This points to the possibility that Sir2 may be modified by \[Beta\]-1,3-glucan, or \[Beta\]-1,3-glucan is linked to Sir2 through interacting chromatin proteins. Since the catalytic activity of Gas1 is required for telomeric silencing and Gas1 and Sir2 physically interact, a model is proposed in which carbohydrate post-translational modification of chromatin components provides a new regulatory element that may be critical for chromatin function. Other proteins contributing to steps upstream of Gas1 in carbohydrate modification pathways are also newly identified as necessary for silencing. These include Kre6, another Sir2- interacting protein and a \[Beta\]-1,6-glucan synthase, Fks1, a \[Beta\]-1,3-glucan synthase, and Pmt1, a mannosyltransferase. The initial genetic characterization of KRE6, FKS1, and PMT1 demonstrates that these genes are also required for telomeric silencing and further reinforces the potentially crucial roles of GAS1 and carbohydrate modification in transcriptional silencing.