| The establishment, maintenance, and inheritance of silent chromatin have been extensively studied. Less investigated are the determinants that constrain its linear spread along the chromatin fiber and the manner by which it regulates gene transcription. The studies described in this dissertation have focused on elucidating how Sir-mediated silencing regulates gene transcription and what determinants may affect the linear diffusion of Sir complex along chromatin template in the yeast Saccharomyces cerevisiae.;Similar strategies were employed to investigate the mechanism by which SIR modulates gene activation. Such investigations revealed that, by three criteria—namely nucleosome density, histone acetylation and methylation state, and occupancy of the histone variant Htz1—transcriptional activation of SIR-silenced hsp82 is independent of its associated chromatin state. Moreover, I demonstrate that recruitment of 5'-capping enzyme, but not that of Pol II, tightly correlates with the activated transcription level, indicating SIR-mediated silencing acts primarily on elongation phase of transcription in limiting gene activation. Finally, I show that the presence of Sir proteins and Htz1 is not mutually exclusive and that Htz1 alone is not sufficient to prevent Sir2/3/4 complex spread. My findings cast doubt on the generality of the “histone code” in regulating eukaryotic gene transcription.;Chromatin immunoprecipitation (ChIP) and Northern analysis were utilized to monitor recruitment of selected transcription factors and expression states at the natural targets of Sir2. In combination with analysis at HMRE -silenced hsp82, such studies revealed that, for basal silencing, SIR-mediated silent chromatin is permissive to both pre-initiation complex (PIC) assembly and transcription initiation. By contrast, occupancy of factors critical to both mRNA capping and Pol II elongation is virtually abolished. In agreement, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2, and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. |
