Abstract
The silencing of HM loci is mediated by the silent information regulator (SIR) complex.\r Post-translational modifications of the N-terminal tail of histones H3 or H4 are implicated to\r play an important role in the SIR transcriptional silencing pathway. In order to quantify to what\r extent silencing is disrupted upon histone N-terminal tail truncation, we measure transcriptional\r activity in individual yeast cells with fluorescent reporter genes inserted at HML and HMR. We\r first tested the efficiency of the fluorescent reporter system by adding Nicotinamide, a Sir2\r inhibitor, into a culture of vegetatively growing budding yeast and measured the fluorescent\r signal coming from the nucleus. We observed a significant increase in the fluorescent intensity\r after 2hrs suggesting desilencing of heterochromatin loci takes 1-2 cell divisions. Next, we used\r modified histone H3 and H4 genes to observe their effects on transcriptional silencing in HM\r loci. We observed that an extra copy of histone H3 gene that carries a deletion of the first 32\r amino acids does not affect the silencing of HM loci. In addition, cells having an extra copy of\r the histone H4 genes with the N-terminal truncated via deletion of the first 16 amino acids, alters\r silencing at the HMR locus but does not effect at HML. We also observed that cells carrying\r extra copies of histone H4 with point mutations H4K16R and H4K16Q experience a difference\r in transcriptional activity at the HM Loci