Biswas Moumita, Maqani Nazif, Rai Ragini, Kumaran Srikala P, Iyer Kavitha R, Sendinc Erdem, Smith Jeffrey S, Laloraya Shikha
Department of Biochemistry, Indian Institute of Science, C. V. Raman Ave., Bangalore KA 560012, India.
Mol Cell Biol. 2009 May;29(10):2889-98. doi: 10.1128/MCB.00728-08. Epub 2009 Mar 16.
In Saccharomyces cerevisiae, transcriptional silencing occurs at the cryptic mating-type loci (HML and HMR), telomeres, and ribosomal DNA (rDNA; RDN1). Silencing in the rDNA is unusual in that polymerase II (Pol II) promoters within RDN1 are repressed by Sir2 but not Sir3 or Sir4. rDNA silencing unidirectionally spreads leftward, but the mechanism of limiting its spreading is unclear. We searched for silencing barriers flanking the left end of RDN1 by using an established assay for detecting barriers to HMR silencing. Unexpectedly, the unique sequence immediately adjacent to RDN1, which overlaps a prominent cohesin binding site (CARL2), did not have appreciable barrier activity. Instead, a fragment located 2.4 kb to the left, containing a tRNA(Gln) gene and the Ty1 long terminal repeat, had robust barrier activity. The barrier activity was dependent on Pol III transcription of tRNA(Gln), the cohesin protein Smc1, and the SAS1 and Gcn5 histone acetyltransferases. The location of the barrier correlates with the detectable limit of rDNA silencing when SIR2 is overexpressed, where it blocks the spreading of rDNA heterochromatin. We propose a model in which normal Sir2 activity results in termination of silencing near the physical rDNA boundary, while tRNA(Gln) blocks silencing from spreading too far when nucleolar Sir2 pools become elevated.
在酿酒酵母中，转录沉默发生在隐蔽的交配型位点（HML和HMR）、端粒以及核糖体DNA（rDNA；RDN1）。rDNA中的沉默现象较为特殊，因为RDN1内的聚合酶II（Pol II）启动子受Sir2抑制，但不受Sir3或Sir4抑制。rDNA沉默单向向左扩散，但其扩散限制机制尚不清楚。我们通过一种已建立的检测HMR沉默障碍的方法，寻找RDN1左端两侧的沉默障碍。出乎意料的是，紧邻RDN1的独特序列，与一个突出的黏连蛋白结合位点（CARL2）重叠，却没有明显的障碍活性。相反，位于左侧2.4 kb处的一个片段，包含一个tRNA(Gln)基因和Ty1长末端重复序列，具有强大的障碍活性。该障碍活性依赖于tRNA(Gln)的Pol III转录、黏连蛋白Smc1以及SAS1和Gcn5组蛋白乙酰转移酶。当SIR2过表达时，该障碍的位置与rDNA沉默的可检测极限相关，在该位置它会阻止rDNA异染色质的扩散。我们提出了一个模型，其中正常的Sir2活性导致在物理rDNA边界附近沉默终止，而当核仁Sir2池升高时，tRNA(Gln)会阻止沉默扩散得太远。
