Department of Biology, University of Rochester, Rochester, New York 14627, USA.
J Biol Chem. 2010 Mar 5;285(10):7525-36. doi: 10.1074/jbc.M109.016360. Epub 2010 Jan 4.
Saccharomyces cerevisiae Esc2p is a member of a conserved family of proteins that contain small ubiquitin-like modifier (SUMO)-like domains. It has been implicated in transcriptional silencing and shown to interact with the silencing protein Sir2p in a two-hybrid analysis. However, little is known about how Esc2p regulates the structure of silent chromatin. We demonstrate here that ESC2 differentially regulates silent chromatin at telomeric, rDNA, and HM loci. Specifically, ESC2 is required for efficient telomeric silencing and Sir2p association with telomeric silent chromatin and for silencing and maintenance of silent chromatin structure at rDNA. On the other hand, ESC2 negatively regulates silencing at HML and HMR and destabilizes HML silent chromatin without affecting Sir2p association with chromatin. We present evidence that Esc2p is associated with both transcriptionally silent and active loci in the genome, and the abundance of Esc2p is not correlated with the chromatin state at a particular locus. Using affinity pull-down analyses, we show that Esc2p and Sir2p interact in vivo, and recombinant Esc2p and Sir2p interact directly. Moreover, we dissect Esc2p and identify a putative SUMO-binding motif that is necessary and sufficient for interacting with Sir2p and SUMO and is required for the function of Esc2p in transcriptional silencing.
酿酒酵母 Esc2p 是一种含有小泛素样修饰物 (SUMO) 样结构域的保守蛋白家族成员。它被认为与转录沉默有关,并在双杂交分析中显示与沉默蛋白 Sir2p 相互作用。然而,关于 Esc2p 如何调节沉默染色质的结构知之甚少。我们在这里证明 ESC2 可在端粒、rDNA 和 HM 位点上差异调节沉默染色质。具体来说,ESC2 是端粒沉默和 Sir2p 与端粒沉默染色质结合所必需的,并且对于 rDNA 上沉默染色质的沉默和维持是必需的。另一方面,ESC2 负调控 HML 和 HMR 的沉默,并在不影响 Sir2p 与染色质结合的情况下破坏 HML 沉默染色质。我们提供的证据表明,Esc2p 与基因组中转录沉默和活跃的基因座都有关联,并且特定基因座上 Esc2p 的丰度与染色质状态无关。通过亲和下拉分析,我们表明 Esc2p 和 Sir2p 在体内相互作用,并且重组的 Esc2p 和 Sir2p 直接相互作用。此外,我们对 Esc2p 进行了剖析,并鉴定出一个假定的 SUMO 结合基序,该基序对于与 Sir2p 和 SUMO 相互作用以及 Esc2p 在转录沉默中的功能是必需和充分的。