裂殖酵母转录共激活复合物SAGA促进异染色质形成的机制

Heterochromatin is a heavily condensed form of chromatin. Genes inside the heterochromatin are commonly silenced. Heterochromatin contributes to genome stability and regulates differentiated gene expressions during development. Formation of heterochromatin depends on the heterochromatin formation factors and repetitive sequences. SAGA is a transcriptional co-activator complex and is highly conserved in eukaryotes. During our previous studies, we showed that certain subunit of SAGA is required for the genome stability, and deletion of certain SAGA subunit impairs heterochromatin silencing. These results suggest SAGA complex promotes heterochromatin formation, but underlying mechanism is unclear (BMC Genomics,2012,13:662;J Cell Sci,2014,127:4024)。In this project, we will analyze the genetic relationships between SAGA and classic heterochromatin formation pathways, by constructing double deletion mutants and functional complementation assay. We will identify SAGA-mediated transcriptional and post-transcriptional regulations of heterochromatin formation factors and repetitive sequences through RNA-seq and other methods. We will identify the heterochromatin formation factors that directly interact with SAGA by TAP/LC-MS and other methods, and validate the contribution of thus interactions to heterochromatin formation. This project will unveil a novel mechanism by which a transcriptional co-activator complex promotes heterochromatin formation. Our study will provide valuable hints to heterochromatin study in higher eukaryotes.

异染色质高度凝聚，其区域内的基因处于沉默状态。异染色质是维持基因组稳定性和调控发育时期基因差异表达的基础。异染色质的形成依赖于异染色质形成因子和重复序列。SAGA是真核生物中保守的转录共激活复合物。我们前期研究中发现裂殖酵母SAGA亚基参与维持基因组稳定，SAGA亚基的突变破坏异染色质基因沉默,提示SAGA促进异染色质形成，但机制不明(BMC Genomics,2012,13:662;J Cell Sci,2014,127:4024)。本课题将通过构建双突变体和功能回补，遗传分析SAGA与异染色质形成通路之间的关系；利用RNA-seq等研究SAGA通过调控异染色质形成因子和重复序列的转录加工来促进异染色质形成的机制；利用TAP/LC-MS等鉴定SAGA通过直接结合异染色质形成因子来调控异染色质的机制。课题将揭示转录共激活复合物促进异染色质形成的新机制，为高等生物的异染色质研究提供重要线索。

异染色质高度凝聚，其区域内的基因处于沉默状态。异染色质是维持基因组稳定性和调控发育时期基因差异表达的基础。SAGA是真核生物中保守的转录共激活复合物。Sgf73是SAGA复合物的核心亚基。本项目发现Sgf73的缺失引起着丝粒异染色质区域基因沉默的缺陷、H3K9甲基化水平的下降和RNA聚合酶II水平的上升。Sgf73对基因沉默的调控不依赖SAGA的转录活性。Sgf73可以与RNAi的关键效应复合物RITS的亚基直接结合。Sgf73的缺失会导致RITS复合物不稳定，从而影响了异染色质的建立。本研究发现了第一个RITS的稳定因子，同时也揭示了SAGA复合物亚基促进异染色质形成的全新机制。.SAGA复合物的酶学亚基Gcn5与Set3的双突变引起了异染色质的严重缺陷。提示Set3可能也参与了异染色质的形成。Set3是转录复合物Set3C的核心亚基。set3+的单突变会造成异染色质的功能轻微受损。Set3调控异染色质的功能依赖于其PHD结构域，但与SET结构域和Set3C复合物的其他亚基无关。Set3不定位与着丝粒区域， 但定位于CLRC复合物核心亚基Clr4和Rik1的启动子。CLRC复合物负责组蛋白H3K9的甲基化，该修饰是异染色质形成的基础。Set3促进了Clr4和Rik1的转录和表达。过量表达Clr4或Rik1可以补偿set3突变体的缺陷。本研究揭示了一条通过调控异染色质核心效应因子的转录水平来微调异染色质功能的新途径。.Epe1是维持异染色质边界的重要因子，与Sgf73存在遗传关系。本项目发现Epe1参与了对于细胞分解代谢的调控。在外界应急条件下，如低糖，高盐，高氧化压力，Epe1促进了AMPK的alpha亚基Ssp2的磷酸化，进而激活了AMPK通路。本研究发现了Epe1在异染色质调控功能以外的新功能，揭示了异染色质调控因子的功能的多样性。