PDF(Pubmed)
Abstract：
Epigenetic control of gene expression is crucial for maintaining gene regulation. Sin3 is an evolutionarily conserved repressor protein complex mainly associated with histone deacetylase (HDAC) activity. A large number of proteins are part of Sin3/HDAC complexes, and the function of most of these members remains poorly understood. SAP25, a previously identified Sin3A associated protein of 25 kDa, has been proposed to participate in regulating gene expression programs involved in the immune response but the exact mechanism of this regulation is unclear. SAP25 is not expressed in HEK293 cells, which hence serve as a natural knockout system to decipher the molecular functions uniquely carried out by this Sin3/HDAC subunit. Using molecular, proteomic, protein engineering, and interaction network approaches, we show that SAP25 interacts with distinct enzymatic and regulatory protein complexes in addition to Sin3/HDAC. While the O-GlcNAc transferase (OGT) and the TET1 /TET2/TET3 methylcytosine dioxygenases have been previously linked to Sin3/HDAC, in HEK293 cells, these interactions were only observed in the affinity purification in which an exogenously expressed SAP25 was the bait. Additional proteins uniquely recovered from the Halo-SAP25 pull-downs included the SCF E3 ubiquitin ligase complex SKP1/FBXO3/CUL1 and the ubiquitin carboxyl-terminal hydrolase 11 (USP11), which have not been previously associated with Sin3/HDAC. Finally, we use mutational analysis to demonstrate that distinct regions of SAP25 participate in its interaction with USP11, OGT/TETs, and SCF(FBXO3).) These results suggest that SAP25 may function as an adaptor protein to coordinate the assembly of different enzymatic complexes to control Sin3/HDAC-mediated gene expression.
摘要：
基因表达的表观遗传控制对于维持基因调控至关重要。Sin3是一种进化上保守的抑制蛋白复合物，主要与组蛋白脱乙酰酶（HDAC）活性有关。大量蛋白质是Sin3/HDAC复合物的一部分，和功能的大多数这些成员仍然知之甚少。SAP25，一种先前鉴定的25kDa的Sin3A相关蛋白，已被提议参与调节参与免疫反应的基因表达程序，但这种调节的确切机制尚不清楚。SAP25在HEK293细胞中不表达，因此，它可以作为天然敲除系统来破译由该Sin3/HDAC亚基独特地执行的分子功能。使用分子，蛋白质组学，蛋白质工程，和交互网络方法，除了Sin3/HDAC外，我们还显示SAP25与不同的酶和调节蛋白复合物相互作用。虽然O-GlcNAc转移酶（OGT）和TET1/TET2/TET3甲基胞嘧啶双加氧酶先前已与Sin3/HDAC连接，在HEK293细胞中，这些相互作用仅在以外源表达的SAP25为诱饵的亲和纯化中观察到。从Halo-SAP25下拉法中独特回收的其他蛋白质包括SCFE3泛素连接酶复合物SKP1/FBXO3/CUL1和泛素羧基末端水解酶11（USP11），以前没有与Sin3/HDAC相关联。最后,我们使用突变分析来证明SAP25的不同区域参与其与USP11，OGT/TETs的相互作用，和SCF(FBXO3)。）这些结果表明，SAP25可能充当衔接蛋白，以协调不同酶复合物的组装，以控制Sin3/HDAC介导的基因表达。
