E2F1 Transcription Factor Regulates O-linked N-acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Expression
E2F1 Transcription Factor Regulates O-linked N-acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Expression
复制标题
DOI：
10.1074/jbc.m115.677534
复制
发表时间：
2015-12-25
影响因子：
4.8
通讯作者：
Jones, Steven P.
中科院分区：
文献类型：
--
作者：
Muthusamy, Senthilkumar;Hong, Kyung U.;Jones, Steven P.
O-GlcNAc转移酶（OGT）和O-GlcNAc酶（OGA）控制的蛋白质O-GlcNAc化已成为一种重要的翻译后修饰，可能是多种疾病的因素。直到最近，人们认为OGT/OGA蛋白的表达相对恒定。包括我们在内的几个研究小组已经表明，OGT和/或OGA表达在几种病理情况下发生变化，但调节这些酶表达的顺式和反式元件基本上仍未被探索。在这里，我们使用基于PCR的测定来分析最小启动子，并利用计算机模拟来提名Ogt（即OGT蛋白的基因）和Mgea 5（即OGA蛋白的基因）中的几个候选转录因子结合位点。我们注意到在两个启动子中有多个E2 F结合位点的共有序列。我们在人和小鼠细胞中进行了染色质免疫沉淀，发现E2 F1与两种启动子中的候选E2 F结合位点结合。在HEK 293细胞中，我们过表达E2 F1，这显著降低了OGT和MGEA 5的表达。相反，E2 F1缺陷小鼠成纤维细胞Ogt和Mgea 5表达增加。在已知的E2 F1结合伴侣中，我们质疑是否可能涉及视网膜母细胞瘤1（Rb 1）。Rb 1缺陷小鼠胚胎成纤维细胞显示Ogt和Mgea 5表达水平增加，但Rb 1缺陷细胞中E2 F1过表达并不改变Ogt和Mgea 5表达，表明Rb 1是E2 F1介导的抑制所需的。总之，这项工作确定和验证了小鼠Ogt和Mgea 5基因的启动子元件。具体而言，E2 F1负调控Ogt和Mgea 5的表达在Rb 1蛋白依赖性的方式。
Protein O-GlcNAcylation, which is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), has emerged as an important posttranslational modification that may factor in multiple diseases. Until recently, it was assumed that OGT/OGAprotein expression was relatively constant. Several groups, including ours, have shown that OGT and/or OGA expression changes in several pathologic contexts, yet the cis and trans elements that regulate the expression of these enzymes remain essentially unexplored. Here, we used a reporter-based assay to analyze minimal promoters and leveraged in silico modeling to nominate several candidate transcription factor binding sites in both Ogt (i.e. the gene for OGT protein) and Mgea5 (i.e. the gene for OGA protein). We noted multiple E2F binding site consensus sequences in both promoters. We performed chromatin immunoprecipitation in both human and mouse cells and found that E2F1 bound to candidate E2F binding sites in both promoters. In HEK293 cells, we overexpressed E2F1, which significantly reduced OGT and MGEA5 expression. Conversely, E2F1-deficient mouse fibroblasts had increased Ogt and Mgea5 expression. Of the known binding partners for E2F1, we queried whether retinoblastoma 1 (Rb1) might be involved. Rb1-deficient mouse embryonic fibroblasts showed increased levels of Ogt and Mgea5 expression, yet overexpression of E2F1 in the Rb1-deficient cells did not alter Ogt and Mgea5 expression, suggesting that Rb1 is required for E2F1-mediated suppression. In conclusion, this work identifies and validates some of the promoter elements for mouse Ogt and Mgea5 genes. Specifically, E2F1 negatively regulates both Ogt and Mgea5 expression in an Rb1 protein-dependent manner.