Functional analysis of the TET2/OGT complex in epigenetic modifications

摘要： 表观遗传修饰在基因转录调控中起着重要作用。一个典型的例子是 这些表观遗传修饰是DNA甲基化。DNA的甲基化主要发生在5位 胞嘧啶环在CpG二核苷酸的背景下，由DNA甲基转移酶催化。 基因启动子区域和转录起始点的DNA甲基化调控基因转录。 最近，Tet家族的酶被证明氧化甲基化的胞嘧啶向dna迈进了一步。 去甲基化。这些酶专门将甲基化的胞嘧啶(5mC)主要转化为羟甲基化 胞嘧啶(5hmC)。有趣的是，与DNA去甲基化不同，Tet酶依赖于甲基化的氧化 胞嘧啶不仅与转录激活有关，而且与转录抑制有关。一直以来 报道称，TET1与SIN3A和HDAC1/2形成复合体，参与转录抑制。 为了研究Tet酶依赖的DNA去甲基化的分子机制，我们研究了 用无偏蛋白亲和纯化方法对Tet酶的相关蛋白进行纯化，发现OGT 作为TET2在小鼠ES细胞中的功能伙伴。OGT是唯一一种使用UDP-GlcNAc作为 供体催化蛋白质O-GlcN酰化。我们的初步研究表明，OGT与TET2相互作用形成 一种异源二聚体，靶向染色质，通过TET2在小鼠ES细胞中组蛋白GlcN酰化。基因组- 对TET2和OGT的广泛分析表明，TET2、OGT和OGT依赖的组蛋白GlcN酰化是 与小鼠胚胎干细胞中活跃的基因转录有关。此外，依赖于OGT的组蛋白GlcN酰化 调节组蛋白H_2B泛化，一种活跃的基因转录标记。因此，我们假设 OGT/TET2复合体参与转录激活，并对抗依赖TET1的基因沉默。我们 计划进行以下研究来验证我们的假设。 目的：研究TET2/OGT复合体在H_2B泛素化中的作用。 目的：探讨TET2/OGT复合体在组蛋白乙酰化中的作用。 目的：研究TET1和TET2复合体在转录过程中的功能相互作用 监管。 综上所述，这项拟议的研究将全面检查Tet酶依赖的功能 基因转录。它可能提供新的跨尾组蛋白修饰的分子机制 对转录激活很重要。

Abstract: Epigenetic modifications play an important role in gene transcription regulation. One typical example of these epigenetic modifications is DNA methylation. Methylation of DNA is mainly occurred at the 5 position of the cytosine pyrimidine ring in a CpG dinucleotide context, which is catalyzed by DNA methyltransferases. DNA methylation at gene promoter region and transcription start sites regulates gene transcription. Recently, TET family enzymes were shown to oxidize the methylated cytosine in a step towards DNA demethylation. These enzymes specifically convert methylated cytosine (5mC) mainly into hydroxymethylated cytosine (5hmC). Interestingly, unlike DNA demethylation, TET enzymes-dependent oxidation of methylated cytosine is not only associated with transcription activation but also transcription repression. It has been reported that TET1 forms a complex with SIN3A and HDAC1/2, which is involved in transcription repression. To study the molecular mechanism of TET enzyme-dependent DNA demethylation, we examined the associated proteins of TET enzymes using an unbiased protein affinity purification approach, and found OGT as a functional partner of TET2 in mouse ES cells. OGT is the only enzyme that uses UDP-GlcNAc as the donor to catalyze protein O-GlcNAcylation. Our preliminary study shows that OGT interacts with TET2 to form a heterodimer and is targeted to chromatin for histone GlcNAcylation via TET2 in mouse ES cells. Genome- wide profiling of TET2 and OGT suggests that TET2, OGT and OGT-dependent histone GlcNAcylation are associated with active gene transcription in mouse ES cells. Moreover, OGT-dependent histone GlcNAcylation regulates histone H2B ubiquitiation, an active gene transcription mark. Thus, we hypothesize that the OGT/TET2 complex is involved in transcription activation and counteracts TET1-dependent gene silencing. We plan to perform following studies to test our hypothesis. Aim1: To examine the role of the TET2/OGT complex in H2B ubiquitination. Aim2: To investigate the role of the TET2/OGT complex in histone acetylation. Aim3: To study the functional interaction between the TET1 complex and the TET2 complex in transcription regulation. Taken together, the proposed study will comprehensively examine the function of TET enzyme-dependent gene transcription. It might provide novel molecular mechanisms of trans-tail histone modifications that are important for transcription activation.