Functional analysis of the TET2/OGT complex in epigenetic modifications

描述(由申请人提供)：表观遗传修饰在染色质重塑中起着重要作用。这些表观遗传修饰的一个典型例子是DNA甲基化。在CpG二核苷酸环境中，DNA的甲基化主要发生在胞嘧啶核嘧啶环的5位，这是由DNA甲基转移酶催化的。DNA甲基化改变了染色质的状态，调节了许多分子细胞过程。最近，Tet家族的酶被证明可以氧化甲基化的胞嘧啶，从而实现DNA去甲基化。这些酶特异性地将甲基化胞嘧啶(5mC)主要转化为羟甲基胞嘧啶(5hmC)。有趣的是，与DNA去甲基化不同，Tet酶依赖的甲基化胞嘧啶氧化不仅与转录激活有关，而且与转录抑制有关。据报道，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，我们用无偏蛋白亲和纯化的方法检测了Tet酶的相关蛋白，发现OGT是TET2在小鼠ES细胞中的功能伙伴。OGT是唯一一种以UDP-GlcNAc为供体催化蛋白质O-GlcN酰化的酶。我们的初步研究表明，OGT与TET2相互作用形成异源二聚体，并通过TET2靶向染色质进行组蛋白GlcN酰化。TET2和OGT的全基因组图谱表明，TET2、OGT和OGT依赖的组蛋白GlcNac化与小鼠ES细胞中活跃的基因转录有关。此外，TET2是髓系恶性肿瘤中最常见的突变基因之一，提示在肿瘤发生过程中，基因突变会引起异常的表观遗传修饰。因此，在这一应用中，我们计划研究TET2/OGT复合体在表观遗传调控中的分子机制以及它在肿瘤发生中的作用。这项拟议的研究将揭示一种新的机制，即体细胞突变如何放松对几种类型的表观遗传修饰的调控，这可能导致肿瘤发生。

DESCRIPTION (provided by applicant): Epigenetic modifications play an important role in chromatin remodeling. 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 changes the status of chromatin and regulates numerous molecular cellular processes. 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, TET2 is one of the most frequently mutated genes in myeloid malignancies, suggesting that genetic mutations induce abnormal epigenetic modifications during carcinogenesis. Thus, in this application, we plan to examine the molecular mechanism of the TET2/OGT complex in epigenetic regulation as well as its role in tumorigenesis. The proposed study will reveal a novel mechanism of how somatic mutations deregulate several types of epigenetic modification, which may result tumorigenesis.