Serotonylation of the Histone H3 tail: Uncovering the function of a novel epigenetic mark

项目摘要 组蛋白PTM涉及多种核过程，包括基因调控、DNA修复和细胞凋亡。 染色质结构这些化学装饰品的失调对人类的健康有着巨大的影响。 生物学，并有可能驱动疾病状态。最近，我们发现了一种新的组蛋白PTM， 包括将神经递质5-羟色胺共价添加到组蛋白H3的N-末端尾部。 有趣的是，在令人信服的初步数据中，我们已经表明，5-羟色胺在谷氨酰胺5 由组织转氨酶2（TGM 2）催化。重要的是，TGM 2的错误调节已经 与癌症和神经退行性疾病有关，突出了其重要性。我们的初步数据还 强调了新开发的高度特异性的H3 Q5 ser验证抗体的使用，以表明 在产生阿糖胞苷的生物体中，阿糖胞苷酰化高度富集， 后分化中缝髓母细胞瘤细胞（RN 46 A-B 14）。虽然血清素是通过 虽然已经发现，但它在H3上的作用尚不清楚。这项提案的主要目标是， 打开H3 Q5 ser的功能，并询问是否为动态PTM。 为了做到这一点，我计划采用半合成技术，已开发和优化的穆尔 实验室以化学方式构建含有光交联剂的腺苷酸化单核体。一旦到手，我会 利用它们来捕获与H3 Q5 ser标记密切相互作用的蛋白质，称为“阅读器”。这 实验将作为一个主要的基准，开始了解如何H3 Q5 ser有助于调节 染色质的功能输出。这项研究计划的第二个主要目标是确定H3 Q5 ser是否可以 如果是的话，如何删除？有趣的是，负责血清素沉积的TGM 2， 已知具有脱氨酶活性。如果H3 Q5 ser被TGM 2去除，那么点突变（Gln至Glu）将 留在自己的位置。这种可能性是特别有趣的，因为组蛋白尾部突变的倾向， 癌 总之，这些实验旨在阐明H3 Q5 ser作为新的组蛋白H3 PTM的作用。生成的数据 这项拟议的研究将引起染色质和化学生物学界的巨大兴趣， 阐明了一种新的组蛋白PTM的功能。

PROJECT SUMMARY Histone PTMs are implicated in a variety of nuclear processes including gene regulation, DNA repair and chromatin structure. Misregulation of these chemical decorations have immense consequences for human biology and have the potential to drive disease states. Recently, we have uncovered a new histone PTM that involves the covalent addition of the neurotransmitter, serotonin, on to the N-terminal tail of histone H3. Interestingly, in compelling preliminary data, we have shown that the installation of serotonin onto glutamine 5 is catalyzed by the enzyme tissue transglutaminase 2 (TGM2). Importantly, the misregulation of TGM2 has been implicated in cancer and neurodegenerative disease highlighting its importance. Our preliminary data also highlights the use of a newly developed, highly specific validated antibody for H3Q5ser, to show that serotonylation is highly enriched in serotonin-producing organisms and that the modification is increased in post-differentiated raphe-medulloblastoma cells (RN46A-B14). While the mechanism by which serotonin is added has been uncovered, its role while present on H3 is unknown. The major goal of this proposal is to shed light on the function of H3Q5ser and also interrogate if it is a dynamic PTM. To do this, I plan to employ semisynthetic techniques that have been developed and optimized within the Muir lab to chemically construct serotonylated mononucleosomes containing a photo crosslinker. Once in hand, I will utilize them to capture the proteins that closely interact with the H3Q5ser mark, termed `readers'. This experiment will serve as a major benchmark to begin to understand how H3Q5ser contributes to regulate the functional output of chromatin. The second major goal of this research program is to determine if H3Q5ser can be removed, and if so, how? Interestingly, TGM2, the same enzyme responsible for serotonin deposition, is known to possess deaminase activity. If H3Q5ser is removed by TGM2 then a point mutation (Gln to Glu) would be left in its place. This possibility is particularly interesting giving the propensity of histone tail mutations in cancer. Together, these experiments aim to elucidate the role of H3Q5ser as a new histone H3 PTM. The data generated by the proposed research will be of immense interest to the chromatin and chemical biology community in illuminating the function of a novel histone PTM.