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

项目总结 组蛋白PTMS参与多种核过程，包括基因调控、DNA修复和 染色质结构。这些化学装饰的不当管理对人类造成了巨大的后果 并有可能导致疾病状态。最近，我们发现了一种新的组蛋白PTM 涉及神经递质5-羟色胺在组蛋白H3的N端尾部的共价加成。 有趣的是，在令人信服的初步数据中，我们已经表明，在谷氨酰胺5上安装5-羟色胺 是由组织转谷氨酰胺酶2(TGM2)催化的。重要的是，对TGM2的不当监管一直是 与癌症和神经退行性疾病有关，突出了它的重要性。我们的初步数据也 重点介绍了新开发的针对H3Q5ser的高度特异的有效抗体的使用，以表明 5-羟色胺在产生5-羟色胺的生物体中高度丰富，而且这种修饰在 分化后的中缝髓母细胞瘤细胞(RN46A-B14)。而5-羟色胺的作用机制是 Add已被发现，其在H3上的作用尚不清楚。这项提议的主要目标是摆脱 了解H3Q5ser的功能，并询问它是否是动态PTM。 为了做到这一点，我计划使用在Muir中开发和优化的半合成技术 化学构建含有光交联剂的5-羟色胺基单核糖体的实验室。一旦拿到手，我会 利用它们捕获与H3Q5sers标记密切相互作用的蛋白质，称为“Readers”。这 实验将作为一个主要基准，开始了解H3Q5ser如何有助于规范 染色质的功能输出。该研究计划的第二个主要目标是确定H3Q5ser是否可以 被撤职，如果是，是如何被撤职的？有趣的是，负责5-羟色胺沉积的同一种酶TGM2是 已知具有脱氨酶活性。如果H3Q5ser被TGM2移除，则点突变(Gln到Glu)将 留在原地。考虑到组蛋白尾部突变的倾向，这种可能性特别有趣 癌症。 总之，这些实验旨在阐明H3Q5ser作为一种新的组蛋白H3PTM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界的极大兴趣 阐明了一个新的组蛋白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.