Biochemical characterization of epigenomic markers

遗传信息是由我们的基因及其调控元件携带的，有助于所有生物的正常发育。除了这种遗传密码的重要性，另一层信息也被认为是至关重要的。事实上，表观遗传信息，被定义为不依赖于潜在DNA序列的遗传信息，最近作为许多生物过程的主要调节器浮出水面。DNA支架蛋白（如组蛋白）和DNA本身可以被化学修饰，这种共价改变可以触发特定的分子信号，这些发现已经成为揭示一个复杂、错综复杂的新密码的基石：组蛋白密码。现在已知单个或组合修饰携带对许多分子事件至关重要的关键信息，包括基因转录，蛋白质翻译，DNA修复和许多其他事件。在这些共价修饰中，赖氨酸残基的甲基化是组蛋白最显著的改变之一。然而，尽管它们在细胞核中起着关键作用，但控制组蛋白底物识别和蛋白质赖氨酸甲基转移酶（pkmt）催化的机制还需要进一步研究。我们最近的研究发现，特定的植物PKMT选择性地甲基化组蛋白变体组蛋白H3.1而不是组蛋白H3.3；组蛋白H3的两个亚类已知赋予染色质特异性，这促使我们进一步研究植物pkmt和组蛋白变体之间的相互作用。最近的研究结果表明，植物PKMT在植物的花区和生长中起着关键作用，这也促使我们努力关注植物PKMT；提高了我们的发现可能对农业产生重要影响的希望。在这个提议中，我们打算用x射线晶体学和体外实验来定义SET DOMAIN GROUP8 （SDG8）和SET DOMAIN GROUP2 （SDG2）识别组蛋白的机制基础。在接下来的五年里，我们的目标是（图2）到1)确定控制SDG2和SDG8识别组蛋白H3的分子基础，2)确定这些蛋白质识别的一致氨基酸序列，3)与Yannick Jacob博士（耶鲁大学）合作，测试针对这些酶识别残基的组蛋白突变体对植物的影响，具体目标是识别具有生长优势的突变。增加花的形成，改善防御机制。这个项目符合我们的长期目标，即阐明控制组蛋白共价修饰和表观基因组编写的分子决定因素

The genetic information, which is carried by our genes and their regulatory elements, contribute to the proper development of all living organisms. Despite the importance of this genetic code, an additional layer of information is also known to be vital. In fact, epigenetic information, which is defined as heritable information that do not depend on the underlying DNA sequence, has recently surfaced as a master regulator of many biological processes. The discoveries that DNA scaffolding proteins, such as histones, and DNA itself could be chemically modified and that such covalent alterations could trigger specific molecular signals, have been cornerstone in unraveling a complex, intricate and new code: the histone code. Single or a combination of modifications is now known to carry critical information vital for many molecular events including gene transcription, protein translation, DNA repair and many others. Among those covalent modifications, methylation of lysine residues is amongst the most prominent alteration of histone proteins. However, despite their critical roles in the nucleus, the mechanisms controlling histone substrates recognition and catalysis of protein lysine methyltransferases (PKMTs) require further investigation. Our recent findings that a specific plant PKMT selectively methylates histone variant histone H3.1 and not histone H3.3; two sub-classes of histone H3 known to impart specific properties to chromatin, have motivated us to further investigate the interplay between plant PKMTs and histone variants. Our endeavors to focus on plant PKMT is also motivated by recent findings showing that plant PKMTs play a pivotal role in plant floraison and growth; raising the hope that our findings may have important impact in agriculture. In this proposal, we intend to define the mechanistic basis underlying histone recognition by SET DOMAIN GROUP8 (SDG8) and SET DOMAIN GROUP2 (SDG2) using X-Ray crystallography and in vitro assays. In the next five years, we aim (Fig. 2) to 1) to determine the molecular basis controlling histone H3 recognition by SDG2 and SDG8, 2) determine the consensus amino acid sequences recognized by these proteins and 3), in collaboration with Dr. Yannick Jacob (Yale U.), test the impact of histone mutants targeting residues recognized by these enzymes in plants with the specific goal of identifying mutations conferring growth advantages, increasing floral initiation and improving defense mechanisms. This project is in line with our long-term goals of elucidating the molecular determinants controlling the covalent modification of histone proteins and the writing of the epigenome.**