mDpy-30, intracellular protein transport, and histone methylation

描述（由申请人提供）：本提案的主要目标是研究细胞内区室的协调或通信。具体来说，我们将研究组蛋白修饰机制的亚基是否以及如何协调内体运输和基因转录。 在这一建议中，我们提供的证据表明，组蛋白H3赖氨酸4甲基转移酶复合物（H3 K4 MT），mDpy-30（哺乳动物直系同源的秀丽隐杆线虫Dpy-30蛋白）的亚基，驻留在细胞核和细胞质，后者是丰富的trans-Golgi网络（TGN）。基于我们的初步数据，我们假设mDpy-30及其相关蛋白质的细胞质/TGN池限制特定的粘附/迁移蛋白在其内化后再循环至细胞突起，而核mDpy-30及其相互作用蛋白质通过调节调节内体运输和细胞粘附/迁移的关键基因的转录发挥类似的作用。该假设将通过以下两个目的进行独立检验：目的1旨在利用成像和生物化学方法，使用CIMPR和21整合素作为模型货物蛋白，阐明胞质/TGN mDpy-30及其相关蛋白在内体运输中的作用。目的2以syntaxin 6和21 integrin为模型靶基因，采用实时荧光定量PCR和染色质免疫共沉淀技术研究核内体mDpy-30及其相互作用蛋白对内体再循环和粘附/迁移相关基因表达的调控作用。我们还将采用微阵列方法结合通路分析和定量实时PCR来鉴定mDpy-30调控的其他基因。 这里提出的研究将推进我们对细胞是否以及如何通过一种类型的组蛋白甲基化来协调胞质溶胶中的内体转运与细胞核中的基因调控的理解。关于TGN/内体的功能与核内基因表达之间的关系知之甚少。我们的研究有望为这一问题提供更多的见解。此外，尽管H3 K4甲基化对基因调控至关重要，但这种修饰的生理作用仍然知之甚少。在这方面，我们预计我们提出的研究将阐明H3 K4甲基化或H3 K4 MT在运输/粘附/迁移中的新功能。最后，从这项研究中获得的结果应该为未来的机制研究奠定基础，以破译由mDpy-30及其相关蛋白介导的细胞网络，以控制生理和病理状态下的细胞粘附/迁移。细胞运动是生物体生存的基本过程。在简单的生物体中，细胞运动主要用于交配和寻找食物的行为。在复杂的生物体中，细胞运动是一个严格控制的过程，在许多生理事件中起着至关重要的作用，包括胚胎发育，组织工程，伤口愈合，免疫监视等。细胞运动的失调往往会导致严重的后果，如发育缺陷，血管疾病，免疫缺陷，慢性炎症，以及肿瘤转移。因此，毫不奇怪，这种细胞行为的调节剂作为潜在的治疗靶点引起了极大的兴趣。在这方面，我们的研究也可能为开发与细胞迁移相关的疾病的有效治疗方法提供更多潜在的靶点。 公共卫生相关性：该提案旨在研究蛋白质运输和基因表达的协调。组蛋白的共价修饰允许细胞调节基因的转录，并且异常的组蛋白修饰已经与多种病理状况相关联。组蛋白修饰的一种形式涉及通过组蛋白赖氨酸甲基转移酶复合物甲基化组蛋白H3和H4内的特定赖氨酸残基。我们已经发现，mDpy-30（秀丽隐杆线虫Dpy-30蛋白的哺乳动物直系同源物），组蛋白H3赖氨酸4甲基化机制的亚基，位于细胞核和高尔基体，后者是已知的细胞内的蛋白质分选活动中发挥核心作用。该建议旨在阐明mDpy-30的这种双重定位是否代表了一种细胞机制，以协调蛋白质在高尔基体的转运与细胞核中的基因转录。虽然组蛋白甲基化在染色质结构和功能中的基本作用已经牢固确立，但关于组蛋白甲基化如何与胞质中的其他细胞事件偶联的研究仅处于起步阶段。我们希望我们的研究能够揭示组蛋白甲基化和膜运输之间的联系。

DESCRIPTION (provided by applicant): The broad goal of this proposal is to investigate the coordination or communication of intracellular compartments. Specifically, we will investigate whether and how a subunit of the histone modification machinery coordinates endosomal trafficking and gene transcription. In this proposal we provide evidence that a subunit of the histone H3 lysine 4 methyltransferase complex (H3K4MT), mDpy-30 (the mammalian ortholog of Caenorhabditis elegans Dpy-30 protein), resides in both the nucleus and the cytoplasm, the latter of which is enriched at the trans-Golgi network (TGN). Based on our preliminary data, we hypothesize that the cytoplasmic/TGN pool of mDpy-30 and its associated protein restricts specific adhesion/migration proteins from being recycled to cell protrusions following their internalization, whereas nuclear mDpy-30 and its interacting proteins exert a similar effect by modulating the transcription of key genes that regulate endosomal trafficking and cell adhesion/migration. This hypothesis will be independently tested, as follows, by two aims: Aim 1 is intended to utilize imaging and biochemical methods to elucidate the role of cytoplasmic/TGN mDpy-30 and its associated proteins in endosomal trafficking using CIMPR and 21 integrin as model cargo proteins. Aim 2 employs biochemical tools, quantitative real-time PCR and chromatin immunoprecipitation to investigate the role of nuclear mDpy-30 and its interacting proteins in regulating expression of specific genes involved in endosomal recycling and adhesion/migration using syntaxin 6 and 21 integrin as two model target genes. We will also adopt a microarray approach in combined with the pathway analysis and quantitative real-time PCR to identify other genes regulated by mDpy-30. Research proposed here will advance our understanding of whether and how a cell coordinates the endosomal transport in the cytosol with gene regulation in the nucleus via one type of histone methylation. Little is known about the relationship between the function of TGN/endosomes and the gene expression in the nucleus. Our study is expected to provide more insight into this question. Moreover, although H3K4 methylation is essential for gene regulation, the physiological roles of this modification remain much less understood. In this regard, we anticipate that our proposed research will elucidate a novel function of H3K4 methylation or H3K4MT in trafficking/adhesion/migration. Finally, results obtained from this study should set the stage for future mechanistic studies to decipher the cellular network mediated by mDpy-30 and its associated proteins to control cell adhesion/migration in physiological as well as pathological states. Cell movement is a process fundamental to the viability of organisms. In simple organisms, cell locomotion primarily functions in mating and food searching behaviors. In complex organisms, cell locomotion is a tightly controlled process that plays an essential role in many physiological events, including embryonic development, tissue engineering, wound healing, immune surveillance, etc. The deregulation of cell locomotion often leads to serious consequences such as developmental defects, vascular diseases, immunological deficiency, chronic inflammation, as well as tumor metastasis. Thus, it is of no surprise that regulators of this cell behavior are of great interest as potential therapeutic targets. In this aspect, our research might also provide more potential targets for the development of effective treatments for diseases associated with cell migration. PUBLIC HEALTH RELEVANCE: This proposal seeks to investigate the coordination of protein trafficking and gene expression. Covalent modifications of histones allow a cell to modulate the transcription of genes and aberrant histone modifications have been associated with a variety of pathological conditions. One form of histone modification involves the methylation of specific lysine residues within histones H3 and H4 by histone lysine methyltransferase complexes. We have found that, mDpy-30 (the mammalian ortholog of Caenorhabditis elegans Dpy-30 protein), a subunit of the histone H3 lysine 4 methylation machinery, resides at both the nucleus and the Golgi apparatus, the latter of which is known to play a central role in protein sorting activities within a cell. This proposal aims to elucidate whether this dual localization of mDpy-30 represents a cellular mechanism to coordinate protein transport at the Golgi apparatus with gene transcription in the nucleus. While a fundamental role of histone methylation in chromatin structure and function has been firmly established, the research on how histone methylation is coupled to other cellular events in the cytosol is only in its infancy. We expect that our studies will unmask a link between histone methylation and membrane trafficking.