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.
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.
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