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Research Summary

Almost all of the functions in a cell or organism can trace their beginnings to the cell nucleus with the turn on of a gene, processing of its RNA, and subsequent transport of that RNA into the cytoplasm where it is synthesized into a protein. Yet very little is known about how these events are spatially and temporally coordinated within the context of the nucleus to allow for the regulation of accurate gene expression; or how they go awry resulting in various diseases. For example, while changes in nuclear organization have long been used as markers for the identification of cancer cells, relatively little is known about the direct cause or effect of these changes. 

The focus of my laboratory centers around understanding the spatial organization of gene expression in living cells. While much information is available from in vitro studies as to the factors that are involved in gene expression, much less is known about the spatial and temporal dynamics of these factors and their substrates within the context of the living cell. My laboratory has taken a molecular/cell biological approach to elucidate the intranuclear dynamics and signals involved in the movement of transcription and RNA processing factors from storage/modification sites to sites of active transcription. Our studies are focused in two main arenas: gene expression and nuclear organization.

First, we have recently developed an inducible system to directly visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of gene array (200 copies) stably integrated into a euchromatic region (1p36) in human U2OS cells. In the transcriptionally silent state the array exhibits all of the features characteristic of heterochromatin including association with HP1, histone H3 methylated at lysine 9, as well as the presence of the H3 lysine 9 histone methyltransferase, Suv39h1. Using this system we have been able to examine the dynamics of the locus as well as the loss and recruitment of proteins critical to silencing and gene expression. We have observed nascent RNA as early as 5 minutes post-induction and mRNPs leaving the locus and moving through the nucleoplasm. This system has allowed us to assess the spatial and temporal aspects of gene expression within the context of the living cell. Ongoing studies are using this system to examine the recruitment of the transcription/RNA processing machinery and to study the processes of silencing and DNA replication in living cells.

Our second approach has focused upon a nuclear organelle first identified by Hewson Swift in 1959 and termed interchromatin granule clusters or nuclear speckles. These structures are enriched in pre-mRNA splicing factors and several transcription factors, including the large subunit of RNA polymerase II. Each mammalian cell nucleus contains 30-50 of these clusters and each cluster is composed of several hundred granules each measuring 20-25nm in diameter. Using GFP/splicing factor fusion proteins we have shown that these clusters are dynamic in living cells and that splicing factors are recruited from these clusters to sites of transcription. In addition, we have shown that the mechanism involved in the recruitment is hyperphosphorylation of the splicing factors by specific kinases that reside in this nuclear region. Most interestingly, we have found that the carboxy terminal domain of the large subunit of RNA polymerase II also plays a role in the recruitment process, perhaps by acting as a staging ground for the factors involved in various aspects of RNA processing. Ongoing studies in the laboratory are aimed at understanding how the approximately 100 proteins needed for expression of a single gene are recruited to the site of transcription and what proteins may be involved in forming complexes at the IGCs for subsequent recruitment to transcription sites.

To complement our cell biological studies we have taken a biochemical approach and purified IGCs from mouse liver nuclei. Using mass spectrometry we have identified 136 proteins that are bona fide constituents of the IGCs and, in addition, we have identified 25 new proteins. We are currently employing motif analysis on the new proteins to identify potentially interesting motifs that may provide clues as to the function of these proteins. It is now clear that in addition to encoding proteins the genome also encodes for many different classes of RNAs that are likely to play central roles in regulating gene expression. We have previously determined that although IGCs are not active transcription sites they contain a stable population of poly(A)⁺ RNA. We have recently purified this RNA population and have constructed a library. We are in the process of screening potential candidates for structural and/or stable polyadenylated RNAs that may have roles in nuclear structure and/or in regulating gene expression. 

In summary, our interdisciplinary approach to studying nuclear structure/function will establish the in vivo spatial and temporal parameters necessary for efficient gene expression and will serve as the basis from which to study alterations in nuclear structure/function that are related to various diseases.