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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 on understanding the spatial organization and regulation 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 cell/molecular biological approach to elucidate the organization and 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 a multi-component live-cell gene expression system whereby we are able to visualize a stably integrated regulatable genetic locus, and follow in real-time, transcription of that locus, including visualization of its mRNA and protein products in living cells. The system is composed of a 200-copy array stably integrated into a euchromatic region (1p36) in human U2OS cells. Although it was known that the production of proteins based on the information stored in DNA involves dynamic interactions among many molecules that carry out the processes of transcription, RNA splicing and export, and translation, it was never before possible to simultaneously track all of the products of transcription and translation as they are produced and move throughout living cells. Using this system we were able to detect specific events that transform the architecture of chromatin from a transcriptionally silent state to an actively transcribed state. In addition, we were able to visualize and track individual mRNPs from their transcription site throughout the cell. We have observed nascent RNA as early as 5 minutes post-induction and mRNPs leaving the locus and moving through the nucleoplasm by a mechanism consistent with diffusion. This system has revealed fundamental information about how genes are switched on and off in the context of living cells and is currently being used in many laboratories to address a variety of fundamental questions. Ongoing studies in our laboratory are using this system to examine the recruitment of members of the gene expression/silencing machinery. In addition, we are studying the exchange of critical factors at the site of this specific gene locus as the locus enters into and exits from cell division.

A second focus of my laboratory is to identify novel mechanisms of regulating gene expression with the ultimate goal of developing new approaches to understand and treat disease. Recent large-scale studies of the human and mouse genomes have revealed that although there are approximately 22,000 protein-coding genes in human and in mouse, significantly larger portions of both genomes are transcribed. Such analyses suggest that protein-coding genes alone are not sufficient to account for the complexity of higher eukaryotic organisms. It is estimated that approximately 98% of the transcriptional output of the human genome represents RNA that does not encode protein and therefore either these non-coding RNAs are largely useless transcripts or they are fulfilling a wide range of unexpected functions in eukaryotic biology. We are focusing our efforts on a class of non-coding RNAs that are large (>1 kb) and retained in the nuclei of cells. We suggest that within this class of large non-coding RNAs will be found a diverse group of key regulatory molecules that will provide significant insight into basic cellular functions, developmental regulation, and disease. Such non-coding RNAs may play a much more crucial role in physiological and pathological processes than currently anticipated. For example, mutations in non-coding regulatory RNAs may give rise to some of the phenotypes associated with specific diseases by altering gene expression at a specific locus or at a more global level. We are currently pursuing the function of several of these nuclear retained non-coding RNAs and are developing screens to elucidate others.