THE CELL BIOLOGY OF GENOMES

Tom Misteli
National Cancer Institute, NIH, Bethesda, MD 20892
mistelit@mail.nih.gov

Genomes are expressed in the context of the architectural framework of the cell nucleus.  While we have learnt much about the linear sequence of genomes and the molecular nature of key components involved in gene expression, little is known about how genomes are expressed in their natural environment of the cell nucleus.  To understand genome function vivo, two fundamental questions must be addressed: How are genomes spatially organized within the cell nucleus and how do cellular gene expression machines such as transcription complexes interact with chromatin in vivo?  Modern imaging methods enable us to address both questions. 

Genomes are organized in the interphase cell nucleus in the form of chromosomes.  The spatial arrangement of chromosomes within the nucleus is now known to be non-random.  Chromosomes occupy preferential radial positions with respect to the center of the nucleus and they are positioned in particular patterns relative to each other (1).  While a direct function of spatial positioning in regulation of gene expression remains elusive, the relative positioning of chromosomes and gene loci has an important role in genome stability and formation of chromosomal translocations as observed in cancer cells (2,3).  We have proposed that proximal positioning of chromosomes increases their probability of forming translocations.  In support, we have found preferential proximal positioning of translocation-prone gene loci and chromosomes in mouse and human systems in comparison to non-translocating loci (2,3).  Consistent with a role of spatial proximity in determining translocation partners, we have recently found evidence for tissue specificity of spatial genome organization within the interphase nucleus.  Efforts to uncover the molecular mechanisms of chromosome positioning are now underway. 

In order to address how proteins interact with the genome in intact living cells we use in vivo imaging methods.  Live-cell imaging allows us to visualize proteins in their native environment and application of computational approaches enables us to extract quantitative information about biophysical properties of factors acting on genomes.  We have demonstrated that binding of many proteins to native chromatin is transient and that many proteins have dwell times on chromatin on the order of seconds (4, 5).  Together with their ability to rapidly diffuse throughout the nucleus, these observations indicate that chromatin proteins find their binding sites by three-dimensional scanning of the genome space (6-8).  Rapid exchange dynamics are also a hallmark of structural proteins in heterochromatin and the dynamic nature of these proteins is consistent with a stochastic mechanism for the establishment and maintenance of chromatin domains (5).  We propose that chromatin proteins constitute a dynamic interaction network and that dynamic competition plays a critical role in determining global and local chromatin states.  This property is likely crucial for accurate gene regulation and to maintain high plasticity in genome expression. 

The desire to understand how genomes are spatially organized in vivo and how molecular machines translate the genome information in intact living cells is a logical extension of genome sequencing projects.  The complementary information from sequencing efforts and the emerging cell biological understanding of genomes is leading towards an integrated view of genome function and promises to reveal novel principles of cellular organization and function.  Uncovering the molecular mechanisms of genome function in the context of nuclear architecture will be critical in the successful development of gene therapy approaches, cloning strategies and control of stem cell differentiation.

References:

1. Parada, L.A. and Misteli, T. (2002) Trends Cell Biol. 12, 425-432
2. Parada, L.A., McQueen, P. G., Munson, P.J and Misteli, T. (2002) Current Biology. 1692-1697
3. Roix, J.J., McQueen, P.G., Munson, P. J., Parada, L.A. and Misteli, T. (2003) Nature Genetics, 34, 287-291
4. Misteli, T., Gunjan, A., Hock, R., Bustin, M. and Brown, D.T. (2000) Nature. 408, 877-881
5. Cheutin, T., McNairn, A.J., Jenuwein, T., Gilbert, D.M., Singh, P.B. and Misteli, T. (2003) Science. 299, 721-725
6. Phair, R. D. and Misteli, T. (2000) Nature. 404, 604-609
7. Misteli, T. (2001) Science. 291, 843-847
8. Dundr, M., Hoffman-Rohrer, U., Hu, Q., Grummt, I., Rothblum, L.I, Phair, R.D. and Misteli, T. (2002) Science. 298, 1623-1626