Spatio-temporal dynamics of genomic organization and function in the mammalian cell nucleusIntroductionNuclear zoning of replication and transcription and the coordination of genomic functionComputer image analysis of higher order genomic functionSpatio-temporal dynamics of chromatin organization in living cellsDynamics of DNA replication sites in living cellsSummaryAcknowledgmentsReferencesAdvan. Enzyme Regul. 45 (2005) 17–26Spatio-temporal dynamics of genomicorganization and function in the mammaliancell nucleusRonald Berezneya,, Kishore S. Malyavanthama, Artem Plissa,Sambit Bhattacharyab, Raj AcharyacaDepartment of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260,USAbDepartment of Computer Science and Engineering, State University of New York at Buffalo, Buffalo,New York 14260, USAcDepartment of Computer Science and Engineering, The Pennsylvania State University, University Park,PA 16802, USAIntroductionRecent developments in microscopy and computer imaging have led to a new viewof the cell nucleus based on genomic function. It is now known that genomicfunctions and factors which mediate these functions including DNA replication andtranscription, RNA transcript tracks and splicing factors are compartmentalized inthe cell nucleus in discrete domains (Berezney et al., 1995; Nickerson et al., 1995;Berezney, 2002; van Driel et al., 2003). Moreover, development of humanchromosome-specific DNA ‘‘paints’’ have demonstrated that the functional genomeitself is arranged into discrete chromosome specific territories in the interphasenucleus (Chevret et al., 2000; Cremer and Cremer 2001; Cremer et al., 2001; Paradaet al., 2003).The association of genomic function with the nuclear matrix, the structuralfoundation of the cell nucleus (Ma et al., 1998, 1999; Wei et al., 1998; Volpi et al.,ARTICLE IN PRESSwww.elsevier.com/locate/advenzreg0065-2571/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.advenzreg.2005.02.013Corresponding author.2000), provides another dimension for understanding nuclear architecture (Berezney,2002; Berezney et al., 1995; Berezney et al., 2000; Cremer et al., 2001, 2004). Asreported in numerous studies (Berezney and Coffey, 1974, 1976, 1977; Berezney,1979, 1984, 1991; Berezney et al., 1995; Pienta and Coffey, 1984), nuclear matrixarchitecture is maintained following extraction of chromatin from isolated nuclei orin cells grown on coverslips. While the precise relationship between chromosometerritories and the nuclear matrix remains to be resolved, it has been reported thatchromosome territorial organization is maintained following high salt extractionfor nuclear matrix and is disrupted following progressive treatment with RNase(Ma et al., 1999). Disruption of chromosomal territorial organization coincideswith disruption and extraction of the internal structure of the nuclear matrix.This suggests an important role of the nuclear matrix in maintaining terri-torial organiz ation of the chromatin in the cell nuc leus (Ma et al., 1999; Berezney,2002)These findings support the view that the cell nucleus is remarkably well-ordered inboth genomic spatial organization (nuclear matrix architecture and chromosometerritories) and genomic function. They also provide the foundation for a new andexciting field in gene expression and regulation in which the role of nucleararchitecture in the organization and function of genes and gene products is underinvestigation (Volpi et al., 2000; Stein et al., 1999, 2000, 2003, 2004).This paper will focus on recent advances of our research group concerning thespatio-temporal organiz ation and dynamics of functional chromatin domains in themammalian cell nucleus. Critical to these studies are the development of computerimaging approaches that enable a rigorous analysis of the spatial organization of thefunctional genomic sites and their dynamics in living cells.Nuclear zoning of replication and transcription and the coordination of genomicfunctionWhile the findings of discrete nuclear domains of replication, transcription andRNA splicing have provided a foundation for studying the relationships of nuclearform and function, many questions remain unanswered and are providing the basisfor current and future experimenta tion. One fundamental question is: Are the sites ofDNA replication or transcription ‘‘randomly’’ arranged in the nucleus or are therehigher levels of organization above the individual sites? We have addressed thisquestion by simultaneously labeling replication and transcription sites in permea-bilized mammalian cells and applying three–dimensional (3-D) microscopy andcomputer imaging techniques. Our results demonstrated for the first time thatreplication and transcription sites are clustered into separate higher order domainsor ‘‘zones’’ in the cell nucleus (Wei et al., 1998). Since replication sites contain anaverage of 1 mbp DNA (Ma et al., 1998; Jackson and Pombo, 1998; Berezneyet al., 2000) and each replication zone likely contains a dozen or more individualreplication sites, the chromatin contained within these replication zones must behuge (410 mbp) and may represent a fundamental level for the coordinationof replication timing among multiple genes and gene families (Hatton et al., 1988;ARTICLE IN PRESSR. Berezney et al. / Advan. Enzyme Regul. 45 (2005) 17–2618Dhar et al., 1988; Selig et al., 1992; Kitsberg et al., 1993). Similarly, individualtranscription sites are also believed to contain multiple genes and their higher orderarrangement into separate transcription zones may be the structural expression oftranscriptional programming in the cell.This analysis was performed in early S-phase, when discrete sites of bothreplication and transcription are readily observed (Wei et al., 1998). Since activelytranscribed genes are preferentially replicated in early S-phase (Goldman et al.,1984), these finding have important implications for our understanding of thecoordination of replication and transcription of genes. What is suggested is that hugeregions of chromatin are ‘‘recruited’’ for replication or transcription at a specificmoment in S-phase. At another moment of time (e.g., 1 h later in S-phase) anothergroup of replication zones would be active. This implies a dynamic process of re-zoning (Wei et al., 1998; Berezney and Wei, 1998; Cook, 1998) in which regions inthe nucleus previously commissioned for
View Full Document