Chromatin Dynamics in Living Cells: Identification of Oscillatory Motion ARTEM PLISS, 1 KISHORE S. MALYAVANTHAM, 1,2 SAMBIT BHATTACHARYA, 3 AND RONALD BEREZNEY 1 * 1 Department of Biological Sciences, University at Buffalo, The State University of New York, Buffalo, New York 2 Department of Pathology and Anatomical Sciences, School of Medicine, University at Buffalo, The State University of New York, Buffalo, New York 3 Department of Mathematics and Computer Science, Fayetteville State University, Fayetteville, North Carolina Genomic DNA in mammalian cells is organized into 1 Mbp chromatin domains (ChrD) which represent the basic structural units for DNA compaction, replication, and transcription. Remarkably, ChrD are highly dynamic and undergo both translational movement and configurational changes. In this study, we introduce an automated motion tracking analysis to measure, both in 2D and 3D, the linear displacement of early, mid and late S-phase replicated ChrD over short time periods (<1 sec). We conclude that previously identified large-scale transitions in the spatial position and configuration of chromatin, originate from asymmetric oscillations of the ChrD detectable in fractions of a second. The rapid oscillatory motion correlates with the replication timing of the ChrD with early S replicated ChrD showing the highest levels of motion and late S-phase chromatin the lowest. Virtually identical levels of oscillatory motion were detected when ChrD were measured during active DNA replication or during inhibition of transcription with DRB or a-amanitin. While this motion is energy independent, the oscillations of early S and mid S, but not late S replicated chromatin, are reduced by cell permeabilization. This suggests involvement of soluble factors in the regulation of chromatin dynamics. The DNA intercalating agent actinomycin D also significantly inhibits early S-labeled chromatin oscillation. We propose that rapid asymmetric oscillations of <1 sec are the basis for translational movements and configurational changes in ChrD previously detected over time spans of minutes–hours, and are the result of both the stochastic collisions of macromolecules and specific molecular interactions. J. Cell. Physiol. 228: 609–616, 2013. ß 2012 Wiley Periodicals, Inc. Arguably, the most efficient and physiologically relevant approach for visualizing large populations of native chromatin in live cells is based on the fluorescent labeling of chromatin during DNA replication (Zink and Cremer, 1998; Zink et al., 2003; Berezney et al., 2005; Pliss et al., 2009; Maya-Mendoza et al., 2010). This approach yields a pattern with either early, mid or late S-phase replicated DNA arranged into relatively uniform chromatin domains (ChrD) (Jackson and Pombo, 1998; Ma et al., 1998; Koberna et al., 2005). These ChrD are megamolecular complexes comprised of 1 Mbp of DNA plus hundreds to thousands of different proteins and RNA molecules associated by various mechanisms. Independent electron microscopic and super-resolution light microscopic studies revealed a nearly identical average size of individual ChrD, measuring 110–120 nm, regardless of their replication timing (Baddeley et al., 2010; Koberna et al., 2005). Upon completion of DNA synthesis, the organization of ChrD into particular replication patterns as well as their replication timing is strictly maintained till the end of interphase and into subsequent cell cycles (Ma et al., 1998). This indicates the fundamental role of ChrD as basic units for the regulation of DNA compaction and replication. Remarkably, the fluorescently labeled ChrD, as well as exogenous DNA loci inserted into the genome, undergo both linear motion and configurational transformations (Marshall et al., 1997; Zink et al., 1998; Pliss et al., 2009; Olivares-Chauvet et al., 2011). However, little is understood about the functional significance of chromatin dynamics and regulatory mechanisms that govern this process. While early experiments reported that the dynamics of chromatin was passive and resembled constrained Brownian motion (Marshall et al., 1997), further studies focusing on the positioning of specific DNA sequences in the cell nucleus, revealed very complex behavior of chromatin that does not correspond entirely with diffusion and indicates the existence of underlying mechanisms regulating chromatin motion. For instance, changes were demonstrated in the intranuclear position and configuration of fluorescently stained exogenous DNA loci in response to transcription activation (Tumbar et al., 1999; Tumbar and Belmont, 2001) or at the onset of DNA replication (Li et al., 1998). Moreover, the motion of chromatin can be coordinated for even larger regions of the genome as was shown for the major histocompatibility complex containing several Mbp of DNA that loops out of its chromosome territory in response to transcription upregulation with interferon-gamma (Volpi et al., 2000). In addition, translational movement of chromatin in the cell nuclear space can be regulated with striking precision, bringing distantly located genes to the same transcription site or nuclear Additional supporting information may be found in the online version of this article. Contract grant sponsor: National Institutes of Health; Contract grant number: GM 072131. Artem Pliss’s present address is The Institute for Lasers, Photonics, and Biophotonics, University at Buffalo, The State University of New York, Buffalo, NY 14260-3000. Kishore Malyavantham’s present address is IMMCO Diagnostics, Inc., 60 Pineview Drive, Buffalo, NY 14228. *Correspondence to: Ronald Berezney, Department of Biological Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14260. E-mail: berezney@buffalo.edu Manuscript Received: 21 July 2012 Manuscript Accepted: 31 July 2012 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 8 August 2012. DOI: 10.1002/jcp.24169 ORIGINAL RESEARCH ARTICLE 609 Journal of Journal of Cellular Physiology Cellular Physiology ß 2012 WILEY PERIODICALS, INC.