Matrin 3 (matr3), an abundant protein of the internal nuclear matrix, has been linked to a variety of functional events. As a step toward defining its multifunctional nature, we have studied the association of matr3 with chromosome territories and identified potential interacting proteins. A similar staining pattern of matr3 was observed in fixed WI38 fibroblast cells and in live HeLa cells using a matr3-GFP construct. Matr3 was detected throughout autosomal and the active X chromosome territories. Conversely, matr3 was strikingly excluded from the inactive X chromosome as well as within both the perinuclear and perinucleolar heterochromatin. Yeast two hybrid analysis identified matr3 interactions with 33 unique nuclear localized proteins and also revealed its propensity for self association. A majority of these proteins are involved in RNA metabolism and chromatin remodeling while others function in protein translation, DNA replication/repair and apoptosis. Further analysis of a selection of these proteins and scaffold attachment factor A (SAFA) by co-localization and co-immunoprecipitation experiments using HeLa cells confirmed their interactions with matr3.
Higher order chromatin organization in concert with epigenetic regulation is a key process that determines gene expression at the global level. The organization of dynamic chromatin domains and their associated protein factors is intertwined with nuclear function to create higher levels of functional zones within the cell nucleus. As a step towards elucidating the organization and dynamics of these functional zones, we have investigated the spatial proximities among a constellation of functionally related sites that are found within euchromatic regions of the cell nucleus including: HP1g, nascent transcript sites (TS), active DNA replicating sites in early S-phase (PCNA) and RNA polymerase II sites. We report close associations among these different sites with proximity values specific for each combination. Analysis of matrin 3 and SAF-A sites demonstrates that these nuclear matrix proteins are highly proximal with the functionally related sites as well as to each other and display closely aligned and overlapping regions following application of the minimal spanning tree (MST) algorithm to visualize higher order network-like patterns. Our findings suggest that multiple factors within the nuclear microenvironment collectively form higher order combinatorial arrays of function. We propose a model for the organization of these functional neighborhoods which takes into account the proximity values of the individual sites and their spatial organization within the nuclear architecture. J. Cell. Biochem. 105: 391-403, 2008. ß 2008 KEY WORDS: RNA POLYMERASE II SITES; HP1g SITES; REPLICATION SITES; TRANSCRIPTION SITES; CELL NUCLEUS; PROLIFERATING CELL NUCLEAR ANTIGEN; NUCLEAR MATRIX; MATRIN 3; SAF-A; COMPUTER IMAGE SEGMENTATION; PROXIMITY ANALYSIS; PATTERN RECOGNITION IMAGE ANALYSIS; MINIMAL SPANNING TREE NETWORKS D espite significant advances in molecular biology and biochemistry, our understanding of the nucleus is at its infancy. The genome is more than a linear sequence of DNA [Misteli, 2007] and is segmented into chromosomes which occupy distinct territories in the interphase cell nucleus [Cremer et al., 2001]. Chromatin within these chromosomes is arranged into multiple levels of hierarchical organization from the nucleosomal 10 nm arrays to the 30 nm fibers to chromatin loops and higher order domains [Berezney, 2002;Cremer et al., 2006;Razin et al., 2007]. Although the eukaryotic nucleus is devoid of any internal membranes, it introduces an incredible level of complexity and several layers of control, which regulate the genomic functions and increase the efficiency of processivity and enzyme regulatory mechanisms. Moreover, the compartmentalization of genomic functions and factors that mediate these functions suggests a high level of structural organization [Berezney et al., 1996;Strouboulis Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 105: 391-403 (2008)
391Abbreviations used: DAPI, 4 0 ,6-diamidino-2-phenylindole; HP1g, heterochromatin protein 1, gamma; MST, minimal span...
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