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First published online 14 September 2004
doi: 10.1242/jcs.01229

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Quantitative signature for architectural organization of regulatory factors using intranuclear informatics

¹ Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0106, USA
² Program in Molecular Medicine, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0106, USA

View larger version (51K): [in a new window]   Fig. 1. Conceptual framework for the quantitation of subnuclear organization by intranuclear informatics. Four main groups of parameters, selected on the basis of inherent biological variability, are examined. Parameters that describe domain quantity and nuclear size comprise group 1 (upper left panel). Group 1 includes: number of domains and domain density. Parameters that describe domain size and variability comprise group 2 (upper right panel). Group 2 includes: domain size mean, median, standard deviation, variance, skewness, kurtosis, coefficient of variation, and index of dispersion. Parameters that describe the domain spatial randomness, which is based on domain nearest-neighbor distances, comprise group 3 (lower left panel). Group 3 includes: domain nearest-neighbor mean, median, standard deviation, variance, skewness, kurtosis, coefficient of variation, index of dispersion, domain density, nearest-neighbor distance mean and variance expected for a random distribution, ratios between actual and expected mean and variance, and the standard error in the nearest-neighbor distances. Parameters that characterize the radial position of domains comprise group 4 (lower right panel). Group 4 includes: mean perimeter radius, mean domain radius, and mean relative domain radius.

View larger version (64K): [in a new window]   Fig. 2. Post-mitotic restoration of the spatially ordered Runx subnuclear organization is functionally conserved. ROS 17/2.8 osteosarcoma cells (top panel) were subjected to in situ immunofluorescence microscopy for endogenous Runx2. Runx2 is distributed at punctate subnuclear domains throughout the interphase and telophase nucleus (lower panel). Subnuclear organization parameters were computed from deconvoluted images for Runx2 for interphase nuclei (I), and both progeny telophase nuclei, denoted at random as telophase nucleus 1 (T1) or telophase nucleus 2 (T2). A color map has been applied to the standardized data assigning red to higher values and green to lower values (see supplementary material). Each increment of one reflects one (row) standard deviation (inner left and right panels). ANOVA was performed to assess the significance of observed differences between T1, T2, and I nuclei. Asterisks indicate statistically significant differences based on a 0.05 level with correction for false discovery rate. Bonferroni's multiple comparison tests were use to determine which nuclei differed significantly at a 0.05 level. In each case significant differences were observed between telophase (T1,T2) and interphase nuclei (I), but differences were not observed between telophase nuclei. Overall mean Clark and Evans statistics (Ro/Re) were 1.4 for Runx2, indicating a non-random organization with spatial order. Bar, 10 µm.

View larger version (54K): [in a new window]   Fig. 3. Mutation of NMTS alters the interphase Runx subnuclear organization. Point mutations within the Runx2 NMTS were generated using PCR-mediated mutagenesis. Deconvoluted images were analyzed of whole cells (HeLa) expressing either HA-tagged wild-type Runx2, an HA-tagged C-terminal deletion or one of the four HA-tagged NMTS point mutants. As shown, each of these mutants and wild-type Runx exhibits a punctate subnuclear distribution (top panel). Standardized mean subnuclear organization data for the indicated proteins are shown (right panel). A color map has been applied to the standardized values assigning red to higher values and green to lower values (see supplementary material). Using a repeated-measure analysis of variance (ANOVA) we detect significant differences at a 0.05 level in 17 of 25 parameters measured, as indicated by asterisks. Bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 4. Discrimination between wild-type Runx2 and NMTS mutants on the basis of domain size, packing, and spatial randomness. To understand the subnuclear organization of the wild-type Runx protein and the five mutants, we analyzed factors scores, which reflect the sum of standardized subnuclear organization parameters multiplied by respective factor loadings. Factor scores assign a value to each of the unobservable factors (factor A: domain size properties, factor B: domain packing, and factor C: domain spatial randomness). Using the data acquired from the 330 nuclear image sections, we computed factor scores for wild-type and each of the mutants and analyzed star-plots of these scores on three axes (see supplementary material). The center of the star-plot has a value of -0.5, the end of each axis has a value of 0.5, and the mid-point on each axis is zero; these values are in standardized units. The three mean factor scores for each protein define the points of a filled triangle that has been drawn to illustrate the similarities and differences among each of the proteins. Based upon the shape of each of the filled triangles, we can discriminate two groups of domain organizations: one comprised of the wild-type Runx2 protein along with the Y407A and R398A mutants and a second group containing Y433A, Y428A, and the functionally compromised Runx2-C mutant. Differences in the shape of the triangles highlight the selective alterations in subnuclear organization as a consequence of NMTS mutations.

View larger version (67K): [in a new window]   Fig. 5. The subnuclear organization of Runx domains is linked with subnuclear targeting, biological function and disease. In order to determine the extent to which the subnuclear organization of each mutant differs from wild-type we performed hierarchical cluster analysis using the Euclidean distance matrix and complete linkage. Cluster organization is illustrated using a dendrogram. Subnuclear organization data is presented in a compressed form with a color map as described in Fig. 3. As shown, there are two main clusters: one including wild-type and one including the Runx2-C protein, which does not contain the NMTS. We find a clear parallel between this cluster analysis and our factor analysis, particularly with respect to the clustering of Runx2-C with Y433A and Y428A. This parallel lends strength to the observed clusters. Shown at the bottom is a symbolic representation of the extent to which each protein associates with the nuclear matrix as determined by biochemical fractionation and western blot analysis, i.e. ranging from `+++' (associated) for wild-type to `-' (no association for Runx2-C) (Zaidi et al., 2001; Choi et al., 2001) (our unpublished observations). We find a correlation between subnuclear organization and nuclear matrix association. The schematic below indicates whether a protein will promote differentiation or is involved in disease (i.e. cleidocranial dysplasia) [yes, no, or not determined (ND)].