Mitotic partitioning and selective reorganization of tissue-specific transcription factors in progeny cells

Abstract

Postmitotic gene expression requires restoration of nuclear organization and assembly of regulatory complexes. The hematopoietic and osteogenic Runx (Cbfa/AML) transcription factors are punctately organized in the interphase nucleus and provide a model for understanding the subnuclear organization of tissue-specific regulatory proteins after mitosis. Here we have used quantitative in situ immunofluorescence microscopy and quantitative image analysis to show that Runx factors undergo progressive changes in cellular localization during mitosis while retaining a punctate distribution. In comparison, the acetyl transferase p300 and acetylated histone H4 remain localized with DNA throughout mitosis while the RNA processing factor SC35 is excluded from mitotic chromatin. Subnuclear organization of Runx foci is completely restored in telophase, and Runx proteins are equally partitioned into progeny nuclei. In contrast, subnuclear organization of SC35 is restored subsequent to telophase. Our results show a sequential reorganization of Runx and its coregulatory proteins that precedes restoration of RNA processing speckles. Thus, mitotic partitioning and spatiotemporal reorganization of regulatory proteins together render progeny cells equivalently competent to support phenotypic gene expression.

Fig. 1.

Runx proteins partition equivalently in progeny cells following cell division. Jurkat lymphoma cells (A) or ROS 17/2.8 osteosarcoma cells (B) were subjected to in situ immunofluorescence microscopy. Runx1 and Runx2 were detected by rabbit polyclonal antibodies followed by the incubation of cells with secondary antibodies conjugated with Alexa 488 fluorochrome. Both Runx1 and Runx2 were distributed at punctate subnuclear foci throughout the interphase nucleus (Upper). A quantitative image analysis was applied to determine the relative levels of Runx in nuclei of the telophase cells (n = 10; Lower). We defined a PC that reflects the ratio of integrated signal intensities between progeny nuclei. Both Runx proteins exhibited a PC equivalent to that of DNA, demonstrating that these factors are equally segregated in progeny cells after cell division. Student's t test was performed to assess the significance of observed differences.

Fig. 2.

Runx foci dynamically redistribute during mitosis. Endogenous Runx2 and tubulin were visualized by in situ immunofluorescence in synchronized ROS 17/2.8 cells at indicated stages of mitosis. Runx2 foci shown in deconvoluted images (marked by dotted boxes) in Left are distributed throughout the cell in prophase. This distribution of Runx2 foci changes to predominantly extra-chromosomal in metaphase and anaphase. In telophase, Runx2 foci appear to colocalize with DNA. Tubulin shows characteristic staining throughout mitosis and serves as a marker to identify mitotic stages. The DNA boundaries from 4′,6-diamidino-2-phenylindole images are drawn as white dotted lines. Specificity is demonstrated by in situ preparations in which the primary antibody incubation was omitted (bottom panel).

Fig. 3.

Some of Runx2 foci associate with chromosomes throughout mitosis. Images showing mitotic redistribution of Runx2 foci were subjected to quantitative image analysis. Intensity profiles (center panel in each mitotic stage) of images shown on the left were generated by using metamorph imaging software. A scatter plot between the signal intensities of Runx2 (y axis) and DNA (x axis) indicates that Runx2 is associated with DNA in interphase and telophase although this association decreases during prophase-metaphase and anaphase. A subset of Runx2 foci (indicated by arrows) is associated with chromosomes during all stages of mitosis. The red line demarcates the level above which all pixels correspond to Runx foci. The bar at the bottom right represents the pseudocolor map for image intensity.

Fig. 4.

Runx2 foci are equally segregated to progeny nuclei with restoration of subnuclear organization during telophase. We performed a quantitative image analysis on telophase and G₂ nuclei to assess the number and size of Runx2 domains in parent and progeny cells. The analysis was carried out by image deconvolution (A, left-most panels; for details, see Materials and Methods) followed by image thresholding and binarization to define Runx2 domains (A, right panels). The domain number, size, and spatial organization were then calculated in each of the telophase (designated as T1 and T2) and G₂ nuclei analyzed, and mean values are displayed as bar graphs (n = 10). Asterisks indicate statistically significant differences between G₂ and telophase nuclei. The error bars represent SEM. We found double the number of Runx2 domains in G₂ nuclei compared with telophase nuclei, while the domain size and spatial distribution remain the same. Conversely, we find equal numbers of SC35 foci, but these foci are smaller and exhibit a different spatial distribution than G₂ nuclei.

Fig. 5.

Chromatin-modifying factor p300 and nucleosomal protein histone H4, but not RNA processing factor SC35, show colocalization with DNA during mitotic progression. Chromatin-modifying factor p300 (A Left), RNA processing protein SC35 (A Right), and tetra-acetylated histone H4 (B) were detected by in situ immunofluorescence in synchronized ROS 17/2.8 cells at indicated stages of mitosis. Histone H4 and p300 show a constitutive DNA localization throughout mitosis, while SC35 is excluded from DNA.

Fig. 6.

Sequential redistribution of nuclear proteins involved in RNA synthesis and processing after cell division. The relationship between DNA and different nuclear proteins during mitosis was confirmed by Pearson correlation analysis. The correlation between image intensities reflects the colocalization of different nuclear proteins with DNA during mitotic progression. Whereas histone H4 and p300 are colocalized to DNA throughout mitosis, SC 35 is excluded. Runx2, in comparison, shows a gradual decrease in chromosomal localization until cells enter anaphase. In telophase, Runx2 exhibits a restoration of interphase subnuclear distribution. Asterisks indicate correlations that are significantly different from interphase.