In eukaryotes, the interaction of DNA with proteins and supramolecular complexes involved in gene expression is controlled by the dynamic organization of chromatin inasmuch as it defines the DNA accessibility. Here, the nuclear distribution of microinjected fluorescein-labeled dextrans of 42 kDa to 2.5 MDa molecular mass was used to characterize the chromatin accessibility in dependence on histone acetylation. Measurements of the fluorescein-dextran sizes were combined with an image correlation spectroscopy analysis, and three different interphase chromatin condensation states with apparent pore sizes of 16-20 nm, 36-56 nm and 60-100 nm were identified. A reversible change of the chromatin conformation to a uniform 60-100 nm pore size distribution was observed upon increased histone acetylation. This result identifies histone acetylation as a central factor in the dynamic regulation of chromatin accessibility during interphase. In mitotic chromosomes, the chromatin exclusion limit was 10-20 nm and independent of the histone acetylation state.

Telomerase-negative tumor cells maintain their telomeres via an alternative lengthening of telomeres (ALT) mechanism.This process involves the association of telomeres with promyelocytic leukemia nuclear bodies (PML-NBs). Here, the mobility of both telomeres and PML-NBs as well as their interactions were studied in human U2OS osteosarcoma cells, in which the ALT pathway is active. A U2OS cell line was constructed that had lac operator repeats stably integrated adjacent to the telomeres of chromosomes 6q, 11p, and 12q. By fluorescence microscopy of autofluorescent LacI repressor bound to the lacO arrays the telomere mobility during interphase was traced and correlated with the telomere repeat length. A confined diffusion model was derived that describes telomere dynamics in the nucleus on the time scale from seconds to hours. Two telomere groups were identified that differed with respect to the nuclear space accessible to them. Furthermore, translocations of PML-NBs relative to telomeres and their complexes with telomeres were evaluated. Based on these studies, a model is proposed in which the shortening of telomeres results in an increased mobility that could facilitate the formation of complexes between telomeres and PML-NBs. INTRODUCTIONTelomeres are specialized chromatin structures at the end of linear chromosomes, in which repetitive DNA sequences (5Ј-TTAGGG-3Ј in vertebrates) associate into a nucleoprotein complex (de Lange et al., 2006). This complex-the "telosome"-protects the chromosome ends from degradation and genomic rearrangement and includes the "shelterin" proteins TRF1, TRF2, and POT1 that directly recognize the TTAGGG repeat sequence (de Lange, 2005;Bertuch and Lundblad, 2006;Croy and Wuttke, 2006;Blasco, 2007). Because of incomplete DNA synthesis at the chromosome ends, 50 -200 base pairs of telomeric DNA are lost during each replication cycle (Harley et al., 1990;Martens et al., 2000). After ϳ60 -80 cell divisions telomere repeats are shortened from a typical initial length of 10 -15 kb in human cells to ϳ5 kb and below, which triggers cell senescence or apoptosis (Harley et al., 1990;Martens et al., 2000;Blasco, 2007). Accordingly, tumor cells need to compensate the loss of their telomere repeats in order to sustain an unlimited proliferative potential. In most cases this is accomplished by reactivating telomerase, a reverse transcriptase that synthesizes telomeric repeats at the chromosome ends (Greider and Blackburn, 1985;Chan and Blackburn, 2004;Collado et al., 2007;Johnson and Broccoli, 2007). However, a fraction of ϳ10 -15% of tumors is able to maintain their telomeres in the absence of telomerase activity. This process has been designated as alternative lengthening of telomeres (ALT; Bryan et al., 1995;Neumann and Reddel, 2006;Johnson and Broccoli, 2007). In yeast and mammals, it has been shown that the ALT mechanism involves homologous recombination events between telomere repeats (Dunham et al., 2000;Kass-Eisler and Greider, 2000;Lundblad, 2002;Muntoni and Reddel, 2005). It is characte...

Promyelocytic leukemia (PML) and Cajal bodies are mobile subnuclear organelles, which are involved in activities like RNA processing, transcriptional regulation, and antiviral defense. A key parameter in understanding their biological functions is their mobility. The diffusion properties of PML and Cajal bodies were compared with a biochemically inactive body formed by aggregates of murine Mx1 by using single-particle tracking methods. The artificial Mx1-yellow fluorescent protein body showed a very similar mobility compared with PML and Cajal bodies. The data are described quantitatively by a mechanism of nuclear body movement consisting of two components: diffusion of the body within a chromatin corral and its translocation resulting from chromatin diffusion. This finding suggests that the body mobility reflects the dynamics and accessibility of the chromatin environment, which might target bodies to specific nuclear subcompartments where they exert their biological function.C ajal and promyelocytic leukemia (PML) bodies are essential components of the nucleus that are thought to contain activities for RNA processing, transcriptional regulation, and antiviral defense. For understanding their biological functions, parameters have to be identified that characterize their mobility and lead to a localization of Cajal and PML bodies at their target sites in the nucleus. Whereas Cajal bodies are often found associated to several different small nuclear RNA and small nucleolar RNA gene loci as well as histone gene loci (1-5), PML bodies are frequently located in the MHC gene region (6). For both nuclear bodies, different types of movements were described and assigned to distinct subgroups of Cajal and PML bodies (7-12). The analysis of a baby hamster kidney cell line revealed that a fraction of PML bodies moved over longer distances and in an energy-dependent manner. These faster moving bodies were not observed in HeLa cells (8). In a detailed study of Cajal bodies in HeLa cells, an anomalous diffusion behavior and an ATP-and transcription-dependent association with chromatin was reported (7).Here, the mobility of PML and Cajal bodies has been compared with the nuclear body-like structures formed by Mx1 protein fused to yellow fluorescent protein (YFP). Mx proteins are IFN-induced GTPases of the dynamin super family involved in defense mechanisms against RNA viruses (13). The murine Mx1 protein, 72 kDa in size, has a natural nuclear localization sequence sequence and displays a nuclear body-like distribution (14,15). In contrast to the Mx1 WT protein, the Mx1-YFP construct studied has no antiviral activity as tested with influenza and Thogoto virus infection and formed crystals (S. Stertz and O. Haller, personal communication). The diffusion properties of this bona fide biologically inert body were used as a reference to identify principles determining the mobility of bodies within the nucleus. Cell Culture and Transfection. HeLa cells (ATCC CCL-2) were cultured on 12-mm glass coverslips for immunofluorescence or 35-...