Dynamic organization of chromosomes in the mammalian cell nucleus

Uncovering the motifs of a higher order nuclear architecture and its implications on nuclear function has raised increasing interest in the past decade. The nucleus of higher eukaryotes is considered to display a highly dynamic interaction of DNA and protein factors. There is an emerging view that there are hierarchical levels of gene regulation, reaching from epigenetic modifications at the DNA- and histone level to a higher order functional nuclear topology, in the context of which gene-activating and -repressing processes influence the gene expression profile of an individual cell beyond the sequence information of the DNA. The present work focuses on the analysis of the dynamic aspects of higher order nuclear architecture in living cells. As a prerequisite, an in vivo replication labeling strategy was developed, that enabled the simultaneous visualization of early and mid-to-late replicating chromatin as well as single chromosome territories on the basis of a labeling/segregation approach. The presented scratch replication labeling protocol combines a high labeling efficiency with reduced “damaging” effects and can be successfully applied to a number of adherently growing cell lines, including primary human fibroblasts. In addition, a live cell observation system was developed that facilitates time-lapse confocal (4D) microscopy over elongated time periods which made it possible to follow a complete cell cycle or more. To address possible long-range movements of chromosome territories (CTs) during an entire interphase, fluorescence labeling of a small number of CTs was performed in living HeLa cells stably expressing histone H2B-GFP. This was achieved by in vivo scratch replication labeling with fluorescent nucleotides. Labeled cells were cultivated for several cell cycles until labeled chromatids had segregated. Such cells were followed by time-lapse confocal microscopy over time-scales of up to 20 hours covering major parts or the complete cell cycle. Positional changes of the intensity gravity centers of labeled CTs in the order of several µm were observed in early G1, thereafter, the positions remained within a range of ~1 µm till the end of G2. In conclusion, CT arrangements were highly constrained from mid G1 to late G2 / early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. More extended movements observed in early G1 might play a role when CTs “home in” to establish a non-random radial CT arrangement. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase whereas unbleached chromosome segments were often observed to become located at distant sites in the metaphase plates. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus, indicating that CT neighborhoods were not preserved during mitosis. The variability of CT neighborhoods during clonal growth was further confirmed by 3D-FISH experiments. A series of experiments of a more preliminary character looked at the influence of the nuclear lamina in constraining and determining a higher order nuclear architecture by selectively interacting with mid-to-late replicating chromatin. Simultaneous immunodetection of lamin B on two-color replication labeled neuroblastoma cell nuclei revealed specific attachment of the mid-to-late replicating chromatin compartment not only along the periphery but also inside the nucleus along invaginations of the lamina. 4D-live cell observation of lamin C-GFP expressing CHO cells with mid-to-late replicating chromatin labeled simultaneously revealed concomitant movements of replication foci attached to lamin invaginations. Moreover, a functional essay was employed which uses injection of a dominant negative lamin A mutant protein (ΔNLA) to cause a reversible disruption of the nuclear lamina. Initial results point to concomitant distortion of the mid-to-late replication pattern and a preferential attachment of the respective chromatin sites to partially disrupted lamin B (as compared to lamin A and nuclear pore complex). Finally, a model is presented on the chromosome positioning in mammalian nuclei depending on cell cycle and nuclear shape

Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages

Whether chromosomes maintain their nuclear positions during interphase and from one cell cycle to the next has been controversially discussed. To address this question, we performed long-term live-cell studies using a HeLa cell line with GFP-tagged chromatin. Positional changes of the intensity gravity centers of fluorescently labeled chromosome territories (CTs) on the order of several μm were observed in early G1, suggesting a role of CT mobility in establishing interphase nuclear architecture. Thereafter, the positions were highly constrained within a range of ∼1 μm until the end of G2. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase, whereas the contiguity of unbleached chromosome segments was lost to a variable extent, when the metaphase plate was formed. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus. We conclude that CT arrangements were stably maintained from mid G1 to late G2/early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. The variability of CT neighborhoods during clonal growth was further confirmed by chromosome painting experiments

DNA choreography: correlating mobility and organization of DNA across different resolutions from loops to chromosomes

The dynamics of DNA in the cell nucleus plays a role in cellular processes and fates but the interplay of DNA mobility with the hierarchical levels of DNA organization is still underexplored. Here, we made use of DNA replication to directly label genomic DNA in an unbiased genome-wide manner. This was followed by live-cell time-lapse microscopy of the labeled DNA combining imaging at different resolutions levels simultaneously and allowing one to trace DNA motion across organization levels within the same cells. Quantification of the labeled DNA segments at different microscopic resolution levels revealed sizes comparable to the ones reported for DNA loops using 3D super-resolution microscopy, topologically associated domains (TAD) using 3D widefield microscopy, and also entire chromosomes. By employing advanced chromatin tracking and image registration, we discovered that DNA exhibited higher mobility at the individual loop level compared to the TAD level and even less at the chromosome level. Additionally, our findings indicate that chromatin movement, regardless of the resolution, slowed down during the S phase of the cell cycle compared to the G1/G2 phases. Furthermore, we found that a fraction of DNA loops and TADs exhibited directed movement with the majority depicting constrained movement. Our data also indicated spatial mobility differences with DNA loops and TADs at the nuclear periphery and the nuclear interior exhibiting lower velocity and radius of gyration than the intermediate locations. On the basis of these insights, we propose that there is a link between DNA mobility and its organizational structure including spatial distribution, which impacts cellular processes

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs

Restricted mobility of Dnmt1 in preimplantation embryos: implications for epigenetic reprogramming

BACKGROUND: Mouse preimplantation development is characterized by both active and passive genomic demethylation. A short isoform of the prevalent maintenance DNA methyltransferase (Dnmt1S) is found in the cytoplasm of preimplantation embryos and transiently enters the nucleus only at the 8-cell stage. RESULTS: Using GFP fusions we show that both the long and short isoforms of Dnmt1 localize to the nucleus of somatic cells and the cytoplasm of preimplantation embryos and that these subcellular localization properties are independent of phosphorylation. Importantly, photobleaching techniques and salt extraction revealed that Dnmt1S has a very restricted mobility in the cytoplasm, while it is highly mobile in the nucleus of preimplantation embryos. CONCLUSION: The restricted mobility of Dnmt1S limits its access to DNA and likely contributes to passive demethylation and epigenetic reprogramming during preimplantationdevelopment

Super-resolution structured illumination microscopy: past, present and future.

Structured illumination microscopy (SIM) has emerged as an essential technique for three-dimensional (3D) and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualization and image processing methods. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'

DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required

Myb-binding Protein 1a (Mybbp1a) Regulates Levels and Processing of Pre-ribosomal RNA

Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes

Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y

The expression of a new histone variant H3.Y increases during cellular stress to regulate cell cycle progression and gene expression

Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation

Postreplicative maintenance of genomic methylation patterns was proposed to depend largely on the binding of DNA methyltransferase 1 (Dnmt1) to PCNA, a core component of the replication machinery. We investigated how the slow and discontinuous DNA methylation could be mechanistically linked with fast and processive DNA replication. Using photobleaching and quantitative live cell imaging we show that Dnmt1 binding to PCNA is highly dynamic. Activity measurements of a PCNA-binding-deficient mutant with an enzyme-trapping assay in living cells showed that this interaction accounts for a 2-fold increase in methylation efficiency. Expression of this mutant in mouse dnmt1−/− embryonic stem (ES) cells restored CpG island methylation. Thus association of Dnmt1 with the replication machinery enhances methylation efficiency, but is not strictly required for maintaining global methylation. The transient nature of this interaction accommodates the different kinetics of DNA replication and methylation while contributing to faithful propagation of epigenetic information