Mammalian chromosomes are organized in structural and functional domains of 0.1–10 Mb, which are characterized by high self-association frequencies in the nuclear space and different contact probabilities with nuclear sub-compartments. They exhibit distinct chromatin modification patterns, gene expression levels and replication timing. Recently, nucleolus-associated chromosomal domains (NADs) have been discovered, yet their precise genomic organization and dynamics are still largely unknown. Here, we use nucleolus genomics and single-cell experiments to address these questions in human embryonic fibroblasts during replicative senescence. Genome-wide mapping reveals 1,646 NADs in proliferating cells, which cover about 38% of the annotated human genome. They are mainly heterochromatic and correlate with late replicating loci. Using Hi-C data analysis, we show that interactions of NADs dominate interphase chromosome contacts in the 10–50 Mb distance range. Interestingly, only minute changes in nucleolar association are observed upon senescence. These spatial rearrangements in subdomains smaller than 100 kb are accompanied with local transcriptional changes. In contrast, large centromeric and pericentromeric satellite repeat clusters extensively dissociate from nucleoli in senescent cells. Accordingly, H3K9me3-marked heterochromatin gets remodelled at the perinucleolar space as revealed by immunofluorescence analyses. Collectively, this study identifies connections between the nucleolus, 3D genome structure, and cellular aging at the level of interphase chromosome organization.

The importance of human herpesvirus 6 (HHV-6) species as human pathogens is increasingly appreciated. However, we do not understand how infection is controlled in healthy virus carriers, and why control fails in patients with disease. Other persistent viruses are under continuous surveillance by antigen-specific T cells, and specific T-cell repertoires have been well characterized for some of them. In contrast, knowledge on HHV-6-specific T-cell responses is limited, and missing for CD8 + T cells. Here we identify CD8 + T-cell responses to HHV-6B, the most widespread HHV-6 species, in healthy virus carriers. HHV-6B-specific CD8 + T-cell lines and clones recognized HLA-A2-restricted peptides from the viral structural proteins U54 and U11, and displayed various antigenspecific antiviral effector functions. These CD8 + T cells specifically recognized HHV-6B-infected primary CD4 + T cells in an HLA-restricted manner, produced antiviral cytokines, and killed infected cells, whereas HHV-6A-infected cells were not recognized. Thus, HHV-6B-specific CD8 + T cells are likely to contribute to control of infection, overcoming the immunomodulatory effects exerted by the virus. Potentially, HHV-6-associated disease could be addressed by active or passive immunotherapy that reconstitutes virusspecific CD8 + T-cell responses. Keywords: CD8+ T cells r human herpesvirus 6B r infectious diseases r virology IntroductionHuman herpesvirus 6 (HHV-6) species are widespread pathogens, and more than 90% of humans are seropositive. The two species HHV-6A and HHV-6B have close sequence homology but differ in their epidemiology and pathogenicity [1,2]. Primary infection with HHV-6B, the more widespread species, usually takes place in early childhood and is often associated with a self-limiting illness known as three-day fever or exanthema subitum [3,4]. After primary infection, HHV-6 remains in a latent state in its immunocompetent host [5], and occasional reactivations are normally asymptomatic. However, HHV-6 (in most cases HHV-6B) can reactivate in immunosuppressed individuals. HHV-6B reactivation is observed in 40-50% of patients receiving stem cell transCorrespondence: Dr. Andreas Moosmann e-mail: andreas.moosmann@helmholtz-muenchen.de plantation, and viral reactivation is associated with delirium and cognitive decline, severe encephalitis, graft-versus-host disease, transplant failure, and overall mortality [6][7][8][9]. Apart from the immunosuppressed host, HHV-6 has been involved in a variety of diseases involving the CNS, such as febrile seizures, encephalitis, epilepsy, and multiple sclerosis [2,10]. Healthy humans frequently carry a number of other persistent viruses that may reactivate under immunosuppression, including the herpesvirus family members Epstein-Barr virus (EBV) and cytomegalovirus (CMV) [11]. It is assumed that these viruses are under continuous control by antigen-specific T cells, and viral reactivation results from a deficiency in virus-specific T cells caused by therapy-related immunosuppression [11]. In accorda...

Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is the first key step of ribosome biogenesis. While the molecular mechanisms of rRnA transcription regulation have been elucidated in great detail, the functional organization of the multicopy rRnA gene clusters (rDnA) in the nucleolus is less well understood. Here we apply super-resolution 3D structured illumination microscopy (3D-SiM) to investigate the spatial organization of transcriptionally competent active rDnA chromatin at size scales well below the diffraction limit by optical microscopy. We identify active rDNA chromatin units exhibiting uniformly ring-shaped conformations with diameters of ~240 nm in mouse and ~170 nm in human fibroblasts, consistent with rDNA looping. The active rDNA chromatin units are clearly separated from each other and from the surrounding areas of rRnA processing. Simultaneous imaging of all active genes bound by pol i and the architectural chromatin protein Upstream Binding transcription factor (UBf) reveals a random spatial orientation of regular repeats of rDnA coding sequences within the nucleoli. these observations imply rDnA looping and exclude potential formation of systematic spatial assemblies of the well-ordered repetitive arrays of transcription units. collectively, this study uncovers key features of the 3D organization of active rDNA chromatin units and their nucleolar clusters providing a spatial framework of nucleolar chromatin organization at unprecedented detail.

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Mammalian chromosomes are organized in structural and functional domains of 0.1-10 Mb, which are characterized by high self-association frequencies in the nuclear space and different contact probabilities with nuclear sub-compartments. They exhibit distinct chromatin modification patterns, gene expression levels and replication timing. Recently, nucleolus-associated chromosomal domains (NADs) have been discovered, yet their precise genomic organization and dynamics are still largely unknown. Here, we use nucleolus genomics and single-cell experiments to address these questions in human embryonic fibroblasts during replicative senescence. Genome-wide mapping reveals 1,646 NADs in proliferating cells, which cover about 38% of the annotated human genome. They are mainly heterochromatic and correlate with late replicating loci. Using Hi-C data analysis, we show that interactions of NADs dominate interphase chromosome contacts in the 10-50 Mb distance range. Interestingly, only minute changes in nucleolar association are observed upon senescence. These spatial rearrangements in subdomains smaller than 100 kb are accompanied with local transcriptional changes. In contrast, large centromeric and pericentromeric satellite repeat clusters extensively dissociate from nucleoli in senescent cells. Accordingly, H3K9me3-marked heterochromatin gets remodelled at the perinucleolar space as revealed by immunofluorescence analyses. Collectively, this study identifies connections between the nucleolus, 3D genome structure, and cellular aging at the level of interphase chromosome organization.

The nuclear distribution of eu- and heterochromatin is nonrandom, heterogeneous, and dynamic, which is mirrored by specific spatiotemporal arrangements of histone posttranslational modifications (PTMs). Here we describe a semiautomated method for the analysis of histone PTM localization patterns within the mammalian nucleus using confocal laser scanning microscope images of fixed, immunofluorescence stained cells as data source. The ImageJ-based process includes the segmentation of the nucleus, furthermore measurements of total fluorescence intensities, the heterogeneity of the staining, and the frequency of the brightest pixels in the region of interest (ROI). In the presented image analysis pipeline, the perinucleolar chromatin is selected as primary ROI, and the nuclear periphery as secondary ROI.

Chromatin is unevenly distributed within the eukaryote nucleus and it contributes to the formation of morphologically and functionally distinct substructures, called chromatin domains and nuclear bodies. Here we describe an approach to assess specific chromatin features, the histone posttranslational modifications (PTMs), of the largest nuclear sub-compartment, the nucleolus. In this chapter, methods for the isolation of nucleolus-associated chromatin from native or formaldehyde-fixed cells and the effect of experimental procedures on the outcome of mass spectrometry analysis of histone PTMs are compared.