Characterization of nucleolus-associated chromatin domains during cellular aging and upon genetic inactivation of the Upstream Binding Factor protein

The nucleus of mammalian cells consists of several structural and functional domains, of which the nucleolus is the largest one. Although its primary function in the synthesis of ribosomal RNA and early steps of ribosome assembly is well characterized and its role in stress sensing, cell cycle regulation and RNA modification has been revealed, the involve- ment of the nucleolus in genome organization is less well understood. Nucleolus-associated chromosomal domains (NADs) were initially described independently by two different lab- oratories, providing a snapshot of the nucleolus-associated genome in HeLa and HT1080 human cancer cells (N emeth et al., 2010; van Koningsbruggen et al., 2010). These inves- tigations showed that NADs represent mainly a specific heterochromatin compartment. However, NADs have not been mapped in normal diploid cells and other species than human. Moreover, the dynamics of the nucleolus-associated chromatin during diverse cel- lular processes such as cellular aging, or after inhibition of nucleolar transcription remained largely unknown. Here, the effect of different nucleolus isolation protocols on the mapping of NADs and on the identification of histone post-translational modifications (PTMs) by masss spectrom- etry were monitored first. The results revealed robust, comparable mappability of NADs under various conditions, but differences in the maintaining of PTMs depending on the nucleolus isolation procedure. Next, the dynamics of nucleolus-associated chromatin was investigated during replicative senescence in IMR90 human embryonic fibroblasts. In vitro human diploid fibroblasts stop dividing after a certain number of cell divisions (Hayflick, 1965), a process called replicative senescence. During this process the nuclear architecture and nucleolar morphology get largely rearranged (Bridger et al., 2000; Mehta et al., 2007; Hein et al., 2011). The nucleolar hallmarks of senescent cells include increased size and fu- sion of nucleoli, furthermore chromatin and DNA methylation changes of ribosomal DNA (rDNA) (Hein et al., 2011). In the course of this study the nucleolus association of chromo- somal domains was mapped using comparative genomic hybridization on high-resolution microarrays. The initial analyses suggest that NADs are stable units and their genomic maps show only minor differences between the population of proliferating and senescent IMR90 cells. Three-dimensional analysis of nucleolar morphology provided quantitative data about the increase of nucleolar volume in senescent IMR90 cells and the accompa- nying decrease of their number per cell. Immunostaining of histone PTMs in proliferating and senescent IMR90 cells revealed that the distribution of a marker for constitutive het- erochromatin, trimethylation of the lysine 9 of histone H3 (H3K9me3), exhibits the most remarkable changes at the nucleoli during cellular aging. In senescent cells this histone-modification was depleted in the perinucleolar space, whereas a notable alteration in its distribution, but only a minor change in its relative amount was visible at the nuclear periphery, another characteristic site of heterochromatin accumulation. Immuno-FISH ex- periments revealed firstly that the localization of rDNA arrays with respect to nucleoli does not change remarkably during cellular senescence, secondly that senescence-associated dis- tension of satellites goes together with their disassociation from nucleoli, moreover that the localization of selected genomic regions frequently, but not completely corresponds to the nucleolus association states, which were observed in high-throughput genomic analyses. Finally, nucleolus-associated DNA was isolated from mouse embryonic fibroblast cells, in which inhibition of ribosomal RNA transcription was achieved by induced depletion of the Upstream Binding Factor (UBF) protein, an essential factor of nucleolar RNA polymerase I transcription in vivo (Hamdane et al., 2014). Here again data were collected in high-resolution comparative genomic hybridization experiments for future bioinformatic analyses. In addition, 3D immuno-FISH experiments were performed to monitor large-scale alterations of nuclear and nucleolar architecture. The results illustrated, that the rDNA tends to form discrete foci, which get condensed and co-localized with major satellite repeat clusters at the centromeres. In summary, in this study nucleoli were isolated from different human and mouse cells and their DNA and protein content was subjected to high-throughput genomic and mass spectrometry analyses, respectively. In addition, the dynamics of the nucleolus-associated chromatin was investigated in replicative senescence in human embryonic fibroblasts. The results shed new light on the role of the nucleolus in genome organization during these processes and provide a basis for further investigations.