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Smeets, Daniel (2013): Analysis of the Barr body with super-resolution microscopy: implications for a structural role of Xist RNA in mammalian X chromosome inactivation. Dissertation, LMU München: Fakultät für Biologie



X chromosome inactivation (XCI) in female mammalian cells is an ideal model system to study the relationship of epigenetic regulation and higher-order chromatin structure. However, light microscopic studies of chromosomal organization have long been limited by the diffraction barrier of optical resolution. Super-resolution 3D-structured illumination microscopy (3D-SIM) – one of several recent techniques that circumvent this limitation – enables multicolor optical sectioning of entire cells with eightfold-improved volumetric resolution compared to conventional fluorescence imaging methods. In the present work, 3D-SIM has been applied to analyze higher-order chromatin structure of the Barr body in mammalian nuclei, a characteristic hallmark of XCI, with yet unprecedented detail. First, the increased resolution prompted to reappraise the potential detrimental effect of the DNA-FISH procedure on chromatin structure. Comparative analyses revealed slight deteriorations at the resolution level of 3D-SIM, especially within more decondensed euchromatin sites within the nuclear interior. In contrast, overall nuclear morphology and the nuclear envelope as well as heterochromatic sites in general maintained well preserved. The results suggest that DNA-FISH studies can benefit from a combination with super-resolution microscopy. In particular, when keeping in mind the current developments of the FISH technique with increasingly small and higher-complexity probes. The compact shape of the Barr body led to the assumption of a contribution of this special higher-order chromatin structure to the establishment and maintenance of the silenced state in the inactive X chromosome (Xi). However, a confirmation of this view has always been hampered by the restrictions of conventional light microscopy. In this work, the 3D chromosomal organization of the Xi and autosomes has been investigated with 3D-SIM in various human and mouse somatic cells and in mouse embryonic stem cell (ESC) lines. The precise subchromosomal localization of a variety of factors involved in XCI in different developmental states was qualitatively and quantitatively assessed utilizing combined immunofluorescence, EdU- pulse and RNA-/DNA-FISH labeling protocols and novel data analysis tools customized for the special requirements of 3D-SIM. The results demonstrate that all autosomes are made of a three-dimensional interconnected network of chromatin domains (CDs, or topology associated domains, TADs) of highly-variable shape and dynamics. CDs/TADs are comprised of a compacted chromatin core enriched with repressive marks, which is collectively proposed to be the functionally passive chromatin compartment (PNC). This PNC is surrounded by a 50 – 150 nm locally defined, less compacted perichromatin region (PR) that is enriched with active histone modifications and pervaded by a three-dimensional interchromatin (IC) network. The PR and the IC are collectively referred to as being the functionally relevant active nuclear compartment (ANC) that harbors all major nuclear processes, including transcription and replication. 3D-SIM data revealed that the Barr body maintains this principle compartmentalization and that it is still pervaded by a narrow ANC network, which is able to fulfill its functional role as a hub for replication or rarely occurring expression of XCI-escape genes. Live-cell super-resolution imaging on HeLa H2B-GFP cells confirmed that the observed chromatin features do not reflect fixation artifacts. Xist RNA, the key factor of XCI, has been found to be preferentially located as distinct discernible foci within the ANC throughout the entire volume of the Barr body. Here, it is tightly associated with a Xi-specific form of the nuclear matrix protein SAF-A, which confirms a previously suggested role for this Xi-enriched protein in Xist RNA spreading. In contrast, Xist RNA shows no spatial correlation with repressive Xi-enriched histone marks that are found within compacted chromatin sites. This specific localization of Xist RNA reflects an intrinsic feature as it is already present during early spreading in differentiating female ESCs, where it precedes chromatin compaction concomitant with RNA Polymerase II exclusion. Its localization is further confirmed in a male ESC line carrying an inducible Xist transgene on an autosome, but where Xist RNA fails to form a true autosomal Barr body, which is less compacted and maintains transcriptional activity. Last, Xist RNA shows no direct association with PRC2, the mediator of H3K27me3, which is in contrast to the generally believed direct recruitment model of PRC2 to the Xi by Xist RNA. The data collected in this work reflects further support and a refinement of the not unequivocally accepted CT-IC (chromosome territory - interchromatin compartment) model of higher-order chromosome architecture. In addition, a first attempt has been made to integrate these findings with a recently growing number of studies using chromosome conformation capturing (3C)-based techniques and to complement them on the single-cell level. Finally, a novel model for Xist RNA function in XCI is presented, which proposes a sequence-independent structural role for gene silencing and the formation of a repressive chromatin compartment.