Summary of novel insights about nuclear architecture gained by 3D-SIM

which is hampered in the autosomal context and thus leads to the observable incomplete silencing effects.

novel chromosome conformation capture techniques, but also with theoretical in silico models about chromatin organization based on known chromatin features and dynamics (for reviews see Dekker et al., 2013; Marti-Renom and Mirny, 2011; Mirny, 2011). Here, diameters of individual ~500 kb CDs/TADs have been determined by a simple model calculation to be on average ~360 nm². Compacted CD/TAD cores are enriched in repressive chromatin modifications (like H3K27me3 or macroH2A1) and are surrounded by less condensed chromatin, probably containing small-scale chromatin loops and harboring essential nuclear processes like transcription and replication (Figure 5-1, B). This functional region is enriched with active histone marks like H3K4me3 in autosomal chromatin, which has not been resolvable yet with conventional microscopy. How the compacted cores of individual CDs/TADs are organized remains highly speculative today, and has therefore not be addressed any further here, but it will surely become a field of intensive investigation in the nearer future. The entire nucleus is also pervaded by another, by definition DNA-free, IC channel network, which occasionally forms larger IC-lacunae, known to contain macromolecular complexes like splicing speckles or PML bodies. Here, the PR and IC are collectively referred to as the ANC. It could be shown that the ANC is ultimately leading towards nuclear pores and that it does not reflect a fixation artifact, thus implying and reinforcing its functional importance. The ANC is found to be enriched with SAF-A, thus linking the presence of a suggested nuclear matrix with this functional chromatin compartment. A narrowed, but still present, ANC is also clearly visible within the Barr body. Although, in the Barr body larger IC-lacunae are very rare, they can still be observed occasionally and coincide with active histone marks and active transcription, thus possibly harbor XCI escape genes. The collapsed ANC of the Barr body is still able to fulfill its functional role as a hub for replication or rarely occurring expression of escape genes, which leads to a widening of this preformed channel system, and which can even be chemically inducible by e.g. hyperosmotic conditions.

Xist RNA has been shown to be preferentially located within this narrow ANC of the Barr body, where it strongly colocalizes with SAF-A. This interaction is very likely to cause a Xi-specific

2 According to recent 3C-based studies, an average CD/TAD is comprised of ~500 kb of DNA, which according to the CT-IC model and confirmed by the 3D-SIM data in this thesis would be organized in all kinds of compaction states (Bickmore and van Steensel, 2013; Dixon et al., 2012; Lieberman-Aiden et al., 2009; Nora et al., 2012).

The entire Barr body has a volume of about 8 – 10 µm³ (roughly 2 x 2 x 2 µm in C2C12 cells). Given the total genomic size of ~170 Mbp of the mouse X chromosome results in an average volume of ~0.0235 µm³ per CD/TAD. Assuming a sphere-like shape, results in a diameter of ~360 nm per CD/TAD. It is reasonable to assume a sphere-like shape here as this sphere would not be homogenous, but also include all possible ANC regions, because the initial volume value has been deduced from the total Xi volume, which equally includes all possible compaction states. Of note, this value is just an average number, which might vary substantially in vivo and between individual CDs/TADs. However, this value is far above the resolvable limit of 3D-SIM and the fact that no regularly shaped entities are observable within chromatin stainings implies that individual CDs/TADs are highly variable and dynamic structures, which are moreover interconnected throughout the nucleus. This calculation is not supposed to provide an exact number, but rather give a rough estimate of size scales for discussions and future experiments.

translational modification of SAF-A, which may induce its polymerization and thus act as an anchor system for the sequence-independent spreading of Xist RNA throughout the Xi. This post-translational modification may also explain the observed enrichment of SAF-A:GFP at the Xi (Fackelmayer et al., 1994; Hasegawa et al., 2010; Helbig and Fackelmayer, 2003). The fact that Xist RNA is not distributed evenly but as distinct foci may also help to explain its role in XCI. An intriguing explanation is the accumulation of Xist RNA at non-repetitive sites, possibly reflecting genic sites or regulatory elements, which has already been hypothesized in studies on metaphase spreads or in more recent high-throughput epigenomic studies (Duthie et al., 1999; Marks et al., 2009; Pinter et al., 2012). Here, it could block the access for the transcription machinery, hence, offering a novel mechanism for Xist RNA-induced gene silencing. Another (parallel) mode of action might be the induction of locally constrained chromatin compaction, which in turn mediates the recruitment of other silencing machinery. This is also in line with the observation that Xist RNA is not directly colocalizing with PRC2, as previously assumed, and that PRC2 may rather be recruited through indirect effects, like the Xist RNA-induced chromatin compaction (Brockdorff, 2013; Cerase et al., 2013, submitted; Yuan et al., 2012).

Lastly, a first attempt has been made to integrate the findings of this work into a refined model of higher-order chromatin architecture of both Barr body and autosomal chromatin at the nucleosomal level (see Figure 5-1, C). There is still a vast lack of understanding how higher-order chromatin structure is organized within the interphase nucleus between the level of the 10 nm DNA-fiber and chromosome territories. This is mainly due to historical methodological limitations of EM on the one side and conventional light microscopy on the other side. Recent developments in surpassing the resolution limit of light, like the 3D-SIM data in this work, but also improvement of sequencing depth in 3C-based techniques or advancements in theoretical physical modeling of chromatin properties have started to close this gap. It will need further attempts in the future to integrate all these different areas to be able to truly understand how chromatin is organized on the nucleosomal level within CDs/TADs and how their structure and dynamics are able to fulfill the various needs like genome integrity, transcriptional regulation, fast and reliable access to specific genomic loci or rapid and accurate replication.

Figure 5-1 In-scale summary about the organization of Barr body and autosomal chromatin together with distribution of Xi-specific chromatin marks and other factors including a hypothetical outlook. (A) Single optical z-section of a DAPI-stained C2C12 nucleus. White boxes display typical Barr body and neighboring autosomal chromatin. Arrowheads point at nuclear pores. (B) Summary of nuclear architecture of Barr body and autosomal chromatin including the distribution of some of the analyzed factors in this work. Scheme is drawn to scale using the marked areas in (A) as a reference at the resolution achievable with 3D-SIM. Dotted circle indicates the ~ 360 nm average diameter of a 500 kb CD/TAD as determined by theoretical calculations. Its diameter is above the level of 3D-SIM resolution almost by factor of 3. (C) Hypothetical model view about the chromatin architecture at 4 exemplary nuclear sites drawn roughly to scale. (Inset 1)

Decondensed sites harboring escape genes can potentially emerge throughout the nucleus. Individual chromatin loops, enriched with active histone marks, can reach out of the compacted chromatin environment and be transcribed in the PR region, where they have also access to the nuclear ANC. (Inset 2) Typical site of repressed Barr body chromatin. Only very small-scale and still compacted chromatin loops, enriched with repressive histone marks, reach out of the compacted CD/TAD core into the ANC. The ANC network is only very narrow and can occasionally disappear completely when opposing chromatin loops come together. Neighboring or opposing chromatin loops do not overlap or intermingle to minimize detrimental entanglement effects. However, IC channels are prone to emerge and widen again upon need, e.g. during replication or via osmotic induction (HCC). (Inset 3) Xist RNA spreads along Xi-specific SAF-A, which probably polymerizes within the Barr body, either as consequence or prerequisite of Xist RNA interaction. Xist RNA undergoes multimerization and potentially accumulates at genic sites throughout the Barr body, where it blocks the access of the transcription machinery and thus contributes to the maintenance of Xi-linked gene silencing. It may also have a functional role in causing or maintaining chromatin compaction, which is a prerequisite for the accumulation of repressive chromatin modifications.

(Inset 4) Autosomal chromatin is comprised of interconnected CDs/TADs of variable shape and highly dynamic. Individual CDs/TADs cannot be separated visually. Compacted cores of CDs/TADs are enriched with repressive histone marks and surrounded by decondensed chromatin enriched in active chromatin marks and harboring nuclear processes like transcription and replication. Autosomal chromatin is, unlike the Barr body, pervaded by wider IC lacunae, which are known to carry large macromolecular complexes like splicing speckles or PML bodies, and are ultimately leading towards the nuclear pores. Figure adapted from (Smeets et al., 2013b, submitted).

The data obtained with 3D-SIM presented in this thesis, substantiated and expanded the current CT-IC model of chromatin architecture that is mainly based on observations with conventional microscopy and EM, but has not been commonly accepted because of these methodological constraints. 3D-SIM data presented here has also provided a basis to make first promising attempts to integrate and complement high-throughput data of 3C-based whole-population studies on chromatin architecture with findings and model views based on the single-cell level. Lastly, 3D-SIM probably uncovered a yet unknown functional role of Xist RNA in gene silencing and maintaining the Barr body structure, which will contribute to the understanding of XCI reflecting a fundamental process in mammalian epigenetic regulation and chromatin architecture and will probably lead to a reappraisal of commonly accepted models about Xist RNA and its role in XCI.

6 Appendix