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"Quantitative 3D-SIM imaging of chromatin domain organisation"

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Genome function in higher eukaryotes depends on the context of a hierarchical chromatin organisation. Recent genome-wide contact probability (HiC) maps have highlighted distinct ~1Mb sized topologically associated domains (TADs) that may serve as fundamental subunits important for genome stability and regulation of gene expression. A deeper understanding of how chromatin folds into higher-order domains to create a functional landscape for transcription remained elusive due to the lack of appropriate high-resolution single-cell analysis methods. By live and fixed cell super-resolution 3D structured illumination microscopy (3D-SIM) we have been able to resolve a dynamic 3D landscape, consisting of a heterogeneous network of 300-700-nm-wide chromatin domain clusters, that are approximately in the size scale of TADs confirmed by FISH . This network is co-aligned with a DNA -free interchromatin network of similar dimension leading to nuclear pores. Applying a custom ‘deep content’ quantitative imaging workflow we systematically mapped the spatial distribution of a wide range of epigenetic marker and structural proteins. We find transcriptionally active/permissive chromatin marks as well as cohesin and CTCF highly enriched at decondensed domain surfaces exposed to interchromatin space, whereas repressive chromatin marks are located towards the interior of domains. This correlation between nano-scale chromatin conformation and epigenetic marks/states persists after ablation of cohesin function, is enhanced upon biophysical perturbation, such as ATP depletion and induced chromatin hyper-condensation, and temporarily lost in post-replicative chromatin. Our findings support a model of a higher-order chromatin architecture on the size level of TADs that creates distinct functional environments through the action of biophysical forces other than cohesin-CTCF mediated looping interaction, that may contribute to regulate genome function through physical accessibility.

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