Seminar Series
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Date:
Time: Location: Speaker: Title: |
01 Nov 2016
1-2pm LTE, Hicks Building Dr Lothar Schermelleh, Department of Biochemistry, University of Oxford Functional chromatin organisation analysed by multidimensional super-resolution imaging |
Abstract: 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 topological 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 illusive due to the lack of appropriate high-resolution single-cell analysis methods.
Using super-resolution 3D structured illumination microscopy (3D-SIM) we have been able to resolve a 3D landscape of 200-500nm wide chromatin domain clusters co-aligned with a DNA-free interchromatin network leading to nuclear pores. We present our recent advances in developing and applying a custom ‘deep-content’ quantitative 3D-SIM imaging workflow to systematically map the spatial distribution of a range of epigenetic marker and interacting factors identified by proteomic screenings on the size-scale of TADs. We find transcriptionally active/permissive chromatin marks highly enriched at decondensed domain surfaces exposed to interchromatin space, whereas repressive chromatin marks are located towards the interior of domains. Furthermore, by combining 3D-SIM with localisation microscopy and single particle tracking we are now able to study molecular relationships and dynamic properties of key structural regulators, such as cohesin subunits, in the context of a 3D chromatin landscape thus laying the foundations to further explore sub-TAD structure-function at unprecedented detail. Our findings support a model of higher-order chromatin architecture harbouring distinct functional environments that contribute to regulate genome function through physical accessibility.
Using super-resolution 3D structured illumination microscopy (3D-SIM) we have been able to resolve a 3D landscape of 200-500nm wide chromatin domain clusters co-aligned with a DNA-free interchromatin network leading to nuclear pores. We present our recent advances in developing and applying a custom ‘deep-content’ quantitative 3D-SIM imaging workflow to systematically map the spatial distribution of a range of epigenetic marker and interacting factors identified by proteomic screenings on the size-scale of TADs. We find transcriptionally active/permissive chromatin marks highly enriched at decondensed domain surfaces exposed to interchromatin space, whereas repressive chromatin marks are located towards the interior of domains. Furthermore, by combining 3D-SIM with localisation microscopy and single particle tracking we are now able to study molecular relationships and dynamic properties of key structural regulators, such as cohesin subunits, in the context of a 3D chromatin landscape thus laying the foundations to further explore sub-TAD structure-function at unprecedented detail. Our findings support a model of higher-order chromatin architecture harbouring distinct functional environments that contribute to regulate genome function through physical accessibility.
