Expansion microscopy allows high resolution single cell analysis of epigenetic readers.

Interactions between epigenetic readers and histone modifications play a pivotal role in gene expression regulation and aberrations can enact etiopathogenic roles in both developmental and acquired disorders like cancer. Typically, epigenetic interactions are studied by mass spectrometry or chromatin immunoprecipitation sequencing. However, in these methods, spatial information is completely lost. Here, we devise an expansion microscopy based method, termed Expansion Microscopy for Epigenetics or ExEpi, to preserve spatial information and improve resolution. We calculated relative co-localization ratios for two epigenetic readers, lens epithelium derived growth factor (LEDGF) and bromodomain containing protein 4 (BRD4), with marks for heterochromatin (H3K9me3 and H3K27me3) and euchromatin (H3K36me2, H3K36me3 and H3K9/14ac). ExEpi confirmed their preferred epigenetic interactions, showing co-localization for LEDGF with H3K36me3/me2 and for BRD4 with H3K9/14ac. Moreover addition of JQ1, a known BET-inhibitor, abolished BRD4 interaction with H3K9/14ac with an IC50 of 137 nM, indicating ExEpi could serve as a platform for epigenetic drug discovery. Since ExEpi retains spatial information, the nuclear localization of marks and readers was determined, which is one of the main advantages of ExEpi. The heterochromatin mark, H3K9me3, is located in the nuclear rim whereas LEDGF co-localization with H3K36me3 and BRD4 co-localization with H3K9/14ac occur further inside the nucleus.

Post-mitotic BET-induced reshaping of integrase quaternary structure supports wild-type MLV integration.

The Moloney murine leukemia virus (MLV) is a prototype gammaretrovirus requiring nuclear disassembly before DNA integration. In the nucleus, integration site selection towards promoter/enhancer elements is mediated by the host factor bromo- and extraterminal domain (BET) proteins (bromodomain (Brd) proteins 2, 3 and 4). MLV-based retroviral vectors are used in gene therapy trials. In some trials leukemia occurred through integration of the MLV vector in close proximity to cellular oncogenes. BET-mediated integration is poorly understood and the nature of integrase oligomers heavily debated. Here, we created wild-type infectious MLV vectors natively incorporating fluorescent labeled IN and performed single-molecule intensity and Förster resonance energy transfer experiments. The nuclear localization of the MLV pre-integration complex neither altered the IN content, nor its quaternary structure. Instead, BET-mediated interaction of the MLV intasome with chromatin in the post-mitotic nucleus reshaped its quaternary structure.

Evaluation of Direct Grafting Strategies via Trivalent Anchoring for Enabling Lipid Membrane and Cytoskeleton Staining in Expansion Microscopy.

Super-resolution fluorescence microscopy is a key tool in the elucidation of biological fine structures, providing insights into the distribution and interactions of biomolecular complexes down to the nanometer scale. Expansion microscopy is a recently developed approach for achieving nanoscale resolution on a conventional microscope. Here, biological samples are embedded in an isotropically swollen hydrogel. This physical expansion of the sample allows imaging with resolutions down to the tens-of-nanometers. However, because of the requirement that fluorescent labels are covalently bound to the hydrogel, standard, small-molecule targeting of fluorophores has proven incompatible with expansion microscopy. Here, we show a chemical linking approach that enables direct, covalent grafting of a targeting molecule and fluorophore to the hydrogel in expansion microscopy. We show application of this series of molecules in the antibody-free targeting of the cell cytoskeleton and in an example of lipid membrane staining for expansion microscopy. Furthermore, using this trivalent linker strategy, we demonstrate the benefit of introducing fluorescent labels post-expansion by visualizing an immunostaining through fluorescent oligonucleotide hybridization after expanding the polymer. Our probes allow different labeling approaches that are compatible with expansion microscopy.

Fluorescence Photobleaching as an Intrinsic Tool to Quantify the 3D Expansion Factor of Biological Samples in Expansion Microscopy.

Four years after its first report, expansion microscopy (ExM) is now being routinely applied in laboratories worldwide to achieve super-resolution imaging on conventional fluorescence microscopes. By chemically anchoring all molecules of interest to the polymer meshwork of an expandable hydrogel, their physical distance is increased by a factor of ∼4-5× upon dialysis in water, resulting in an imprint of the original sample with a lateral resolution up to 50-70 nm. To ensure a correct representation of the original spatial distribution of the molecules, it is crucial to confirm that the expansion is isotropic, preferentially in all three dimensions. To address this, we present an approach to evaluate the local expansion factor within a biological sample and in all three dimensions. We use photobleaching to introduce well-defined three-dimensional (3D) features in the cell and, by comparing the size and shape pre- and postexpansion, these features can be used as an intrinsic ruler. In addition, our method is capable of pointing out sample distortions and can be used as a quality control tool for expansion microscopy experiments in biological samples.