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Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin
Irene Nepita, Simonluca Piazza, Martina Ruglioni, Sofia Cristiani, Emanuele Bosurgi, Tiziano Salvadori, Giuseppe Vicidomini, Alberto Diaspro, Marco Castello, Paolo Bianchini, Barbara Storti and Ranieri Bizzarri
chromatin topology; polycomb proteins; PRC1; PRC2; BMI1; EZH2; RING1b; Image Scanning Microscopy; super-resolution microscop
Super-resolution microscopy has been recently applied to understand the 3D topology of chromatin at an intermediated genomic scale (kilobases to a few megabases), as this corresponds to a sub-diffraction spatial scale crucial for the regulation of gene transcription. In this context, polycomb proteins are very renowned gene repressors that organize into the multiprotein complexes Polycomb Repressor Complex 1 (PRC1) and 2 (PRC2). PRC1 and PRC2 operate onto the chromatin according to a complex mechanism, which was recently recapitulated into a working model. Here, we present a functional colocalization study at 100–140 nm spatial resolution targeting PRC1 and PRC2 as well as the histone mark H3K27me3 by Image Scanning Microscopy (ISM). ISM offers a more flexible alternative to diffraction-unlimited SRMs such as STORM and STED, and it is perfectly suited to investigate the mesoscale of PRC assembly. Our data suggest a partially simultaneous effort of PRC1 and PRC2 in locally shaping the chromatin topology.