Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin

Authors:

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

Keywords:

chromatin topology; polycomb proteins; PRC1; PRC2; BMI1; EZH2; RING1b; Image Scanning Microscopy; super-resolution microscop

Abstract:

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.

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