# Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin
###### 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 Regressor 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 H3K27 me3 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. chromatic topology; polycomb proteins; PRC1; PRC2; BMI1; EZH2; RING1b; Image Scanning Microscopy; super-resolution microscopy +
Footnote †: journal: Medical and Molecular Pathology, Critical Care Medicine, University of Pisa, via Roma 65, 56126 Pisa, Italy
known as BMI1, is a crucial molecular player in cell differentiation and cancers [6; 7; 8]. vPRC1 has been demonstrated to possess an enhanced ubiquitination activity with respect to cPRC1 [9]. Notably, PRC1 complexes are endowed with multiple functions by the presence of ancillary scaffold proteins [10; 11]. PRC2 trimethylates histone 3 on lysine 27 (H3K27 me3) via its enzymatic unit EZH (or its paralog EZH, more abundant in resting cells) in combination with the PRC2 scaffold proteins SUZ2 and EED [5]. A further subdivision of PRC2 into PRC2.1 and PRC2.2 (with PRC2.2 endowed with higher catalytic activity) stems from the different ancillary proteins that participate in the complexes [12]. The working model of PcG repressing activity assumes a coordinated interplay of PRC1 and PRC2 along the genomic regions that must be silenced [3]. At first, vPRC1.1 (containing PCGF1) is tethered at non-methylated CG-rich sequences (CGIs) by the ancillary protein KDM2B. PRC1.1 deposits the H2 AK119 ub mark, which in turn recruits PRC2, which activates H3 methylation. The latter modification allosterically activates PRC2 to expand the methylation region, and a similar mechanism also operates for vPRC1 from ubiquitinated H2A foci. Finally, the H3K27 me3 mark is used for docking cPRC1 by the ancillary protein CBXs. cPRC1, through the Sterile Alpha Motif (SAM) of PHC1-3 proteins, activates the physical crosslinking of distal chromatin sequences [13]. Of note, a few details on PcG repressing activities came from super-resolution microscopy (SRM) studies, mostly by STORM imaging combined with multilabel Fluorescence In Situ Hybridization [14; 15; 16]. These studies complemented chromosome capture studies in recapitulating PcG-modulated 3D topologies of chromatin molecules at nanoscale resolution [16]. Yet, some aspects of PRC1 and PRC2 activities are still obscure. For instance, it is unclear whether the activity of vPRC1, PRC2, and cPRC1 is temporally sequential or, vice-versa, some of these protein complexes operate on the same DNA strands at the same time. We recently questioned this aspect by investigating the functional colocalization of cPRC1 component PCGF4 (aka: BMI1) with H2 AK119 ub by confocal microscopy in a model epithelial (i.e., differentiated) cell line [17]. Functional colocalization was expressed by the Pearson's coefficient \(R\), which quantifies the degree of correlated variability between two acquisition channels and thereby samples the functional association between the two fluorescent partners through their stoichiometric correlation [18]. Remarkably, statistically significant positive \(R\) values were found between H2 AK119 ub and BMI1, albeit the concentrations of these two proteins were highly correlated at the whole nuclear level [17].