Protocol for visualizing the chromatin assembly properties of epigenetic protein complexes via an HTM module-mediated artificial tethering system

Abstract

The detailed chromatin assembly processes for many epigenetic regulatory complexes are largely unknown. Here, we present a protocol utilizing heterochromatin-targeting module (HTM) module-mediated chromatin tethering followed by microscopy-based visualization to detect the recruitment priority between two components in Polycomb repressive complex 1 (PRC1). Moreover, we detail procedures for detecting the resultant histone-modifying activities of PRC1 using immunofluorescence (IF) analyses. This approach allows directly visualization of the on-chromatin assembly of the histone-modifying complexes of interest in live cells. For complete details on the use and execution of this protocol, please refer to Cheng et al.¹.

Keywords: biotechnology and bioengineering; cell biology; molecular biology.

Graphical abstract

Figure 1

Schematic diagram for an HTM-mediated artificial tethering system and its potential applications (A) This system can be used to detect the main factor for complex assembly and the corresponding enzymatic activities. (a) When CBX7 in PRC1 is tethered with HTM, the endogenous factor(s) being recruited and the resultant de novo histone modification(s) can detected via IF assays, while the EGFP-fused exogenous factor(s) can be directly detected via EGFP/mCherry colocalization analyses. (b) Similar HTM assays are applied to check the other PRC1 component(s) for their capability to facilitate complex assembly. (B) The other potential applications of this system. (a) The assay can be used to verify whether a transcription factor of interest can directly recruit the epigenetic regulators to chromatin. (b) This assay can be applied to confirm the interaction relationship between different regulatory complexes. (c) The assay also facilitates to dissect the functional domains involved in these protein-protein interaction events.

Figure 2

Quantitative analyses of the HTM-tethered complex assembly The confocal images for the co-localization of the mCherry-HTM-CBX7 (a core component in PRC1 with the H3K27me3 reader activity) and the endogenous RING1B (a core component in PRC1 with the catalytic activity for H2AK119ub) were analyzed (top panel). Chromatin was labeled via Hoechst staining. The intensity of various fluorescence signals across the randomly selected regions (shown as the line segments) was scanned and statistically analyzed using ImageJ software (bottom panel) to assess the working mechanism of the recruitment and assembly of functional PRC1 on chromatin (Refer to Step 30).

Figure 3

Calculation of the co-localization proportion to evaluate the complex assembly performance of the individual PRC1 component The proportion of the endogenous RING1B-containing foci that were co-localized with HTM-CBX7 was quantitatively analyzed using ImageJ software.

Figure 4

System verification and the identification of the main component for PRC1 assembly by the HTM-mediated chromatin tethering (A) The effectiveness of this experimental system was verified by testing the distribution of the foci formed by mCherry-HTM and their co-localization with H3K9me3 or with an irrelevant PRC1 component RING1B and its catalytic product H2AK119ub (negative controls). (B) The recruitment of PRC1 components, RING1B and PHC2, and the resultant H2AK119ub by the chromatin tethering of CBX7 was detected and analyzed. (C) The recruitment of CBX7 by the chromatin tethering of RING1B or PHC2 was detected and analyzed. The confocal images were shown in (a) of each part, (scale bars, 2 μm). The intensity of various fluorescence signals across the randomly selected regions (shown as the yellow dashed line segments in a) was scanned and plotted in a curve graph to demonstrate the colocalization status of the indicated factors or corresponding histone modifications (b). The proportion of the co-localized foci to the HTM-tethered foci was quantified by ImageJ (number of ROIs = 5) and shown in the bar graph (c). Data were represented as mean ± SD.