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Updated: Jul 2, 2025
Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC
Published on: May 9, 2020
The apparent loss of PRC2 chromatin occupancy as an artifact of RNA depletion
Evan Healy1, Qi Zhang2, Emma H Gail1
1Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia.
Abstract:
RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favor and against this idea. RNase treatment of chromatin is commonly used to study RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (ChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase Polycomb repressive complex 2 (PRC2). This led to suggestions of an "RNA bridge" between PRC2 and chromatin. Here, we show that RNase A treatment during ChIP causes the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome-wide. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results emphasize substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay.
Insights
RNase A treatment during ChIP assays can distort results by altering DNA representation, invalidating prior conclusions about RNA
Area of Science:
- Molecular Biology
- Epigenetics
- Genomics
Background:
- RNA's role in recruiting chromatin modifiers is debated.
- RNase treatment is used to study RNA-mediated regulation, but its limitations are unclear.
- RNase A treatment in ChIP assays suggested an RNA bridge between Polycomb repressive complex 2 (PRC2) and chromatin.
Purpose of the Study:
- To investigate the limitations of RNase A treatment in chromatin immunoprecipitation (ChIP) assays.
- To re-evaluate the role of RNA in Polycomb repressive complex 2 (PRC2) chromatin occupancy.
Main Methods:
- Chromatin immunoprecipitation (ChIP) assays with and without RNase A treatment.
- Genome-wide analysis of H3K27me3 and PRC2 occupancy.
- DNA sequencing and analysis of targeted and non-targeted chromatin regions.
Main Results:
- RNase A treatment during ChIP caused an apparent genome-wide loss of facultative heterochromatin, including PRC2 and H3K27me3.
- This apparent loss resulted from a gain of DNA from non-targeted chromatin, not a true loss of heterochromatin.
- The study identified a significant bias in DNA sequencing due to RNase A treatment.
Conclusions:
- RNase A treatment during ChIP assays has substantial limitations for mapping RNA-dependent chromatin occupancy.
- Previous conclusions regarding PRC2's RNA-dependent chromatin association, based on RNase A treatment, are invalidated.
- The study highlights the need for careful consideration of enzymatic treatments in epigenetic studies.
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