The apparent loss of PRC2 chromatin occupancy as an artifact of RNA depletion
Evan Healy,
Qi Zhang,
Emma H. Gail,
Samuel C. Agius,
Guizhi Sun,
Michael Bullen,
Varun Pandey,
Partha Pratim Das,
Jose M. Polo,
Chen Davidovich
Affiliations
Evan Healy
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia
Qi Zhang
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia; South Australian immunoGENomics Cancer Institute (SAiGENCI), Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia; EMBL-Australia at SAiGENCI, Adelaide, SA, Australia
Emma H. Gail
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia
Samuel C. Agius
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia
Guizhi Sun
Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Wellington Road, Clayton, VIC 3800, Australia
Michael Bullen
Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia
Varun Pandey
Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia
Partha Pratim Das
Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Wellington Road, Clayton, VIC 3800, Australia; Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia
Jose M. Polo
Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Wellington Road, Clayton, VIC 3800, Australia; Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia; Adelaide Centre for Epigenetics and South Australian immunoGENomics Cancer Institute, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA, Australia
Chen Davidovich
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia; EMBL-Australia, Clayton, VIC, Australia; Corresponding author
Summary: 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.
