Poster Presentation Australasian RNA Biology and Biotechnology Association 2024 Conference

The apparent loss of PRC2 chromatin occupancy as an artifact of RNA depletion (#135)

Evan Healy 1 , Qi Zhang 1 2 , Emma H Gail 1 , Samuel C Agius 1 , Guizhi Sun 3 , Michael Bullen 4 , Varun Pandey 4 , Partha Pratim Das 3 4 , Jose M Polo 3 4 5 , Chen Davidovich 1 6
  1. Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia
  2. South Australian immunoGENomics Cancer Institute (SAiGENCI), Faculty of Health and Medical Sciences, University of Adelaide, Adelaide., Adelaide, South Australia, Australia
  3. Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Wellington Road, Clayton, VIC, Australia
  4. Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC, Australia
  5. Adelaide Centre for Epigenetics and South Australian immunoGENomics Cancer Institute, Faculty of Health and Medical Sciences, , The University of Adelaide, Adelaide, SA, Australia, Adelaide, South Australia, Australia
  6. EMBL-Australia, Clayton, VIC, Australia

RNA has been implicated in the recruitment of chromatin modifiers, with previous studies providing evidence in favour as well as against this idea. The histone methyltransferase Polycomb repressive complex 2 (PRC2) has been proposed to interact with RNA, but there is intense ongoing debate as to the extent and functional consequences of this interaction. RNase A treatment during chromatin immunoprecipitation (RNase-ChIP or rChIP-seq) reduces chromatin occupancy of PRC2, with this data supporting a model whereby RNA facilitates the recruitment of PRC2 to chromatin via an RNA ‘bridge’.

Here we show that rChIP-seq leads to the apparent loss of all facultative heterochromatin, including PRC2 and its H3K27me3 mark genome-wide. We track this artificial displacement of PRC2 to a gain of DNA from non-targeted chromatin following RNA degradation. This DNA is then sequenced at the expense of antibody-targeted DNA, thereby reducing specific ChIP-seq signals, despite the factors in question—PRC2 and H3K27me3—remaining on chromatin. Our results imply that the RNA bridge model has been supported based on an experimental artifact. More positively, our results suggest a crucial role for RNA in solubilising chromatin which should be considered when developing new experimental approaches in this area. Collectively, our results point to substantial technical limitations with the usage of RNase treatment of chromatin during ChIP, but also imply broader roles for RNA in stabilising chromatin structure, beyond direct interactions with chromatin modifiers.