Using the novel reCUT&RUN method to investigate how treatment impacts the bivalent histone modification profile and gene expression of HER2+/ER+ and HER2+/ER- breast cancer cells

Introduction:The impact that treatment has on histone methylation marks in diseases such as Human Epidermal growth factor Receptor 2 (HER2) positive breast cancer remains uncharacterized. Two histone 3 trimethylations, the transcription promoting mark at lysine 4 (H3K4me3) mark, and the transcription repressing mark at lysine 27 (H3K27me3) simultaneously create a phenomenon called bivalency. Understanding how these epigenetic marks change under the pressure of treatments like trastuzumab could help us understand how the cells adapt to external stimuli.Methods:To study this impact, we developed a novel technique to identify the true co-location of both bivalent marks by adapting the Cleavage Under Targets & Releasing Under Nuclease (CUT&RUN) protocol into what we call reCUT&RUN. We used reCUT&RUN to characterize bivalency in HER2 positive (HER2+) breast cancer and how the bivalency status changes after the treatment with trastuzumab and estradiol (E2). We applied this method to HER2+/ER+ and HER2+/ER- cell lines and used RNA-seq to characterize how these changes in response to treatment impact gene expression.Results:We found that there was between 20-60% overlap of genes identified as bivalent using reCUT&RUN with those identified as being bivalent using standard CUT&RUN, and that the reCUT&RUN marked genes has significantly lower gene expression (p.adj < 2.3x10-18) than genes identified as having both marks using CUT&RUN. We also show that both trastuzumab and estrogen treatment impacts these histone modifications, both on a global and pathway level (p.adj < 0.05), specifically in genes marked with reCUT&RUN. Last, we found a significant overrepresentation of H3K4me3 marked genes among the genes that were significantly differentially expressed under each treatment condition.Conclusion:In this study, we are the first to show that histone modifications can change under treatment conditions through our reCUT&RUN protocol. In addition, we also show that H3K4me3 plays a role in which genes have the potential to be influenced by treatment.