Virtual Oral Presentation Australian Epigenetics Alliance Conference 2022

TEM-seq: Ultrasensitive Multiomic Platform for Epitope-Targeted DNA Methylation Mapping (#42)

Chaithanya Ponnaluri 1 , Vishnu S Udayakumar 2 , Pierre-Olivier Estève 1 , Louise Williams 1 , Bryan Venters 2 , Martis W. Cowles 2 , Michael Keogh 2 , Sriharsa Pradhan 1
  1. New England Biolabs Australia, Melbourne, VIC, Australia
  2. Epicypher, Boston, US

Vertebrate gene expression is regulated by the integration of DNA methylation (DNAme) and other chromatin features, including histone post-translational modifications (PTMs), chromatin remodelers, and transcription factors. However, our current understanding of how DNA methylation co-occurs (and coordinates) with the chromatin features to control gene expression is limited by a lack of reliable genomic tools. Here we have combined quantitative CUT&RUN technology (Skene and Henikoff, 2017) to isolate epitope targeted DNA followed by unbiased DNA methylation analysis using NEB’s enzymatic methyl-seq (EM-seq, Vaisvila et al., 2021) approach to generate truly quantitative data using dramatically reduced cell input and sequencing depth. EM-seq utilizes the enzymatic conversion of methyl-DNA (5mC and 5hmC) and provides a much-warranted alternative to bisulfite sequencing (BS, bisulfite is a harsh chemical treatment that introduces DNA breaks and systemic biases) to generate high resolution, unbiased DNA methylation profiles with 10-fold less sample input (vs. BS) thus saving valuable experimental materials and time (>10-fold saving on each parameter). We have used various cell lines (MCF7 and K562) to study genome wide PTM and TF targeted DNA methylation on chromatin. For example, we found H3K4me3 at active promoters and H3K27me3 at inactive genes. As expected, these features contained low levels of DNAme. By contrast, H3K36me3 mapped to active gene bodies, which contained high levels of DNAme. We also observed selected ChAPs (chromatin associated proteins) at their expected locations: e.g., many BRD4 peaks were found at active promoters with low levels of DNAme. Interestingly, in some cases, we observed low DNAme at promoters in close proximity to highly methylated domains associated with gene repression.

 

 

  1. References:  Peter J Skene Steven Henikoff (2017) An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites eLife 6:e21856. Vaisvila R, Ponnaluri VKC, Sun Z, Langhorst BW, Saleh L, Guan S, Dai N, Campbell MA, Sexton BS, Marks K, Samaranayake M, Samuelson JC, Church HE, Tamanaha E, Corrêa IR Jr, Pradhan S, Dimalanta ET, Evans TC Jr, Williams L, Davis TB. Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome Res. 2021 Jun 17;31(7):1280–1289.