Abstract 5371: Effect of DNA methyltransferase inhibitor 5-azacitidine on 3D chromatin structure measured by Hi-C

Abstract

Abstract Therapies targeting epigenetic modifiers such as the DNA methyltransferase (DNMT) inhibitor, 5-azacytidine (Aza), are used in treating hematologic malignancies and also show promising results in subsets of solid tumors. The molecular mechanisms for these results, however, are not fully understood. Nonetheless, a number of clinical studies using combinations of epigenetic therapies plus chemo or immunotherapy to enhance the tumor response are ongoing. HCT-116, a colon cancer cell line genetically defined by microsatellite instability (MSI), resulting in a highly mutated, yet primarily diploid genome, has been routinely used to study epigenetic mechanisms and response to DNMT inhibition. In a previous study, we integrated a combination of data types including DNA methylation, DNA accessibility, histone modifications and transcriptomic data that revealed strong correlations between epigenomic changes and gene expression following Aza treatment. However, for some genes, we could not predict expression changes using these unidimensional, region-specific analysis methods. As others have demonstrated, genes do not work as single, isolated units, but rather interact with distal regulatory elements. These regulatory elements, often several Mb away can control gene expression through physical interactions such as bending and looping. We reason that in addition to direct demethylation of the immediate regulatory elements within the gene, Aza treatment can affect long-range chromosomal communications. In order to further understand the chromatin remodeling effect of Aza treatment, we performed high resolution genome-wide chromosome conformation capture (Hi-C) followed by NGS. HCT-116 cells were treated with DMSO or 1 μM Aza for 42 and 96 hours. Hi-C and RNA-Seq data was analyzed and integrated with other datatypes. The results showed that AZA treatment alters topologically associating domains (TAD), with the formation of new TAD boundaries and disappearance of others. The genome was then separated into 25K base pair consecutive bins, with each bin marked as type A (active) or type B (inactive). We observed bin switching following AZA treatment and differential loop formations (e.g. promoter-enhancer). Bins that switched from B to A at both time points were found enriched in genes that are related to acute phase response, interferon pathway, and cancer including PI3KCB, DDX58, CD274 and CDKN2A. Our findings using Hi-C were found to be in agreement with previous results using alternative experimental methods, which identified Aza and CTCF as the top upstream regulators of these genes. Our data also suggests that overall changes in 3D chromatin activities measured by Hi-C could be a better predictor of transcriptional regulation compared to H2K27me3 and H3K4me3 alone, particularly in situations where there are no significant changes in those marks but where changes in gene expression exist. Citation Format: Yuchen Vincent Bai, John Whitaker, Emanuele Palescandolo, Vinod Krishna, Vipul Bhargava, Satya Saxena, Xiang Yao, David Pocalyko, Kurt Bachman. Effect of DNA methyltransferase inhibitor 5-azacitidine on 3D chromatin structure measured by Hi-C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5371. doi:10.1158/1538-7445.AM2017-5371