Multiomic Epigenetic Analysis Turns Short Stories into Epic Tales
March 9, 2022
Table of Contents:
Epigenetics has moved well past a simple understanding of a single epigenetic layer of control at genomic regions of interest, thanks to advances in many techniques and the application of “multomics”. We can now analyze genome-wide histone modification patterns, transcription factor binding profiles, chromatin accessibility profiles, three-dimensional chromosomal conformation, and DNA methylation dynamics combined with transcriptomic analyses and associated analytical platforms. A range of fascinating studies have begun to show how integrating multiple next-generation techniques at different epigenetic levels has supported the construction of multifactorial, epitranscriptomic maps. These tools can reveal the underlying mechanics controlling gene expression and how they may influence normal and/or pathological biological processes.
Here we highlight a range of studies that utilize multiomics-based analyses of critical genomic elements, where researchers have combined methods like ATAC-Seq, RNA-Seq, DNA Methylation and MeDIP-Seq, and ChIP-Seq for analysis of many types of cells and tissues. These studies each support the benefit of a multiomic approach for the ultimate view of epigenetic processes in normal development, disease, and cancer.
Cardiomyocyte Development – Getting to the Heart of the Matter
In a key example of multiomics in a cardiovascular development model, Kranzhofer et al. sought deep insight into the epigenetic dynamics of newborn and adult mouse cardiomyocytes (the cells responsible for generating contractile force in the heart) to understand why these all-important cells lose their proliferative capacity upon cell maturation, which takes place soon after birth. Simultaneous analysis using 5-hydroxymethylcytosine sequencing (hMeDIP-Seq), 5-methylcytosine sequencing (MeDIP-Seq), RNA-Seq, and chromatin immunoprecipitation (ChIP)-Seq (for H3 lysine 27 acetylation [H3K27ac]) in cardiomyocytes isolated from newborn and adult mice allowed the authors to demonstrate the positive association of the 5-hmC modification with subsequent loss of DNA methylation in gene bodies of genes displaying upregulated expression (Mb and Pdk4 genes). Overall, this multiomics-based study helped better define the epigenetic control of gene expression during the maturation of mouse cardiomyocytes, which may contribute to future therapeutic epigenetic interventions that “switch on” non-proliferative cardiomyocytes to repair damaged tissues following myocardial infarction in human patients.
Embryonic Stem Cells - Interdependent Layers that Influence Cell Fate
In this work, Brinkman et al. employed ChIP-bisulfite-seq to quantitatively assess DNA methylation patterns associated with the histone modifications H3 lysine 27 trimethylation (H3K27me3) in mouse embryonic stem cells (pluripotent stem cells derived from the inner cell mass of early-stage pre-implantation embryos). Studying both epigenetic modifications across the genome helped the authors to understand the interdependency between DNA methylation and H3K27me3; fascinatingly, this study revealed that H3K27me3 and DNA methylation co-existed throughout the genome except for CpG islands (DNA methylation-sensitive regions that usually form regulatory regions for associated genes), where the two modifications displayed mutual exclusivity (CpG islands associated with the Plekha2/Htra4 and Lmx1b/C130021I20Rik genes). The authors of this multiomics-based study anticipated that similar research could provide more insight into the complex composition of different chromatin forms and their biological roles in embryonic stem cells, thereby supporting advanced differentiation protocols that generate therapeutically relevant cell types at high efficiency.
Multiomic-based Epigenetic Analysis of Plants – Blooming Great!
A study by Zhong et al. sought to take advantage of multiomic technology to understand how DNA methylation impacted chromatin accessibility, higher-order genome organization, and gene expression. However, instead of mouse or human cells, the authors sought to explore this fascinating link in the plant Arabidopsis thaliana (a popular model organism in genetics). The authors integrated DNA methylation data with chromatin accessibility profiling (via assay for transposase-accessible chromatin using sequencing [ATAC-Seq]) and higher-order chromosome conformation profiles (via high-throughput chromosome conformation capture [Hi-C] sequencing) (Figure 6 of Zhong et al.). Fascinating insights from this study included the finding that increased chromatin accessibility did not always lead to increased gene transcription and the suggestion that DNA methylation may impact chromatin structure by other mechanisms. Overall, the authors discovered fascinating links between specific DNA methylation patterns, chromatin accessibility, and three-dimensional genome architecture and revealed that DNA methylation directly impacts chromatin structure in this important model system.
Multiomic Analyses of Disease – Revealing Novel Mechanisms & Treatment Targets for Cancer
Heller et al. combined DNA methylation analysis with ChIP-Seq, ATAC-Seq, and RNA-Seq to describe the dysregulation of a critical regulatory mechanism – the CDK6-mediated regulation of DNA methylation at CpG islands – in cells from patients with acute lymphoblastic leukemia (ALL). Their multi-omics-based approach highlighted how CDK6 (overexpressed in many cancer types) controlled multiple levels of regulation, including classical gene regulation and epigenetic regulation. One fascinating part of this study integrated ATAC-Seq and ChIP-Seq for CDK6 to show how CDK6 binds to the Dntm3b gene to increase chromatin accessibility, promote gene expression, and, consequently, induce genome-wide DNA methylation (Figure 2 of Heller et al.); meanwhile, the induced loss of CDK6 expression reduces chromatin accessibility, represses Dnmt3b expression, and reduces DNA methylation levels. Overall, the discovery of CDK6 as a regulator of DNA methylation in cancer cells supports the exploration of small molecule modifiers of CDK6 activity as a potentially interesting anti-ALL therapy.
Multiomic Applications in Neuroblastoma
Neuroblastoma, a cancer that develops in immature nerve cells, involves the amplification of the MYCN gene in 1 in 6 cases, with the supernumerary gene copies commonly found on highly rearranged, extrachromosomal circular DNA particles. Helmsauer et al. undertook a multiomics-based analysis of the MYCN locus via ChIP-seq for H3K27ac and H3 lysine 4 monomethylation (H3K4me1), ATAC-Seq, and circular chromosome conformation capture (4C) (Figure 3 of Helmsauer et al.) in neuroblastoma cell lines and integrated these findings with data regarding the MYCN amplicon structure derived from short-read and Nanopore sequencing. Overall, this exciting study reported that “ectopic enhancer hijacking” drives MYCN expression, which helps to explain the observed structural diversity and the epigenetic regulation associated with MYCN amplification.
Blood Cancers – Approaches Published and Featured in a Webinar!
The final examples of the multiomics-based evaluation of disease involve multiple research papers (Beekman et al., Ott et al., and Rendiero et al.) on chronic lymphocytic leukemia (CLL), which formed the basis of a recent webinar from Active Motif (“Epigenetic Tools for Blood Cancer Research”). These CLL-based studies hoped to explore the disease-associated altered epigenome and regulatory networks, define the relationship between the genetic and epigenetic architecture. Their work seeks to identify potential therapeutic targets through an understanding of the basis of disease heterogeneity. As part of their study, Beekman et al. evaluated CLL-associated gene loci with ATAC-Seq, DNA methylation profiling, and RNA-seq in primary CLL and normal cells (FMOD and TCF4 gene loci, Figure 3). Similarly, Ott et al. employed H3K27ac ChIP-seq, ATAC-seq, and transcription-factor binding site profiling to understand disease-associated dynamics at super enhancer loci in CLL cells (Figures 1-3). Finally, Rendiero et al. profiled CLL-associated gene loci with RNA-Seq, ATAC-Seq, and ChIPmentation (ChIP-sequencing library preparation using Tn5-mediated tagmentation) for H3K4me1, H3K27ac, and H3K27me3 in CLL cells (Rendiero et al.; ZNF667 and ZBTB20 gene loci, Figure 3F). Overall, these studies contributed significantly to our understanding of disease associated epigenetic mechanisms, helped to identify CLL subtypes, and provided scope for epigenetic therapies for most common leukemia in adults, which include the possible application of inhibitors of the bromodomain and extra-terminal domain (BET) protein family of epigenetic “readers”.
Single-cell Multiomic Analyses – Doubling up Your Data
While the epigenetic techniques discussed above often use millions of cells as input and integrate datasets from different experiments using different cell samples, the rarity of certain cell samples (including patient samples) represents a significant obstacle to multiomics-based evaluations. Active Motif now provides a solution by launching a new service called “Single-Cell Multiome,” a technique developed by 10X Genomics that combines single-cell (sc)ATAC-seq with scRNA-seq. Swanson et al. provided an example of this approach’s potential; the authors created a droplet-based multiomics platform that supports simultaneous scRNA-Seq, scATAC-Seq, and protein abundance in thousands of single cells that they called TEA-seq (transcriptomics, epitopes, and accessibility). The authors hoped that similar multimodal single-cell assays could support the identification of cell type-specific gene regulation and expression in rare populations of phenotypically defined cell types. Ultimately, single-cell multiomics can eliminate more laborious methods that require isolation strategies such as FACS or magnetic sorting, which may alter the cell’s biology due to sample handling itself, giving a true look into the individual cell’s epigenetic signature.
About the author
Stuart P. Atkinson, Ph.D.
Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!
Contact Stuart on Twitter with any questions