Combinations of old favorites can be even tastier than the sum of their parts. Who doesn’t leave peanut butter and jam, chips and dip, and DNA methylation and chromatin assays? While they sound appetizing, those last two aren’t quite as easy to get together.
Many assays have been developed in the past to combine ChIP and bisulfite sequencing; however, they have often required large amounts of input DNA, and high sequencing costs. This has made combinatorial profiling small groups of cells, or single cells, impossible. A new group of techniques have been developed to get around these issues and offer an improved characterization of chromatin features. In this special feature, we highlight four new and exciting combinatorial DNA methylation/chromatin methods.
Methyl-ATAC-seq and ATAC-Me: A Multiomic Assault on the Epigenome
Methyl-ATAC seq (mATAC-seq) and ATAC-Me are both combine ATAC-seq and DNA methylation assays. mATAC-seq was developed in the lab of Paul D. Soloway at Cornell University and ATAC-Me was developed in lab of Emily Hodges at Vanderbilt University. These talented teams wanted to avoid the need to infer where hits overlap when comparing DNA methylation and chromatin accessibility (ATAC-seq) data from two separate sets of cells. The protocol is a killer combination of ATAC-seq followed by bisulfite sequencing:
- Tagmentation is performed on nuclei using a Tn5 transposase loaded with methylated oligonucleotides, which protects the incorporated adapters from deamination during bisulfite treatment
- Tagmented DNA is end-repaired, purified, bisulfite-converted, amplified, and sequenced
- The resulting ATAC-seq peaks are not only a measure of chromatin accessibility, but also simultaneously offer the C->T conversions in their sequence that reflect DNA methylation level
By combining these methods with simultaneous RNA isolation, expression data from the cells can also be integrated. Both of these methods provide streamlined solutions for combing ATAC-seq and DNA methylation experiments to not only save time, money (less sequencing needed), and precious samples, but also provide greater certainty that these features occur at the same genomic positions in the same samples.
The methods are particularly useful for identifying narrow windows where DNA methylation, chromatin state, and expression are not as tightly linked as ones thought. The ATAC-Me paper shows that DNA methylation, chromatin accessibility, and DNA methylation can be uncoupled, where they change independently of one another during early development, with gene expression being linked to chromatin state and not DNA methylation.
EpiMethylTag: Dealers Choice
EpiMethylTag was developed by the lab of Jane Skok at New York University to address the challenges associated with characterizing DNA methylation at transcription factor (TF) binding sites. Many TFs are DNA methylation dependent, but limitations in old methods have meant few genome-wide studies have been performed to assess this. EpiMethylTag combines ATAC-seq or ChIPmentation with bisulfite conversion (M-ATAC or M-ChIP, respectively). Here’s how its done:
- Tagmentation using Tn5 is performed on nuclear lysates (M-ATAC) or during chromatin immunoprecipitation (M-ChIP)
- DNA is purified, bisulfite converted, PCR amplified, and sequenced
- As in mATAC-seq above, the amount of a sequence that is present (ATAC-seq or ChIP-seq peak) is a measure of chromatin openness/TF binding, while the sequence itself provides DNA methylation information
EpiMethylTag is a fast, low-input, approach useful for smaller cell populations. It is ideal for determining the DNA methylation status of transcription factor binding sites. DNA methylation rarely occurs at TF binding sites and open chromatin; however, Dr. Skok’s group found some peaks where this occurs. These highly accessible, highly methylated regions also show transcriptional activity, high H3K4me1, low H3K4me3, and low H3K27ac. The biological role of these “poised promoters” remains an exciting mystery.
Go tag all the details in Genome Biology, November 2019
scNMT-seq: Singling in on Multiomic Insight
A full ATAC on chromatin isn’t the only approach. scNMT-seq (single-cell nucleosome, methylation, and transcription sequencing) is a triple threat developed by the labs of Wolf Reik (Babraham Institute, UK) and Oliver Stegle (EMBL-EBI, UK). scNMT-seq uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing (an adapted form of NOME-seq). By using “no signal” as an indication of closed chromatin, the low sample input from single cells is not problematic. Here’s how it works:
- A GpC methyltransferase labels accessible DNA in sorted single-cells
- RNA and DNA are physically separated
- RNA is subjected to library preparation and sequencing using Smart-seq2
- DNA is treated to library perpetration and single-cell bisulfite sequencing (scBS-seq), where CpG and GpC methylation provide information about DNA methylation and nucleosome occupancy, respectively
This group used scNMT-seq to find novel interactions of these three molecular layers at individual loci. Finally, they also journeyed down the epigenetic landscape and examined differentiating mESCs, where they discovered distinct dynamics in the coupling of all three molecular layers at developmentally relevant regions.
For all of these techniques, being able to work from the input sample in a major step forward, bypassing the need to assume marks are actually co-occurring. Each of these four approaches can make the quest to understand how epigenetic marks are served up together easier for everyone.