Staying in your own neighborhood can be nice, but sometimes you need to go on a trip and see different scenery. Chromatin is a lot like that—things can look very different along a DNA strand that bends and folds. That’s why two separate teams have come up with multi-omics methods that go the whole distance on single DNA molecules.
BIND&MODIFY Combines Local Methylation with Long Reads
Sometimes, ChIP-seq-based methods stop short of your goals. They only analyze and read small fragments, and they lose the context of the epigenetic modifications—only providing information for regions brought down with antibodies. The results are local, not global.
So, the Chong Tang’s team (BGI Genomics) and other labs in China and Denmark (University of Chinese Academy of Sciences, University of Copenhagen) got together and developed BIND&MODIFY to travel along a single chromatin fiber and gather intel on histone and DNA methylation, as well as accessibility to transcription factors in cancer cells.
Similar to CUT&TAG, the new method labels DNA via a protein of interest, and brings in an enzyme. The big difference, though, is that BIND&MODIFY doesn’t require cutting the DNA. Long story short:
- Add an antibody to the protein of interest
- Next, add pA-M.EcoGII, a recombinant protein that includes a methyltransferase, which gets tethered to the antibodies
- Sprinkle in some S-adenosylmethionine to induce non-specific local adenine methylation
- Perform nanopore sequencing on the whole strand
- Run the data through a base-calling algorithm
The results with BIND&MODIFY were similar to those of conventional ChIP-seq, and the new method allowed the teams to the assess H3K9me3/H3K37me3 and CTCF status in breast cancer cell lines. With more global, long-range sequencing, they found H3K27me3 along the boundary of centromeres and not in the core, something that might have been missed with shorter reads. The researchers could also phase the genome, looking at alleles contributed by maternal or paternal genomes, and deduce interactions between distant cis-regulators.
NOMe-HiC Methylates and Ligates to Reach for Long-Range Spatial Interactions
Meanwhile, spinning the globe around to the States, Yaping Liu and Li Wang brought together their groups (University of Cincinnati and Xavier University) to create NOMe-HiC, which assesses SNPs, DNA methylation, chromatin accessibility, chromatin conformation dynamics, and the transcriptome—all in one technique.
Transcription factors can have a long reach, grabbing onto cis-regulatory elements from farther down the DNA strand. But again, conventional methods with methyltransferases that could get at this spatial proximity information, such as NOMe-seq, only read short stretches. SMAC-seq uses longer-read nanopore sequencing, but can’t tell cis- and trans-effects apart. So, leaping off from their NOMe-seq and Methyl-HiC strategies, the team’s NOMe-HiC goes long on a single DNA molecule with these steps:
- Crosslink with formaldehyde, then cut the genome with DpnII
- Ligate the nearby fragments to each other, capturing longer-range interactions
- Use GpC methyltransferase to investigate chromatin accessibility
- Reverse the crosslinks and then do total RNA-seq to get at the transcriptome
- Do bisulfite conversion and pair-end sequencing on the DNA
- Analyze the data with an improved Bis-SNP computational method
Results from NOMe-HiC were similar to those from many separate methods, including Hi-C and Methyl-HiC. The method picked up on coordinated epigenetic statuses for some genomic regions that were nearby in space, but far away on the linear strand in two human cell lines. They could also determine the allele-specific methyltransferase footprint at long-range.
Next Stop on Your Research Trip: Single Molecule Multi-omics
Multi-omics techniques are currently taking off, efficiently providing unprecedented levels of data all in one shot. And methods like BIND&MODIFY and NOMe-HiC that take a long-range, single-molecule approach will let researchers go where they have never gone before, getting a more global DNA modification picture to help them better understand what’s really going on in disease states.
Take the long and winding road with BIND&MODIFY (Genome Biology, March 2023) and NOMe-HiC (Genome Biology, March 2023).