Spatial DNA methylation profiling

Spatial DNA methylation profiling refers to technologies that combine the measurements of DNA methylation and the spatial information of cells. These experimental technologies are used to gather insight into these distinct patterns and how they can impact gene regulation in individual cells, from visualizing the overall pattern of specific methylation marks within tissues, to identifying the location of these methylation marks both in the genome and within a tissue. DNA methylation is one of multiple major epigenetic factors that influence genetic expression. Traditional bulk and newer single-cell methylation assays measure methylation levels but lose important information about where cells reside, thus losing a level of analysis on outside influences on cells. By incorporating spatial information, researchers can use spatial DNA methylation profiling to investigate the influence of epigenetic regulation across various tissue environments and how it varies between them. Key methods include imaging-based approaches like antibody staining of methylation marks followed by visualization using microscopy, and sequencing-based methods like assigning unique barcodes to DNA or tissue dissection followed by traditional methylation sequencing approaches. The technologies are still being developed and tested, and although these methods vary in resolution, interpretation, and throughput, they promise to enable a more comprehensive understanding of tissue biology and its influences on development and disease.

Background

DNA methylation DNA methylation is a direct chemical modification to the DNA molecule that influences mechanisms such as cell differentiation as well as gene regulation. The amount of DNA methylation within the genome varies to mediate cell-specific processes, meaning that gene expression differs within and across tissues, despite having the same DNA sequence. DNA methylation is catalysed by a family of proteins called DNA methyltransferases, which work by transferring a methyl group from an S-adenyl methionine to the fifth carbon of a cytosine to form a 5-methylcytosine (5mC). DNA methylation accounts for only ~1% of nucleic acids in the human genome, with the majority occurring on cytosines preceding a guanine, creating a CpG site. Methylation is found more in intergenic regions to silence transposable and viral elements that have inserted themselves into the human genome over time. CpG islands are associated with promoters and transcription factors, where methylation can cause stable silencing of gene expression. DNA methylation of the gene body acts differently, being associated with active transcription, not silencing. Functionally, DNA methylation plays a role in:

Silencing transposable and viral elements Tissue-specific gene regulation Genomic imprinting X-chromosome inactivation

Early DNA methylation assays DNA methylation was discovered in mammals almost as early as DNA was identified, with Rolin Hotchkiss discovering modified cytosine or 5-methylcytosine (5mC). But, 5mC was reported in bacteria in 1925 by Johnson and Coghill. 5mC was shown to be found across a variety of species and cell types in the 1970s, and further studies showed it lagged behind DNA replication in 1971. Initial assays used restriction enzymes to see where DNA methylation occurred and if any changes occurred. Early DNA methylation assays did not show what methylation was associated with, but they understood it was associated with a change in cell identity. Once it was understood to have a function in bacteria, other types of assays were used to explore the methylation content using mass spectrometry. A culmination of these assays and studies into DNA methylation concluded that 5mC plays a role in gene expression. In the 1990s, bisulfite sequencing was invented, allowing for bases in nucleic acids to be detected, which led to microarrays being created in the 2000s to have large-scale, high-throughput studies of specific CpG sites.

Bulk DNA Methylation sequencing Bulk DNA methylation sequencing is similar to bulk single-cell sequencing, taking an average of measurements. In this, measurements are of the level of 5mC across a population of cells, showing averages of methylation, to understand the current epigenetic regulation and gene expression in a specific tissue. Typically, bulk DNA methylation analysis uses bisulfite conversion or nanopore sequencing technologies to measure methylation. Some of these analyses have been used to identify abnormal methylation patterns associated with cancer.

Single-Cell Methylation sequencing In recent years, many fields have begun utilizing single-cell technologies, ranging from single-cell sequencing to single-cell ATAC-seq. Following this technology boom, single-cell methylation studies have been produced; some of these are Q-RRBS, snmC-seq, and scMspJI. These kits and assays are not as powerful - meaning rapid, cost-effective, and simultaneous - as Bulk DNA methylation or other standard methylation analyses; however, they have started to be used to analyse and profile rare cells and disease states, to capture the underlying differences in rarer cell types that are missed in bulk analyses that average methylation across a population of cells.

Scientific principles

Preservation of spatial information in analyses Tissues are organised into structured microenvironments within the body. They are composed of diverse cell types, extracellular components, and signalling gradients. Certain elements of spatial information influence DNA methylation, like cell lineage, environmental exposures, cell-cell interactions, and disease microenvironments. Conventional assays cannot determine the ways epigenetic differences are influenced by physical location, which has been linked to differences in disease severity. Preserving spatial information allows for better analysis of methylation states and allows linkage between physical states and methylation states.

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