Single-Cell Epigenomics: Changes to Genomes without DNA Alterations

Humans have similar DNA sequences. Any two humans can share 99% of the same genome sequence. Nonetheless, the 1% difference contributes a large portion of what makes a human unique. Moreover, researchers have discovered differences independent of a person’s genome sequence that affect their traits. These differences are known as epigenetics. Here, diverse human cell types modify their DNA using molecular groups. 

These changes are not spurious. Aberrant gene modifications have been linked with cardiovascular disease and cancer. Tools that profile these modifications can therefore spur new approaches to treat disease beyond a person’s genome.

The repertoire of alterations also varies between individual cells. Massive heterogeneity in single-cell profiles exists. These changes affect how stem cells develop and how tumors proliferate. Discovering these minute differences encouraged researchers to extend existing epigenetics protocols to profile epigenomes with single-cell resolution. The growing amount of PubMed publications on epigenetics further reflects this growing trend.

In this article, we will discuss epigenetics, their role in health and disease, and their characterization at the single-cell level.

What Is Epigenetics?

A mammalian cell’s genome contains strings of nucleotides that are wound into proteins called histones. The resulting structure, chromatin, packages the DNA inside nuclei. The repeating chromatin units are then known as the nucleosome. The study of any modifications to the nucleic acid and histone proteins within the nucleosome — independent of DNA sequence — is known as epigenetics. Researchers first considered epigenetics when they identified X chromosome inactivation in mice in the absence of DNA arrangements. Since then, scientists have identified a wide range of these modifications that affect which and how much genes are expressed. Each of them shares several traits:

• Dynamic: Epigenetic modifications can change over time. These include the kinds of modifications and when they are present. The dynamic nature of epigenetic modifications enables cell populations to adopt diverse cell states by progressively changing their transcriptional activity. The transient nature of epigenetic modifications also has health effects. For instance, epigenetic changes can occur as people age and have been linked with aging.
• Heritable: Epigenetic changes can also be passed down from parent to child without affecting the sequences of different loci. Researchers first learned of this possibility as genetics alone did not explain the heritability of various phenotypes. Although the embryo reverses some of these modifications, other modifications can be passed down from generation to generation.
• Reversible: The epigenetic modifications are also reversible. Many enzymes can add or remove modifications to the nucleosome, affecting the regions of open chromatin present within a chromosome. It’s this trait that determines which loci regulatory elements can access.

Types of Epigenetic Modifications

Cells can modify genes through several mechanisms. Each of these affects chromatin accessibility, gene expression, and cell identity. Here are some of the epigenetic modifications that exist:

• DNA methylation: The methylome comprises the methylated DNA regions within a cell’s nucleus. Methylation occurs when methyl groups are added to cytosine residues in a DNA sequence. First discovered in the 1920s, scientists since have expended efforts to determine their relevance to DNA. Methylated nucleotides prevent transcription factors from binding to DNA promoters, facilitating gene regulation. DNA methylation also prevents mobile genetic elements from transposing from one location to another. With their modes of action, changes in methylation affect clinical outcomes. Disruptions in DNA methylation have been observed among cancer cells, both when too much or too little methylation occurs.

• Histone modifications: Epigenetic modifications can also take place in proteins. Histones are proteins that condense DNA within the nucleus by winding it. Like other proteins, histones can undergo post-translational modifications, affecting their function and interactions with DNA. Multiple chemical groups can be found in a single histone, and many groups can modify a histone protein. For example, histone acetylation loosens higher-order chromatin structure, making DNA promoters more accessible for transcription. The diverse histone modifications increase the variability of genome conformations and affect gene expression.
• Noncoding RNA: The central dogma of genetics teaches that genetic information flows from DNA into RNA before leading to protein synthesis. Since then, researchers have also discovered that not all RNA has to translate into a functional protein; these transcripts are called noncoding RNA (ncRNA). ncRNAs are transcripts that are as short as 20 bp, as long as more than 200 bp, that control gene and protein expression. Through single-cell RNA sequencing and bioinformatics tools, researchers have generated datasets that classify ncRMA into two categories: housekeeping ncRNAs and regulatory ncRNAs. The former are abundantly and universally expressed in cells, regulating critical cellular functions. The latter controls gene expression, comprising many classes that inhibit transcription and translation.
• Enhancers: Enhancers are short DNA regions between coding genes that increase gene expression. Transcription factors activate the enhancers either upstream or downstream of the gene. Dysregulation of these enhancer sequences has been associated with Alzheimer’s Disease and other cognitive issues. Alterations in enhancer activity also affect cardiovascular disease onset.

Conducting a Single-Cell Epigenomics Study

Many kinds of epigenetic modifications that affect a cell’s differentiation and generate cell-type-specific phenotypes exist. Scientists have since learned to characterize the variability in a cell’s epigenome by developing high-throughput profiling tools. Additionally, researchers have fine-tuned these tools to become single-cell multi-omics approaches. By using features such as barcoding and cell enrichment, these techniques have helped generate single-cell genome-wide epigenetics profiles. Some of these tools include:

• Bisulfite sequencing: Bisulfite sequencing is a foundational tool for identifying variants based on DNA methylation profiles. The core of bisulfite sequencing lies in deaminating unmodified cytosine residues with bisulfite. While the unmethylated cytosines are sequenced as thymines, the methylated sequences are read as cytosines. In single-cell bisulfite sequencing, single cells are isolated into separate wells, with the extracted DNA being processed to generate indexed sequencing libraries. These indexes enable multiplexing to identify subtle methylation differences in individual cells.
• Single-cell chromatin mapping (ChIP-Seq): Single-cell chromatin mapping comprises tools that characterize the DNA sequences associated with histones. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is the first method to map these modifications. It begins by precipitating the DNA-bound proteins with an antibody and characterizing the DNA sequences bound to those proteins. Researchers have also extended ChIP-Seq for single-cell research at high resolution. Known as single-cell chIP-seq, the extension integrates microfluidics, DNA barcoding, and high-throughput sequencing, to separate single cells and sequence their DNA.
• Single-cell chromatin mapping (Single-cell ATAC-Seq): Researchers such as Jason D. Buenrostro have also expanded on chromatin mapping with the single-cell analysis of accessible chromatin (scATAC-Seq). In single-cell ATAC-Seq, chromatin is exposed to Tn5 transposases that fragment the chromatin and insert PCR barcodes to be amplified with specific adapter sequences. This assay ultimately determines which regions of DNA have more open chromatin formations. Known as chromatin accessibility, this technique allows researchers to know which regions are available for transcription factors to initiate gene expression. Chromatin mapping can also map post-translational modifications in histone proteins.
• Single-cell Hi-C sequencing (Hi-C): Hi-C represents another tool for characterizing chromatin organization. It is a sequencing technique that captures chromatin interactions within the nuclei of eukaryotic cells. The basic protocol features fixing the chromatin using cross-linkers such as formaldehyde (see this Excedr article to learn about fixation) before the DNA is cut through restriction digestion. After digesting the DNA, the DNA located close to the histone proteins is prepared for sequencing. The technique can be integrated with short- and long-read genome sequencing technologies to find structural variants in chromatin that arise from genomes rearranging themselves.

Many of the tools listed here have already shed important insights into cell activity in the human body. For instance, the Darren Cusanovich lab modified scATAC-seq to identify enhancers to identify tissue-specific processes that occur as embryos are being developed.

Epigenetics in Therapy

Epigenetics plays an important role in regulating cell activity in vivo. As such, researchers have applied the epigenomic profiling tools we discussed to study many kinds of diseases. For the following diseases, epigenetic modifications have played crucial roles:

• Cancer: Cancer is a multifaceted disease that invokes many biochemical mechanisms, from mutations to aberrant gene expression. Researchers, however, have also identified changes in epigenetic profiles among individual cancer cells. For instance, the Greenleaf Lab developed single-cell ATAC-seq to measure and map the effects of perturbing transcription factor levels on chromatin accessibility. To fine-tune transcription factor activity, the lab used CRISPR-based techniques to knock down protein activity to specific levels. Doing so helped Greenleaf identify regulatory elements behaving distinctly in cancer cells. Histone modifications have also been implicated in cancer progression, such as acetylation (labeled H4K16) in histone protein 4.
• Autoimmune diseases: Epigenetic modifications have also been associated with an array of autoimmune diseases. Through extensive computational analysis, researchers identified aberrant DNA methylation profiles in immune cells among patients with systemic autoimmune rheumatic diseases (SARDs). For instance, reduced DNA methylation in T cells and increased histone methylation in T cell chromatin in systemic lupus erythematosus (SLE) have been observed.
• Other diseases: Epigenetics also plays an important role in developmental biology. Their modifications can work together with genes to dictate the developmental processes that occur as a person develops. Mutations to genes encoding histone-modifying proteins can cause developmental disorders such as Rubinstein-Taybi syndrome (characterized by broad and angulated thumbs, moderate-to-severe intellectual disability, and distinctive facial features).

Excedr Leases the Equipment You Need for Single-Cell Epigenomics

A single-cell epigenetics study can be a time-consuming and costly process given the complex pipelines associated with them. Our brand-agnostic approach allows us to provide you with the exact equipment you need to study epigenetics at the single-cell level. Two technologies are especially relevant for studying the DNA and proteins involved with epigenetic modifications. They are:

• Next-generation sequencers: Next-generation sequencers facilitate DNA profiling by determining the order of nucleotides within a DNA fragment. All major companies, from Illumina to PacBio, offer sequencing platforms that can characterize epigenetic modifications in DNA. Short-read sequencers such as Illumina’s NextSeq sequencers are well-established for bisulfite sequencing, where PCR amplification is achieved with a DNA polymerase that can tolerate uracil residues to distinguish methylated and unmethylated DNA.
• Mass spectrometers: Mass spectrometers determine protein structure by delineating the molecular masses of the constituent peptide sequences or the ionized proteins directly. Mass spectrometers started as a tool for studying protein composition in cell suspensions and complex samples. In single-cell epigenomics, mass spectrometers are most useful for identifying post-translational modifications in histone proteins. Nonetheless, mass spectrometry has also been adapted for profiling single-cell proteomes.

If you’re interested in acquiring NGS and mass spectrometry equipment, leasing is a cost-effective and flexible option. The most significant benefit besides getting the equipment you need into your lab is the ability to reduce upfront costs and retain cash, thus extending your cash runway and preserving working capital for other areas.

Improve Your Epigenomic Profiling Methods with New Equipment

Genetics has been a key driver of the diverse traits that comprise human life. Even so, human cells can demonstrate varying phenotypes when genetic differences are absent. Many of these modifications manifest as molecular groups that modify nucleic acids and histone proteins. With so many kinds of modifications, scientists have developed high-throughput single-cell tools to profile them all in single cells. By using next-generation sequencers and mass spectrometers, researchers have identified many modifications associated with health and disease and shed valuable insights into the diversity of cellular life.

Excedr’s leasing program can help you study the epigenome at the single-cell level. We can lease the equipment your lab needs to study the epigenetic modifications within individual cells. From reducing upfront costs to extending cash runway, speak with our team today to learn exactly how we can help.