Epigenetic regulators play a crucial role in establishing and maintaining gene expression states. To date, the main efforts to study cellular heterogeneity have focused on elucidating the variable nature of the chromatin landscape. Specific chromatin organisation is fundamental for normal organogenesis and developmental homeostasis and can be affected by different environmental factors. The latter can lead to detrimental alterations in gene transcription, as well as pathological conditions such as cancer. Epigenetic marks regulate the transcriptional output of cells. Centromeres are chromosome structures that are epigenetically regulated and are crucial for accurate segregation. The advent of single-cell epigenetic profiling has provided finer analytical resolution, exposing the intrinsic peculiarities of different cells within an apparently homogenous population. In this review, we discuss recent advances in methodologies applied to epigenetics, such as CUT&RUN and CUT&TAG. Then, we compare standard and emerging single-cell techniques and their relevance for investigating human diseases. Finally, we describe emerging methodologies that investigate centromeric chromatin specification and neocentromere formation.
In 1942, for the first time, Waddington coined the term ‘epigenetic’ as the branch of biology that studies the causal interactions between genes and their cellular products and implements the phenotype.
In past decades, the concept of epigenetic regulation has evolved, thanks to technological advances that have revolutionised the investigation of biological phenomena. Currently, epigenetics is defined as the science that studies stable and potentially heritable changes in gene expression and the phenotype occurring without alterations in the DNA sequence.
The main epigenetic mechanisms include DNA methylation, histone modifications and non-coding RNAs (ncRNAs). These mechanisms are dynamically regulated in response to developmental and environmental stimuli; thus, they establish feedback in the control of several biological processes such as gene expression, genome architecture, growth, apoptosis, alternative splicing, DNA repair and ultimately, evolution.
Most often, DNA methylation is found in association with silent chromatin states and typically takes the form of methylation of cytosine; such a modification mainly occurs on CpG islands, which are typically found in regulatory regions such as enhancers and promoters of genes or in repetitive DNA sequences.
Histone modifications (methylation, acetylation, phosphorylation, ubiquitination and many others) are key regulatory modifications resulting in changes in transcription, DNA replication and chromosome condensation. Generally, acetylation is associated with euchromatin, a general term for transcriptionally active chromatin states, while methylation may be found in both euchromatin or heterochromatin (transcriptionally inactive) regions depending on the specific lysine/arginine residues that are modified.
Non-coding RNAs (ncRNAs) are a heterogeneous class of regulatory molecules derived from genes that are transcribed but not translated into proteins. They can be further classified in small-interfering RNAs (siRNAs), microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and long non-coding RNAs (lncRNAs) which have key roles in many biological processes.
Epigenetic modifications are continuously and dynamically modulated, especially by environmental factors. Indeed, the main benefit they provide lies in the ability to ‘fine-tune’ gene expression in line with progressive ontogenic and environmental changes. Thus, epigenetic markers are preferred developmental indicators, being more stable, accurate and specific than transcriptional markers.
Human health is impacted by epigenetics, and disruption in the correct balance of open and closed chromatin states can result in the onset of epigenetic machinery disorders. Growing evidence support that epigenetic machinery may profoundly affect human health.
Mendelian disorders of the epigenetic machinery (MDEMs) are genetic diseases caused by mutations in genes coding for epigenetic factors and are often associated with intellectual disability, revealing that epigenetic mechanisms are very crucial for normal neurological development.
In addition to monogenic diseases, epigenetic mechanisms are significantly involved in multifactorial diseases such as neurodegenerative disorders, metabolic dysfunction and cancer progression, although it is unclear the degree by which epigenetic alterations are the cause or the consequence of pathogenesis. For example, epigenetic variability between cancer cells that may arise in response to environmental stimuli may exacerbate cell-to-cell heterogeneity and contribute to cancer progression and resistance to therapy. For many years, cell variability has been hard to quantify due to the lack of specific methodologies. For instance, chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a major technique to profile chromatin accessibility and DNA–protein interactions in a population of cells but is not sufficiently sensitive to quantify epigenetic features at the level of single cells. In recent years, single-cell sequencing techniques have been developed and applied to the study of epigenetics. For instance, Bolhaqueiro and colleagues have developed a methodology to analyse single-cell DNA sequencing data to reveal genome changes and chromosomal aberrations, such as duplication or deletions, to explore cell-to-cell variability in a heterogeneous cancer cell population.
Chromosomal instability (CIN) is a hallmark of many types of cancer. However, how CIN contributes to cancer progression remains to be elucidated. CIN is characterised by loss or rearrangement of genetic material during cell division, resulting in aneuploidy, chromosome structural alterations and segregation errors. Thus, CIN can increase genetic heterogeneity between tumour cells. A key structure for chromosome segregation is a chromatin region called the ‘centromere’. Centromeres recruit the kinetochore complex, a proteinaceous structure that provides the physical attachment to the microtubules of the mitotic spindle. Any alteration in the architecture of the centromere can result in cell cycle defects, leading to chromosomal instability. Indeed, the overexpression of CENP-A, the main epigenetic centromere marker, has been reported in several types of tumours, and it correlates with poor prognosis for patients. The molecular consequences of CENP-A overexpression in cancer cells are still unclear. However, CENP-A overexpression drives chromosomal instability and aneuploidy, as shown in a cancer cell line and in a xenograft mouse model.
Epigenetic marks can affect the cells’ transcriptional output, and consequently, their function. Therefore, understanding their effects on single-cell resolution can determine variations that occur in cell fate and function. Owing to this need, new techniques for studying single-cell epigenomics have been developed and previously deepened in some respects. In summary, although epigenetic profiling of cell populations has contributed new insights on the role that epigenetic marks have on cellular function, understanding the effects of these marks at single-cell resolution is needed to appreciate cell-to-cell variability in fate determination and function in health and disease.
In this review, we discuss single-cell methodologies that have recently emerged and that may provide new impetus in achieving the goals of ‘personalised medicine’. We present a critical review of such techniques examining strengths, challenges and limitations and offering insight in the epigenetic field into molecular components of the genome and its functional output.
Over the last few years, single-cell sequencing (SCS) has emerged as a powerful set of technologies applied to a multitude of biological questions. Before the widespread adoption of single-cell genomics, RNA (in the form of cDNA) or (genomic) DNA sequencing was performed on nucleic acids isolated from whole tissue. Tissues are made of different cell types that are difficult to separate. Moreover, even within a single cell type, the population is heterogeneous, which in large part is due to the stochastic accumulation of mutations introduced during DNA replication. In this regard, physiological functions in health and disease are distinguished by the interplay between cells, but traditional sequencing technologies afford low resolution to the problem. Conversely, SCS can detect heterogeneity among individual cells and can lead to the discovery of new cell types (or sub-types), rebuilding the cell development trajectory. Generally, SCS is used to analyse gene expression, to identify events of sister chromatin exchange and to define the methylation status of genes of interest. For instance, SCS applied on post-mortem brain tissue has allowed studying the cellular composition of some isolated neurons and their involvement in some neurodegenerative diseases or brain lesions. SCS technologies have been applied in stem, neuronal or glial cells to study the epigenetic profiles of open chromatin and investigate its role in the pathogenesis of diseases. In combination with single-cell immune profiling, SCS also allows obtaining immune profiling with the possibility to characterise new immune cell types and states. From this point of view, SCS can aid in identifying new drug targets and verify whether they work as expected.
In light of the potential impact on several diseases’ development, epigenetics is a powerful field to study all mechanisms and risk factors contributing to their progression. Indeed, in an organism, the epigenome is normally established at
