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Review
. 2021 Aug 16;22(16):8809.
doi: 10.3390/ijms22168809.

Emerging Single-Cell Technological Approaches to Investigate Chromatin Dynamics and Centromere Regulation in Human Health and Disease

Laura Leo  1 , Nunzia Colonna Romano  1
Affiliations
Review

Emerging Single-Cell Technological Approaches to Investigate Chromatin Dynamics and Centromere Regulation in Human Health and Disease

Laura Leo et al. Int J Mol Sci. .

Abstract

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.

Keywords: centromere; chromatin regulation; emerging technologies; epigenetics; epigenetics in human health & disease; single-cell epigenetics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of the main epigenetic mechanisms: DNA methylation, histone post-translational modifications (PMTs) and non-coding RNAs. In the nucleus, chromatin is organised in peculiar chromosome structures composed of DNA wrapped around nucleosomes. Nucleosomes are formed by four histones that can be modified in their N-terminal tails to open or compact chromatin, for example, acetylation, phosphorylation and methylation. DNA methylation usually occurs at 5′-position cytosine residues on CpG islands. Non-coding RNAs can be transcribed but not translated in a protein playing a crucial role in epigenetic regulation of the genome. All epigenetic modifications work together to determine an epigenetic code to regulate chromatin organisation and gene expression.
Figure 2
Figure 2
Overview of ChIP-seq and sequencing variants. ChIP-Seq combines chromatin immunoprecipitation with DNA sequencing to show specific transcription factor binding sites. DNase-seq reveals regions of open chromatin after DNase I endonuclease digestion as also in MNAse-seq, in which Micrococcal nuclease digests DNA to create a sequencing library. Additionally, ATAC-seq is used to study chromatin accessibility genome-wide in DNase I hypersensitive sites, in which an engineered Tn5 transposase cleaves DNA that is tagmented with primer DNA sequences to NGS sequencing. This method appears as complementary technique to DNAse-seq, MNase-seq and FAIRE-seq. In FAIRE-seq, used to detect the nucleosome-free regions of the genome, the chromatin is crosslinked with formaldehyde in vivo, sheared by sonication and phenol-chloroform extracted to NGS sequencing (created with biorender.com).
Figure 3
Figure 3
Enzyme-tethering analysis. DamID is used to map the binding sites of DNA and chromatin-binding proteins and consists of fusing DNA methyltransferase to a protein of interest (usually a transcription factor), which methylates adenine in GATC sequences. The genome is digested by DpnI, which cuts only methylated GATCs, that are amplified in PCR assay and subsequently analysed. In ChEC-seq analysis, Micrococcal Nuclease (MNase) cleaves DNA after Ca2+ addition, allowing chromatin-associated protein profiling. ChIC-seq procedure is based on binding of the primary antibody (AB) to chromatin-associated protein of interest and on subsequent fusion of protein pA-MN to the secondary AB that produces DNA fragments used for sequencing library creation and data analysis (created with biorender.com).
Figure 4
Figure 4
Graphic illustration of the principal technological approaches for epigenetic chromatin profiling. ChIP-seq is a well-used method to profile DNA–protein interactions. In this method, after crosslink and DNA fragmentation, a hybridisation step with a specific antibody against a protein of interest is needed. After DNA extraction, library preparation can be performed. CUT&RUN is an enzyme-tethering strategy that, after in situ incubation with a specific antibody, makes use of a micrococcal nuclease fusion protein (pA-MNase) for DNA fragment generation. CUT&TAG is the most recent methodology introduced for epigenome features profiling. As with CUT&RUN, it begins with an antibody incubation but exploits the transposome Tn5-transposase-protein A (pA-Tn5) fusion protein loaded with sequencing adapters for tagmentation and sequencing.
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