Chromatin Accessibility
The genome DNA of eukaryotes is not naked, but rather it is bound to histones in the cell nucleus, forming nucleosomes, the basic structural unit of chromatin. The nucleosome core is composed of 147 bp DNA, which wraps a globular protein octamer in a left-handed superhelical manner, and the octamer is composed of two molecules each of the four core histones H2A, H2B, H3, and H4. Each core histone has a DNA-binding domain and a disordered N-terminal tail. The core particles of nucleosomes are connected by linker DNA of approximately 10-80 bp in length, together with histone H1, forming a beads-on-a-string chromatin fiber. The chromatin fibers are highly folded and compressed to form a helical chromosomal structure. These highly compacted chromosomal structures need to expose specific DNA sequences during replication and transcription, in order to allow transcription factors and other regulatory elements to bind to them. The characteristic that enables access to regulatory elements, such as promoters, enhancers, insulators, and silencers, and the proximity of cis-regulatory elements and trans-acting factors, is called chromatin accessibility, also known as open chromatin regions.
Chromatin Accessibility Assay
To study the relationship between chromatin structure and its function in different cellular biological processes, the analysis of active open regions is required. The current methods for studying chromatin accessibility mainly combine enzyme digestion or physicochemical methods with next-generation sequencing techniques to detect open or protected regions of chromatin. Commonly used methods include DNaseI hypersensitive site sequencing (DNase-seq), micrococcal nuclease sequencing (MNase-seq), formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq), and assay for targeting accessible-chromatin with high-throughput sequencing (ATAC-seq).
Fig.1 Schematic diagram of current chromatin accessibility assays performed with typical experimental conditions (Tsompana., Buck M.J. 2014).
1. MNase-seq
MNase is used to digest the naked DNA region between nucleosomes, release nucleosomes from chromatin, and then enrich and sequence the DNA fragments bound to nucleosomes. Mapping the position of nucleosomes, which indirectly reflects regions of chromatin accessibility.
2. DNase-seq
Genomic DNA is non-specifically digested and cleaved using the nucleic acid endonuclease
DNase I. DNA sequences bound to nucleosomes are not cleaved by DNase I. It is because of this feature that information on open chromatin can be obtained by identifying the DNA fragments that are left behind after DNase I digestion.
3. FAIRE-seq
Genomic DNA is covalently bound to the chromatin protein through the cross-linking agent formaldehyde, and the chromatin is cut into fragments by ultrasonic waves. After phenol-chloroform extraction, the nucleosomes wrapped with DNA are distributed at the junction of the two phases, while the nucleosomes without nuclei are distributed in the water phase. Open chromatin regions can be identified by sequencing analysis of DNA in the aqueous phase.
4. ATAC-seq
A Tn5 transposase preloaded with a DNA adapter is used to digest the genome and label the cleaved DNA fragments. Due to steric hindrance, most DNA tag sequences are integrated into open regions of chromatin. It is the first choice for analyzing genome-wide chromatin accessibility in recent years.
Chromatin Accessibility in Gene Regulation
The regulation of gene expression can be achieved by altering the topological structure of chromatin or chromatin modifications. When the binding strength between histones and DNA increases, chromatin condenses into a closed conformation and prevents the access of transcription factors to DNA, leading to gene repression. Conversely, when the binding strength between histones and DNA decreases, chromatin decondensed into an open conformation, allowing transcription factors to approach DNA and leading to transcription activation.
As chromatin morphology and function change, the epigenetic state of cells and gene expression profiles undergo dynamic changes that continuously adapt to environmental variations. Modulating the accessibility of chromatin can regulate gene expression associated with morphogenesis and lineage specification. This dynamic reshaping of chromatin is closely related to embryonic development, cellular aging, tumor development, immunity, and cell fate determination. Additionally, the dynamic structure of chromatin is also related to human health. By gaining a comprehensive understanding of chromatin accessibility at the whole genome level, it is possible to decipher the regulatory elements involved in gene transcription regulation, providing new insights into the mechanisms of diseases.
References
- Tsompana.; Buck M.J. Chromatin accessibility: a window into the genome. Epigenetics & Chromatin. 2014, 7(1):1-6.
- Klemm S.L.; et al. Chromatin accessibility and the regulatory epigenome. Nature Reviews Genetics. 2019, 20(4): 207-20.
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