Mind the gap: Epigenetic regulation of chromatin accessibility in plants

Abstract

Chromatin plays a crucial role in genome compaction and is fundamental for regulating multiple nuclear processes. Nucleosomes, the basic building blocks of chromatin, are central in regulating these processes, determining chromatin accessibility by limiting access to DNA for various proteins and acting as important signaling hubs. The association of histones with DNA in nucleosomes and the folding of chromatin into higher-order structures are strongly influenced by a variety of epigenetic marks, including DNA methylation, histone variants, and histone post-translational modifications. Additionally, a wide array of chaperones and ATP-dependent remodelers regulate various aspects of nucleosome biology, including assembly, deposition, and positioning. This review provides an overview of recent advances in our mechanistic understanding of how nucleosomes and chromatin organization are regulated by epigenetic marks and remodelers in plants. Furthermore, we present current technologies for profiling chromatin accessibility and organization.

Figure 1.

Chromatin accessibility and compaction across multiple levels. The fundamental unit of chromatin is the nucleosome, which results from the interaction between DNA and histones. Both of these components can undergo epigenetic modifications, such as histone variants and PTMs, in addition to DNA methylation. In euchromatic regions, chromatin organization facilitates access to the transcriptional machinery, aided by the actions of chromatin remodelers and modifiers that establish specific configurations and modifications to promote transcription. In contrast, facultative and constitutive heterochromatic regions, characterized by marks like H3K27me3 and H3K9me2, among others, adopt a condensed conformation that restricts access to DNA and transcription. Chromatin also forms higher-order structures organized by structural proteins, including loops, topologically associated domains (TADs) that bring functionally related regions in close proximity, and compartments A and B, which are formed by large clusters of euchromatin and heterochromatin, respectively. Lastly, chromosomes occupy distinct and exclusive territories within the nucleus. The circles connecting the chromatin loops represent structural proteins involved in higher-order chromatin associations. Created with BioRender.com.

Figure 2.

Current methods to profile chromatin accessibility and conformation in plants. A) FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing): Chromatin crosslinked with formaldehyde is fragmented through sonication, and the accessible (or nucleosome-free) DNA is isolated using a phenol-chloroform–based phase separation, and then used for library preparation. FAIRE-seq stands out as a simple and cost-effective, non-enzymatic method that enables the genome-wide characterization of open chromatin regions (Baum et al. 2020). B) MNase-seq: A brief enzymatic incubation of chromatin with Micrococcal Nuclease (MNase) digests internucleosomal DNA, allowing the isolation of nucleosome-bound DNA. Subsequently, the DNA corresponding to mononucleosomes (147 bp) is isolated, purified, and then used for library preparation. This method enables precise identification of nucleosome positions. By using different enzyme concentrations or incubation times, one can distinguish stable nucleosomes from those with high turnover, known as fragile nucleosomes (Zhang and Jiang 2018). Variants of this method, such as MNase-defined cistrome-Occupancy Analysis sequencing (MOA-seq) and MNase hypersensitivity sequencing (MH-seq), employ milder MNase treatments to recover subnucleosomal DNA fragments that represent TF footprints (Tao et al. 2020; Zhao et al. 2020; Savadel et al. 2021). C) ATAC-seq: The hyperactive Tn5 transposase simultaneously cleaves and attaches sequencing adapters to accessible DNA, expediting library construction after DNA isolation and size selection. Tagmentation has led to shorter, more streamlined protocols with reduced input requirements (enabling single-cell studies), making ATAC-seq the gold standard for characterizing highly accessible chromatin regions today (Bajic et al. 2018). Recent advances in single-cell technologies have led to the development of single-cell ATAC-seq protocols in plants (scATAC-seq) (Dorrity et al. 2021; Marand et al. 2021). Additionally, recent protocols like Single-cell combinatorial fluidic indexing ATAC-seq (scifi-ATAC-seq) aim to enhance throughput (Zhang et al. 2023b). Moreover, the Cleavage Under Targets and Tagmentation (CUT&Tag) method, which coupled the Tn5-based tagmentation with antibody-specific chromatin targeting, can be used to study chromatin accessibility over specific chromatin regions (Henikoff et al. 2020). D) Hi-C: Crosslinked chromatin is digested, biotinylated, and then re-ligated in a manner that exclusively forms ligation products from fixed DNA-DNA interactions. These DNA hybrids are subsequently sheared and purified by pulling down biotin before library construction (Pérez-de Los Santos et al. 2022). A variant of this method, known as Open Chromatin Enrichment and Network Hi-C (OCEAN-C), integrates Hi-C and FAIRE-seq protocols to identify interactions between open chromatin regions in species with large genomes, such as wheat (Yuan et al. 2022). Combined INTACT and Hi-C protocols have recently been employed to isolate cells from the endosperm and leaves, profiling their 3D organization (Yadav et al. 2021). Created with BioRender.com.

Figure 3.

Epigenetic regulation of chromatin accessibility. A) DNA methylation exerts a negative influence on chromatin accessibility by impeding TF binding to their targets and promoting the recruitment of reader complexes that lead to gene silencing and chromatin compaction. B) Histone PTMs exert both direct and indirect effects on nucleosome stability and chromatin compaction. Histone acetylation, catalyzed by HATs and removed by HDACs, reduces histone tail-DNA affinities, promoting an open conformation. Additionally, acetylated histones recruit complexes such as the SWI/SNF remodeler complex to further loosen chromatin. For example, the BAS SWI/SNF complex incorporates multiple Bromodomain readers in the BRM and BRD subunits. In the absence of acetylation, histone tails interact more closely with DNA, restricting access. Histone ubiquitination, catalyzed by HUBs and removed by DUBs, adds bulky moieties that decrease interactions between chromatin fibers. While H2Bub is associated with active transcription, H2Aub is recognized by the PRC2 polycomb complex, leading to gene silencing and chromatin compaction. Histone methylation, catalyzed by HMTs and removed by DMTs, can be associated with gene expression and an open chromatin conformation, as exemplified in the cartoon by the H3K4me3-mediated binding of the ARID5-containing CRAF ISWI complex. Conversely, methylation at other residues can lead to gene silencing and chromatin compaction, exemplified in the cartoon by the H3K27me3-mediated recruitment of the Polycomb PRC1 complex. C) Histone variants exert distinct effects on nucleosome stability, DNA accessibility, and chromatin condensation. Among them, H2A.Z has the strongest negative impact on nucleosome stability. H2A.W decreases the accessibility to entry and exit nucleosomal DNA. H1 facilitates the condensation of the same chromatin fiber, while H2A.W promotes compaction by bridging distinct chromatin fibers. Additionally, the histone variant H2B.8 promotes chromatin condensation via phase separation. D) Different families of ATP-dependent chromatin remodelers perform a wide range of functions on the nucleosomal landscape, broadly categorized into nucleosome sliding, eviction, exchange, maturation, and spacing. Created with BioRender.com.