Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner

SUMMARYLinker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we have identified a new function of histone H1 in chromatin structure and transcription using a range of genomic approaches. We show that histone H1-depleted cells accumulate nascent non-coding RNAs on chromatin, suggesting that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accumulated non-coding transcripts have reduced levels of m6A modification and cause replication-transcription conflicts. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism and DNA replication to maintain genome performance.

The Expanding Constellation of Histone Post-Translational Modifications in the Epigenetic Landscape

The emergence of a nucleosome-based chromatin structure accompanied the evolutionary transition from prokaryotes to eukaryotes. In this scenario, histones became the heart of the complex and precisely timed coordination between chromatin architecture and functions during adaptive responses to environmental influence by means of epigenetic mechanisms. Notably, such an epigenetic machinery involves an overwhelming number of post-translational modifications at multiple residues of core and linker histones. This review aims to comprehensively describe old and recent evidence in this exciting field of research. In particular, histone post-translational modification establishing/removal mechanisms, their genomic locations and implication in nucleosome dynamics and chromatin-based processes, as well as their harmonious combination and interdependence will be discussed.

MNase Digestion Protection Patterns of the Linker DNA in Chromatosomes

The compact nucleosomal structure limits DNA accessibility and regulates DNA-dependent cellular activities. Linker histones bind to nucleosomes and compact nucleosomal arrays into a higher-order chromatin structure. Recent developments in high throughput technologies and structural computational studies provide nucleosome positioning at a high resolution and contribute to the information of linker histone location within a chromatosome. However, the precise linker histone location within the chromatin fibre remains unclear. Using monomer extension, we mapped core particle and chromatosomal positions over a core histone-reconstituted, 1.5 kb stretch of DNA from the chicken adult β-globin gene, after titration with linker histones and linker histone globular domains. Our results show that, although linker histone globular domains and linker histones display a wide variation in their binding affinity for different positioned nucleosomes, they do not alter nucleosome positions or generate new nucleosome positions. Furthermore, the extra ~20 bp of DNA protected in a chromatosome is usually symmetrically distributed at each end of the core particle, suggesting linker histones or linker histone globular domains are located close to the nucleosomal dyad axis.

Effect of A-tract-mediated linker histone orientation on trinucleosome compaction

Linker histones (LH) have been shown to preferentially bind to AT-rich DNA, and specifically to A-tracts, contiguous stretches of adenines. Recently, using spFRET (single pair Foerster/Fluorescence Resonance Energy Transfer), we showed that the globular domain (gH) of Xenopus laevis H1.0b LH orients towards A-tracts present on the linker-DNA (L-DNA) in LH:mononucleosome complexes. We investigated the impact of this A-tract mediated orientation of the gH on the compaction of higher-order structures by studying trinucleosomes as minimal models for chromatin. Two 600 bp DNA sequences were constructed containing three Widom 601 core sequences separated by about 40 bp linkers and A-tracts inserted on either the outer or the inner L-DNAs flanking the 1st and the 3rd Widom 601 sequences. The two inner L-DNAs were fluorescently labelled at their midpoints. Trinucleosomes were reconstituted using the doubly-labelled 600 bp DNA, core histone octamers and the full-length H1.0b LH. SpFRET was performed for a range of NaCl concentrations. While the LH compacted the trinucleosomes, surprisingly, the extent of compaction was similar for trinucleosomes with A-tracts either on the two outer or on the two inner L-DNAs. Modeling constrained by the FRET efficiency suggests that the trinucleosomes adopt a zig-zagged conformation with the 1st and 3rd nucleosomes stacked on top of each other. Even though we expect that the gH of neighbouring (1st and 3rd) LHs are oriented towards the A-tracts, our models suggest that they are not sufficiently close to dimerize and affect compaction. Thus, despite differences in A-tract placements, the LH compacts trinucleosomes similarly.

A glitch in the snitch: the role of linker histone H1 in shaping the epigenome in normal and diseased cells

Histone H1s or the linker histones are a family of dynamic chromatin compacting proteins that are essential for higher-order chromatin organization. These highly positively charged proteins were previously thought to function solely as repressors of transcription. However, over the last decade, there is a growing interest in understanding this multi-protein family, finding that not all variants act as repressors. Indeed, the H1 family members appear to have distinct affinities for chromatin and may potentially affect distinct functions. This would suggest a more nuanced contribution of H1 to chromatin organization. The advent of new technologies to probe H1 dynamics in vivo , combined with powerful computational biology, and in vitro imaging tools have greatly enhanced our knowledge of the mechanisms by which H1 interacts with chromatin. This family of proteins can be metaphorically compared to the Golden Snitch from the Harry Potter series, buzzing on and off several regions of the chromatin, in combat with competing transcription factors and chromatin remodellers, thereby critical to the epigenetic endgame on short and long temporal scales in the life of the nucleus. Here, we summarize recent efforts spanning structural, computational, genomic and genetic experiments which examine the linker histone as an unseen architect of chromatin fibre in normal and diseased cells and explore unanswered fundamental questions in the field.

Changes in Chromatin Organization Eradicate Cellular Stress Resilience to UVA/B Light and Induce Premature Aging

Complex interactions among DNA and nuclear proteins maintain genome organization and stability. The nuclear proteins, particularly the histones, organize, compact, and preserve the stability of DNA, but also allow its dynamic reorganization whenever the nuclear processes require access to it. Five histone classes exist and they are evolutionarily conserved among eukaryotes. The linker histones are the fifth class and over time, their role in chromatin has been neglected. Linker histones interact with DNA and the other histones and thus sustain genome stability and nuclear organization. Saccharomyces cerevisiae is a brilliant model for studying linker histones as the gene for it is a single-copy and is non-essential. We, therefore, created a linker histone-free yeast strain using a knockout of the relevant gene and traced the way cells age chronologically. Here we present our results demonstrating that the altered chromatin dynamics during the chronological lifespan of the yeast cells with a mutation in ARP4 (the actin-related protein 4) and without the gene HHO1 for the linker histone leads to strong alterations in the gene expression profiles of a subset of genes involved in DNA repair and autophagy. The obtained results further prove that the yeast mutants have reduced survival upon UVA/B irradiation possibly due to the accelerated decompaction of chromatin and impaired proliferation. Our hypothesis posits that the higher-order chromatin structure and the interactions among chromatin proteins are crucial for the maintenance of chromatin organization during chronological aging under optimal and UVA-B stress conditions.

Complete depletion of Arabidopsis linker histones impairs the correlations among chromatin compartmentalization, DNA methylation and gene expression

In eukaryotic cells, linker histone H1 anchors in and out ends of nucleosome DNA to promote chromatin to fold into the 30 nm fiber. However, if H1 plays a role in coordinating the three-dimensional (3D) chromatin architecture, DNA methylation, and transcriptional regulation is not clear. We engineered H1 knockout mutants in Arabidopsis thaliana which shows pleiotropic phenotypes. Using High-throughput Chromosome Conformation Capture (Hi-C), we found that H1 complete depletion dampens inter- and intra-chromosomal interactions, as well as intra- and inter-chromosomal arm interactions. MNase accessibility assays followed by sequencing (MNase-seq) showed that the nucleosome density decreases in centromeric regions and increases in chromosome arms. In contrast, DNA methylation level in CHG and CHH contexts increases in centromeric regions and decreases in chromosome arms as revealed by whole genome bisulfite sequencing (WGBS) in h1 mutant. Importantly, the functional link between DNA methylation and gene transcription is defected, and the extensive switches between chromatin compartment A and B are uncoupled from genome-wide DNA methylation and most of gene transcriptions upon H1 depletion. These results suggested that linker histone H1 works as linkers among chromatin compartmentalization, DNA methylation and transcription.

LEAFY, a Pioneer Transcription Factor in Plants: A Mini-Review

A subset of eukaryotic transcription factors (TFs) possess the ability to reprogram one cell type into another. Genes important for cellular reprograming are typically located in closed chromatin, which is covered by nucleosomes. Pioneer factors are a special class of TFs that can initially engage their target sites in closed chromatin prior to the engagement with, opening of, or modification of the sites by other factors. Although many pioneer factors are known in animals, a few have been characterized in plants. The TF LEAFY (LFY) acts as a pioneer factor specifying floral fate in Arabidopsis. In response to endogenous and environmental cues, plants produce appropriate floral inducers (florigens). During the vegetative phase, LFY is repressed by the TERMINAL FLOWER 1 (TFL1)–FD complex, which functions as a floral inhibitor, or anti-florigen. The florigen FLOWERING LOCUS T (FT) competes with TFL1 to prevent the binding of the FD TF to the LFY locus. The resulting FT–FD complex functions as a transient stimulus to activate its targets. Once LFY has been transcribed in the appropriate spatiotemporal manner, LFY binds to nucleosomes in closed chromatin regions. Subsequently, LFY opens the chromatin by displacing H1 linker histones and recruiting the SWI/SNF chromatin-remodeling complex. Such local changes permit the binding of other TFs, leading to the expression of the floral meristem identity gene APETALA1. This mini-review describes the latest advances in our understanding of the pioneer TF LFY, providing insight into the establishment of gene expression competence through the shaping of the plant epigenetic landscape.