Doxorubicin Impacts the Chromatin Binding of Hmgb1, Histone H1 and Retinoic Acid Receptor

Doxorubicin (Dox), a widely used anticancer DNA-binding drug, affects chromatin in multiple ways, and these effects contribute to both its efficacy and dose-limiting side-effects, especially cardiotoxicity. Here we studied the Dox effects on the chromatin binding of the architectural proteins high mobility group B1 (HMGB1) and the linker histone H1, and the transcription factor retinoic acid receptor (RARα) by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS), in live cells. At lower drug concentrations, Dox increased the binding of HMGB1 to DNA while decreasing the binding of the linker histone H1. At higher doses that correspond to the peak plasma concentrations reached in chemotherapy, Dox reduced the binding of HMGB1 as well. This biphasic effect is interpreted in terms of a hierarchy of competition between the ligands involved and Dox-induced local conformational changes of nucleosome-free DNA. When combined, FRAP and FCS mobility data suggest that Dox decreases the overall binding of RARα to DNA, an effect that was only partially overcome by agonist binding. The intertwined interactions described likely contribute to the effects as well as side-effects of Dox.

Mechanisms of Nucleosome Reorganization by PARP1

Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair, chromatin organization and transcription. During transcription initiation, PARP1 interacts with gene promoters where it binds to nucleosomes, replaces linker histone H1 and participates in gene regulation. However, the mechanisms of PARP1-nucleosome interaction remain unknown. Here, using spFRET microscopy, molecular dynamics and biochemical approaches we identified several different PARP1-nucleosome complexes and two types of PARP1 binding to mononucleosomes: at DNA ends and end-independent. Two or three molecules of PARP1 can bind to a nucleosome depending on the presence of linker DNA and can induce reorganization of the entire nucleosome that is independent of catalytic activity of PARP1. Nucleosome reorganization depends upon binding of PARP1 to nucleosomal DNA, likely near the binding site of linker histone H1. The data suggest that PARP1 can induce the formation of an alternative nucleosome state that is likely involved in gene regulation and DNA repair.

Uncharged Components of Single-Stranded DNA Modulate Liquid–Liquid Phase Separation With Cationic Linker Histone H1

Liquid–liquid phase separation (LLPS) of proteins and DNAs has been recognized as a fundamental mechanism for the formation of intracellular biomolecular condensates. Here, we show the role of the constituent DNA components, i.e., the phosphate groups, deoxyribose sugars, and nucleobases, in LLPS with a polycationic peptide, linker histone H1, a known key regulator of chromatin condensation. A comparison of the phase behavior of mixtures of H1 and single-stranded DNA-based oligomers in which one or more of the constituent moieties of DNA were removed demonstrated that not only the electrostatic interactions between the anionic phosphate groups of the oligomers and the cationic residues of H1, but also the interactions involving nucleobases and deoxyriboses (i) promoted the generation of spherical liquid droplets via LLPS as well as (ii) increased the density of DNA and decreased its fluidity within the droplets under low-salt conditions. Furthermore, we found the formation of non-spherical assemblies with both mobile and immobile fractions at relatively higher concentrations of H1 for all the oligomers. The roles of the DNA components that promote phase separation and modulate droplet characteristics revealed in this study will facilitate our understanding of the formation processes of the various biomolecular condensates containing nucleic acids, such as chromatin organization.

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.

Site-specific ubiquitylation acts as a regulator of linker histone H1

Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.

The interactome of site-specifically acetylated linker histone H1

Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by posttranslational modifications (PTMs) is rather limited. In this study, we report on the generation of site-specifically mono- and di-acetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification-mass spectrometry (AP-MS) and show that site-specific acetylation results in overlapping, but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD+-dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together our data suggests that site-specific acetylation of H1.2 plays a role in modulating protein-protein interactions.

The histone variant H2A.W and linker histone H1 co-regulate heterochromatin accessibility and DNA methylation

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.

Single-stranded nucleic acid sensing and coacervation by linker histone H1

The linker histone H1 is the most abundant group of eukaryotic chromatin-binding proteins. The mechanism underlying the diverse physiological functions of H1 remains unclear. Here we used single-molecule fluorescence and force microscopy to observe the behavior of H1 on DNA under different tensions. Unexpectedly, we found that H1 coalesces around nascent ssDNA. Molecular dynamics simulations revealed that multivalent and transient interactions between H1 and ssDNA mediate their phase separation. We further showed that longer and unpaired nucleic acids result in more viscous, gel-like H1 droplets. Finally, we imaged H1 puncta in cells under normal and stressed conditions and observed that RPA and H1 occupy separate nuclear regions. Overall, our results provide a new perspective to understanding the role of H1 in genome organization and maintenance.