Discovery of NSD2 non-histone substrates and design of a super-substrate.

The human protein lysine methyltransferase NSD2 catalyzes dimethylation at H3K36. It has very important roles in development and disease but many mechanistic features and its full spectrum of substrate proteins are unclear. Using peptide SPOT array methylation assays, we investigate the substrate sequence specificity of NSD2 and discover strong readout of residues between G33 (-3) and P38 (+2) on H3K36. Unexpectedly, we observe that amino acid residues different from natural ones in H3K36 are preferred at some positions. Combining four preferred residues led to the development of a super-substrate which is methylated much faster by NSD2 at peptide and protein level. Molecular dynamics simulations demonstrate that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. To investigate the substrate spectrum of NSD2, we conducted a proteome wide search for nuclear proteins matching the specificity profile and discovered 22 peptide substrates of NSD2. In protein methylation studies, we identify K1033 of ATRX and K819 of FANCM as NSD2 methylation sites and also demonstrate their methylation in human cells. Both these proteins have important roles in DNA repair strengthening the connection of NSD2 and H3K36 methylation to DNA repair.

Deep learning-based spectroscopic single-molecule localization microscopy.

Significance: Spectroscopic single-molecule localization microscopy (sSMLM) takes advantage of nanoscopy and spectroscopy, enabling sub-10 nm resolution as well as simultaneous multicolor imaging of multi-labeled samples. Reconstruction of raw sSMLM data using deep learning is a promising approach for visualizing the subcellular structures at the nanoscale. Aim: Develop a novel computational approach leveraging deep learning to reconstruct both label-free and fluorescence-labeled sSMLM imaging data. Approach: We developed a two-network-model based deep learning algorithm, termed DsSMLM, to reconstruct sSMLM data. The effectiveness of DsSMLM was assessed by conducting imaging experiments on diverse samples, including label-free single-stranded DNA (ssDNA) fiber, fluorescence-labeled histone markers on COS-7 and U2OS cells, and simultaneous multicolor imaging of synthetic DNA origami nanoruler. Results: For label-free imaging, a spatial resolution of 6.22 nm was achieved on ssDNA fiber; for fluorescence-labeled imaging, DsSMLM revealed the distribution of chromatin-rich and chromatin-poor regions defined by histone markers on the cell nucleus and also offered simultaneous multicolor imaging of nanoruler samples, distinguishing two dyes labeled in three emitting points with a separation distance of 40 nm. With DsSMLM, we observed enhanced spectral profiles with 8.8% higher localization detection for single-color imaging and up to 5.05% higher localization detection for simultaneous two-color imaging. Conclusions: We demonstrate the feasibility of deep learning-based reconstruction for sSMLM imaging applicable to label-free and fluorescence-labeled sSMLM imaging data. We anticipate our technique will be a valuable tool for high-quality super-resolution imaging for a deeper understanding of DNA molecules' photophysics and will facilitate the investigation of multiple nanoscopic cellular structures and their interactions.

To slide or not to slide: key role of the hexasome in chromatin remodeling revealed.

Hexasomes are non-canonical nucleosomes that package DNA with six instead of eight histones. First discovered 40 years ago as a consequence of transcription, two near-atomic-resolution cryo-EM structures of the hexasome in complex with the chromatin remodeler INO80 have now started to unravel its mechanistic impact on the regulatory landscape of chromatin. Loss of one histone H2A-H2B dimer converts inactive nucleosomes into distinct and favorable substrates for ATP-dependent chromatin remodeling.

Angle between DNA linker and nucleosome core particle regulates array compaction revealed by individual-particle cryo-electron tomography.

The conformational dynamics of nucleosome arrays generate a diverse spectrum of microscopic states, posing challenges to their structural determination. Leveraging cryogenic electron tomography (cryo-ET), we determine the three-dimensional (3D) structures of individual mononucleosomes and arrays comprising di-, tri-, and tetranucleosomes. By slowing the rate of condensation through a reduction in ionic strength, we probe the intra-array structural transitions that precede inter-array interactions and liquid droplet formation. Under these conditions, the arrays exhibite irregular zig-zag conformations with loose packing. Increasing the ionic strength promoted intra-array compaction, yet we do not observe the previously reported regular 30-nanometer fibers. Interestingly, the presence of H1 do not induce array compaction; instead, one-third of the arrays display nucleosomes invaded by foreign DNA, suggesting an alternative role for H1 in chromatin network construction. We also find that the crucial parameter determining the structure adopted by chromatin arrays is the angle between the entry and exit of the DNA and the corresponding tangents to the nucleosomal disc. Our results provide insights into the initial stages of intra-array compaction, a critical precursor to condensation in the regulation of chromatin organization.

Impact of Combinatorial Histone Modifications on Acetyllysine Recognition by the ATAD2 and ATAD2B Bromodomains.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C.

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a 'pincer-like' conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

High-Speed Atomic Force Microscopy Reveals the Nucleosome Sliding and DNA Unwrapping/Wrapping Dynamics of Tail-less Nucleosomes.

Each nucleosome contains four types of histone proteins, each with a histone tail. These tails are essential for the epigenetic regulation of gene expression through post-translational modifications (PTMs). However, their influence on nucleosome dynamics at the single-molecule level remains undetermined. Here, we employed high-speed atomic force microscopy to visualize nucleosome dynamics in the absence of the N-terminal tail of each histone or all of the N-terminal tails. Loss of all tails stripped 6.7 base pairs of the nucleosome from the histone core, and the DNA entry-exit angle expanded by 18° from that of wild-type nucleosomes. Tail-less nucleosomes, particularly those without H2B and H3 tails, showed a 10-fold increase in dynamics, such as nucleosome sliding and DNA unwrapping/wrapping, within 0.3 s, emphasizing their role in histone-DNA interactions. Our findings illustrate that N-terminal histone tails stabilize the nucleosome structure, suggesting that histone tail PTMs modulate nucleosome dynamics.

Epigenetic marks uniquely tune the material properties of HP1α condensates.

Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1α) with DNA contributes to the formation of condensed chromatin states. HP1α localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1α and dictating emergent functions of its condensates remains to be understood. Here, we leverage a reductionist system, composed of modified and unmodified histone H3 peptides, HP1α, and DNA, to examine the contribution of specific epigenetic marks to phase behavior of HP1α. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1α condensates, whereas peptides containing unmodified residues or bearing the transcriptional activation mark H3K4me3 are incompatible with HP1α phase separation. Using fluorescence microscopy and rheological approaches, we further demonstrate that H3K9me3 histone peptides modulate the dynamics and viscoelastic network properties of HP1α condensates in a concentration-dependent manner. Additionally, in cells exposed to uniaxial strain, we find there to be a decreased ratio of nuclear H3K9me3 to HP1α. These data suggest that HP1α-DNA condensates are viscoelastic materials, whose properties may provide an explanation for the dynamic behavior of heterochromatin in cells and in response to mechanostimulation.

Production of a Monoclonal Antibody for Histone H2b Isoform H2b3b.

H2b3b is one of the histone H2b isoforms that differs from canonical H2b by five to six amino acids. Previously, we identified H3t as the testis-specific histone H3 variant located in histone cluster 3, which is also the site of H2b3b. In this study, we produced monoclonal antibodies against H2b3b, using the iliac rat lymph node method for rat antibody and the immunochamber method for rabbit antibody. Immunoblot analysis confirmed that our antibodies could specifically discriminate between H2b3b and canonical H2b. Moreover, immunostaining revealed colocalization with a testicular stem cell marker, Plzf, but not with a meiotic marker, Sycp. This indicated that H2b3b is expressed in spermatogenic cells before meiosis. Our monoclonal antibodies enable further studies to reveal specific functions of H2b3b during spermatogenesis. We also hope that the established method will lead to the production of antibodies that can identify other H2b isoforms.

Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site.

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs)1,2. However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.

Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

Sequence Dependence in Nucleosome Dynamics.

The basic packaging unit of eukaryotic chromatin is the nucleosome that contains 145-147 base pair duplex DNA wrapped around an octameric histone protein. While the DNA sequence plays a crucial role in controlling the positioning of the nucleosome, the molecular details behind the interplay between DNA sequence and nucleosome dynamics remain relatively unexplored. This study analyzes this interplay in detail by performing all-atom molecular dynamics simulations of nucleosomes, comparing the human α-satellite palindromic (ASP) and the strong positioning "Widom-601" DNA sequence at time scales of 12 µs. The simulations are performed at salt concentrations 10-20 times higher than physiological salt concentrations to screen the electrostatic interactions and promote unwrapping. These microsecond-long simulations give insight into the molecular-level sequence-dependent events that dictate the pathway of DNA unwrapping. We find that the "ASP" sequence forms a loop around SHL ± 5 for three sets of simulations. Coincident with loop formation is a cooperative increase in contacts with the neighboring N-terminal H2B tail and C-terminal H2A tail and the release of neighboring counterions. We find that the Widom-601 sequence exhibits a strong breathing motion of the nucleic acid ends. Coincident with the breathing motion is the collapse of the full N-terminal H3 tail and formation of an α-helix that interacts with the H3 histone core. We postulate that the dynamics of these histone tails and their modification with post-translational modifications (PTMs) may play a key role in governing this dynamics.

Histone variant H2A.Z and linker histone H1 influence chromosome condensation in Saccharomyces cerevisiae.

Chromosome condensation is essential for the fidelity of chromosome segregation during mitosis and meiosis. Condensation is associated both with local changes in nucleosome structure and larger-scale alterations in chromosome topology mediated by the condensin complex. We examined the influence of linker histone H1 and variant histone H2A.Z on chromosome condensation in budding yeast cells. Linker histone H1 has been implicated in local and global compaction of chromatin in multiple eukaryotes, but we observe normal condensation of the rDNA locus in yeast strains lacking H1. However, deletion of the yeast HTZ1 gene, coding for variant histone H2A.Z, causes a significant defect in rDNA condensation. Loss of H2A.Z does not change condensin association with the rDNA locus or significantly affect condensin mRNA levels. Prior studies reported that several phenotypes caused by loss of H2A.Z are suppressed by eliminating Swr1, a key component of the SWR complex that deposits H2A.Z in chromatin. We observe that an htz1Δ swr1Δ strain has near-normal rDNA condensation. Unexpectedly, we find that elimination of the linker histone H1 can also suppress the rDNA condensation defect of htz1Δ strains. Our experiments demonstrate that histone H2A.Z promotes chromosome condensation, in part by counteracting activities of histone H1 and the SWR complex.

Trimethyllysine Reader Proteins Exhibit Widespread Charge-Agnostic Binding via Different Mechanisms to Cationic and Neutral Ligands.

In the last 40 years, cation-π interactions have become part of the lexicon of noncovalent forces that drive protein binding. Indeed, tetraalkylammoniums are universally bound by aromatic cages in proteins, suggesting that cation-π interactions are a privileged mechanism for binding these ligands. A prominent example is the recognition of histone trimethyllysine (Kme3) by the conserved aromatic cage of reader proteins, dictating gene expression. However, two proteins have recently been suggested as possible exceptions to the conventional understanding of tetraalkylammonium recognition. To broadly interrogate the role of cation-π interactions in protein binding interactions, we report the first large-scale comparative evaluation of reader proteins for a neutral Kme3 isostere, experimental and computational mechanistic studies, and structural analysis. We find unexpected widespread binding of readers to a neutral isostere with the first examples of readers that bind the neutral isostere more tightly than Kme3. We find that no single factor dictates the charge selectivity, demonstrating the challenge of predicting such interactions. Further, readers that bind both cationic and neutral ligands differ in mechanism: binding Kme3 via cation-π interactions and the neutral isostere through the hydrophobic effect in the same aromatic cage. This discovery explains apparently contradictory results in previous studies, challenges traditional understanding of molecular recognition of tetraalkylammoniums by aromatic cages in myriad protein-ligand interactions, and establishes a new framework for selective inhibitor design by exploiting differences in charge dependence.

Decoding Chromatin Ubiquitylation: A Chemical Biology Perspective.

Since Strahl and Allis proposed the "language of covalent histone modifications", a host of experimental studies have shed light on the different facets of chromatin regulation by epigenetic mechanisms. Initially proposed as a concept for controlling gene transcription, the regulation of deposition and removal of histone post-translational modifications (PTMs), such as acetylation, methylation, and phosphorylation, have been implicated in many chromatin regulation pathways. However, large PTMs such as ubiquitylation challenge research on many levels due to their chemical complexity. In recent years, chemical tools have been developed to generate chromatin in defined ubiquitylation states in vitro. Chemical biology approaches are now used to link specific histone ubiquitylation marks with downstream chromatin regulation events on the molecular level. Here, we want to highlight how chemical biology approaches have empowered the mechanistic study of chromatin ubiquitylation in the context of gene regulation and DNA repair with attention to future challenges.

Regulation of adipogenesis by histone methyltransferases.

Epigenetic regulation is a critical component of lineage determination. Adipogenesis is the process through which uncommitted stem cells or adipogenic precursor cells differentiate into adipocytes, the most abundant cell type of the adipose tissue. Studies examining chromatin modification during adipogenesis have provided further understanding of the molecular blueprint that controls the onset of adipogenic differentiation. Unlike histone acetylation, histone methylation has context dependent effects on the activity of a transcribed region of DNA, with individual or combined marks on different histone residues providing distinct signals for gene expression. Over half of the 42 histone methyltransferases identified in mammalian cells have been investigated in their role during adipogenesis, but across the large body of literature available, there is a lack of clarity over potential correlations or emerging patterns among the different players. In this review, we will summarize important findings from studies published in the past 15 years that have investigated the role of histone methyltransferases during adipogenesis, including both protein arginine methyltransferases (PRMTs) and lysine methyltransferases (KMTs). We further reveal that PRMT1/4/5, H3K4 KMTs (MLL1, MLL3, MLL4, SMYD2 and SET7/9) and H3K27 KMTs (EZH2) all play positive roles during adipogenesis, while PRMT6/7 and H3K9 KMTs (G9a, SUV39H1, SUV39H2, and SETDB1) play negative roles during adipogenesis.

The role of cryptic ancestral symmetry in histone folding mechanisms across Eukarya and Archaea.

Histones compact and store DNA in both Eukarya and Archaea, forming heterodimers in Eukarya and homodimers in Archaea. Despite this, the folding mechanism of histones across species remains unclear. Our study addresses this gap by investigating 11 types of histone and histone-like proteins across humans, Drosophila, and Archaea through multiscale molecular dynamics (MD) simulations, complemented by NMR and circular dichroism experiments. We confirm and elaborate on the widely applied "folding upon binding" mechanism of histone dimeric proteins and report a new alternative conformation, namely, the inverted non-native dimer, which may be a thermodynamically metastable configuration. Protein sequence analysis indicated that the inverted conformation arises from the hidden ancestral head-tail sequence symmetry underlying all histone proteins, which is congruent with the previously proposed histone evolution hypotheses. Finally, to explore the potential formations of homodimers in Eukarya, we utilized MD-based AWSEM and AI-based AlphaFold-Multimer models to predict their structures and conducted extensive all-atom MD simulations to examine their respective structural stabilities. Our results suggest that eukaryotic histones may also form stable homodimers, whereas their disordered tails bring significant structural asymmetry and tip the balance towards the formation of commonly observed heterotypic dimers.

The 80th Threonine Residue of Histone H3 Is Important for Maintaining HM Silencing in Saccharomyces cerevisiae.

Gene expression in eukaryotic cells is intricately regulated by chromatin structure and various factors, including histone proteins. In Saccharomyces cerevisiae, transcriptionally silenced regions, such as telomeres and homothallic mating (HM) loci, are essential for genome stability and proper cellular function. We firstly observed the defective HM silencing in alanine substitution mutant of 80th threonine residue of histone H3 (H3T80A). To identify which properties in the H3T80 residue are important for the HM silencing, we created several substitution mutants of H3T80 residue by considering the changed states of charge, polarity, and structural similarity. This study reveals that the structural similarity of the 80th position of H3 to the threonine residue, not the polarity and charges, is the most important thing for the transcriptional silencing in the HM loci.

Using NMR diffusion data to validate MD models of disordered proteins: Test case of N-terminal tail of histone H4.

MD simulations can provide uniquely detailed models of intrinsically disordered proteins (IDPs). However, these models need careful experimental validation. The coefficient of translational diffusion Dtr, measurable by pulsed field gradient NMR, offers a potentially useful piece of experimental information related to the compactness of the IDP's conformational ensemble. Here, we investigate, both experimentally and via the MD modeling, the translational diffusion of a 25-residue N-terminal fragment from histone H4 (N-H4). We found that the predicted values of Dtr, as obtained from mean-square displacement of the peptide in the MD simulations, are largely determined by the viscosity of the MD water (which has been reinvestigated as a part of our study). Beyond that, our analysis of the diffusion data indicates that MD simulations of N-H4 in the TIP4P-Ew water give rise to an overly compact conformational ensemble for this peptide. In contrast, TIP4P-D and OPC simulations produce the ensembles that are consistent with the experimental Dtr result. These observations are supported by the analyses of the 15N spin relaxation rates. We also tested a number of empirical methods to predict Dtr based on IDP's coordinates extracted from the MD snapshots. In particular, we show that the popular approach involving the program HYDROPRO can produce misleading results. This happens because HYDROPRO is not intended to predict the diffusion properties of highly flexible biopolymers such as IDPs. Likewise, recent empirical schemes that exploit the relationship between the small-angle x-ray scattering-informed conformational ensembles of IDPs and the respective experimental Dtr values also prove to be problematic. In this sense, the first-principle calculations of Dtr from the MD simulations, such as demonstrated in this work, should provide a useful benchmark for future efforts in this area.