Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag).

Individual analysis of the epigenome of preimplantation embryos is useful for characterizing each embryo and for investigating the effects of environmental factors on their epigenome. However, it is difficult to analyze genome-wide epigenetic modifications, especially histone modifications, in a large number of single embryos due to the small number of cells and the complexity of the analysis methods. To solve this problem, we further modified the CUT&Tag method, which can analyze histone modifications in a small number of cells, such that the embryo is handled as a cell mass in the reaction solutions in the absence of the solid-phase magnetic beads that are used for antibody and enzyme reactions in the conventional method (NON-TiE-UP CUT&Tag; NTU-CAT). By using bovine blastocysts as a model, we showed that genome-wide profiles of representative histone modifications, H3K4me3 and H3K27me3, could be obtained by NTU-CAT that are in overall agreement with the conventional chromatin immunoprecipitation-sequencing (ChIP-seq) method, even from single embryos. However, this new approach has limitations that require attention, including false positive and negative peaks and lower resolution for broad modifications. Despite these limitations, we consider NTU-CAT a promising replacement for ChIP-seq with the great advantage of being able to analyze individual embryos.

Heparin-Functionalized Adsorbents Eliminate Central Effectors of Immunothrombosis, including Platelet Factor 4, High-Mobility Group Box 1 Protein and Histones.

Inflammation and thrombosis are closely intertwined in numerous disorders, including ischemic events and sepsis, as well as coronavirus disease 2019 (COVID-19). Thrombotic complications are markers of disease severity in both sepsis and COVID-19 and are associated with multiorgan failure and increased mortality. Immunothrombosis is driven by the complement/tissue factor/neutrophil axis, as well as by activated platelets, which can trigger the release of neutrophil extracellular traps (NETs) and release further effectors of immunothrombosis, including platelet factor 4 (PF4/CXCL4) and high-mobility box 1 protein (HMGB1). Many of the central effectors of deregulated immunothrombosis, including activated platelets and platelet-derived extracellular vesicles (pEVs) expressing PF4, soluble PF4, HMGB1, histones, as well as histone-decorated NETs, are positively charged and thus bind to heparin. Here, we provide evidence that adsorbents functionalized with endpoint-attached heparin efficiently deplete activated platelets, pEVs, PF4, HMGB1 and histones/nucleosomes. We propose that this elimination of central effectors of immunothrombosis, rather than direct binding of pathogens, could be of clinical relevance for mitigating thrombotic complications in sepsis or COVID-19 using heparin-functionalized adsorbents.

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5'-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.

Genome information processing by the INO80 chromatin remodeler positions nucleosomes.

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

Histone Purification Combined with High-Resolution Mass Spectrometry to Examine Histone Post-Translational Modifications and Histone Variants in Caenorhabditis elegans.

Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA-related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post-translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)-based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co-occurring PTMs and the discovery of novel PTMs become feasible. The success of MS-based approaches relies substantially on obtaining pure and well-preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom-up MS, and then evaluate the resulting data by a C. elegans-adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large-scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom-up mass spectrometry analysis of histone PTMs and histone variants.

Detecting protein and post-translational modifications in single cells with iDentification and qUantification sEparaTion (DUET).

While technologies for measuring transcriptomes in single cells have matured, methods for measuring proteins and their post-translational modification (PTM) states in single cells are still being actively developed. Unlike nucleic acids, proteins cannot be amplified, making detection of minute quantities from single cells difficult. Here, we develop a strategy to detect targeted protein and its PTM isoforms in single cells. We barcode the proteins from single cells by tagging them with oligonucleotides, pool barcoded cells together, run bulk gel electrophoresis to separate protein and its PTM isoform and quantify their abundances by sequencing the oligonucleotides associated with each protein species. We used this strategy, iDentification and qUantification sEparaTion (DUET), to measure histone protein H2B and its monoubiquitination isoform, H2Bub, in single yeast cells. Our results revealed the heterogeneities of H2B ubiquitination levels in single cells from different cell-cycle stages, which is obscured in ensemble measurements.

Structure of a single-chain H2A/H2B dimer.

Chromatin is the complex assembly of nucleic acids and proteins that makes up the physiological form of the eukaryotic genome. The nucleosome is the fundamental repeating unit of chromatin, and is composed of ∼147 bp of DNA wrapped around a histone octamer formed by two copies of each core histone: H2A, H2B, H3 and H4. Prior to nucleosome assembly, and during histone eviction, histones are typically assembled into soluble H2A/H2B dimers and H3/H4 dimers and tetramers. A multitude of factors interact with soluble histone dimers and tetramers, including chaperones, importins, histone-modifying enzymes and chromatin-remodeling enzymes. It is still unclear how many of these proteins recognize soluble histones; therefore, there is a need for new structural tools to study non-nucleosomal histones. Here, a single-chain, tailless Xenopus H2A/H2B dimer was created by directly fusing the C-terminus of H2B to the N-terminus of H2A. It is shown that this construct (termed scH2BH2A) is readily expressed in bacteria and can be purified under non-denaturing conditions. A 1.31 Šresolution crystal structure of scH2BH2A shows that it adopts a conformation that is nearly identical to that of nucleosomal H2A/H2B. This new tool is likely to facilitate future structural studies of many H2A/H2B-interacting proteins.

Quick and facile preparation of histone proteins from the green microalga Chlamydomonas reinhardtii and other photosynthetic organisms.

The development of universal, broadly applicable methods for histone extraction from animal cells and tissues has unlocked the ability to compare these epigenetic-influencing proteins across tissue types, healthy and diseased states, and cancerous versus normal cells. However, for plants and green algae, a quick and easily implemented histone extraction method has yet to be developed. Here, we report an optimized method that provides a unified approach to extract histones for the green microalgal species Chlamydomonas reinhardtii and Scenedesmus dimorphus as well as for maize (corn) leaf tissue. Histone extraction methods include treatment with high salt concentrations and acidification. Preparations of nuclei can be made in ∼3.5 h and histones extracted in ∼3.5 h either immediately or nuclei may be frozen and histone proteins can be later extracted without a change in histone PTM patterns. To examine the efficiency of the new methods provided, we performed both qualitative and quantitative analysis of salt and acid-extracted whole histone proteins (SAEWH) via SDS-PAGE gel electrophoresis and intact protein mass spectrometry. SDS-PAGE analysis indicated that histone yields decrease when using walled Chlamydomonas strains relative to cell-wall-less mutants. Using top-down mass spectrometry (TDMS) for intact protein analysis, we confirmed the presence of H4K79me1 in multiple algal species; however, this unique modification was not identified in corn leaf tissue and has not been reported elsewhere. TDMS measurements of SAEWH extracts also revealed that oxidation which occurs during the histone extraction process does not increase with exposure of harvested algal cells, their nuclei and the extracted histone samples to light.

Dynamics of the nucleosomal histone H3 N-terminal tail revealed by high precision single-molecule FRET.

Chromatin compaction and gene accessibility are orchestrated by assembly and disassembly of nucleosomes. Although the disassembly process was widely studied, little is known about the structure and dynamics of the disordered histone tails, which play a pivotal role for nucleosome integrity. This is a gap filling experimental FRET study from the perspective of the histone H3 N-terminal tail (H3NtT) of reconstituted mononucleosomes. By systematic variation of the labeling positions we monitored the motions of the H3NtT relative to the dyad axis and linker DNA. Single-molecule FRET unveiled that H3NtTs do not diffuse freely but follow the DNA motions with multiple interaction modes with certain permitted dynamic transitions in the µs to ms time range. We also demonstrate that the H3NtT can allosterically sense charge-modifying mutations within the histone core (helix α3 of histone H2A (R81E/R88E)) resulting in increased dynamic transitions and lower rate constants. Those results complement our earlier model on the NaCl induced nucleosome disassembly as changes in H3NtT configurations coincide with two major steps: unwrapping of one linker DNA and weakening of the internal DNA - histone interactions on the other side. This emphasizes the contribution of the H3NtT to the fine-tuned equilibrium between overall nucleosome stability and DNA accessibility.

Outer Membrane Protease OmpT-Based Strategy for Simplified Analysis of Histone Post-Translational Modifications by Mass Spectrometry.

Histone modifications play an important role in regulating transcriptional gene expression and chromatin processes in eukaryotes. Increasing researches proved that aberrant post-translational modifications (PTMs) of histones is associated with many diseases. However, MS-based identification and quantification of histone PTMs are still challenging. Although classic chemical derivatization in conjunction with trypsin digestion is widely used for histone PTMs analysis in a bottom-up strategy, several side reactions have been observed in practice. In this work, outer membrane protease T (OmpT) was utilized as a protease for direct histone proteolysis and generated appropriate lengths of histone peptides for retention on reversed-phase chromatography. The powerful and unique tolerance of OmpT for modified lysines and arginines was demonstrated and can be quantitatively described for the first time, making it useful for detecting natural modifications. Using the optimized digestion conditions, we succeeded in identifying 121 histone marks from HEK293T cells, 42 of which were previously unreported. Additionally, histone H3 PTMs were quantitatively profiled in the KMS11 multiple myeloma cells and NSD2 selective knockout KMS11cells, revealing that NSD2 was of high specificity on H3K36 dimethylation. Histone chemical derivatizations are not required in our strategy, showing a remarkable strength over the conventional trypsin-based workflow.

Biochemical and structural analyses of the nucleosome containing human histone H2A.J.

Histone H2A.J, a histone H2A variant conserved in mammals, may function in the expression of genes related to inflammation and cell proliferation. In the present study, we purified the human histone H2A.J variant and found that H2A.J is efficiently incorporated into the nucleosome in vitro. H2A.J formed the stable nucleosome, which accommodated the DNA ends. Mutations in the H2A.J-specific residues did not affect the nucleosome stability, although the mutation of the H2A.J Ala40 residue, which is conserved in some members of the canonical H2A class, reduced the nucleosome stability. Consistently, the crystal structure of the H2A.J nucleosome revealed that the H2A.J-specific residues, including the Ala40 residue, did not affect the nucleosome structure. These results provide basic information for understanding the function of the H2A.J nucleosome.

Expression and phase separation potential of heterochromatin proteins during early mouse development.

In most eukaryotes, constitutive heterochromatin is associated with H3K9me3 and HP1α. The latter has been shown to play a role in heterochromatin formation through liquid-liquid phase separation. However, many other proteins are known to regulate and/or interact with constitutive heterochromatic regions in several species. We postulate that some of these heterochromatic proteins may play a role in the regulation of heterochromatin formation by liquid-liquid phase separation. Indeed, an analysis of the constitutive heterochromatin proteome shows that proteins associated with constitutive heterochromatin are significantly more disordered than a random set or a full nucleome set of proteins. Interestingly, their expression begins low and increases during preimplantation development. These observations suggest that the preimplantation embryo is a useful model to address the potential role for phase separation in heterochromatin formation, anticipating exciting research in the years to come.

High-Throughput Quantitative Top-Down Proteomics: Histone H4.

Proteins physiologically exist as "proteoforms" that arise from one gene and acquire additional function by post-translational modifications (PTM). When multiple PTMs coexist on single protein molecules, top-down proteomics becomes the only feasible method of characterization; however, most top-down methods have limited quantitative capacity and insufficient throughput to truly address proteoform biology. Here we demonstrate that top-down proteomics can be quantitative, reproducible, sensitive, and high throughput. The proteoforms of histone H4 are well studied both as a challenging proteoform identification problem and due to their essential role in the regulation of all eukaryotic DNA-templated processes. Much of histone H4's function is obfuscated from prevailing methods due to combinatorial mechanisms. Starting from cells or tissues, after an optimized protein purification process, the H4 proteoforms are physically separated by on-line C3 chromatography, narrowly isolated in MS1 and sequenced with ETD fragmentation. We achieve more than 30 replicates from a single 35-mm tissue culture dish by loading 55 ng of H4 on column. Parallelization and automation yield a sustained throughput of 12 replicates per day. We achieve reproducible quantitation (average biological Pearson correlations of 0.89) of hundreds of proteoforms (about 200-300) over almost six orders of magnitude and an estimated LLoQ of 0.001% abundance. We demonstrate the capacity of the method to precisely measure well-established changes with sodium butyrate treatment of SUM159 cells. We show that the data produced by a quantitative top-down method can be amenable to parametric statistical comparisons and is capable of delineating relevant biological changes at the full proteoform level.

Nucleosome and ubiquitin position Set2 to methylate H3K36.

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification deposited by the Set2 methyltransferases. Recent findings show that over-expression or mutation of Set2 enzymes promotes cancer progression, however, mechanisms of H3K36me are poorly understood. Set2 enzymes show spurious activity on histones and histone tails, and it is unknown how they obtain specificity to methylate H3K36 on the nucleosome. In this study, we present 3.8 Å cryo-EM structure of Set2 bound to the mimic of H2B ubiquitinated nucleosome. Our structure shows that Set2 makes extensive interactions with the H3 αN, the H3 tail, the H2A C-terminal tail and stabilizes DNA in the unwrapped conformation, which positions Set2 to specifically methylate H3K36. Moreover, we show that ubiquitin contributes to Set2 positioning on the nucleosome and stimulates the methyltransferase activity. Notably, our structure uncovers interfaces that can be targeted by small molecules for development of future cancer therapies.

Histone chaperone exploits intrinsic disorder to switch acetylation specificity.

Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.

Harnessing the HDAC-histone deacetylase enzymes, inhibitors and how these can be utilised in tissue engineering.

There are large knowledge gaps regarding how to control stem cells growth and differentiation. The limitations of currently available technologies, such as growth factors and/or gene therapies has led to the search of alternatives. We explore here how a cell's epigenome influences determination of cell type, and potential applications in tissue engineering. A prevalent epigenetic modification is the acetylation of DNA core histone proteins. Acetylation levels heavily influence gene transcription. Histone deacetylase (HDAC) enzymes can remove these acetyl groups, leading to the formation of a condensed and more transcriptionally silenced chromatin. Histone deacetylase inhibitors (HDACis) can inhibit these enzymes, resulting in the increased acetylation of histones, thereby affecting gene expression. There is strong evidence to suggest that HDACis can be utilised in stem cell therapies and tissue engineering, potentially providing novel tools to control stem cell fate. This review introduces the structure/function of HDAC enzymes and their links to different tissue types (specifically bone, cardiac, neural tissues), including the history, current status and future perspectives of using HDACis for stem cell research and tissue engineering, with particular attention paid to how different HDAC isoforms may be integral to this field.

Imaging Flow Cytometry Quantifies Neural Genome Dynamics.

Somatic mosaicism is a common consequence of normal development. DNA repair is simply not perfect, and each cell's genome incurs continuous DNA damage as a consequence of transcription, replication, and other cell biological stressors. Brain somatic mosaicism is particularly noteworthy because the vast majority of an individual's neurons are with that individual for life and neural circuits give rise directly to behavioral phenotypes. Brain somatic mosaicism, now revealed and tractable due to advances in single cell 'omic approaches, has emerged as an intriguing and unexplored aspect of neuronal diversity. Furthermore, the study of DNA damage during early neurodevelopment, when the rate of mutagenesis is high, is the perfect starting point to understand the origins of brain mosaicism. Flow cytometry is a highly efficient technique to study cell cycle and intracellular proteins of interest, particularly those related to DNA damage, but it lacks the high resolution of microscopy to examine the localization of these proteins. In this study, we outline a novel single-cell approach to quantify DNA double-strand break (DNA DSB) dynamics during early human neurodevelopment by applying imaging flow cytometry (IFC) to human-induced pluripotent stem cell-derived neural progenitor cells (NPCs) undergoing neurogenesis. We establish an increase of DNA DSBs by quantifying γH2AX foci in mildly stressed NPCs using various single-cell approaches in addition to IFC including fluorescent microscopy, conventional flow cytometry, and measuring DNA DSBs with the comet assay. We demonstrate the dose-dependent sensitive detection of γH2AX foci through IFC and reveal the dynamics of DNA DSBs in proliferating and differentiating neural cells in early neurogenesis. © 2019 International Society for Advancement of Cytometry.

Aptamer functionalized magnetic graphene oxide nanocomposites for highly selective capture of histones.

The binding coverage of aptamer was an important restricted factor for aptamer-based affinity enrichment strategy for capturing target molecules. Herein, we designed and prepared aptamer functionalized graphene oxide based nanocomposites (GO/NH2 -NTA/Fe3 O4 /PEI/Au), and the coverage density of aptamer was high to 33.1 nmol/mg. The high aptamer coverage density was contributed to the large surface area of graphene oxide. The successive modification of Nα,Nα-Bis(carboxymethyl)-L-lysine, magnetic nanoparticles, polyethylenimine, and Au nanoparticles ensured the histone purification with fast speed and high purity. Histones could be captured rapidly and specifically from nucleoproteins by our aptamer based purification strategy, while traditional acid-extraction could not specifically enrich histones. Compared with traditional acid-extraction method, rapid and efficient discovery of histones and their post-translational modifications, such as several kinds of methylation at H3.1K9 and H3.1K27, were achieved confidently. It demonstrated that our aptamer functionalized magnetic graphene oxide nanocomposites have a great potential for histone analysis.

Chromatin Characterization in Xenopus laevis Cell-Free Egg Extracts and Embryos.

Xenopus laevis development is marked by accelerated cell division solely supported by the proteins maternally deposited in the egg. Oocytes mature to eggs with concomitant transcriptional silencing. The unique maternal chromatin state contributing to this silencing and subsequent zygotic activation is likely established by histone posttranslational modifications and histone variants. Therefore, tools for understanding the nature and function of maternal and embryonic histones are essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Here we describe protocols for isolating pronuclear sperm chromatin from Xenopus egg extracts and hydroxyapatite-based histone purification from this chromatin. The histones purified through this method can be directly assembled into chromatin through in vitro assembly reactions, providing a unique opportunity to biochemically dissect the effect of histone variants, histone modifications, and other factors in chromatin replication and assembly. We also describe how to isolate chromatin from staged embryos and analyze the proteins to reveal dynamic developmental histone modifications. Finally, we present protocols to measure chromatin assembly in extracts, including supercoiling and micrococcal nuclease assays. Using these approaches, analysis of maternal and zygotic histone posttranslational modifications concomitant with cell-cycle and developmental transitions can be tested.