Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation.

Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.

Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells.

The placenta is a highly evolved, specialized organ in mammals. It differs from other organs in that it functions only for fetal maintenance during gestation. Therefore, there must be intrinsic mechanisms that guarantee its unique functions. To address this question, we comprehensively analyzed epigenomic features of mouse trophoblast stem cells (TSCs). Our genome-wide, high-throughput analyses revealed that the TSC genome contains large-scale (>1-Mb) rigid heterochromatin architectures with a high degree of histone H3.1/3.2-H3K9me3 accumulation, which we termed TSC-defined highly heterochromatinized domains (THDs). Importantly, depletion of THDs by knockdown of CAF1, an H3.1/3.2 chaperone, resulted in down-regulation of TSC markers, such as Cdx2 and Elf5, and up-regulation of the pluripotent marker Oct3/4, indicating that THDs maintain the trophoblastic nature of TSCs. Furthermore, our nuclear transfer technique revealed that THDs are highly resistant to genomic reprogramming. However, when H3K9me3 was removed, the TSC genome was fully reprogrammed, giving rise to the first TSC cloned offspring. Interestingly, THD-like domains are also present in mouse and human placental cells in vivo, but not in other cell types. Thus, THDs are genomic architectures uniquely developed in placental lineage cells, which serve to protect them from fate reprogramming to stably maintain placental function.

H3 K27M and EZHIP Impede H3K27-Methylation Spreading by Inhibiting Allosterically Stimulated PRC2.

Diffuse midline gliomas and posterior fossa type A ependymomas contain the recurrent histone H3 lysine 27 (H3 K27M) mutation and express the H3 K27M-mimic EZHIP (CXorf67), respectively. H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3 lysine 27 trimethylation (H3K27me3) levels; however, residual peaks of H3K27me3 remain at CpG islands (CGIs) through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with PRC2 that is allosterically activated by H3K27me3 at CGIs and impede its spreading. Moreover, H3 K27M oncohistones reduce H3K27me3 in trans, independent of their incorporation into the chromatin. Although EZHIP is not found outside placental mammals, expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved mechanism. Our results provide mechanistic insights for the retention of residual H3K27me3 in tumors driven by H3 K27M and EZHIP.

DNA polymerase epsilon binds histone H3.1-H4 and recruits MORC1 to mediate meiotic heterochromatin condensation.

Heterochromatin is essential for genomic integrity and stability in eukaryotes. The mechanisms that regulate meiotic heterochromatin formation remain largely undefined. Here, we show that the catalytic subunit (POL2A) of Arabidopsis DNA polymerase epsilon (POL ε) is required for proper formation of meiotic heterochromatin. The POL2A N terminus interacts with the GHKL adenosine triphosphatase (ATPase) MORC1 (Microrchidia 1), and POL2A is required for MORC1's localization on meiotic heterochromatin. Mutations affecting the POL2A N terminus cause aberrant morphology of meiotic heterochromatin, which is also observed in morc1. Moreover, the POL2A C-terminal zinc finger domain (ZF1) specifically binds to histone H3.1-H4 dimer or tetramer and is important for meiotic heterochromatin condensation. Interestingly, we also found similar H3.1-binding specificity for the mouse counterpart. Together, our results show that two distinct domains of POL2A, ZF1 and N terminus bind H3.1-H4 and recruit MORC1, respectively, to induce a continuous process of meiotic heterochromatin organization. These activities expand the functional repertoire of POL ε beyond its classic role in DNA replication and appear to be conserved in animals and plants.

The in vivo Interaction Landscape of Histones H3.1 and H3.3.

Chromatin structure, transcription, DNA replication, and repair are regulated via locus-specific incorporation of histone variants and posttranslational modifications that guide effector chromatin-binding proteins. Here we report unbiased, quantitative interactomes for the replication-coupled (H3.1) and replication-independent (H3.3) histone H3 variants based on BioID proximity labeling, which allows interactions in intact, living cells to be detected. Along with a significant proportion of previously reported interactions detected by affinity purification followed by mass spectrometry, three quarters of the 608 histone-associated proteins that we identified are new, uncharacterized histone associations. The data reveal important biological nuances not captured by traditional biochemical means. For example, we found that the chromatin assembly factor-1 histone chaperone not only deposits the replication-coupled H3.1 histone variant during S-phase but also associates with H3.3 throughout the cell cycle in vivo. We also identified other variant-specific associations, such as with transcription factors, chromatin regulators, and with the mitotic machinery. Our proximity-based analysis is thus a rich resource that extends the H3 interactome and reveals new sets of variant-specific associations.

Characterization of histone chaperone MCM2 as a key regulator in arsenic-induced depletion of H3.3 at genomic loci.

Arsenic exposure is associated with an increased risk of many cancers, and epigenetic mechanisms play a crucial role in arsenic-mediated carcinogenesis. Our previous studies have shown that arsenic exposure induces polyadenylation of H3.1 mRNA and inhibits the deposition of H3.3 at critical gene regulatory elements. However, the precise underling mechanisms are not yet understood. To characterize the factors governing arsenic-induced inhibition of H3.3 assembly through H3.1 mRNA polyadenylation, we utilized mass spectrometry to identify the proteins, especially histone chaperones, with reduced binding affinity to H3.3 under conditions of arsenic exposure and polyadenylated H3.1 mRNA overexpression. Our findings reveal that the interaction between H3.3 and the histone chaperon protein MCM2 is diminished by both polyadenylated H3.1 mRNA overexpression and arsenic treatment in human lung epithelial BEAS-2B cells. The increased binding of MCM2 to H3.1, resulting from elevated H3.1 protein levels, appears to contribute to the reduced availability of MCM2 for H3.3. To further investigate the role of MCM2 in H3.3 deposition during arsenic exposure and H3.1 mRNA polyadenylation, we overexpressed MCM2 in BEAS-2B cells overexpressing polyadenylated H3.1 or exposed to arsenic. Our results demonstrate that MCM2 overexpression attenuates H3.3 depletion at several genomic loci, suggesting its involvement in the arsenic-induced displacement of H3.3 mediated by H3.1 mRNA polyadenylation. These findings suggest that changes in the association between histone chaperone MCM2 and H3.3 due to polyadenylation of H3.1 mRNA may play a pivotal role in arsenic-induced carcinogenesis.

Novel histone modifications in microglia derived from a mouse model of chronic pain.

As the resident immune cells in the central nervous system, microglia play an important role in the maintenance of its homeostasis. Dysregulation of microglia has been associated with the development and maintenance of chronic pain. However, the relevant molecular pathways remain poorly defined. In this study, we used a mass spectrometry-based proteomic approach to screen potential changes of histone protein modifications in microglia isolated from the brain of control and cisplatin-induced neuropathic pain adult C57BL/6J male mice. We identified several novel microglial histone modifications associated with pain, including statistically significantly decreased histone H3.1 lysine 27 mono-methylation (H3.1K27me1, 54.8% of control) and H3 lysine 56 tri-methylation (7.5% of control), as well as a trend suggesting increased H3 tyrosine 41 nitration. We further investigated the functional role of H3.1K27me1 and found that treatment of cultured microglial cells for 4 consecutive days with 1-10 µM of NCDM-64, a potent and selective inhibitor of lysine demethylase 7A, an enzyme responsible for the demethylation of H3K27me1, dose-dependently elevated its levels with a greater than a two-fold increase observed at 10 µM compared to vehicle-treated control cells. Moreover, pretreatment of mice with NCDM-64 (10 or 25 mg/kg/day, i.p.) prior to cisplatin treatment prevented the development of neuropathic pain in mice. The identification of specific chromatin marks in microglia associated with chronic pain may yield critical insight into the contribution of microglia to the development and maintenance of pain, and opens new avenues for the development of novel nonopioid therapeutics for the effective management of chronic pain.

Evaluation of plasma nucleosome concentrations in dogs with a variety of common cancers and in healthy dogs.

BACKGROUND: Cell free DNA, in the form of nucleosomes, is released into circulation during apoptosis and necrosis in a variety of diseases. They are small fragments of chromosomes that are composed of DNA wrapped around a histone core made of four duplicate histone proteins forming an octamer. The nucleosome compartment is a relatively uninvestigated area of circulating tumor biomarkers in dogs. The objectives of this study were to quantify and better characterize nucleosome concentrations in 528 dogs with various common malignancies and compare them to 134 healthy dogs. RESULTS: The sensitivity of increased circulating nucleosome concentrations for the detection of cancer in all dogs was 49.8% with a specificity of 97% with an area under the curve of 68.74%. The top 4 malignancies detected by the test included lymphoma, hemangiosarcoma, histiocytic sarcoma and malignant melanoma. The malignancies least likely to be detected were soft tissue sarcomas, osteosarcoma and mast cell tumors. CONCLUSIONS: A variety of tumor types may cause increased nucleosome concentrations in dogs. Tumors of hematopoietic origin are most likely to cause elevations and local tumors such as soft tissue sarcomas are least likely to cause elevations in plasma nucleosome concentrations.

The Role of the TSK/TONSL-H3.1 Pathway in Maintaining Genome Stability in Multicellular Eukaryotes.

Replication-dependent histone H3.1 and replication-independent histone H3.3 are nearly identical proteins in most multicellular eukaryotes. The N-terminal tails of these H3 variants, where the majority of histone post-translational modifications are made, typically differ by only one amino acid. Despite extensive sequence similarity with H3.3, the H3.1 variant has been hypothesized to play unique roles in cells, as it is specifically expressed and inserted into chromatin during DNA replication. However, identifying a function that is unique to H3.1 during replication has remained elusive. In this review, we discuss recent findings regarding the involvement of the H3.1 variant in regulating the TSK/TONSL-mediated resolution of stalled or broken replication forks. Uncovering this new function for the H3.1 variant has been made possible by the identification of the first proteins containing domains that can selectively bind or modify the H3.1 variant. The functional characterization of H3-variant-specific readers and writers reveals another layer of chromatin-based information regulating transcription, DNA replication, and DNA repair.

The effects of fiscal policy on households during the COVID-19 pandemic: Evidence from Thailand and Vietnam.

The economic crisis created by the COVID-19 pandemic induced many governments to provide financial assistance to households. Using representative consumer surveys conducted during the pandemic in 2020, we examine the effects of this fiscal policy instrument on households in two emerging economies, Thailand and Vietnam. Our paper contributes to the literature by studying how consumer sentiment and durable spending relate to receiving government financial support and the underlying transmission channels for these responses. We find that financial support to households is related to more positive consumer sentiment and increases in actual and planned durable spending, while also being correlated with a more optimistic macroeconomic outlook, higher trust in the government, and higher personal well-being.

New efficient synthesis of polysubstituted 3,4-dihydroquinazolines and 4H-3,1-benzothiazines through a Passerini/Staudinger/aza-Wittig/addition/nucleophilic substitution sequence.

A new efficient synthesis of polysubstituted 3,4-dihydroquinazolines and 4H-3,1-benzothiazines via sequential Passerini/Staudinger/aza-Wittig/addition/nucleophilic substitution reaction has been developed. The three-component Passerini reactions of 2-azidobenzaldehydes 1, benzoic acid (2), and isocyanides 3 produced the azide intermediates 4, which were treated sequentially with triphenylphosphine, isocyanates (or CS2), and secondary amines to give polysubstituted 3,4-dihydroquinazolines 8 and 4H-3,1-benzothiazines 11 in good overall yields through consecutive Passerini/Staudinger/aza-Wittig/addition/nucleophilic substitution reactions.

Characterizing circulating nucleosomes in the plasma of dogs with hemangiosarcoma.

BACKGROUND: Nucleosomes consist of DNA wrapped around a histone octamer core like thread on a spool to condense DNA as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death, chromatin fragmentation and release of nucleosomes into the blood. The Nu.Q™ platform measures circulating nucleosomes in the blood of humans that result from disease and has been used to detect and identify cancer even at early stages. The objectives of this study are to quantify and better characterize nucleosomes in dogs with various stages of hemangiosarcoma (HSA) using this ELISA-based assay. Samples from 77 dogs with a confirmed diagnosis of hemangiosarcoma and 134 healthy controls were utilized for this study. The HSA samples were recruited from the Texas A&M University Small Animal Clinic (TAMU-SAC) or purchased from biobanks. All control samples were recruited from the TAMU-SAC. RESULTS: Dogs with hemangiosarcoma had a 6.6-fold increase in their median plasma nucleosome concentrations compared to controls (AUC 92.9 %). Elevated nucleosome concentrations were seen at all stages of disease and nucleosome concentrations increased with the stage of the disease. CONCLUSIONS: Plasma nucleosome concentrations are a reliable way to differentiate dogs with hemangiosarcoma from healthy dogs. Further testing is underway to better characterize cancer associated HSA circulating nucleosomes and optimize future diagnostics for canine HSA detection.

Histone H3K27M Mutation in Brain Tumors.

Histones form chromatin and play a key role in the regulation of gene expression. As an epigenetic information form, histone modifications such as methylation, phosphorylation, acetylation, and ubiquitination are closely related to the regulation of genes. In the last two decades, cancer scientists discovered that some histone modifications, including acetylation and methylation, are perturbed in cancer diseases. Recurrent histone mutations, which hinder histone methylation and are implicated in oncogenesis, are recently identified in several cancer disease and called oncohistones. Well-known oncohistones, with mutations on both H3.1 and H3.3, include H3K36M in chondroblastoma, H3K27M in glioma, and H3G34 mutations that exist in bone cancers and gliomas. Oncohistone expression can lead to epigenome/transcriptome reprogramming and eventually to oncogenesis. The H3K27M, H3G34V/R, and H3K36M histone mutations can lead to the substitution of amino acid(s) at or near a lysine residue, which is a methylation target. H3K27M characteristically exists in diffuse intrinsic pontine glioma (pediatric DIPG), and its expression can cause a global decrease of the methylation of histone at the lysine residue. Uncovering the molecular mechanisms of H3K27M-driven tumorigenesis has recently led to the identification of some potential therapeutic targets in diffuse intrinsic pontine glioma. In this chapter, we will review and summarize recent studies on the H3K27M-driven tumorigenic mechanisms and properties and the role of H3.1K27M and H3.3K27M oncohistones in brain tumors.

Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes.

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18 -StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.

Synthesis, Spectroscopic Studies and Keto-Enol Tautomerism of Novel 1,3,4-Thiadiazole Derivative Containing 3-Mercaptobutan-2-one and Quinazolin-4-one Moieties.

In this study, a novel 1,3,4-thiadiazole derivative containing 3-mercaptobutan-2-one and quinazolin-4-one moieties (Compound 3) is synthesized by the coupling of 2-amino-1,3,4-thiadiazole-5-(3-mercaptobutan-2-one) (Compound 1) with 2-Phenyl-4H-3,1-benzoxazin-4-one (Compound 2) in one molecule moiety. Compound 3 is found to exist as two types of intra-molecular hydrogen bonding with keto-enol tautomerism characters, which is further confirmed using FTIR, 1H-NMR, 13C-NMR, mass spectrometer, and UV-Visible spectra. The 1H-NMR and UV-Visible spectra of Compound 3 are investigated in different solvents such as methanol, chloroform, and DMSO. Compound 3 exhibits keto-enol tautomeric forms in solvents with different percentage ratios depending on the solvent polarity. The 1H-NMR and UV-Visible spectral results show that Compound 3 favors the keto over the enol form in polar aprotic solvents such as DMSO and the enol over the keto form in non-polar solvents such as chloroform. The 13C-NMR spectrum gives two singles at δ 204.5 ppm, due to ketonic carbon, and δ 155.5 ppm, due to enolic carbon, confirming the keto-enol tautomerism of Compound 3. Furthermore, the molecular ion at m/z 43 and m/z 407 in the mass spectrum of Compound 3 and fragmentation mechanisms proposed reveal the existence of the keto and enol forms, respectively.

H3.1 K36M mutation in a congenital-onset soft tissue neoplasm.

We describe a patient who presented with a congenital soft tissue lesion initially diagnosed as infantile fibromatosis at 15 days of age. Unusually, the mass demonstrated malignant progression leading to death at 20 months of age. Biological progression to malignancy is not known to occur in fibromatosis, and fibrosarcoma is not known to progress from a benign lesion. Whole-exome sequencing of the tumor identified a driver mutation in histone H3.1 at lysine (K)36. Our findings support the link between oncohistones and infantile soft tissue tumors and provide additional evidence for the oncogenic effects of p.K36M in H3 variants.

Experimental evidence for previously unclassified calcium phosphate structures in the casein micelle.

1H-31P Cross-polarization magic angle spinning (CP-MAS) measurements of 40-d-old Mozzarella cheese and 20 mM EDTA-treated casein micelles revealed that each sample had immobile phosphorus with the same spectral pattern, which did not match that of native casein micelles. To identify the immobile phosphorus bodies, 1H-31P CP-MAS spectra and cross-polarization kinetics measurements were undertaken on native casein micelles, EDTA-chelated casein micelles, and reference samples of ß-casein and hydroxyapatite. The results showed that the immobile phosphorus bodies in the mature Mozzarella cheese had the following characteristics: they are immobile phosphoserine residues (not colloidal calcium phosphate); they are not the product of phosphoserine to colloidal calcium phosphate bridging; the phosphate is complexed to calcium; their rigidity is localized to a phosphorus site; their rigidity and bond coupling is unaffected by protein hydration; and the immobile bodies share a narrow range of bond orientations. Combining these observations, the best explanation of the immobile phosphorus bodies is that bonding structures of phosphorus-containing groups and calcium exist within the casein micelle that are not yet clearly classified in the literature. The best candidate is a calcium-bridged phosphoserine-to-phosphoserine linkage, either intra- or inter-protein.

Time-dependent stabilization of the +1 nucleosome is an early step in the transition to stable cold-induced repression of FLC.

In vernalized Arabidopsis, the extent of FLC repression and promotion of flowering are correlated with the length of winter (low temperature exposure), but how plants measure the duration of winter is unknown. Repression of FLC occurs in two phases: establishment and maintenance. This study investigates the early events in the transition between establishment and maintenance of repression. Initial repression was rapid but transient; within 24 h of being placed at low temperatures FLC transcription was reduced by 40% and repression was complete after 5 days in the cold. The extent to which repression was maintained depended on the length of the cold treatment. Occupancy of the +1 nucleosome in FLC chromatin increased in a time-dependent manner over a 4-week low temperature treatment concomitant with decreased histone acetylation and increased trimethylation of histone H3 lysine 27 (H3K27me3). Mutant analyses showed that increased nucleosome occupancy occurred independent of histone deacetylation and increased H3K27me3, suggesting that it is an early step in the switch between transient and stable repression. Both altered histone composition and deacetylation contributed to increased nucleosome occupancy. The time-dependency of the steps required for the switch between transient and stable repression suggests that the duration of winter is measured by the chromatin state at FLC. A chromatin-based switch is consistent with finding that each FLC allele in a cell undergoes this transition independently.

Control and role of pH in peptide-lipid interactions in oriented membrane samples.

To understand the molecular mechanisms of amphiphilic membrane-active peptides, one needs to study their interactions with lipid bilayers under ambient conditions. However, it is difficult to control the pH of the sample in biophysical experiments that make use of mechanically aligned multilamellar membrane stacks on solid supports. HPLC-purified peptides tend to be acidic and can change the pH in the sample significantly. Here, we have systematically studied the influence of pH on the lipid interactions of the antimicrobial peptide PGLa embedded in oriented DMPC/DMPG bilayers. Using solid-state NMR (31P, 2H, 19F), both the lipid and peptide components were characterized independently, though in the same oriented samples under typical conditions of maximum hydration. The observed changes in lipid polymorphism were supported by DSC on multilamellar liposome suspensions. On this basis, we can present an optimized sample preparation protocol and discuss the challenges of performing solid-state NMR experiments under controlled pH. DMPC/DMPG bilayers show a significant up-field shift and broadening of the main lipid phase transition temperature when lowering the pH from 10.0 to 2.6. Both, strongly acidic and basic pH, cause a significant degree of lipid hydrolysis, which is exacerbated by the presence of PGLa. The characteristic re-alignment of PGLa from a surface-bound to a tilted state is not affected between pH of 7 to 4 in fluid bilayers. On the other hand, in gel-phase bilayers the peptide remains isotropically mobile under acidic conditions, displays various co-existing orientational states at pH7, and adopts an unknown structural state at basic pH.

1H and 31P Magnetic Resonance Spectroscopic Metabolomic Imaging: Assessing Mitogen-Activated Protein Kinase Inhibition in Melanoma.

The MAPK signaling pathway with BRAF mutations has been shown to drive the pathogenesis of 40-60% of melanomas. Inhibitors of this pathway's BRAF and MEK components are currently used to treat these malignancies. However, responses to these treatments are not always successful. Therefore, identifying noninvasive biomarkers to predict treatment responses is essential for personalized medicine in melanoma. Using noninvasive 1H magnetic resonance spectroscopy (1H MRS), we previously showed that BRAF inhibition reduces lactate and alanine tumor levels in the early stages of effective therapy and could be considered as metabolic imaging biomarkers for drug response. The present work demonstrates that these metabolic changes observed by 1H MRS and those assessed by 31P MRS are also found in preclinical human melanoma models treated with MEK inhibitors. Apart from 1H and 31P MRS, additional supporting in vitro biochemical analyses are described. Our results indicate significant early metabolic correlations with response levels to MEK inhibition in the melanoma models and are consistent with our previous study of BRAF inhibition. Given these results, our study supports the potential clinical utility of noninvasive MRS to objectively image metabolic biomarkers for the early prediction of melanoma's response to MEK inhibition.