BACKGROUND: Diffuse midline glioma, H3 K27-altered (DMG) is a fatal tumour that arises in the midline structures of the brain. When located in the pons, it is more commonly referred to as diffuse intrinsic pontine glioma (DIPG). DMG/DIPG is usually diagnosed when children are < 10 years, and it has a median overall survival of < 12 months after diagnosis. Radiological imaging is still the gold standard for DIPG diagnosis while the use of biopsy procedures led to our knowledge on its biology, such as with the identification of the canonical histone H3K27M mutation. However, the need to improve survival encourages the development of non-invasive, fast and inexpensive assays on biofluids for optimizing molecular diagnoses in DMG/DIPG. Here, we propose a rapid, new, imaging and epigenetics-based approach to diagnose DMG/DIPG in the plasma of paediatric patients. METHODS: A total of 20 healthy children (mean age: 10.5 years) and 24 children diagnosed with DMG/DIPG (mean age: 8.5 years) were recruited. Individual histones (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2), histone dimers and nucleosomes were assayed in biofluids by means of a new advanced flow cytometry ImageStream(X)-adapted method. RESULTS: We report a significant increase in circulating histone dimers and tetramers (macroH2A1.1/H2B versus control: p value < 0.0001; macroH2A1.2/H2B versus control: p value < 0.0001; H2A/H2B versus control: p value < 0.0001; H3/H4 versus control: p value = 0.008; H2A/H2B/H3/H4 versus control: p value < 0.0001) and a significant downregulation of individual histones (H2B versus control: p value < 0.0001; H3 versus control: p value < 0.0001; H4 versus control: p value < 0.0001). Moreover, histones were also detectable in the cerebrospinal fluid (CSF) of patients with DMG/DIPG and in the supernatant of SF8628, OPBG-DIPG002 and OPBG-DIPG004 DMG/DIPG cell lines, with patterns mostly similar to each other, but distinct compared to blood plasma. CONCLUSIONS: In summary, we identified circulating histone signatures able to detect the presence of DMG/DIPG in biofluids of children, using a rapid and non-invasive ImageStream(X)-based imaging technology, which may improve diagnosis and benefit the patients.

• MeSH
• difuzní intrinsický pontinní gliom genetika   diagnóza   krev   MeSH
• dítě MeSH
• epigeneze genetická MeSH
• gliom genetika   diagnóza   krev   patologie   diagnostické zobrazování   MeSH
• histony * genetika   metabolismus   krev   MeSH
• lidé MeSH
• mladiství MeSH
• mutace MeSH
• nádorové biomarkery krev   MeSH
• nádory mozkového kmene genetika   diagnóza   krev   diagnostické zobrazování   patologie   metabolismus   MeSH
• předškolní dítě MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Introduction: Pediatric brain tumours (PBT) are one of the most common malignancies during childhood, with variable severity according to the location and histological type. Certain types of gliomas, such a glioblastoma and diffuse intrinsic pontine glioma (DIPG), have a much higher mortality than ependymoma and medulloblastoma. Early detection of PBT is essential for diagnosis and therapeutic interventions. Liquid biopsies have been demonstrated using cerebrospinal fluid (CSF), mostly restricted to cell free DNA, which display limitations of quantity and integrity. In this pilot study, we sought to demonstrate the detectability and robustness of cell free histones in the CSF. Methods: We collected CSF samples from a pilot cohort of 8 children with brain tumours including DIPG, medulloblastoma, glioblastoma, ependymoma and others. As controls, we collected CSF samples from nine children with unrelated blood malignancies and without brain tumours. We applied a multichannel flow imaging approach on ImageStream(X) to image indiviual histone or histone complexes on different channels. Results: Single histones (H2A, macroH2A1.1, macroH2A1.2 H2B, H3, H4 and histone H3 bearing the H3K27M mutation), and histone complexes are specifically detectable in the CSF of PBT patients. H2A and its variants macroH2A1.1/macroH2A1/2 displayed the strongest signal and abundance, together with disease associated H3K27M. In contrast, mostly H4 is detectable in the CSF of pediatric patients with blood malignancies. Discussion: In conclusion, free histones and histone complexes are detectable with a strong signal in the CSF of children affected by brain tumours, using ImageStream(X) technology and may provide additive diagnostic and predictive information.

• Publikační typ
• časopisecké články MeSH

Aim: Human induced pluripotent stem cells (iPSCs) are inefficiently derived from somatic cells by overexpression of defined transcription factors. Overexpression of H2A histone variant macroH2A1.1, but not macroH2A1.2, leads to increased iPSC reprogramming by unclear mechanisms. Materials & methods: Cleavage under targets and tagmentation (CUT&Tag) allows robust epigenomic profiling of a low cell number. We performed an integrative CUT&Tag-RNA-Seq analysis of macroH2A1-dependent orchestration of iPSCs reprogramming using human endothelial cells. Results: We demonstrate wider genome occupancy, predicted transcription factors binding, and gene expression regulated by macroH2A1.1 during reprogramming, compared to macroH2A1.2. MacroH2A1.1, previously associated with neurodegenerative pathologies, specifically activated ectoderm/neural processes. Conclusion: CUT&Tag and RNA-Seq data integration is a powerful tool to investigate the epigenetic mechanisms occurring during cell reprogramming.

• MeSH
• endoteliální buňky metabolismus   MeSH
• histony * metabolismus   MeSH
• indukované pluripotentní kmenové buňky * metabolismus   MeSH
• lidé MeSH
• přeprogramování buněk genetika   MeSH
• sekvenování transkriptomu MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Onemocnění jater je obrovskou zdravotní zátěží po celém světě. Mezi hlavní rizikové faktory patří obezita, alkohol a virová hepatitida. Jejich patologickým vyústěním potom může být i ničivý hepatocelulární karcinom (HCC), v současné době rezistentní vůči chemoterapii. Chemoterapeutické léky indukují buněčnou senescenci, zatímco samotný epigenetický mechanismus zůstává nejasný. Histon makroH2A1.1, varianta histonu H2A, je marker senescence, ale účastní se také opravných procesů souvisejících s poškozením DNA. Podle našich nedávných zjištění existuje epigenetická vazba mezi makroH2A1.1 a karcinogenezí jater. Důvodem je skutečnost, že nadměrná exprese tohoto proteinu, typická pro rakovinu jater, způsobuje tzv. únik HCC buněk z DNA de-methylačního účinku léčiva decitabinu. Nové hypo-methylačního léčivo, guadecitabin, však vykazuje mimořádnou aktivitu při léčbě rakoviny jater. Proto je naším záměrem porozumět, jak toto léčivo překonává pro-tumorigenní, anti-senescentní a chemorezistentní účinky proteinu makroH2A1.1 a zabraňuje tak progresi onemocnění jater.; Liver diseases are a tremendous health burden worldwide. Obesity, in addition to alcohol and viral hepatitis, are major risks for liver disease, the pathological endpoint of which is the devastating hepatocellular carcinoma (HCC), which is currently refractory to chemotherapy. Chemotherapeutic drugs induce cellular senescence, but the epigenetic basis underlying these therapeutic effects remains unclear. The histone macroH2A1.1, a variant of histone H2A, is a marker of senescence and it is also implicated in DNA damage repair. Recently, we found an epigenetic link between macroH2A1.1 and liver carcinogenesis, whereby overexpression of this protein, which occurs in liver cancer, triggers an escape from the effect of the DNA demethylating anti-cancer agent decitabine. However, guadecitabine, a novel generation hypomethylating agent, demonstrated a superior activity against liver cancer. We propose to study here how this new hypomethylating agent can overcome the pro-tumorigenic, anti-senescence and chemoresistant effect of macroH2A1.1, preventing the progression of liver diseases.

• MeSH
• chemorezistence MeSH
• epigenomika MeSH
• hepatocelulární karcinom genetika   terapie   MeSH
• histony MeSH
• karcinogeneze MeSH
• protinádorové látky MeSH
• stárnutí buněk MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• genetika, lékařská genetika
• hepatologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.

• MeSH
• DNA MeSH
• endoteliální buňky metabolismus   MeSH
• histony * metabolismus   MeSH
• indukované pluripotentní kmenové buňky * metabolismus   MeSH
• lidé MeSH
• myši MeSH
• oprava DNA MeSH
• protein XRCC1 genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Gene expression and epigenetic processes in several brain regions regulate physiological processes such as cognitive functions and social behavior. MacroH2A1.1 is a ubiquitous variant of histone H2A that regulates cell stemness and differentiation in various organs. Whether macroH2A1.1 has a modulatory role in emotional behavior is unknown. Here, we employed macroH2A1.1 knock-out (-/- ) mice to perform a comprehensive battery of behavioral tests, and an assessment of hippocampal synaptic plasticity (long-term potentiation) accompanied by whole hippocampus RNA sequencing. MacroH2A1.1-/- mice exhibit a stunningly enhancement both of sociability and of active stress-coping behavior, reflected by the increased social behavior in social activity tests and higher mobility time in the forced swim test, respectively. They also display an increased hippocampal synaptic plasticity, accompanied by significant neurotransmission transcriptional networks changes. These results suggest that systemic depletion of histone macroH2A1.1 supports an epigenetic control necessary for hippocampal function and social behavior.

• MeSH
• adaptace psychologická MeSH
• chování zvířat * MeSH
• hipokampus cytologie   MeSH
• histony klasifikace   genetika   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• neuroplasticita fyziologie   MeSH
• psychický stres MeSH
• regulace genové exprese MeSH
• sociální chování MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Background: Gene expression in eukaryotic cells can be governed by histone variants, which replace replication-coupled histones, conferring unique chromatin properties. MacroH2A1 is a histone H2A variant containing a domain highly similar to H2A and a large non-histone (macro) domain. MacroH2A1, in turn, is present in two alternatively exon-spliced isoforms: macroH2A1.1 and macroH2A1.2, which regulate cell plasticity and proliferation in a remarkably distinct manner. The N-terminal and the C-terminal tails of H2A histones stem from the nucleosome core structure and can be target sites for several post-translational modifications (PTMs). MacroH2A1.1 and macroH2A1.2 isoforms differ only in a few amino acids and their ability to bind NAD-derived metabolites, a property allegedly conferring their different functions in vivo. Some of the modifications on the macroH2A1 variant have been identified, such as phosphorylation (T129, S138) and methylation (K18, K123, K239). However, no study to our knowledge has analyzed extensively, and in parallel, the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting, which could facilitate the understanding of their distinct biological functions in health and disease. Methods: We used a mass spectrometry-based approach to identify the sites for phosphorylation, acetylation, and methylation in green fluorescent protein (GFP)-tagged macroH2A1.1 and macroH2A1.2 expressed in human hepatoma cells. The impact of selected PTMs on macroH2A1.1 and macroH2A1.2 structure and function are demonstrated using computational analyses. Results: We identified K7 as a new acetylation site in both macroH2A1 isoforms. Quantitative comparison of histone marks between the two isoforms revealed significant differences in the levels of phosphorylated T129 and S170. Our computational analysis provided evidence that the phosphorylation status in the intrinsically disordered linker region in macroH2A1 isoforms might represent a key regulatory element contributing to their distinct biological responses. Conclusions: Taken together, our results report different PTMs on the two macroH2A1 splicing isoforms as responsible for their distinct features and distribution in the cell.

• Publikační typ
• časopisecké články MeSH

BACKGROUND: Although metabolic associate fatty liver disease (MAFLD) is associated with obesity, it can also occur in lean patients. MAFLD is more aggressive in lean patients compared to obese patients, with a higher risk of mortality. Specific biomarkers to diagnose differentially lean or overweight MAFLD are missing. Histones and nucleosomes are released in the bloodstream upon cell death. Here, we propose a new, fast, imaging and epigenetics based approach to investigate the severity of steatosis in lean MAFLD patients. RESULTS: A total of 53 non-obese patients with histologically confirmed diagnosis of MAFLD were recruited. Twenty patients displayed steatosis grade 1 (0-33%), 24 patients with steatosis grade 2 (34-66%) and 9 patients with steatosis grade 3 (67-100%). The levels of circulating nucleosomes were assayed using enzyme-linked immunosorbent assay, while individual histones or histone dimers were assayed in serum samples by means of a new advanced flow cytometry ImageStream(X)-adapted method. Circulating nucleosome levels associated poorly with MAFLD in the absence of obesity. We implemented successfully a multi-channel flow methodology on ImageStream(X), to image single histone staining (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2). We report here a significant depletion of the levels of histone variants macroH2A1.1 and macroH2A1.2 in the serum of lean MAFLD patients, either individually or in complex with H2B. CONCLUSIONS: In summary, we identified a new circulating histone signature able to discriminate the severity of steatosis in individuals with lean MAFLD, using a rapid and non-invasive ImageStream(X)-based imaging technology.

• MeSH
• dospělí MeSH
• histony krev   MeSH
• hubenost krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metabolické nemoci krev   komplikace   MeSH
• stupeň závažnosti nemoci MeSH
• ztučnělá játra krev   komplikace   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Epigenetic regulation is important in hematopoiesis, but the involvement of histone variants is poorly understood. Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis. MacroH2A1.1 is a histone H2A variant that negatively correlates with the self-renewal capacity of embryonic, adult, and cancer stem cells. MacroH2A1.1 is a target of the frequent U2AF1 S34F mutation in MDS. The role of macroH2A1.1 in hematopoiesis is unclear. RESULTS: MacroH2A1.1 mRNA levels are significantly decreased in patients with low-risk MDS presenting with chromosomal 5q deletion and myeloid cytopenias and tend to be decreased in MDS patients carrying the U2AF1 S34F mutation. Using an innovative mouse allele lacking the macroH2A1.1 alternatively spliced exon, we investigated whether macroH2A1.1 regulates HSC homeostasis and differentiation. The lack of macroH2A1.1 decreased while macroH2A1.1 haploinsufficiency increased HSC frequency upon irradiation. Moreover, bone marrow transplantation experiments showed that both deficiency and haploinsufficiency of macroH2A1.1 resulted in enhanced HSC differentiation along the myeloid lineage. Finally, RNA-sequencing analysis implicated macroH2A1.1-mediated regulation of ribosomal gene expression in HSC homeostasis. CONCLUSIONS: Together, our findings suggest a new epigenetic process contributing to hematopoiesis regulation. By combining clinical data with a discrete mutant mouse model and in vitro studies of human and mouse cells, we identify macroH2A1.1 as a key player in the cellular and molecular features of MDS. These data justify the exploration of macroH2A1.1 and associated proteins as therapeutic targets in hematological malignancies.

• MeSH
• buněčná diferenciace MeSH
• chromozomální delece MeSH
• down regulace * MeSH
• epigeneze genetická MeSH
• haploinsuficience MeSH
• hematopoetické kmenové buňky chemie   cytologie   MeSH
• histony genetika   MeSH
• lidé MeSH
• lidské chromozomy, pár 5 genetika   MeSH
• makrocytární anemie genetika   MeSH
• místa sestřihu RNA MeSH
• modely nemocí na zvířatech MeSH
• mutace MeSH
• myelodysplastické syndromy genetika   MeSH
• myši MeSH
• sekvenční analýza RNA MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Histone variants confer chromatin unique properties. They have specific genomic distribution, regulated by specific deposition and removal machineries. Histone variants, mostly of canonical histones H2A, H2B and H3, have important roles in early embryonic development, in lineage commitment of stem cells, in the converse process of somatic cell reprogramming to pluripotency and, in some cases, in the modulation of animal aging and life span. MacroH2A1 is a variant of histone H2A, present in two alternatively exon-spliced isoforms macroH2A1.1 and macroH2A1.2, regulating cell plasticity and proliferation, during pluripotency and tumorigenesis. Furthermore, macroH2A1 participates in the formation of senescence-associated heterochromatic foci (SAHF) in senescent cells, and multiple lines of evidence in genetically modified mice suggest that macroH2A1 integrates nutritional cues from the extracellular environment to transcriptional programs. Here, we review current molecular evidence based on next generation sequencing data, cell assays and in vivo models supporting different mechanisms that could mediate the function of macroH2A1 in health span and life span. We will further discuss context-dependent and isoform-specific functions. The aim of this review is to provide guidance to assess histone variant macroH2A1 potential as a therapeutic intervention point.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH