BACKGROUND: Obesity has tremendous impact on the health systems. Its epigenetic bases are unclear. 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. Their role in adipose tissue plasticity is unknown. RESULTS: Here, we show evidence that macroH2A1.1 protein levels in the visceral adipose tissue of obese humans positively correlate with BMI, while macroH2A1.2 is nearly absent. We thus introduced a constitutive GFP-tagged transgene for macroH2A1.2 in mice, and we characterized their metabolic health upon being fed a standard chow diet or a high fat diet. Despite unchanged food intake, these mice exhibit lower adipose mass and improved glucose metabolism both under a chow and an obesogenic diet. In the latter regimen, transgenic mice display smaller pancreatic islets and significantly less inflammation. MacroH2A1.2 overexpression in the mouse adipose tissue induced dramatic changes in the transcript levels of key adipogenic genes; genomic analyses comparing pre-adipocytes to mature adipocytes uncovered only minor changes in macroH2A1.2 genomic distribution upon adipogenic differentiation and suggested differential cooperation with transcription factors. MacroH2A1.2 overexpression markedly inhibited adipogenesis, while overexpression of macroH2A1.1 had opposite effects. CONCLUSIONS: MacroH2A1.2 is an unprecedented chromatin component powerfully promoting metabolic health by modulating anti-adipogenic transcriptional networks in the differentiating adipose tissue. Strategies aiming at enhancing macroH2A1.2 expression might counteract excessive adiposity in humans.

• Keywords
• Adipose tissue, Histone variants, Obesity, macroh2a1.2,
• MeSH
• Adipogenesis MeSH
• Cell Differentiation MeSH
• Cell Line MeSH
• Diet, High-Fat MeSH
• Phenotype MeSH
• Glucose Tolerance Test MeSH
• Histones genetics   metabolism   MeSH
• Body Mass Index MeSH
• Cyclin-Dependent Kinase Inhibitor p21 genetics   metabolism   MeSH
• Insulin metabolism   MeSH
• Liver pathology   MeSH
• Skin pathology   MeSH
• Humans MeSH
• Metabolic Engineering MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Pancreas pathology   MeSH
• Adipose Tissue cytology   metabolism   MeSH
• Uncoupling Protein 1 genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histones MeSH
• Cyclin-Dependent Kinase Inhibitor p21 MeSH
• Insulin MeSH
• macroH2A histone MeSH Browser
• Uncoupling Protein 1 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.

• Keywords
• macroH2A1, mass spectrometry, post-translational modifications,
• Publication type
• Journal Article MeSH

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.

• Keywords
• DNA damage, cell reprogramming, induced pluripotent stem cells, macroH2A1.1,
• MeSH
• DNA MeSH
• Endothelial Cells metabolism   MeSH
• Histones * metabolism   MeSH
• Induced Pluripotent Stem Cells * metabolism   MeSH
• Humans MeSH
• Mice MeSH
• DNA Repair MeSH
• X-ray Repair Cross Complementing Protein 1 genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• Histones * MeSH
• MACROH2A1 protein, human MeSH Browser
• X-ray Repair Cross Complementing Protein 1 MeSH
• XRCC1 protein, human MeSH Browser

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.

• Keywords
• CUT&Tag, iPSCs, induced pluripotent stem cells, macroH2A1, reprogramming, somatic cells,
• MeSH
• Endothelial Cells metabolism   MeSH
• Histones * metabolism   MeSH
• Induced Pluripotent Stem Cells * metabolism   MeSH
• Humans MeSH
• Cellular Reprogramming genetics   MeSH
• RNA-Seq MeSH
• Transcription Factors genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histones * MeSH
• Transcription Factors 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.

• Keywords
• Epigenetics, Histones, ImageStream, Lean MAFLD, Liquid biopsy, Metabolic health,
• MeSH
• Adult MeSH
• Histones blood   MeSH
• Thinness blood   MeSH
• Middle Aged MeSH
• Humans MeSH
• Metabolic Diseases blood   complications   MeSH
• Severity of Illness Index MeSH
• Fatty Liver blood   complications   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histones MeSH

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
• Diffuse Intrinsic Pontine Glioma * genetics   diagnosis   blood   MeSH
• Child MeSH
• Glioma * genetics   diagnosis   blood   MeSH
• Histones * genetics   blood   metabolism   MeSH
• Humans MeSH
• Adolescent MeSH
• Mutation MeSH
• Biomarkers, Tumor blood   MeSH
• Brain Stem Neoplasms * genetics   diagnosis   blood   MeSH
• Nucleosomes metabolism   MeSH
• Child, Preschool MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Histones * MeSH
• Biomarkers, Tumor MeSH
• Nucleosomes 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.

• Keywords
• fasting, histone variant macroH2A1, regeneration, senescence,
• Publication type
• Journal Article MeSH
• Review MeSH

The diagnosis of heart failure with preserved left ventricle ejection fraction (HFpEF) remains a challenge, with score-based algorithms showing varying diagnostic performance and biomarkers sometimes inconclusive. This study aimed to examine whether circulating histones and histone complexes, which recently emerged as robust biomarkers of inflammation and stroke, show distinct profiles in plasma from healthy individuals, HF with reduced EF (HFrEF), and HFpEF patients. We evaluated the plasma histone profile of 30 sex/age-matched healthy individuals, 22 HFpEF and 25 HFrEF prior any therapeutic intervention. ImageStreamX-based detection approach was used to measure the levels of circulating particles positive for core histones H2A, H2B, H3, H4, histone variants macroH2A1.1 and macroH2A1.2. While we found increased levels of most of the histones and histone complexes in both HFpEF and HFrEF patients, H2A was significantly elevated only in HFpEF, compared to healthy individuals (p-value = 0.002) and to HFrEF (p-value = 0.00008). In line with these findings, H2A showed positive correlation with EF (r = 0.493). We identified a plasma histone profile able to detect HF and differentiate between HFpEF and HFrEF using a high throughput and imaging flow cytometry-adapted liquid biopsy.

• Keywords
• Heart failure, Histones, Liquid biopsy, Plasma, Preserved ejection fraction, Reduced ejection fraction,
• Publication type
• Journal Article 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.

• Keywords
• epigenetics, histones, imaging, liquid biopsy, pediatric brain tumors,
• Publication type
• Journal Article MeSH

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in children and adolescents, increasing the risk of its progression toward nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. There is an urgent need for noninvasive early diagnostic and prognostic tools such as epigenetic marks (epimarks), which would replace liver biopsy in the future. We used plasma samples from 67 children with biopsy-proven NAFLD, and as controls we used samples from 20 children negative for steatosis by ultrasound. All patients were genotyped for patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain containing 7 (MBOAT7), and klotho-β (KLB) gene variants, and data on anthropometric and biochemical parameters were collected. Furthermore, plasma cell-free DNA (cfDNA) methylation was quantified using a commercially available kit, and ImageStream(X) was used for the detection of free circulating histone complexes and variants. We found a significant enrichment of the levels of histone macroH2A1.2 in the plasma of children with NAFLD compared to controls, and a strong correlation between cfDNA methylation levels and NASH. Receiver operating characteristic curve analysis demonstrated that combination of cfDNA methylation, PNPLA3 rs738409 variant, coupled with either high-density lipoprotein cholesterol or alanine aminotransferase levels can strongly predict the progression of pediatric NAFLD to NASH with area under the curve >0.87. Conclusion: Our pilot study combined epimarks and genetic and metabolic markers for a robust risk assessment of NAFLD development and progression in children, offering a promising noninvasive tool for the consistent diagnosis and prognosis of pediatric NAFLD. Further studies are necessary to identify their pathogenic origin and function.

• MeSH
• Child MeSH
• Histones genetics   MeSH
• Humans MeSH
• Lipase genetics   MeSH
• Membrane Proteins genetics   MeSH
• DNA Methylation genetics   MeSH
• Adolescent MeSH
• Non-alcoholic Fatty Liver Disease * diagnosis   MeSH
• Pilot Projects MeSH
• Cell-Free Nucleic Acids * metabolism   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histones MeSH
• Lipase MeSH
• Membrane Proteins MeSH
• Cell-Free Nucleic Acids * MeSH