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Diagnoses of primary malignant mesenchymal brain tumors are a challenge for pathologists. Here, we report the case of a 52-year-old man with a primary brain tumor, histologically diagnosed as a high-grade glioma, not otherwise specified (NOS). The patient underwent two neurosurgeries in several months, followed by radiotherapy and chemotherapy. We re-examined the tumor samples by methylome profiling. Methylome analysis revealed an epi-signature typical of a primary intracranial sarcoma, DICER1-mutant, an extremely rare tumor. The diagnosis was confirmed by DNA sequencing that revealed a mutation in DICER1 exon 25. DICER1 mutations were not found in the patient's blood cells, thus excluding an inherited DICER1 syndrome. The methylome profile of the DICER1 mutant sarcoma was then compared with that of a high-grade glioma, a morphologically similar tumor type. We found that several relevant regions were differentially methylated. Taken together, we report the morphological, epigenetic, and genetic characterization of the sixth described case of an adult primary intracranial sarcoma, DICER1-mutant to-date. Furthermore, this case report underscores the importance of methylome analysis to refine primary brain tumor diagnosis and to avoid misdiagnosis among morphologically similar subtypes.

Recessive X-linked disorders may occasionally evolve in clinical manifestations of variable severity also in female carriers. For some of such diseases, the frequency of the symptoms’ appearance during women’s life may be particularly relevant. This phenomenon has been largely attributed to the potential skewness of the X-inactivation process leading to variable phenotypes. Nonetheless, in many cases, no correlation with X-inactivation unbalance was demonstrated. However, methods for analyzing skewness have been mainly limited to Human Androgen Receptor methylation analysis (HUMARA). Recently, the X-inactivation process has been largely revisited, highlighting the heterogeneity existing among loci in the epigenetic state within inactive and, possibly, active X-chromosomes. We reasoned that gene-specific and ultra-deep DNA methylation analyses could greatly help to unravel details of the X-inactivation process and the roles of specific X genes inactivation in disease manifestations. We recently provided evidence that studying DNA methylation at specific autosomic loci at a single-molecule resolution (epiallele distribution analysis) allows one to analyze cell-to-cell methylation differences in a given cell population. We here apply the epiallele analysis at two X-linked loci to investigate whether females show allele-specific epiallelic patterns. Due to the high potential of this approach, the method allows us to obtain clearly distinct allele-specific epiallele profiles.

Anti-viral activities of long-chain inorganic polyphosphates (PolyPs) against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection were investigated. In molecular docking analyses, PolyPs interacted with several conserved angiotensin-converting enzyme (ACE)2 and RNA-dependent RNA polymerase (RdRp) amino acids. We thus tested PolyPs for functional interactionsin vitroin SARS-CoV-2–infected Vero E6, Caco2 and human primary nasal epithelial cells. Immunofluorescence, qPCR, direct RNA sequencing, FISH and Immunoblotting were used to determine virus loads and transcription levels of genomic(g)RNAs and sub-genomic(sg)RNAs. We show that PolyP120 binds to ACE2 and enhances its proteasomal degradation. PolyP120 shows steric hindrance of the genomic Sars-CoV-2-RNA/RdRP complex, to impair synthesis of positive-sense gRNAs, viral subgenomic transcripts and structural proteins needed for viral replication. Thus, PolyP120 impairs infection and replication of Korean and European (containing non-synonymous variants) SARS-CoV-2 strains. As PolyPs have no toxic activities, we envision their use as a nebulised formula for oropharyngeal delivery to prevent infections of SARS-CoV-2 and during early phases of antiviral therapy.

We demonstrated previously that rat tyrosine phosphatase r-PTPeta expression was suppressed in rat and human thyroid neoplastic cells, and that restoration of r-PTPeta expression reverted the malignant phenotype. To investigate the potential of this gene for cancer therapy, we generated an adenovirus carrying the r-PTPeta cDNA (Ad-r-PTPeta). This virus infected human thyroid carcinoma cells and overexpressed the r-PTPeta protein. Overexpression of r-PTPeta significantly inhibited the growth of four thyroid carcinoma cell lines. Cell growth inhibition was associated with down-regulation of extracellular signal-regulated kinase 1/2 activity, with increased levels of the cell-cycle inhibitor p27(kip1) protein and with dephosphorylation of PLCgamma1, a substrate of DEP-1, the human homologue of r-PTPeta. Finally, the growth of xenograft tumors induced in athymic mice by anaplastic thyroid carcinoma ARO cells transduced with the Ad-r-PTPeta virus was drastically reduced. These data suggest that gene therapy based on restoration of PTPeta function has potential in the treatment of human thyroid malignant neoplasias.

In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the his IE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, L-histidinol:NAD+ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxyl-terminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps.

Expression of the galectin-1 (L-14-I) gene, elevated in most differentiated and transformed cell lines, has been studied in cell hybrid systems. Fusion of L-14-I nonproducing rat liver differentiated FAO cells with dedifferentiated rat liver BRL3A cells leads to extinction of liver-specific gene expression while L-14-I mRNA levels remain high. Interspecific hybrids produced by fusion of tumorigenic human osteosarcoma 143TK- with FAO cells show loss of both differentiated functions and tumorigenic phenotype and activation of the FAO L-14-I alleles. Increased expression of rat L-14-I alleles was also observed in human osteosarcoma x rat thyroid cells transient heterokaryons. The data presented here show that expression of the L-14-I gene is subject to dominant positive control and that it correlates with loss of differentiation-specific functions, but it is independent from tumorigenicity. L-14-I activation in FAO cells is achieved by treatment with 5-azacytidine. This result suggests that DNA demethylation is responsible or a prerequisite for L-14-I activation in hybrids.

Background: There is mounting evidence of the presence of chronic stress among children during primary school: girls and boys under the age of 15 years often experience anxiety, irritability and sleeping problems with negative consequences on scholastic climate and the spread of bullying and dropping out of school. The promotion of emotion regulation within school environment through innovative didactic methodologies represents a valuable tool for teachers and parents to reduce emotional distress and associated risk behaviours and to promote wellbeing. Aim: Our research aims to explore the psychological and biological consequences of teaching emotional training in an experimental group of Italian Primary School children. Methods: A sample of pupils (81 children aged between 6 and 8) was divided into an experimental group (33 subjects) and a control group (30 subjects). A further advanced group of 18 subjects, who have experienced the method in the previous school year, was also included. The experimental study lasted one school year (from October 2021 to May 2022). The following psychological tests were administered to all groups: TEC (Test of Emotion Comprehension) to measure the children's different emotional abilities and the Projective test (PT) 'A person in the rain', to identify the coping skills of children in a stressful condition. Morning salivary cortisol, IL-6 and TNF-alpha assays were conducted in all three groups. Psychological and biological tests were administered at the beginning of the study and at the end of the study. Results: The MR-Anova model for TEC score showed that there was not a significant group effect [Fgroup = 2.24, p = 0.114]. Pairwise comparisons showed that mean score significantly increased only in the Experimental group (pB < 0.001) and at the end of the project there was a significant difference between Experimental group and Control group (pB = 0.012). The mean score of PT test increased significantly from baseline to the end of the project for the Experimental group (pB < 0.001) and for the Advanced group (pB = 0.004). At the end of the project, there were significant differences between the Experimental group and the Control group (pB = 0.004) and between the Advanced group and the Control group (pB < 0.001). Salivary cortisol analysis revealed a significant effect between subjects [Fgroup = 9.66; p < 0.001] and significant effects within subjects with the main effect of the time [Ftime = 35.41; p < 0.001] and the significant interaction “time x group” [Ftimexgroup = 3.38; p = 0.040]. Pairwise comparisons showed that cortisol levels decreased significantly over time only in the Experimental group (pB < 0.001). Regarding to IL-6 levels, there was not a significant effect between subjects [Fgroups = 0.0481; p = 0.953]. The mean level decreased significantly for each group from baseline to post project (pB < 0.001). With respect to TNF-alpha levels, the mean levels decreased over time for all groups (pB = 0.006 for Experimental group; pB < 0.001 either for the Advanced or Control group). Conclusion: the results documented in the experimental groups who experienced didactics of emotion for at least one school year show a significant increase in children's ability to cope with reality, stress and anxiety, and an improvement of their emotional competence. Meanwhile, a significant reduction in the amount of salivary cortisol was observed in the experimental group at the end of the scholastic year; meantime a stable reduced amount of salivary cortisol in advanced group throughout the project was also observed. These findings show that an intervention through an emotional education program is able to regulate interpersonal skills and the stress axis response.

Glioblastoma, the most common and heterogeneous tumor affecting brain parenchyma, is dismally characterized by a very poor prognosis. Thus, the search of new, more effective treatments is a vital need. Here, we will review the druggable epigenetic features of glioblastomas that are, indeed, currently explored in preclinical studies and in clinical trials for the development of more effective, personalized treatments. In detail, we will review the studies that have led to the identification of epigenetic signatures, IDH mutations, MGMT gene methylation, histone modification alterations, H3K27 mutations, and epitranscriptome landscapes of glioblastomas, in each case discussing the corresponding targeted therapies and their potential efficacy. Finally, we will emphasize how recent technological improvements permit to routinely investigate many glioblastoma epigenetic biomarkers in clinical practice, further enforcing the hope that personalized drugs, targeting specific epigenetic features, could be in future a therapeutic option for selected patients.

Long-chain polyphosphates inhibit SARS-CoV-2 infection by targeting a host receptor and a viral RNA polymerase., Polyphosphates versus SARS-CoV-2 Long-chain, inorganic polyphosphates (polyPs), which are found in many cells in the blood, have cytoprotective and antiviral activities, particularly against HIV-1 infection. Ferrucci et al. tested the effects of polyPs of various lengths on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. Molecular docking and binding analyses showed that polyPs bound to the host receptor ACE2, which facilitates viral entry, and a viral RNA polymerase required for replication. Both proteins underwent proteasomal degradation in cells incubated with polyP120, the optimal species tested, resulting in inhibition of SARS-CoV-2 replication and a reduced inflammatory response. Given that polyPs have low toxicity, these results suggest that their potential therapeutic use should be further explored., Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO43−) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano– LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2–infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

Simple Summary Cancer is a group of diseases characterized by abnormal cell growth with a high potential to invade other tissues. Genetic abnormalities and epigenetic alterations found in tumors can be due to high levels of DNA damage and repair. These can be transmitted to daughter cells, which assuming other alterations as well, will generate heterogeneous and complex populations. Deciphering this complexity represents a central point for understanding the molecular mechanisms of cancer and its therapy. Here, we summarize the genomic and epigenomic events that occur in cancer and discuss novel approaches to analyze the epigenetic complexity of cancer cell populations. Abstract Cancer evolution is associated with genomic instability and epigenetic alterations, which contribute to the inter and intra tumor heterogeneity, making genetic markers not accurate to monitor tumor evolution. Epigenetic changes, aberrant DNA methylation and modifications of chromatin proteins, determine the “epigenome chaos”, which means that the changes of epigenetic traits are randomly generated, but strongly selected by deterministic events. Disordered changes of DNA methylation profiles are the hallmarks of all cancer types, but it is not clear if aberrant methylation is the cause or the consequence of cancer evolution. Critical points to address are the profound epigenetic intra- and inter-tumor heterogeneity and the nature of the heterogeneity of the methylation patterns in each single cell in the tumor population. To analyze the methylation heterogeneity of tumors, new technological and informatic tools have been developed. This review discusses the state of the art of DNA methylation analysis and new approaches to reduce or solve the complexity of methylated alleles in DNA or cell populations.

Background Fabry disease (FD) is a rare X-linked disease caused by mutations in GLA gene with consequent lysosomal accumulation of globotriaosylceramide (Gb3). Women with FD often show highly heterogeneous symptoms that can manifest from mild to severe phenotype. Main body The phenotypic variability of the clinical manifestations in heterozygous women with FD mainly depends on the degree and direction of inactivation of the X chromosome. Classical approaches to measure XCI skewness might be not sufficient to explain disease manifestation in women. In addition to unbalanced XCI, allele-specific DNA methylation at promoter of GLA gene may influence the expression levels of the mutated allele, thus impacting the onset and the outcome of FD. In this regard, analyses of DNA methylation at GLA promoter, performed by approaches allowing distinction between mutated and non-mutated allele, may be much more informative. The aim of this review is to critically evaluate recent literature articles addressing the potential role of DNA methylation in the context of FD. Although up to date relatively few works have addressed this point, reviewing all pertinent studies may help to evaluate the importance of DNA methylation analysis in FD and to develop new research and technologies aimed to predict whether the carrier females will develop symptoms. Conclusions Relatively few studies have addressed the complexity of DNA methylation landscape in FD that remains poorly investigated. The hope for the future is that ad hoc and ultradeep methylation analyses of GLA gene will provide epigenetic signatures able to predict whether pre-symptomatic female carriers will develop symptoms thus helping timely interventions.

The bidirectional microbiota&ndash, gut&ndash, brain axis has raised increasing interest over the past years in the context of health and disease, but there is a lack of information on molecular mechanisms underlying this connection. We hypothesized that change in microbiota composition may affect brain epigenetics leading to long-lasting effects on specific brain gene regulation. To test this hypothesis, we used Zebrafish (Danio Rerio) as a model system. As previously shown, treatment with high doses of probiotics can modulate behavior in Zebrafish, causing significant changes in the expression of some brain-relevant genes, such as BDNF and Tph1A. Using an ultra-deep targeted analysis, we investigated the methylation state of the BDNF and Tph1A promoter region in the brain and gut of probiotic-treated and untreated Zebrafishes. Thanks to the high resolution power of our analysis, we evaluated cell-to-cell methylation differences. At this resolution level, we found slight DNA methylation changes in probiotic-treated samples, likely related to a subgroup of brain and gut cells, and that specific DNA methylation signatures significantly correlated with specific behavioral scores.

DNA methylation is a heritable epigenetic mark that plays a key role in regulating gene expression. Mathematical modeling has been extensively applied to unravel the regulatory mechanisms of this process. In this study, we aimed to investigate DNA methylation by performing a high-depth analysis of particular loci, and by subsequent modeling of the experimental results. In particular, we performed an in-deep DNA methylation profiling of two genomic loci surrounding the transcription start site of the D-Aspartate Oxidase and the D-Serine Oxidase genes in different samples (n = 51). We found evidence of cell-to-cell differences in DNA methylation status. However, these cell differences were maintained between different individuals, which indeed showed very similar DNA methylation profiles. Therefore, we hypothesized that the observed pattern of DNA methylation was the result of a dynamic balance between DNA methylation and demethylation, and that this balance was identical between individuals. We hence developed a simple mathematical model to test this hypothesis. Our model reliably captured the characteristics of the experimental data, suggesting that DNA methylation and demethylation work together in determining the methylation state of a locus. Furthermore, our model suggested that the methylation status of neighboring cytosines plays an important role in this balance.

Background Programmed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as d-serine and d-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of d-amino acids are found. We recently demonstrated that Ddo, the d-aspartate degradation gene, is actively demethylated to ultimately reduce d-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate d-ser levels during brain development has been poorly investigated to date. Methods We performed comprehensive ultradeep DNA methylation and hydroxymethylation profiling along with mRNA expression and HPLC-based d-amino acids level analyses of genes controlling the mammalian brain levels of d-serine and d-aspartate. DNA methylation changes occurring in specific cerebellar cell types were also investigated. We conducted high coverage targeted bisulfite sequencing by next-generation sequencing and single-molecule bioinformatic analysis. Results We report consistent spatiotemporal modifications occurring at the Dao gene during neonatal development in a specific brain region (the cerebellum) and within specific cell types (astrocytes) for the first time. Dynamic demethylation at two specific CpG sites located just downstream of the transcription start site was sufficient to strongly activate the Dao gene, ultimately promoting the complete physiological degradation of cerebellar d-serine a few days after mouse birth. High amount of 5′-hydroxymethylcytosine, exclusively detected at relevant CpG sites, strongly evoked the occurrence of an active demethylation process. Conclusion The present investigation demonstrates that robust and selective demethylation of two CpG sites is associated with postnatal activation of the Dao gene and consequent removal of d-serine within the mouse cerebellum. A single-molecule methylation approach applied at the Dao locus promises to identify different cell-type compositions and functions in different brain areas and developmental stages.

The tendency of individual CpG sites to be methylated is distinctive, non-random and well-regulated throughout the genome. We investigated the structural and spatial factors influencing CpGs methylation by performing an ultra-deep targeted methylation analysis on human, mouse and zebrafish genes. We found that methylation is not a random process and that closer neighboring CpG sites are more likely to share the same methylation status. Moreover, if the distance between CpGs increases, the degree of co-methylation decreases. We set up a simulation model to analyze the contribution of both the intrinsic susceptibility and the distance effect on the probability of a CpG to be methylated. Our finding suggests that the establishment of a specific methylation pattern follows a universal rule that must take into account of the synergistic and dynamic interplay of these two main factors: the intrinsic methylation susceptibility of specific CpG and the nucleotide distance between two CpG sites. © 2019 Elsevier Inc.

The spatio-temporal regulation of genes involved in the synthesis and degradation of D-serine and D-aspartate such as serine racemase (SR), D-amino acid oxidase (DAO), G72 and D-aspartate oxidase (DDO), play pivotal roles in determining the correct levels of these D-amino acids in the human brain. Here we provide a comprehensive analysis of mRNA expression and DNA methylation status of these genes in post-mortem samples from hippocampus, dorsolateral prefrontal cortex, and cerebellum from patients with schizophrenia and non-psychiatric controls. DNA methylation analysis was performed at an ultradeep level, measuring individual epialleles frequency by single molecule approach. Differential CpG methylation and expression was detected across different brain regions, although no significant correlations were found with diagnosis. G72 showed the highest CpG and non-CpG methylation degree, which may explain the repression of G72 transcription in the brain regions considered here. Conversely, in line with the sustained SR mRNA expression in the analyzed areas, very low methylation levels were detected at this gene’s regulatory regions. Furthermore, for DAO and DDO, our single-molecule methylation approach demonstrated that analysis of epiallele distribution was able to detect differences in DNA methylation representing area-specific methylation signatures, which are likely not detectable with targeted or genome-wide classic methylation analyses.

It is long acknowledged that the N-methyl d-aspartate receptor co-agonist, d-serine, plays a crucial role in several N-methyl d-aspartate receptor-mediated physiological and pathological processes, including schizophrenia. Besides d-serine, another free d-amino acid, d-aspartate, is involved in the activation of N-methyl d-aspartate receptors acting as an agonist of this receptor subclass, and is abundantly detected in the developing human brain. Based on the hypothesis of N-methyl d-aspartate receptor hypofunction in the pathophysiology of schizophrenia and considering the ability of d-aspartate and d-serine to stimulate N-methyl d-aspartate receptor-dependent transmission, in the present work we assessed the concentration of these two d-amino acids in the post-mortem dorsolateral prefrontal cortex and hippocampus of patients with schizophrenia and healthy subjects. Moreover, in this cohort of post-mortem brain samples we investigated the spatiotemporal variations of d-aspartate and d-serine. Consistent with previous work, we found that d-aspartate content was selectively decreased by around 30% in the dorsolateral prefrontal cortex, but not in the hippocampus, of schizophrenia-affected patients, compared to healthy subjects. Interestingly, such selective reduction was associated to greater (around 25%) cortical activity of the enzyme responsible for d-aspartate catabolism, d-aspartate oxidase. Conversely, no significant changes were found in the methylation state and transcription of DDO gene in patients with schizophrenia, compared to control individuals, as well as in the expression levels of serine racemase, the major enzyme responsible for d-serine biosynthesis, which also catalyzes aspartate racemization. These results reveal the potential involvement of altered d-aspartate metabolism in the dorsolateral prefrontal cortex as a factor contributing to dysfunctional N-methyl d-aspartate receptor-mediated transmission in schizophrenia., NMDA receptor: Enzyme breaks down ion channel activator in schizophrenic brain Altered metabolism of an amino acid activator of ion channels in the brain could explain dysfunctional nerve signaling in schizophrenia. Researchers in Italy led by Alessandro Usiello from Ceinge Biotecnologie Avanzate and Loredano Pollegioni from the University of Insubria measured the levels of two amino acids—D-aspartate and D-serine—in post-mortem tissues taken from two brain regions of patients with and without schizophrenia. Both amino acids activate the N-methyl D-aspartate receptor, which is known to be less active in people with schizophrenia. The researchers found a mild increase in D-serine levels but a major decrease in D-aspartate in the schizophrenia patients’ dorsolateral prefrontal cortex (DLPFC), a memory and reasoning part of the brain, but not in the hippocampus. They also documented a greater activity of the enzyme responsible for D-aspartate breakdown in the DLPFC.

DNA methylation is often analyzed by reporting the average methylation degree of each cytosine. In this study, we used a single molecule methylation analysis in order to look at the methylation conformation of individual molecules. Using D-aspartate oxidase as a model gene, we performed an in-depth methylation analysis through the developmental stages of three different mouse tissues (brain, lung, and gut), where this gene undergoes opposite methylation destiny. This approach allowed us to track both methylation and demethylation processes at high resolution. The complexity of these dynamics was markedly simplified by introducing the concept of methylation classes (MCs), defined as the number of methylated cytosines per molecule, irrespective of their position. The MC concept smooths the stochasticity of the system, allowing a more deterministic description. In this framework, we also propose a mathematical model based on the Markov chain. This model aims to identify the transition probability of a molecule from one MC to another during methylation and demethylation processes. The results of our model suggest that: 1) both processes are ruled by a dominant class of phenomena, namely, the gain or loss of one methyl group at a time; and 2) the probability of a single CpG site becoming methylated or demethylated depends on the methylation status of the whole molecule at that time.

We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases. We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.

The endogenous NMDA receptor (NMDAR) agonist d-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral d-aspartate levels is due to the concomitant onset of d-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic d-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of d-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free d-aspartate. SIGNIFICANCE STATEMENT: The enzyme d-aspartate oxidase (DDO) catalyzes the degradation of the NMDA receptor agonist, d-aspartate. In the brain, DDO is expressed only during postnatal life, thus reducing the embryonic storage of d-aspartate and keeping this d-amino acid at low levels during adulthood. Although the presence of DDO in mammals is long established, its biological role in the brain and the mechanism regulating its expression are still unclear. Here, we found that Ddo promoter demethylation enables the postnatal expression of Ddo. Moreover, persistent suppression of Ddo expression leads to persistent spillover of extracellular d-aspartate and produces precocious cell death in the mouse brain, thus suggesting a key role for DDO in preventing early neurodegeneration triggered by excessive NMDA receptor stimulation. The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that D-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of D-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate.

We used subtractive library screening to identify the changes that occur in gene expression during thyroid cell neoplastic transformation. Complementary DNA from normal thyroid cells (HTC 2) was subtracted from a complementary DNA library constructed from a human thyroid papillary carcinoma cell line. The library was screened for genes upregulated in human thyroid papillary carcinoma cell line cells, and several cDNA clones were isolated. One of these clones has a sirtuin core and high homology with the human silent information regulator protein family. This clone, designated ‘SIR-T8’, was overexpressed in human thyroid carcinoma cell lines and tissues, but not in adenomas. The human SIR-T8 protein has a molecular weight of 39 kDa and is primarily located in the cytoplasm under the nuclear membrane. The SIR-T8 gene is located on chromosome 17q25-1. British Journal of Cancer (2002) 86, 917–923. DOI: 10.1038/sj/bjc/6600156 www.bjcancer.com © 2002 Cancer Research UK

Modifications at the N-terminal tails of nucleosomal histones a re re quired for effici ent transcription in vivo. W e a nalyzed how H3 histone methylation and demethylation cont rol e xpression of estrog en-responsive g enes and s how t hat a DNA-bound e strog en r ecep tor directs transcription b y p articipa t ing i n be n ding chromati n t o contact the RNA polyme rase II re crui ted t o t he p r omote r. This process i s driven by r eceptor-targeted demethyl atio n o f H 3 l ysine 9 at both enhancer and p romoter sites and is achieved by activation of re sident LSD1 demethylase. Localized d emethylation produces hydrogen peroxide, which modifies the s urrounding DNA and recruits 8-oxog uanine–DNA glycosylase 1and topoisomeraseIIb, t riggering chromatin and DNA c onfo rmational c hanges that are e ssential for e st rogen-induced t ranscr iption. O ur data show a s trategy t hat uses c ontrolled D NA damage and repair to guide prod uctive tra n scription.

Several permanent cell lines of epithelial origin are able to grow in culture in the absence of serum components. It was originally proposed by Temin et al. (1972) that such behaviour was dependent on the production of mitogenic factors (MSA or Multiplication Stimulating Activity) . The most studied system is a rat liver cell line BRL 3A isolated by Coon (1968) . Subsequent studies demonstrated that MSA is the rat equivalent of the insulin like growth factor II (IGF-II) (Rechler et al., 1985), but also showed that the ability of this cell line to grow in serum-free medium is independent of the synthesis and secretion of this polypeptide (Nissley et al., 1977) . We have been investigating this system mainly to understand the biological role and the expression and regulation of the IGF-II gene (Chiariotti et al., 1988). In the course of screening several cDNA libraries derived from the BRL 3A cell line we accidentally isolated some cDNA clones abundantly represented in the stable mRNA population, but not related to IGF-II. Subsequent characterization of these clones lead to the discovery that this mRNA code for a protein of molecular weight 22,800 belonging to the superfamily of ras-related genes (Bucci et al., 1988). In the present study we report some aspects of the organization, structure and expression of this novel putative GTP-binding protein.

We have determined the complete nucleotide sequence of the histidine operons of Escherichia coli and of Salmonella typhimurium. This structural information enabled us to investigate the expression and organization of the histidine operon. The proteins coded by each of the putative histidine cistrons were identified by subcloning appropriate DNA fragments and by analyzing the polypeptides synthesized in minicells. A structural comparison of the gene products was performed. The histidine messenger RNA molecules produced in vivo and the internal transcription initiation sites were identified by Northern blot analysis and S1 nuclease mapping. A comparative analysis of the different transcriptional and translational control elements within the two operons reveals a remarkable preservation for most of them except for the intercistronic region between the first (hisG) and second (hisD) structural genes and for the rho-independent terminator of transcription at the end of the operon. Overall, the operon structure is very compact and its expression appears to be regulated at several levels.

A new member of the ras genes superfamily was isolated from a cDNA library derived from a rat liver cell line (BRL-3A). The predicted 201 amino acids ras-like protein shows 30-35% homology with other members of the ras and ras-related gene products so far described. Conserved features include the GTP-binding and hydrolysis domains and the carboxyl terminal cysteine residues. A protein of the expected size (Mr 23,000) was synthesized in an in vitro transcription-translation system. The BRL-ras gene is present in single copy in the rat genome and is ubiquitously expressed at high levels in all tissues and cell lines examined.

The PATZ1 gene encoding a POZ/AT-hook/Kruppel zinc finger (PATZ) transcription factor, is considered a cancer-related gene because of its loss or misexpression in human neoplasias. As for other POZ/domain and Kruppel zinc finger (POK) family members, the transcriptional activity of PATZ is due to the POZ-mediated oligomer formation, suggesting that it might be not a typical transactivator but an architectural transcription factor, thus functioning either as activator or as repressor depending on the presence of proteins able to interact with it. Therefore, to better elucidate PATZ function, we searched for its molecular partners. By yeast two-hybrid screenings, we found a specific interaction between PATZ and BCL6, a human oncogene that plays a key role in germinal center (GC) derived neoplasias. We demonstrate that PATZ and BCL6 interact in germinal center-derived B lymphoma cells, through the POZ domain of PATZ. Moreover, we show that PATZ is able to bind the BCL6 regulatory region, where BCL6 itself acts as a negative regulator, and to contribute to negatively modulate its activity. Consistently, disruption of one or both Patz1 alleles in mice causes focal expansion of thymus B cells, in which BCL6 is up-regulated. This phenotype was almost completely rescued by crossing Patz1(+/-) with Bcl6(+/-) mice, indicating a key role for Bcl6 expression in its development. Finally, a significant number of Patz1 knock-out mice (both heterozygous and homozygous) also develop BCL6-expressing lymphomas. Therefore, the disruption of one or both Patz1 alleles may favor lymphomagenesis by activating the BCL6 pathway.

The high-mobility group A (HMGA) nonhistone chromosomal proteins HMGA1 and HMGA2 play a role in determining chromatin structure and in regulating the transcription of several genes. High levels of these proteins are characteristic of rapidly dividing cells in embryonic tissue and in tumors. The aim of this study was to determine the role of HMGA1 and HMGA2 throughout mouse spermatogenesis. Northern blot analysis and immunocytochemistry showed HMGA1 and HMGA2 expression during the progression from spermatocyte to spermatid. Interestingly, Western blot analysis with antibodies against the HMGA1 gene product revealed only the HMG1c isoform (27 kDa) in the testis; HMGA1a and HMGA1b were undetectable. These three isoforms are encoded by the HMGA1 gene through alternative splicing. Finally, few spermatids and complete absence of spermatozoa were observed in the testes of HMGA2-null mice, which suggests that the HMGA2 gene plays a critical role in male fertility.

Increasing evidence points to a role for dysfunctional glutamate N-methyl-D-aspartate receptor (NMDAR) neurotransmission in schizophrenia. D-aspartate is an atypical amino acid that activates NMDARs through binding to the glutamate site on GluN2 subunits. D-aspartate is present in high amounts in the embryonic brain of mammals and rapidly decreases after birth, due to the activity of the enzyme D-aspartate oxidase (DDO). The agonistic activity exerted by D-aspartate on NMDARs and its neurodevelopmental occurrence make this D-amino acid a potential mediator for some of the NMDAR-related alterations observed in schizophrenia. Consistently, substantial reductions of D-aspartate and NMDA were recently observed in the postmortem prefrontal cortex of schizophrenic patients. Here we show that DDO mRNA expression is increased in prefrontal samples of schizophrenic patients, thus suggesting a plausible molecular event responsible for the D-aspartate imbalance previously described. To investigate whether altered D-aspartate levels can modulate schizophrenia-relevant circuits and behaviors, we also measured the psychotomimetic effects produced by the NMDAR antagonist, phencyclidine, in Ddo knockout mice (Ddo(-)(/-)), an animal model characterized by tonically increased D-aspartate levels since perinatal life. We show that Ddo(-/-) mice display a significant reduction in motor hyperactivity and prepulse inhibition deficit induced by phencyclidine, compared with controls. Furthermore, we reveal that increased levels of D-aspartate in Ddo(-/-) animals can significantly inhibit functional circuits activated by phencyclidine, and affect the development of cortico-hippocampal connectivity networks potentially involved in schizophrenia. Collectively, the present results suggest that altered D-aspartate levels can influence neurodevelopmental brain processes relevant to schizophrenia.