Maurizio D’Esposito

Epigenetic alteration of microRNAs in DNMT3B-mutated patients of ICF syndrome

Immunodeficiency, Centromeric region instability, Facial anomalies (ICF; OMIM #242860) syndrome, due to mutations in the DNMT3B gene, is characterized by inheritance of aberrant patterns of DNA methylation and heterochromatin defects. Patients show variable agammaglobulinemia and a reduced number of T cells, making them prone to infections and death before adulthood. Other variable symptoms include facial dysmorphism, growth and mental retardation. Despite the recent advances in identifying the dysregulated genes, the molecular mechanisms, which underlie the altered gene expression causing ICF phenotype complexity, are not well understood. Held the recently-shown tight correlation between epigenetics and microRNAs (miRNAs), we searched for miRNAs regulated by DNMT3B activity, comparing cell lines from ICF patients with those from healthy individuals. We observe that eighty-nine miRNAs, some of which involved in immune function, development and neurogenesis, are dysregulated in ICF (LCLs) compared to wild-type cells. Significant DNA hypomethylation of miRNA CpG islands was not observed in cases of miRNA up-regulation in ICF cells, suggesting a more subtle effect of DNMT3B deficiency on their regulation; however, a modification of histone marks, especially H3K27 and H3K4 trimethylation, and H4 acetylation, was observed concomitantly with changes in microRNA expression. Functional correlation between miRNA and mRNA expression of their targets allow us to suppose a regulation either at mRNA level or at protein level. These results provide a better understanding of how DNA methylation and histone code interact to regulate the class of microRNA genes and enable us to predict molecular events possibly contributing to ICF condition.

Lessons from two human chromatin diseases, ICF syndrome and Rett syndrome

Spatial organisation of DNA into chromatin profoundly affects gene expression and function. The recent association of genes controlling chromatin structure to human pathologies resulted in a better comprehension of the interplay between regulation and function. Among many chromatin disorders we will discuss Rett and immunodeficiency, centromeric instability and facial anomalies (ICF) syndromes. Both diseases are caused by defects related to DNA methylation machinery, with Rett syndrome affecting the transduction of the repressive signal from the methyl CpG binding protein prototype, MeCP2, and ICF syndrome affecting the genetic control of DNA methylation, by the DNA methyltransferase DNMT3B. Rather than listing survey data, our aim is to highlight how a deeper comprehension of gene regulatory web may arise from studies of such pathologies. We also maintain that fundamental studies may offer chances for a therapeutic approach focused on these syndromes, which, in turn, may become paradigmatic for this increasing class of diseases.

MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel Mecp2-Deficient Embryonic Stem Cell Reporter Line

The X-linked Mecp2 is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell in vitro differentiation. Therefore, we modified murine Mecp2 deficient (Mecp2 −/y) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in Mecp2 deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of Mecp2 wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found Mecp2 deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of Mecp2 deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state.

Defective Sphingosine-1-phosphate metabolism is a druggable target in Huntington's disease.

Huntington’s disease is characterized by a complex and heterogeneous pathogenic profile. Studies have shown that disturbance in lipid homeostasis may represent a critical determinant in the progression of several neurodegenerative disorders. The recognition of perturbed lipid metabolism is only recently becoming evident in HD. In order to provide more insight into the nature of such a perturbation and into the effect its modulation may have in HD pathology, we investigated the metabolism of Sphingosine-1-phosphate (S1P), one of the most important bioactive lipids, in both animal models and patient samples. Here, we demonstrated that S1P metabolism is significantly disrupted in HD even at early stage of the disease and importantly, we revealed that such a dysfunction represents a common denominator among multiple disease models ranging from cells to humans through mouse models. Interestingly, the in vitro anti-apoptotic and the pro-survival actions seen after modulation of S1P-metabolizing enzymes allows this axis to emerge as a new druggable target and unfolds its promising therapeutic potential for the development of more effective and targeted interventions against this incurable condition.

ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing

Abstract Hypomorphic mutations in DNA-methyltransferase DNMT3B cause majority of the rare disorder Immunodeficiency, Centromere instability and Facial anomalies syndrome cases (ICF1). By unspecified mechanisms, mutant-DNMT3B interferes with lymphoid-specific pathways resulting in immune response defects. Interestingly, recent findings report that DNMT3B shapes intragenic CpG-methylation of highly-transcribed genes. However, how the DNMT3B-dependent epigenetic network modulates transcription and whether ICF1-specific mutations impair this process remains unknown. We performed a transcriptomic and epigenomic study in patient-derived B-cell lines to investigate the genome-scale effects of DNMT3B dysfunction. We highlighted that altered intragenic CpG-methylation impairs multiple aspects of transcriptional regulation, like alternative TSS usage, antisense transcription and exon splicing. These defects preferentially associate with changes of intragenic H3K4me3 and at lesser extent of H3K27me3 and H3K36me3. In addition, we highlighted a novel DNMT3B activity in modulating the self-regulatory circuit of sense-antisense pairs and the exon skipping during alternative splicing, through interacting with RNA molecules. Strikingly, altered transcription affects disease relevant genes, as for instance the memory-B cell marker CD27 and PTPRC genes, providing us with biological insights into the ICF1-syndrome pathogenesis. Our genome-scale approach sheds light on the mechanisms still poorly understood of the intragenic function of DNMT3B and DNA methylation in gene expression regulation.

Impairment of blood-brain barrier is an early event in R6/2 mouse model of Huntington Disease

Blood-brain barrier (BBB) breakdown, due to the concomitant disruption of the tight junctions (TJs), normally required for the maintenance of BBB function, and to the altered transport of molecules between blood and brain and vice-versa, has been suggested to significantly contribute to the development and progression of different brain disorders including Huntington’s disease (HD). Although the detrimental consequence the BBB breakdown may have in the clinical settings, the timing of its alteration remains elusive for many neurodegenerative diseases. In this study we demonstrate for the first time that BBB disruption in HD is not confined to established symptoms, but occurs early in the disease progression. Despite the obvious signs of impaired BBB permeability were only detectable in concomitance with the onset of the disease, signs of deranged TJs integrity occur precociously in the disease and precede the onset of overt symptoms. To our perspective this finding may add a new dimension to the horizons of pathological mechanisms underlying this devastating disease, however much remains to be elucidated for understanding how specific BBB drug targets can be approached in the future.

MECP2 Duplication Syndrome: Evidence of Enhanced Oxidative Stress. A Comparison with Rett Syndrome

Rett syndrome (RTT) and MECP2 duplication syndrome (MDS) are neurodevelopmental disorders caused by alterations in the methyl-CpG binding protein 2 (MECP2) gene expression. A relationship between MECP2 loss-of-function mutations and oxidative stress has been previously documented in RTT patients and murine models. To date, no data on oxidative stress have been reported for the MECP2 gain-of-function mutations in patients with MDS. In the present work, the pro-oxidant status and oxidative fatty acid damage in MDS was investigated (subjects n = 6) and compared to RTT (subjects n = 24) and healthy condition (subjects n = 12). Patients with MECP2 gain-of-function mutations showed increased oxidative stress marker levels (plasma non-protein bound iron, intraerythrocyte non-protein bound iron, F2-isoprostanes, and F4-neuroprostanes), as compared to healthy controls (P ≤ 0.05). Such increases were similar to those observed in RTT patients except for higher plasma F2-isoprostanes levels (P < 0.0196). Moreover, plasma levels of F2-isoprostanes were significantly correlated (P = 0.0098) with the size of the amplified region. The present work shows unique data in patients affected by MDS. For the first time MECP2 gain-of-function mutations are indicated to be linked to an oxidative damage and related clinical symptoms overlapping with those of MECP2 loss-of-function mutations. A finely tuned balance of MECP2 expression appears to be critical to oxidative stress homeostasis, thus shedding light on the relevance of the redox balance in the central nervous system integrity.

Abnormal N-glycosylation pattern for brain nucleotide pyrophosphatase-5 (NPP-5) in Mecp2-mutant murine models of Rett syndrome.

Neurological disorders can be associated with protein glycosylation abnormalities. Rett syndrome is a devastating genetic brain disorder, mainly caused by de novo loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. Although its pathogenesis appears to be closely associated with a redox imbalance, no information on glycosylation is available. Glycoprotein detection strategies (i.e., lectin-blotting) were applied to identify target glycosylation changes in the whole brain of Mecp2 mutant murine models of the disease. Remarkable glycosylation pattern changes for a peculiar 50kDa protein, i.e., the N-linked brain nucleotide pyrophosphatase-5 were evidenced, with decreased N-glycosylation in the presymptomatic and symptomatic mutant mice. Glycosylation changes were rescued by selected brain Mecp2 reactivation. Our findings indicate that there is a causal link between the amount of Mecp2 and the N-glycosylation of NPP-5.

The Role of DNMT3B Mutations in the Pathogenesis of ICF Syndrome

DNA methylation plays an important role in epigenetic signaling, having an impact on gene regulation, chromatin structure, development, and disease. The human genetic disease, called immunodeficiency, centromere instability, facial abnormalities (ICF) syndrome, is one example of the consequence of the impaired setting and maintenance of proper DNA methylation patterns. Here, we review the key properties of the mammalian de novo DNA methyltransferase DNMT3B, whose dysfunction is responsible for the ICF molecular phenotype, and take a closer look at the effects of the reported mutations on its methyltransferase activity. Moreover, we focus on the central role of DNMT3B in the epigenetic signaling network and the key questions still unsolved in the field, such as how this enzyme is targeted to specific genomic regions, leaving some others unmethylated, and how the DNA methylation pattern is modified during development and in response to environmental cues. The emerging models are multifaceted, involving both the intrinsic properties of DNMT3B and the influence of its interaction partners. In this regard, the ICF mutations provide us with a valuable model to understand the molecular properties of this DNA methyltransferase. The current knowledge and the proposed hypothesis about these topics will be summarized.

Non-invasive Prenatal Diagnosis: An Epigenetic Approach to the Detection of Common Fetal Chromosome Disorders by Analysis of Maternal Blood Samples

We describe results obtained by Methylated DNA immunoprecipitation (MeDiP) in combination with high resolution oligonucleotide microarray analysis to enable chromosome-wide identification of DNA methylation patterns in a high-throughput approach. The methylation patterns of chromosomes 13, 18, 21 and the sex chromosomes in female peripheral blood, CVS and placental DNA have been analyzed extensively. Such markers will now be tested with respect to their utility for non-invasive/minimally invasive prenatal diagnosis (NIPD/MIPD) of trisomy 13, 18, and 21 as well as sex chromosome abnormalities (initially XXY, XYY, XXX and XO).