Wnt/ß-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged in vitro culture conditions. Several factors control mESC pluripotency, including Wnt/ß-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/ß-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/ß-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.

Meg3 Non-coding RNA Expression Controls Imprinting by Preventing Transcriptional Upregulation in cis.

Although many long non-coding RNAs (lncRNAs) are imprinted, their roles often remain unknown. The Dlk1-Dio3 domain expresses the lncRNA Meg3 and multiple microRNAs and small nucleolar RNAs (snoRNAs) on the maternal chromosome and constitutes an epigenetic model for development. The domain's Dlk1 (Delta-like-1) gene encodes a ligand that inhibits Notch1 signaling and regulates diverse developmental processes. Using a hybrid embryonic stem cell (ESC) system, we find that Dlk1 becomes imprinted during neural differentiation and that this involves transcriptional upregulation on the paternal chromosome. The maternal Dlk1 gene remains poised. Its protection against activation is controlled in cis by Meg3 expression and also requires the H3-Lys-27 methyltransferase Ezh2. Maternal Meg3 expression additionally protects against de novo DNA methylation at its promoter. We find that Meg3 lncRNA is partially retained in cis and overlaps the maternal Dlk1 in embryonic cells. Combined, our data evoke an imprinting model in which allelic lncRNA expression prevents gene activation in cis.

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

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.

The Large Phenotypic Spectrum of Fabry Disease Requires Graduated Diagnosis and Personalized Therapy: A Meta-Analysis Can Help to Differentiate Missense Mutations.

Fabry disease is caused by mutations in the GLA gene and is characterized by a large genotypic and phenotypic spectrum. Missense mutations pose a special problem for graduating diagnosis and choosing a cost-effective therapy. Some mutants retain enzymatic activity, but are less stable than the wild type protein. These mutants can be stabilized by small molecules which are defined as pharmacological chaperones. The first chaperone to reach clinical trial is 1-deoxygalactonojirimycin, but others have been tested in vitro. Residual activity of GLA mutants has been measured in the presence or absence of pharmacological chaperones by several authors. Data obtained from transfected cells correlate with those obtained in cells derived from patients, regardless of whether 1-deoxygalactonojirimycin was present or not. The extent to which missense mutations respond to 1-deoxygalactonojirimycin is variable and a reference table of the results obtained by independent groups that is provided with this paper can facilitate the choice of eligible patients. A review of other pharmacological chaperones is provided as well. Frequent mutations can have residual activity as low as one-fourth of normal enzyme in vitro. The reference table with residual activity of the mutants facilitates the identification of non-pathological variants.

ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells.

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.

ZFP57 recognizes multiple and closely spaced sequence motif variants to maintain repressive epigenetic marks in mouse embryonic stem cells.

Imprinting Control Regions (ICRs) need to maintain their parental allele-specific DNA methylation during early embryogenesis despite genome-wide demethylation and subsequent de novo methylation. ZFP57 and KAP1 are both required for maintaining the repressive DNA methylation and H3-lysine-9-trimethylation (H3K9me3) at ICRs. In vitro, ZFP57 binds a specific hexanucleotide motif that is enriched at its genomic binding sites. We now demonstrate in mouse embryonic stem cells (ESCs) that SNPs disrupting closely-spaced hexanucleotide motifs are associated with lack of ZFP57 binding and H3K9me3 enrichment. Through a transgenic approach in mouse ESCs, we further demonstrate that an ICR fragment containing three ZFP57 motif sequences recapitulates the original methylated or unmethylated status when integrated into the genome at an ectopic position. Mutation of Zfp57 or the hexanucleotide motifs led to loss of ZFP57 binding and DNA methylation of the transgene. Finally, we identified a sequence variant of the hexanucleotide motif that interacts with ZFP57 both in vivo and in vitro. The presence of multiple and closely located copies of ZFP57 motif variants emerges as a distinct characteristic that is required for the faithful maintenance of repressive epigenetic marks at ICRs and other ZFP57 binding sites.

Fabry_CEP: a tool to identify Fabry mutations responsive to pharmacological chaperones.

Fabry_CEP is a user-friendly web-application designed to help clinicians Choose Eligible Patients for the therapy with pharmacological chaperones. It provides a database and a predictive tool to evaluate the responsiveness of lysosomal alpha-galactosidase mutants to a small molecule drug, namely 1-Deoxy-galactonojirimycin. The user can introduce any missense/nonsense mutation in the coding sequence, learn whether it is has been tested and gain access to appropriate reference literature. In the absence of experimental data structural, functional and evolutionary analysis provides a prediction and the probability that a given mutation is responsive to the drug.

Identification and analysis of conserved pockets on protein surfaces.

BACKGROUND: The interaction between proteins and ligands occurs at pockets that are often lined by conserved amino acids. These pockets can represent the targets for low molecular weight drugs. In order to make the research for new medicines as productive as possible, it is necessary to exploit "in silico" techniques, high throughput and fragment-based screenings that require the identification of druggable pockets on the surface of proteins, which may or may not correspond to active sites. RESULTS: We developed a tool to evaluate the conservation of each pocket detected on the protein surface by CastP. This tool was named DrosteP because it recursively searches for optimal input sequences to be used to calculate conservation. DrosteP uses a descriptor of statistical significance, Poisson p-value, as a target to optimize the choice of input sequences. To benchmark DrosteP we used monomeric or homodimer human proteins with known 3D-structure whose active site had been annotated in UniProt. DrosteP is able to detect the active site with high accuracy because in 81% of the cases it coincides with the most conserved pocket. Comparing DrosteP with analogous programs is difficult because the outputs are different. Nonetheless we could assess the efficacy of the recursive algorithm in the identification of active site pockets by calculating conservation with the same input sequences used by other programs.We analyzed the amino-acid composition of conserved pockets identified by DrosteP and we found that it differs significantly from the amino-acid composition of non conserved pockets. CONCLUSIONS: Several methods for predicting ligand binding sites on protein surfaces, that combine 3D-structure and evolutionary sequence conservation, have been proposed. Any method relying on conservation mainly depends on the choice of the input sequences. DrosteP chooses how deeply distant homologs must be collected to evaluate conservation and thus optimizes the identification of active site pockets. Moreover it recognizes conserved pockets other than those coinciding with the sites annotated in UniProt that might represent useful druggable sites. The distinctive amino-acid composition of conserved pockets provides useful hints on the fundamental principles underlying protein-ligand interaction. AVAILABILITY: http://www.icb.cnr.it/project/drosteppy/

Genetic and epigenetic mutations affect the DNA binding capability of human ZFP57 in transient neonatal diabetes type 1.

In the mouse, ZFP57 contains three classical Cys2His2 zinc finger domains (ZF) and recognizes the methylated TGC(met)CGC target sequence using the first and the second ZFs. In this study, we demonstrate that the human ZFP57 (hZFP57) containing six Cys2His2 ZFs, binds the same methylated sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA interaction. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation.

Therapy of Fabry disease with pharmacological chaperones: from in silico predictions to in vitro tests.

BACKGROUND: Fabry disease is a rare disorder caused by a large variety of mutations in the gene encoding lysosomal alpha-galactosidase. Many of these mutations are unique to individual families. Fabry disease can be treated with enzyme replacement therapy, but a promising novel strategy relies on small molecules, so called "pharmacological chaperones", which can be administered orally. Unfortunately only 42% of genotypes respond to pharmacological chaperones. RESULTS: A procedure to predict which genotypes responsive to pharmacological chaperones in Fabry disease has been recently proposed. The method uses a position-specific substitution matrix to score the mutations. Using this method, we have screened public databases for predictable responsive cases and selected nine representative mutations as yet untested with pharmacological chaperones. Mutant lysosomal alpha galactosidases were produced by site directed mutagenesis and expressed in mammalian cells. Seven out of nine mutations responded to pharmacological chaperones. Nineteen other mutations that were tested with pharmacological chaperones, but were not included in the training of the predictive method, were gathered from literature and analyzed in silico. In this set all five mutations predicted to be positive were responsive to pharmacological chaperones, bringing the percentage of responsive mutations among those predicted to be positive and not used to train the classifier to 86% (12/14). This figure differs significantly from the percentage of responsive cases observed among all the Fabry mutants tested so far. CONCLUSIONS: In this paper we provide experimental support to an "in silico" method designed to predict missense mutations in the gene encoding lysosomal alpha galactosidase responsive to pharmacological chaperones. We demonstrated that responsive mutations can be predicted with a low percentage of false positive cases. Most of the mutations tested to validate the method were described in the literature as associated to classic or mild classic phenotype. The analysis can provide a guideline for the therapy with pharmacological chaperones supported by experimental results obtained in vitro. We are aware that our results were obtained in vitro and cannot be translated straightforwardly into benefit for patients, but need to be validated by clinical trials.

Prediction of the responsiveness to pharmacological chaperones: lysosomal human alpha-galactosidase, a case of study.

BACKGROUND: The pharmacological chaperones therapy is a promising approach to cure genetic diseases. It relies on substrate competitors used at sub-inhibitory concentration which can be administered orally, reach difficult tissues and have low cost. Clinical trials are currently carried out for Fabry disease, a lysosomal storage disorder caused by inherited genetic mutations of alpha-galactosidase. Regrettably, not all genotypes respond to these drugs. RESULTS: We collected the experimental data available in literature on the enzymatic activity of ninety-six missense mutants of lysosomal alpha-galactosidase measured in the presence of pharmacological chaperones. We associated with each mutation seven features derived from the analysis of 3D-structure of the enzyme, two features associated with their thermo-dynamic stability and four features derived from sequence alone. Structural and thermodynamic analysis explains why some mutants of human lysosomal alpha-galactosidase cannot be rescued by pharmacological chaperones: approximately forty per cent of the non responsive cases examined can be correctly associated with a negative prognostic feature. They include mutations occurring in the active site pocket, mutations preventing disulphide bridge formation and severely destabilising mutations. Despite this finding, prediction of mutations responsive to pharmacological chaperones cannot be achieved with high accuracy relying on combinations of structure- and thermodynamic-derived features even with the aid of classical and state of the art statistical learning methods.We developed a procedure to predict responsive mutations with an accuracy as high as 87%: the method scores the mutations by using a suitable position-specific substitution matrix. Our approach is of general applicability since it does not require the knowledge of 3D-structure but relies only on the sequence. CONCLUSIONS: Responsiveness to pharmacological chaperones depends on the structural/functional features of the disease-associated protein, whose complex interplay is best reflected on sequence conservation by evolutionary pressure. We propose a predictive method which can be applied to screen novel mutations of alpha galactosidase. The same approach can be extended on a genomic scale to find candidates for therapy with pharmacological chaperones among proteins with unknown tertiary structures.