Francesca Anselmi

Intragenic DNA methylation prevents spurious transcription initiation

In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.

Molecular modelling of S1 and S2 subunits of SARS coronavirus spike glycoprotein

Abstract The S1 and S2 subunits of the spike glycoprotein of the coronavirus which is responsible for the severe acute respiratory syndrome (SARS) have been modelled, even though the corresponding amino acid sequences were not suitable for tertiary structure predictions with conventional homology and/or threading procedures. An indirect search for a protein structure to be used as a template for 3D modelling has been performed on the basis of the genomic organisation similarity generally exhibited by coronaviruses. The crystal structure of Clostridium botulinum neurotoxin B appeared to be structurally adaptable to human and canine coronavirus spike protein sequences and it was successfully used to model the two subunits of SARS coronavirus spike glycoprotein. The overall shape and the surface hydrophobicity of the two subunits in the obtained models suggest the localisation of the most relevant regions for their activity.

Growth factor stimulation induces cell survival by c-Jun. ATF2-dependent activation of Bcl-XL.

Growth factor stimulation induces c-Jun-dependent survival of primary endothelial cells. However, the mechanism of c-Jun anti-apoptotic activity has not been identified. We here demonstrate that in response to growth factor treatment, primary human endothelial cells as well as mouse fibroblasts respond with an increased expression of c-Jun that forms a complex with ATF2. This complex activates the expression of the anti-apoptotic protein Bcl-XL. By site-directed mutagenesis experiments, we identified two AP-1-binding sites located within the proximal promoter of the Bcl-X gene. Site-directed mutagenesis demonstrated that these AP-1 sites are required for the transcriptional activation of the promoter. Chromatin immunoprecipitation experiments show that in response to growth factor treatment, the heterodimer c-Jun·ATF2 binds to these functional AP-1 sites. Silencing of either c-Jun or ATF2 demonstrated that both nuclear factors are required for the activation of the proximal Bcl-X promoter. Taken together, our experiments provide evidence that growth factor-independent signaling pathways converge in the formation of an active c-Jun·AFT2 dimer, which induces the expression of the anti-apoptotic factor Bcl-XL that mediates a pro-survival response.

TET1 is controlled by pluripotency-associated factors in ESCs and downmodulated by PRC2 in differentiated cells and tissues

Ten-eleven translocation (Tet) genes encode for a family of hydroxymethylase enzymes involved in regulating DNA methylation dynamics. Tet1 is highly expressed in mouse embryonic stem cells (ESCs) where it plays a critical role the pluripotency maintenance. Tet1 is also involved in cell reprogramming events and in cancer progression. Although the functional role of Tet1 has been largely studied, its regulation is poorly understood. Here we show that Tet1 gene is regulated, both in mouse and human ESCs, by the stemness specific factors Oct3/4, Nanog and by Myc. Thus Tet1 is integrated in the pluripotency transcriptional network of ESCs. We found that Tet1 is switched off by cell proliferation in adult cells and tissues with a consequent genome-wide reduction of 5hmC, which is more evident in hypermethylated regions and promoters. Tet1 downmodulation is mediated by the Polycomb repressive complex 2 (PRC2) through H3K27me3 histone mark deposition. This study expands the knowledge about Tet1 involvement in stemness circuits in ESCs and provides evidence for a transcriptional relationship between Tet1 and PRC2 in adult proliferating cells improving our understanding of the crosstalk between the epigenetic events mediated by these factors.

High-throughput single-base resolution mapping of RNA 2′-O-methylated residues

Abstract Functional characterization of the transcriptome requires tools for the systematic investigation of RNA post-transcriptional modifications. 2΄-O-methylation (2΄-OMe) of the ribose moiety is one of the most abundant post-transcriptional modifications of RNA, although its systematic analysis is difficult due to the lack of reliable high-throughput mapping methods. We describe here a novel high-throughput approach, named 2OMe-seq, that enables fast and accurate mapping at single-base resolution, and relative quantitation, of 2΄-OMe modified residues. We compare our method to other state-of-art approaches, and show that it achieves higher sensitivity and specificity. By applying 2OMe-seq to HeLa cells, we show that it is able to recover the majority of the annotated 2΄-OMe sites on ribosomal RNA. By performing knockdown of the Fibrillarin methyltransferase in mouse embryonic stem cells (ESCs) we show the ability of 2OMe-seq to capture 2΄-O-Methylation level variations. Moreover, using 2OMe-seq data we here report the discovery of 12 previously unannotated 2΄-OMe sites across 18S and 28S rRNAs, 11 of which are conserved in both human and mouse cells, and assigned the respective snoRNAs for all sites. Our approach expands the repertoire of methods for transcriptome-wide mapping of RNA post-transcriptional modifications, and promises to provide novel insights into the role of this modification.

c-ABL modulates MAP kinases activation downstream of VEGFR-2 signaling by direct phosphorylation of the adaptor proteins GRB2 and NCK1

Vascular Endothelial Growth Factor-A (VEGF-A) is a key molecule in normal and tumor angiogenesis. This study addresses the role of c-ABL as a novel downstream target of VEGF-A in primary Human Umbilical Vein Endothelial Cells (HUVEC). On the basis of immunoprecipitation experiments, in vitro kinase assay and RNA interference, we demonstrate that VEGF-A induces the c-ABL kinase activity through the VEGF Receptor-2/Phosphatidylinositol-3-Kinase pathway. By treating HUVEC with the specific tyrosine kinase inhibitor STI571 and over-expressing a dominant negative c-ABL mutant, we show that the VEGF-A-activated c-ABL reduces the amplitude of Mitogen-Activated Protein Kinases (ERK1/2, JNKs and p38) activation in a dose-dependent manner by a negative feedback mechanism. By analysis of the adaptor proteins NCK1 and GRB2 mutants we further show that the negative loop on p38 is mediated by c-ABL phosphorylation at tyrosine 105 of the adaptor protein NCK1, while the phosphorylation at tyrosine 209 of GRB2 down-modulates ERK1/2 and JNKs signaling. These findings suggest that c-ABL function is to establish a correct and tightly controlled response of endothelial cells to VEGF-A during the angiogenic process.

Normative Reference Values of Right Heart in Competitive Athletes: A Systematic Review and Meta-Analysis

Training-induced right ventricular (RV) enlargement is frequent in athletes. Unfortunately, RV dilatation is also a common phenotypic expression and one of the diagnostic criteria of arrhythmogenic RV cardiomyopathy (ARVC). The current echocardiographic reference values derived from the general population can overestimate the presence of RV dilatation in athletes. We performed a meta-analysis of the literature to derive the proper reference values for assessing RV enlargement in competitive athletes. We conducted systematic review of English-language studies in the MEDLINE, Scopus, and Cochrane databases investigating RV size and function by echocardiography and by cardiac magnetic resonance (CMR) in competitive athletes. In total, 6,806 and 740 competitive athletes were included for the echocardiographic and CMR quantification of the RV, respectively. In this review, we present normal reference values for RV size and function to be applied in competitive athletes according to the disciplines practiced. The reference ranges reported in this review suggest that physicians should be aware that application of the current recommendations for normal population could be misleading when evaluating athletes. We suggest using these normative reference values, obtained in competitive athletes, to avoid the potential for mistakenly concluding, in this specific population, that RV size or function are abnormal.

Atrial chamber remodelling in healthy pre-adolescent athletes engaged in endurance sports: A study with a longitudinal design. The CHILD study

AIMS Previous studies investigated the exercise-induced adaptation of left (LA) and right atrium (RA) in adults, but little is known about respective changes in the growing heart of children. We aimed to longitudinally investigate the effects of endurance training on biatrial remodelling in preadolescent athletes. METHODS AND RESULTS Ninety-four children (57 endurance athletes, 37 sedentary controls; mean age 10.8±0.2 and 10.2±0.2years, respectively) were evaluated at baseline and after 5months by ECG and by two-dimensional, three-dimensional (3D) and speckle-tracking echocardiography. Athletes were trained at least 10h/week. The resting heart rate was lower in athletes (p=0.046) and decreased further after training (p<0.0001). Neither athletes nor controls had ECG evidence for LA or RA enlargement. At baseline, indexed LA volumes did not differ between groups (p=0.14) but indexed RA dimensions were larger in athletes (p=0.007). After 5months, indexed LA volumes increased in athletes but not in controls (p<0.0001, p=0.29; respectively) while indexed RA volumes increased in both groups (p<0.0001, p=0.018; respectively). At the same time, slight differences in biatrial reservoir and contractile function were found either in athletes, as demonstrated by speckle-tracking echocardiography, but 3D-derived LA and RA ejection fraction remained stable in both groups. CONCLUSION Endurance training influences the growing heart of preadolescent athletes with an additive increase in biatrial size, suggesting that morphological adaptations can occur also in the early phases of the sports career. Training-induced remodelling was associated with a preserved biatrial function, supporting the hypothesis of a physiological remodelling.

RNA structure framework: automated transcriptome-wide reconstruction of RNA secondary structures from high-throughput structure probing data.

SUMMARY The rapidly increasing number of discovered non-coding RNAs makes the understanding of their structure a key feature toward a deeper comprehension of gene expression regulation. Various enzymatic- and chemically- based approaches have been recently developed to allow whole-genome studies of RNA secondary structures. Several methods have been recently presented that allow high-throughput RNA structure probing (CIRS-seq, Structure-seq, SHAPE-seq, PARS, etc.) and unbiased structural inference of residues within RNAs in their native conformation. We here present an analysis toolkit, named RNA Structure Framework (RSF), which allows fast and fully-automated analysis of high-throughput structure probing data, from data pre-processing to whole-transcriptome RNA structure inference. AVAILABILITY AND IMPLEMENTATION RSF is written in Perl and is freely available under the GPLv3 license from http://rsf.hugef-research.org. CONTACT salvatore.oliviero@hugef-torino.org SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Snai1 Promotes ESC Exit from the Pluripotency by Direct Repression of Self‐Renewal Genes

Although much is known about the pluripotency self‐renewal circuitry, the molecular events that lead embryonic stem cells (ESCs) exit from pluripotency and begin differentiation are largely unknown. We found that the zinc finger transcription factor Snai1, involved in gastrulation and epithelial‐mesenchymal transition, is already expressed in the inner cell mass of the preimplantation blastocysts. In ESCs, Snai1 does not respond to TGFβ or BMP4 signaling but it is induced by retinoic acid treatment, which induces the binding, on the Snai1 promoter, of the retinoid receptors RARγ and RXRα, the dissociation of the Polycomb repressor complex 2 which results in the decrease of H3K27me3, and the increase of histone H3K4me3. Snai1 mediates the repression of pluripotency genes by binding directly to the promoters of Nanog, Nr5a2, Tcl1, c‐Kit, and Tcfcp2l1. The transient activation of Snai1 in embryoid bodies induces the expression of the markers of all three germ layers. These results suggest that Snai1 is a key factor that triggers ESCs exit from the pluripotency state and initiate their differentiation processes. Stem Cells 2015;33:742–750