Pancreatic cancer is a very aggressive disease with dismal prognosis; peculiar is the tumor microenvironment characterized by an extensive fibrotic stroma, which favors rapid tumor progression. We previously reported that pancreatic cancer patients have a selective Th2 skew in the anti-carcinoembryonic antigen (CEA) CD4(+) T cell immunity, which correlates with the presence of a predominant GATA-3(+) tumor lymphoid infiltrate. This has negative effects in both effective anti-tumor immunity and further favoring fibrinogenesis. Aim of this study was to evaluate whether the Th2 polarization of CEA-specific CD4(+) T cells from pancreatic cancer patients is stable or can be reverted by immunomodulating cytokines.

Long noncoding RNAs (lncRNAs) are major regulators of physiological and disease-related gene expression, particularly in the central nervous system. Dysregulated lncRNA expression has been documented in several human cancers, and their tissue-specificity makes them attractive candidates as diagnostic/prognostic biomarkers and/or therapeutic agents. Here we show that linc-NeD125, which we previously characterized as a neuronal-induced lncRNA, is significantly overexpressed in Group 4 medulloblastomas (G4 MBs), the largest and least well characterized molecular MB subgroup. Mechanistically, linc-NeD125 is able to recruit the miRNA-induced silencing complex (miRISC) and to directly bind the microRNAs miR-19a-3p, miR-19b-3p and miR-106a-5p. Functionally, linc-NeD125 acts as a competing endogenous RNA (ceRNA) that, sequestering the three miRNAs, leads to de-repression of their targets CDK6, MYCN, SNCAIP, and KDM6A, which are major driver genes of G4 MB. Accordingly, linc-NeD125 downregulation reduces G4 cell proliferation. Moreover, we also provide evidence that linc-NeD125 ectopic expression in the aggressive Group 3 MB cells attenuates their proliferation, migration and invasion.This study unveils the first lncRNA-based ceRNA network in central nervous system tumours and provides a novel molecular circuit underlying the enigmatic Group 4 medulloblastoma.

Exon skipping is an effective strategy for the treatment of many Duchenne Muscular Dystrophy (DMD) mutations. Natural exon skipping observed in several DMD cases can help in identifying novel therapeutic tools. Here, we show a DMD study case where the lack of a splicing factor (Celf2a), which results in exon skipping and dystrophin rescue, is due to a maternally inherited trans-generational epigenetic silencing. We found that the study case and his mother express a repressive long non-coding RNA, DUXAP8, whose presence correlates with silencing of the Celf2a coding region. We also demonstrate that DUXAP8 expression is lost upon cell reprogramming and that, upon induction of iPSCs into myoblasts, Celf2a expression is recovered leading to the loss of exon skipping and loss of dystrophin synthesis. Finally, CRISPR/Cas9 inactivation of the splicing factor Celf2a was proven to ameliorate the pathological state in other DMD backgrounds establishing Celf2a ablation or inactivation as a novel therapeutic approach for the treatment of Duchenne Muscular Dystrophy.

Pancreatic cancer is a very aggressive disease characterized by a marked desmoplasia with a predominant Th2 (GATA-3+) over Th1 (T-bet+) lymphoid infiltrate. We found that the ratio of GATA-3+/T-bet+ tumor-infiltrating lymphoid cells is an independent predictive marker of patient survival. Patients surgically treated for stage IB/III disease with a ratio inferior to the median value had a statistically significant prolonged overall survival, implying an active role for Th2 responses in disease progression. Thymic stromal lymphopoietin (TSLP), which favors Th2 cell polarization through myeloid dendritic cell (DC) conditioning, was secreted by cancer-associated fibroblasts (CAFs) after activation with tumor-derived tumor necrosis factor α and interleukin 1β. TSLP-containing supernatants from activated CAFs induced in vitro myeloid DCs to up-regulate the TSLP receptor (TSLPR), secrete Th2-attracting chemokines, and acquire TSLP-dependent Th2-polarizing capability in vitro. In vivo, Th2 chemoattractants were expressed in the tumor and in the stroma, and TSLPR-expressing DCs were present in the tumor stroma and in tumor-draining but not in nondraining lymph nodes. Collectively, this study identifies in pancreatic cancer a cross talk between tumor cells and CAFs, resulting in a TSLP-dependent induction of Th2-type inflammation which associates with reduced patient survival. Thus, blocking TSLP production by CAFs might help to improve prognosis in pancreatic cancer.

Copper is prevalent in coastal ecosystems due to its use as an algaecide and as an anti-fouling agent on ship hulls. Alteromonas spp. have previously been shown to be some of the early colonizers of copper-based anti-fouling paint but little is known about the mechanisms they use to overcome this initial copper challenge. The main models of copper resistance include the Escherichia coli chromosome-based Cue and Cus systems; the plasmid-based E. coli Pco system; and the plasmid-based Pseudomonas syringae Cop system. These were all elucidated from strains isolated from copper-rich environments of agricultural and/or enteric origin. In this work, copper resistance assays demonstrated the ability of Alteromonas macleodii strains CUKW and KCC02 to grow at levels lethal to other marine bacterial species. A custom database of Hidden Markov Models was designed based on proteins from the Cue, Cus, and Cop/Pco systems and used to identify potential copper resistance genes in CUKW and KCC02. Comparative genomic analyses with marine bacterial species and bacterial species isolated from copper-rich environments demonstrated that CUKW and KCC02 possess genetic elements of all systems, oftentimes with multiple copies, distributed throughout the chromosome and mega-plasmids. In particular, two copies of copA (the key player in cytoplasmic detoxification), each with its own apparent MerR-like transcriptional regulator, occur on a mega-plasmid, along with multiple copies of Pco homologs. Genes from both systems were induced upon exposure to elevated copper levels (100 μM- 3 mM). Genomic analysis identified one of the merR-copA clusters occurs on a genomic island (GI) within the plasmid, and comparative genomic analysis found that either of the merR-copA clusters, which also includes genes coding for a cupredoxin domain-containing protein and an isoprenylcysteine methyltransferase, occurs on a GI across diverse bacterial species. These genomic findings combined with the ability of CUKW and KCC02 to grow in copper-challenged conditions are couched within the context of the genome flexibility of the Alteromonas genus.

Mutations in MECP2 cause several neurological disorders of which Rett syndrome (RTT) represents the best-defined condition. Although mainly working as a transcriptional repressor, MeCP2 is a multifunctional protein revealing several activities, the involvement of which in RTT remains obscure. Besides being mainly localized in the nucleus, MeCP2 associates with the centrosome, an organelle from which primary cilia originate. Primary cilia function as "sensory antennae" protruding from most cells, and a link between primary cilia and mental illness has recently been reported. We herein demonstrate that MeCP2 deficiency affects ciliogenesis in cultured cells, including neurons and RTT fibroblasts, and in the mouse brain. Consequently, the cilium-related Sonic Hedgehog pathway, which is essential for brain development and functioning, is impaired. Microtubule instability participates in these phenotypes that can be rescued by HDAC6 inhibition together with the recovery of RTT-related neuronal defects. Our data indicate defects of primary cilium as a novel pathogenic mechanism that by contributing to the clinical features of RTT might impact on proper cerebellum/brain development and functioning, thus providing a novel therapeutic target.

Changes in the epigenetic regulation of gene expression have a central role in evolution. Here, we extensively profiled a panel of human, chimpanzee, gorilla, orangutan, and macaque lymphoblastoid cell lines (LCLs), using ChIP-seq for five histone marks, ATAC-seq and RNA-seq, further complemented with whole genome sequencing (WGS) and whole genome bisulfite sequencing (WGBS). We annotated regulatory elements (RE) and integrated chromatin contact maps to define gene regulatory architectures, creating the largest catalog of RE in primates to date. We report that epigenetic conservation and its correlation with sequence conservation in primates depends on the activity state of the regulatory element. Our gene regulatory architectures reveal the coordination of different types of components and highlight the role of promoters and intragenic enhancers (gE) in the regulation of gene expression. We observe that most regulatory changes occur in weakly active gE. Remarkably, novel human-specific gE with weak activities are enriched in human-specific nucleotide changes. These elements appear in genes with signals of positive selection and human acceleration, tissue-specific expression, and particular functional enrichments, suggesting that the regulatory evolution of these genes may have contributed to human adaptation.

Long Interspersed Nuclear Elements-1s (L1s) are transposable elements that constitute most of the genome's transcriptional output yet have still largely unknown functions. Here we show that L1s are required for proper mouse brain corticogenesis operating as regulatory long non-coding RNAs. They contribute to the regulation of the balance between neuronal progenitors and differentiation, the migration of post-mitotic neurons and the proportions of different cell types. In cortical cultured neurons, L1 RNAs are mainly associated to chromatin and interact with the Polycomb Repressive Complex 2 (PRC2) protein subunits enhancer of Zeste homolog 2 (Ezh2) and suppressor of zeste 12 (Suz12). L1 RNA silencing influences PRC2's ability to bind a portion of its targets and the deposition of tri-methylated histone H3 (H3K27me3) marks. Our results position L1 RNAs as crucial signalling hubs for genome-wide chromatin remodelling, enabling the fine-tuning of gene expression during brain development and evolution.

Lamins (A/C and B) are major constituents of the nuclear lamina (NL). Structurally conserved lamina-associated domains (LADs) are formed by genomic regions that contact the NL. Lamins are also found in the nucleoplasm, with a yet unknown function. Here we map the genome-wide localization of lamin B1 in an euchromatin-enriched fraction of the mouse genome and follow its dynamics during the epithelial-to-mesenchymal transition (EMT). Lamin B1 associates with actively expressed and open euchromatin regions, forming dynamic euchromatin lamin B1-associated domains (eLADs) of about 0.3 Mb. Hi-C data link eLADs to the 3D organization of the mouse genome during EMT and correlate lamin B1 enrichment at topologically associating domain (TAD) borders with increased border strength. Having reduced levels of lamin B1 alters the EMT transcriptional signature and compromises the acquisition of mesenchymal traits. Thus, during EMT, the process of genome reorganization in mouse involves dynamic changes in eLADs.

Endoribonucleases participate in almost every step of eukaryotic RNA metabolism, acting either as degradative or biosynthetic enzymes. We previously identified the founding member of the Eukaryotic EndoU ribonuclease family, whose components display unique biochemical features and are flexibly involved in important biological processes, such as ribosome biogenesis, tumorigenesis and viral replication. Here we report the discovery of the CG3303 gene product, which we named DendoU, as a novel family member in Drosophila. Functional characterisation revealed that DendoU is essential for Drosophila viability and nervous system activity. Pan-neuronal silencing of dendoU resulted in fly immature phenotypes, highly reduced lifespan and dramatic motor performance defects. Neuron-subtype selective silencing showed that DendoU is particularly important in cholinergic circuits. At the molecular level, we unveiled that DendoU is a positive regulator of the neurodegeneration-associated protein dTDP-43, whose downregulation recapitulates the ensemble of dendoU-dependent phenotypes. This interdisciplinary work, which comprehends in silico, in vitro and in vivo studies, unveils a relevant role for DendoU in Drosophila nervous system physio-pathology and highlights that DendoU-mediated neurotoxicity is, at least in part, contributed by dTDP-43 loss-of-function.

microRNA abundance has been shown to depend on the amount of the microprocessor components or, in some cases, on specific auxiliary co-factors. In this paper, we show that the FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, associated with familial forms of Amyotrophic Lateral Sclerosis (ALS), contributes to the biogenesis of a specific subset of microRNAs. Among them, species with roles in neuronal function, differentiation and synaptogenesis were identified. We also show that FUS/TLS is recruited to chromatin at sites of their transcription and binds the corresponding pri-microRNAs. Moreover, FUS/TLS depletion leads to decreased Drosha level at the same chromatin loci. Limited FUS/TLS depletion leads to a reduced microRNA biogenesis and we suggest a possible link between FUS mutations affecting nuclear/cytoplasmic partitioning of the protein and altered neuronal microRNA biogenesis in ALS pathogenesis.

Although heterochromatin is enriched with repressive traits, it is also actively transcribed, giving rise to large amounts of noncoding RNAs. Although these RNAs are responsible for the formation and maintenance of heterochromatin, little is known about how their transcription is regulated. Here, we show that the Snail1 transcription factor represses mouse pericentromeric transcription, acting through the H3K4 deaminase LOXL2. Since Snail1 plays a key role in the epithelial-to-mesenchymal transition (EMT), we analyzed the regulation of heterochromatin transcription in this process. At the onset of EMT, one of the major structural heterochromatin proteins, HP1α, is transiently released from heterochromatin foci in a Snail1/LOXL2-dependent manner, concomitantly with a downregulation of major satellite transcription. Moreover, preventing the downregulation of major satellite transcripts compromised the migratory and invasive behavior of mesenchymal cells. We propose that Snail1 regulates heterochromatin transcription through LOXL2, thus creating the favorable transcriptional state necessary for completing EMT.

Understanding general principles that safeguard cellular identity should reveal critical insights into common mechanisms underlying specification of varied cell types. Here, we show that SUMO modification acts to stabilize cell fate in a variety of contexts. Hyposumoylation enhances pluripotency reprogramming in vitro and in vivo, increases lineage transdifferentiation, and facilitates leukemic cell differentiation. Suppressing sumoylation in embryonic stem cells (ESCs) promotes their conversion into 2-cell-embryo-like (2C-like) cells. During reprogramming to pluripotency, SUMO functions on fibroblastic enhancers to retain somatic transcription factors together with Oct4, Sox2, and Klf4, thus impeding somatic enhancer inactivation. In contrast, in ESCs, SUMO functions on heterochromatin to silence the 2C program, maintaining both proper H3K9me3 levels genome-wide and repression of the Dux locus by triggering recruitment of the sumoylated PRC1.6 and Kap/Setdb1 repressive complexes. Together, these studies show that SUMO acts on chromatin as a glue to stabilize key determinants of somatic and pluripotent states.

Chromatin regulatory factors (CRFs), are known to be involved in tumorigenesis in several cancer types. Nevertheless, the molecular mechanisms through which driver alterations of CRFs cause tumorigenesis remain unknown. Here, we developed a CRFs Oncomodules Discovery approach, which mines several sources of cancer genomics and perturbaomics data. The approach prioritizes sets of genes significantly miss-regulated in primary tumors (oncomodules) bearing mutations of driver CRFs. We applied the approach to eleven TCGA tumor cohorts and uncovered oncomodules potentially associated to mutations of five driver CRFs in three cancer types. Our results revealed, for example, the potential involvement of the mTOR pathway in the development of tumors with loss-of-function mutations of MLL2 in head and neck squamous cell carcinomas. The experimental validation that MLL2 loss-of-function increases the sensitivity of cancer cell lines to mTOR inhibition lends further support to the validity of our approach. The potential oncogenic modules detected by our approach may guide experiments proposing ways to indirectly target driver mutations of CRFs.

CDKL5 is a kinase with relevant functions in correct neuronal development and in the shaping of synapses. A decrease in its expression or activity leads to a severe neurodevelopmental condition known as CDKL5 deficiency disorder (CDD). CDD arises from CDKL5 mutations that lie in the coding region of the gene. However, the identification of a SNP in the CDKL5 5'UTR in a patient with symptoms consistent with CDD, together with the complexity of the CDKL5 transcript leader, points toward a relevant translational regulation of CDKL5 expression with important consequences in physiological processes as well as in the pathogenesis of CDD. We performed a bioinformatics and molecular analysis of the 5'UTR of CDKL5 to identify translational regulatory features. We propose an important role for structural cis-acting elements, with the involvement of the eukaryotic translational initiation factor eIF4B. By evaluating both cap-dependent and cap-independent translation initiation, we suggest the presence of an IRES supporting the translation of CDKL5 mRNA and propose a pathogenic effect of the C>T -189 SNP in decreasing the translation of the downstream protein.

This study was designed to evaluate the impact of laparoscopic rectal resection on short-term postoperative morbidity and costs.

Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such '2-cell-like-cells' (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

Pluripotent stem cells are thought of as a surrogate of early developmental stages that sustain the capacity to generate all cell types in the body, thereby constituting an invaluable tool to address the mechanisms underlying cellular plasticity. In the mouse, cells resembling totipotent 2-cell-stage embryos (2-cell-like cells) arise at a very low frequency in embryonic stem cell (ESC) cultures. However, the extent to which these early-embryonic-like cells recapitulate the molecular features of the early embryo is unclear. Here, we have undertaken a characterization of some of the metabolic features of early-embryonic-like cells in culture. Our data indicate that early-embryonic-like cells exhibit decreased glycolytic and respiratory activity, lower levels of reactive oxygen species and increased glucose uptake, suggesting a shift of the metabolic programme during 2-cell-like cell reprogramming. Accordingly, we find that 2-cell-like cells can be induced by defined metabolites. Thus, in addition to their transcriptional and chromatin features, 2-cell-like cells recapitulate some of the metabolic features of their in vivo counterpart. Altogether, our work underscores a distinct metabolic state of early-embryonic-like cells and identifies compounds that can induce their emergence in vitro.