RNAi machinery cooperates with SWI/SNF complexes in nucleosome positioning at Transcriptional Start Sites

In Eukaryotes, Argonaute (AGO) proteins have a well-established role in the cytoplasm in post-transcriptional regulation of gene expression in association with different classes of small non-coding RNAs (sRNAs). In plants and yeast, it has been demonstrated that AGO proteins exert a role in the epigenetic regulation of chromatin modifications. Furthermore, AGO2 protein acts also in the nuclei of human cell lines and emerging literature reports that upon the transfection of sRNAs complementary to non-coding promoter transcripts, AGO2 is recruited on target promoters. Previous results in our laboratory demonstrated that AGO2 and SWI/SNF have a physical interaction, which is independent of RNA or DNA, in human cell lines. As SWI/SNF is the major chromatin-remodelling complex in human, these data suggest that AGO2 might participate in the regulation of chromatin plasticity. In eukaryotes, the proper organization of chromatin is essential for the control of gene expression and is achieved through the concerted activity of histone modifications, DNA methylation and nucleosome positioning. The focus of the present thesis has been the development of relevant bioinformatics pipelines for data processing, analysis and visualization, all aiming at dissection of the functional significance of the AGO2-SWI/SNF interaction. Interestingly, this bioinformatics pipeline allowed me to identify a novel class of nuclear AGO2-bound sRNAs arising from genomic regions 150 nt around the Transcription Start Sites (TSS) bound by SWI/SNF (swiRNAs). Furthermore, swiRNAs present a Dicer-dependent processing and show an involvement in nucleosome occupancy at nucleosome +1. These data represent the first description of a molecular mechanism through which AGO2 is involved in nucleosome positioning in mammalian cells

Massive NGS data analysis reveals hundreds of potential novel gene fusions in human cell lines

Background: Gene fusions derive from chromosomal rearrangements and the resulting chimeric transcripts are often endowed with oncogenic potential. Furthermore, they serve as diagnostic tools for the clinical classification of cancer subgroups with different prognosis and, in some cases, they can provide specific drug targets. So far, many efforts have been carried out to study gene fusion events occurring in tumor samples. In recent years, the availability of a comprehensive Next Generation Sequencing dataset for all the existing human tumor cell lines has provided the opportunity to further investigate these data in order to identify novel and still uncharacterized gene fusion events. Results: In our work, we have extensively reanalyzed 935 paired-end RNA-seq experiments downloaded from "The Cancer Cell Line Encyclopedia" repository, aiming at addressing novel putative cell-line specific gene fusion events in human malignancies. The bioinformatics analysis has been performed by the execution of four different gene fusion detection algorithms. The results have been further prioritized by running a bayesian classifier which makes an in silico validation. The collection of fusion events supported by all of the predictive softwares results in a robust set of ∼ 1,700 in-silico predicted novel candidates suitable for downstream analyses. Given the huge amount of data and information produced, computational results have been systematized in a database named LiGeA. The database can be browsed through a dynamical and interactive web portal, further integrated with validated data from other well known repositories. Taking advantage of the intuitive query forms, the users can easily access, navigate, filter and select the putative gene fusions for further validations and studies. They can also find suitable experimental models for a given fusion of interest. Conclusions: We believe that the LiGeA resource can represent not only the first compendium of both known and putative novel gene fusion events in the catalog of all of the human malignant cell lines, but it can also become a handy starting point for wet-lab biologists who wish to investigate novel cancer biomarkers and specific drug targets

Missense mutations of NCPAG gene affect calving ease in Piedmontese cattle: preliminary evidences

A previous genome scan on 323 Piedmontese individuals identified a cluster of 13 SNPs significantly associated with direct calving ease and centred on the three genes LAP3, LCORL and NCAPG in chromosome 6. We investigated missense mutations affecting calving ease in Piedmontese cattle in the identified region using sequences from the whole exome in eight Piedmontese individuals chosen from the extremes of the direct calving ease estimated breeding values distribution for this trait. The present study has not found missense variants in LAP3 and LCORL, while two were identified on NCAPG by three different variant calling methods. Other gene candidates in the same region harbour missense mutations, such as PPM1K, PKD2, SPP1 and MEPE, but both SIFT analysis and chi-square test on frequency of alleles make us hypothesise that NCAPG is the single gene responsible for the trait variation. The two SNPs on NCAPG are in complete linkage disequilibrium in our samples; therefore, further investigations are needed in order to discriminate their role

ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites.

Argonaute (AGO) proteins have a well-established role in post-transcriptional regulation of gene expression as key component of the RNA silencing pathways. Recent evidence involves AGO proteins in mammalian nuclear processes such as transcription and splicing, though the mechanistic aspects of AGO nuclear functions remain largely elusive. Here, by SILAC-based interaction proteomics, we identify the chromatin-remodelling complex SWI/SNF as a novel AGO2 interactor in human cells. Moreover, we show that nuclear AGO2 is loaded with a novel class of Dicer-dependent short RNAs (sRNAs), that we called swiRNAs, which map nearby the Transcription Start Sites (TSSs) bound by SWI/SNF. The knock-down of AGO2 decreases nucleosome occupancy at the first nucleosome located downstream of TSSs in a swiRNA-dependent manner. Our findings indicate that in human cells AGO2 binds SWI/SNF and a novel class of sRNAs to establish nucleosome occupancy on target TSSs

Conditional Inactivation of Limbic Neuropeptide Y-1 Receptors Increases Vulnerability to Diet-Induced Obesity in Male Mice

NPY and its Y1 cognate receptor (Y1R) have been shown to be involved in the regulation of stress, anxiety, depression and energy homeostasis. We previously demonstrated that conditional knockout of Npy1r gene in the excitatory neurons of the forebrain of adolescent male mice (Npy1rrfb mice) decreased body weight growth and adipose tissue and increased anxiety. In the present study, we used the same conditional system to examine whether the targeted disruption of the Npy1r gene in limbic areas might affect susceptibility to obesity and associated disorders during adulthood in response to a 3-week high-fat diet (HFD) regimen. We demonstrated that following HFD exposure, Npy1rrfb male mice showed increased body weight, visceral adipose tissue, and blood glucose levels, hyperphagia and a dysregulation of calory intake as compared to control Npy1r2lox mice. These results suggest that low expression of Npy1r in limbic areas impairs habituation to high caloric food and causes high susceptibility to diet-induced obesity and glucose intolerance in male mice, uncovering a specific contribution of the limbic Npy1r gene in the dysregulation of the eating/satiety balance

Clinical-Genetic Features Influencing Disability in Spastic Paraplegia Type 4: A Cross-sectional Study by the Italian DAISY Network

Background and objectives: Hereditary spastic paraplegias (HSPs) are a group of inherited rare neurologic disorders characterized by length-dependent degeneration of the corticospinal tracts and dorsal columns, whose prominent clinical feature is represented by spastic gait. Spastic paraplegia type 4 (SPG4, SPAST-HSP) is the most common form. We present both clinical and molecular findings of a large cohort of patients, with the aim of (1) defining the clinical spectrum of SPAST-HSP in Italy; (2) describing their molecular features; and (3) assessing genotype-phenotype correlations to identify features associated with worse disability. Methods: A cross-sectional retrospective study with molecular and clinical data collected in an anonymized database was performed. Results: A total of 723 Italian patients with SPAST-HSP (58% men) from 316 families, with a median age at onset of 35 years, were included. Penetrance was 97.8%, with men showing higher Spastic Paraplegia Rating Scale (SPRS) scores (19.67 ± 12.58 vs 16.15 ± 12.61, p = 0.009). In 26.6% of patients with SPAST-HSP, we observed a complicated phenotype, mainly including intellectual disability (8%), polyneuropathy (6.7%), and cognitive decline (6.5%). Late-onset cases seemed to progress more rapidly, and patients with a longer disease course displayed a more severe neurologic disability, with higher SPATAX (3.61 ± 1.46 vs 2.71 ± 1.20, p < 0.001) and SPRS scores (22.63 ± 11.81 vs 12.40 ± 8.83, p < 0.001). Overall, 186 different variants in the SPAST gene were recorded, of which 48 were novel. Patients with SPAST-HSP harboring missense variants displayed intellectual disability (14.5% vs 4.4%, p < 0.001) more frequently, whereas patients with truncating variants presented more commonly cognitive decline (9.7% vs 2.6%, p = 0.001), cerebral atrophy (11.2% vs 3.4%, p = 0.003), lower limb spasticity (61.5% vs 44.5%), urinary symptoms (50.0% vs 31.3%, p < 0.001), and sensorimotor polyneuropathy (11.1% vs 1.1%, p < 0.001). Increasing disease duration (DD) and abnormal motor evoked potentials (MEPs) were also associated with increased likelihood of worse disability (SPATAX score>3). Discussion: The SPAST-HSP phenotypic spectrum in Italian patients confirms a predominantly pure form of HSP with mild-to-moderate disability in 75% of cases, and slight prevalence of men, who appeared more severely affected. Early-onset cases with intellectual disability were more frequent among patients carrying missense SPAST variants, whereas patients with truncating variants showed a more complicated disease. Both longer DD and altered MEPs are associated with worse disability

Parma consensus statement on metabolic disruptors

A multidisciplinary group of experts gathered in Parma Italy for a workshop hosted by the University of Parma, May 16–18, 2014 to address concerns about the potential relationship between environmental metabolic disrupting chemicals, obesity and related metabolic disorders. The objectives of the workshop were to: 1. Review findings related to the role of environmental chemicals, referred to as “metabolic disruptors”, in obesity and metabolic syndrome with special attention to recent discoveries from animal model and epidemiology studies; 2. Identify conclusions that could be drawn with confidence from existing animal and human data; 3. Develop predictions based on current data; and 4. Identify critical knowledge gaps and areas of uncertainty. The consensus statements are intended to aid in expanding understanding of the role of metabolic disruptors in the obesity and metabolic disease epidemics, to move the field forward by assessing the current state of the science and to identify research needs on the role of environmental chemical exposures in these diseases. We propose broadening the definition of obesogens to that of metabolic disruptors, to encompass chemicals that play a role in altered susceptibility to obesity, diabetes and related metabolic disorders including metabolic syndrome

Dissecting the Gene Expression Networks Associated with Variations in the Major Components of the Fatty Acid Semimembranosus Muscle Profile in Large White Heavy Pigs

To date, high-throughput technology such as RNA-sequencing has been successfully applied in livestock sciences to investigate molecular networks involved in complex traits, such as meat quality. Pork quality depends on several organoleptic, technological, and nutritional characteristics, and it is also influenced by the fatty acid (FA) composition of intramuscular fat (IMF). To explore the molecular networks associated with different IMF FA compositions, the Semimembranosus muscle (SM) from two groups of Italian Large White (ILW) heavy pigs divergent for SM IMF content was investigated using transcriptome analysis. After alignment and normalization, the obtained gene counts were used to perform the Weighted Correlation Network Analysis (WGCNA package in R environment). Palmitic and palmitoleic contents showed association with the same gene modules, comprising genes significantly enriched in autophagy, mitochondrial fusion, and mitochondrial activity. Among the key genes related to these FAs, we found TEAD4, a gene regulating mitochondrial activity that seems to be a promising candidate for further studies. On the other hand, the genes comprised in the modules associated with the IMF contents of oleic, n-6, and n-3 polyunsaturated FAs (PUFAs) were significantly enriched in Mitogen-Activated Protein Kinase (MAPK) signaling, in agreement with previous studies suggesting that several MAPK players may have a primary role in regulating lipid deposition. These results give an insight into the molecular cascade associated with different IMF FA composition in ILW heavy pigs. Further studies are needed to validate the results and confirm whether some of the identified key genes may be effective candidates for pork quality