223 results on '"Settore BIO/16"'
Search Results
2. A minimal promoter region of Kit gene recapitulates mast cell differentiation in development, aging and inflammation
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Serena Fuda, Daniela F. Angelini, Ambra Colopi, Eugenia Guida, Angelo Onorato, Paola Grimaldi, Serena Travaglini, Emmanuele A. Jannini, and Susanna Dolci
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Aging ,FceRI ,Settore BIO/16 ,HFD ,Kit ,Gonad ,Developmental Biology ,Mast cell - Published
- 2023
3. The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots
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Teresa Giannattasio, Erika Testa, Ramona Palombo, Lidia Chellini, Flavia Franceschini, Álvaro Crevenna, Petko M. Petkov, Maria Paola Paronetto, and Marco Barchi
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Pharmacology ,Meiosis ,Settore BIO/16 ,Cellular and Molecular Neuroscience ,EWSR1 ,Molecular Medicine ,H3K4me3 ,Cell Biology ,SPO11β ,REC114 ,SPO11α ,Molecular Biology ,XY - Abstract
In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11β and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.
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- 2023
4. Endocannabinoid system and epigenetics in spermatogenesis and testicular cancer
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Marco Barchi, Eugenia Guida, Susanna Dolci, Pellegrino Rossi, and Paola Grimaldi
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paternal inheritance ,Settore BIO/16 ,endocannabinoid system ,spermatogenesis ,testicular germ cell tumor - Published
- 2023
5. The androgen receptor couples promoter recruitment of RNA processing factors to regulation of alternative polyadenylation at the 3' end of transcripts
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Cinzia Caggiano, Marco Pieraccioli, Consuelo Pitolli, Gabriele Babini, Dinghai Zheng, Bin Tian, Pamela Bielli, and Claudio Sette
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Settore BIO/16 - ANATOMIA UMANA ,Male ,Settore BIO/16 ,Tumor ,Settore BIO/11 ,Cleavage And Polyadenylation Specificity Factor ,Prostatic Neoplasms ,Castration-Resistant ,Polyadenylation ,Cell Line ,Androgen ,Prostatic Neoplasms, Castration-Resistant ,Cleavage Stimulation Factor ,Receptors, Androgen ,Cell Line, Tumor ,Receptors ,Benzamides ,Nitriles ,Phenylthiohydantoin ,Genetics ,Humans ,Cell Proliferation - Abstract
Prostate cancer (PC) relies on androgen receptor (AR) signaling. While hormonal therapy (HT) is efficacious, most patients evolve to an incurable castration-resistant stage (CRPC). To date, most proposed mechanisms of acquired resistance to HT have focused on AR transcriptional activity. Herein, we uncover a new role for the AR in alternative cleavage and polyadenylation (APA). Inhibition of the AR by Enzalutamide globally regulates APA in PC cells, with specific enrichment in genes related to transcription and DNA topology, suggesting their involvement in transcriptome reprogramming. AR inhibition selects promoter-distal polyadenylation sites (pAs) enriched in cis-elements recognized by the cleavage and polyadenylation specificity factor (CPSF) complex. Conversely, promoter-proximal intronic pAs relying on the cleavage stimulation factor (CSTF) complex are repressed. Mechanistically, Enzalutamide induces rearrangement of APA subcomplexes and impairs the interaction between CPSF and CSTF. AR inhibition also induces co-transcriptional CPSF recruitment to gene promoters, predisposing the selection of pAs depending on this complex. Importantly, the scaffold CPSF160 protein is up-regulated in CRPC cells and its depletion represses HT-induced APA patterns. These findings uncover an unexpected role for the AR in APA regulation and suggest that APA-mediated transcriptome reprogramming represents an adaptive response of PC cells to HT.
- Published
- 2022
6. Multi-color dSTORM microscopy in Hormad1-/spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure
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Lieke Koornneef, Johan A. Slotman, Esther Sleddens-Linkels, Wiggert A. van Cappellen, Marco Barchi, Attila Tóth, Joost Gribnau, Adriaan B. Houtsmuller, Willy M. Baarends, Developmental Biology, Erasmus MC other, Cell biology, and Pathology
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Male ,Mice, Knockout ,Cancer Research ,Microscopy ,Settore BIO/16 ,Synaptonemal Complex ,Knockout ,Cell Cycle Proteins ,DNA ,Recombinases ,Meiosis ,Mice ,SDG 3 - Good Health and Well-being ,Spermatocytes ,Genetics ,Animals ,Rad51 Recombinase ,Molecular Biology ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics - Abstract
Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.
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- 2022
7. MAPK activation drives male and female mouse teratocarcinomas from late primordial germ cells
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Eugenia Guida, Valentina Tassinari, Ambra Colopi, Federica Todaro, Valeriana Cesarini, Benedetto Jannini, Manuela Pellegrini, Flavia Botti, Gabriele Rossi, Pellegrino Rossi, Emmanuele A. Jannini, and Susanna Dolci
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Male ,Teratocarcinoma ,Mice ,Settore BIO/16 ,Cell Transformation, Neoplastic ,Germ Cells ,Ovary ,Testis ,BRaf V600E ,Oocytes ,Animals ,Female ,Cell Biology - Abstract
Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior.
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- 2022
8. Cannabis and male reproduction: impact on offspring via sperm epigenome
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Paola Grimaldi
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Settore BIO/16 ,biology ,Offspring ,media_common.quotation_subject ,Physiology ,General Medicine ,Cannabis ,Epigenome ,Reproduction ,biology.organism_classification ,Sperm ,media_common - Published
- 2022
9. Extending resolution within a single imaging frame
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Esley Torres-García, Raúl Pinto-Cámara, Alejandro Linares, Damián Martínez, Víctor Abonza, Eduardo Brito-Alarcón, Carlos Calcines-Cruz, Gustavo Valdés-Galindo, David Torres, Martina Jabloñski, Héctor H. Torres-Martínez, José L. Martínez, Haydee O. Hernández, José P. Ocelotl-Oviedo, Yasel Garcés, Marco Barchi, Rocco D’Antuono, Ana Bošković, Joseph G. Dubrovsky, Alberto Darszon, Mariano G. Buffone, Roberto Rodríguez Morales, Juan Manuel Rendon-Mancha, Christopher D. Wood, Armando Hernández-García, Diego Krapf, Álvaro H. Crevenna, and Adán Guerrero
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Settore BIO/16 ,Reading Frames ,Multidisciplinary ,Microscopy, Fluorescence ,General Physics and Astronomy ,Drugs, Generic ,General Chemistry ,General Biochemistry, Genetics and Molecular Biology ,Algorithms ,Imaging ,Fluorescent Dyes - Abstract
The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.
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- 2021
10. Non-Coding RNAs and Splicing Activity in Testicular Germ Cell Tumors
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Pellegrino Rossi, Marco Barchi, Paola Grimaldi, Susanna Dolci, and Pamela Bielli
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0301 basic medicine ,endocrine system ,R-loop ,Science ,Testicular Germ Cell Tumor ,Review ,Biology ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,splicing ,0302 clinical medicine ,lncRNA ,medicine ,splice ,rRNA ,Ecology, Evolution, Behavior and Systematics ,Settore BIO/16 ,Paleontology ,RNA ,Ribosomal RNA ,030104 developmental biology ,Space and Planetary Science ,030220 oncology & carcinogenesis ,RNA splicing ,sncRNA ,Cancer research ,testicular germ cell tumor ,Carcinogenesis ,Function (biology) - Abstract
Testicular germ cell tumors (TGCTs) are the most common tumors in adolescent and young men. Recently, genome-wide studies have made it possible to progress in understanding the molecular mechanisms underlying the development of tumors. It is becoming increasingly clear that aberrant regulation of RNA metabolism can drive tumorigenesis and influence chemotherapeutic response. Notably, the expression of non-coding RNAs as well as specific splice variants is deeply deregulated in human cancers. Since these cancer-related RNA species are considered promising diagnostic, prognostic and therapeutic targets, understanding their function in cancer development is becoming a major challenge. Here, we summarize how the different expression of RNA species repertoire, including non-coding RNAs and protein-coding splicing variants, impacts on TGCTs’ onset and progression and sustains therapeutic resistance. Finally, the role of transcription-associated R-loop misregulation in the maintenance of genomic stability in TGCTs is also discussed.
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- 2021
11. To Be or Not to Be a Germ Cell: The Extragonadal Germ Cell Tumor Paradigm
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Marco Barchi, Susanna Dolci, Carmela Rita Balistreri, Francesca Gioia Klinger, Federica Campolo, Massimo De Felici, De Felici M., Klinger F.G., Campolo F., Balistreri C.R., Barchi M., and Dolci S.
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Epigenomics ,Male ,Pluripotent Stem Cells ,endocrine system ,Cell type ,Transcription, Genetic ,QH301-705.5 ,Population ,Review ,Biology ,germline ,Catalysis ,Germline ,Inorganic Chemistry ,Testicular Neoplasms ,medicine ,primordial germ cells ,Humans ,Epigenetics ,Biology (General) ,Physical and Theoretical Chemistry ,education ,Gonads ,QD1-999 ,Molecular Biology ,Spectroscopy ,education.field_of_study ,Settore BIO/16 ,Organic Chemistry ,EG cells ,Teratoma ,Embryo ,germ cell tumor ,Cell Differentiation ,General Medicine ,Neoplasms, Germ Cell and Embryonal ,medicine.disease ,Computer Science Applications ,Cell biology ,Chemistry ,medicine.anatomical_structure ,Germ Cells ,Extragonadal Germ Cell Tumor ,Germ cell tumors ,Germ cell - Abstract
In the human embryo, the genetic program that orchestrates germ cell specification involves the activation of epigenetic and transcriptional mechanisms that make the germline a unique cell population continuously poised between germness and pluripotency. Germ cell tumors, neoplasias originating from fetal or neonatal germ cells, maintain such dichotomy and can adopt either pluripotent features (embryonal carcinomas) or germness features (seminomas) with a wide range of phenotypes in between these histotypes. Here, we review the basic concepts of cell specification, migration and gonadal colonization of human primordial germ cells (hPGCs) highlighting the analogies of transcriptional/epigenetic programs between these two cell types.
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- 2021
12. Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways
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Cinzia Caggiano, Paola Grimaldi, Teresa Giannattasio, Gioia Cappelletti, Francesca Cavallo, Darren R. Feldman, Pellegrino Rossi, Maria Jasin, and Marco Barchi
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Cancer Research ,DNA repair ,medicine.medical_treatment ,GERM CELL TUMOURS ,lcsh:RC254-282 ,HOMOLOGOUS RECOMBINATION ,Article ,TGCT ,HR ,medicine ,NON HOMOLOGOUS END JOINING ,Protein kinase A ,Cytotoxicity ,DNA-PKcs ,NHEJ ,Cisplatin ,Chemotherapy ,Settore BIO/16 ,Chemistry ,PARP INHIBITORS ,medicine.disease ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,53BP1 ,GERM CELL TUMOURS, CISPLATIN RESISTANCE, DNA REPAIR, HOMOLOGOUS RECOMBINATION, NON HOMOLOGOUS END JOINING, PARP INHIBITORS ,Oncology ,DNA REPAIR ,PARP inhibitor ,Cancer research ,Germ cell tumors ,cisplatin resistance ,medicine.drug ,olaparib (AZD2281) - Abstract
Simple Summary Germ cell tumors are a model of curable solid tumors due to their unique sensitivity to cisplatin-based chemotherapy. Patients are typically young adults, and despite high cure rate, about 20% of them do not achieve remission or relapse, and 50% of them succumb to the disease. The mechanisms behind their resistance to therapy are largely unknown. By using Testicular Germ Cell Tumor (TGCT) cell lines as a model, we investigated the mechanism of acquired resistance to cisplatin. We demonstrated that resistance occurred by a fine modulation of the DNA repair pathway choice. Namely, in resistant cells, repair of double-strand breaks by non-homologous end joining was dampened by the reduced expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs). However, cisplatin-induced damage was repaired efficiently by homologous recombination. Additionally, we demonstrate that pharmacological inhibition of poly (ADP-ribose) polymerase (PARP) combined with cisplatin had an additive/synergistic effect on cisplatin-resistant cells, which represents the proof of concept for introducing PARP inhibitors in salvage therapy. Abstract Despite germ cell tumors (GCTs) responding to cisplatin-based chemotherapy at a high rate, a subset of patients does not respond to treatment and have significantly worse prognosis. The biological mechanisms underlying the resistance remain unknown. In this study, by using two TGCT cell lines that have acquired cisplatin resistance after chronic exposure to the drug, we identified some key proteins and mechanisms of acquired resistance. We show that cisplatin-resistant cell lines had a non-homologous end-joining (NHEJ)-less phenotype. This correlated with a reduced basal expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs) proteins and reduced formation of 53BP1 foci after cisplatin treatment. Consistent with these observations, modulation of 53BP1 protein expression altered the cell line’s resistance to cisplatin, and inhibition of DNA-PKcs activity antagonized cisplatin cytotoxicity. Dampening of NHEJ was accompanied by a functional increase in the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. As a result, cisplatin-resistant cells were more resistant to PARP inhibitor (PARPi) monotherapy. Moreover, when PARPi was given in combination with cisplatin, it exerted an additive/synergistic effect, and reduced the cisplatin dose for cytotoxicity. These results suggest that treatment of cisplatin-refractory patients may benefit from low-dose cisplatin therapy combined with PARPi.
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- 2021
13. The Italian law on body donation: A position paper of the Italian College of Anatomists
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Raffaele De Caro, Rafael, Boscolo-Berto, Marco, Artico, Eugenio, Bertelli, Mario, Cannas, Francesco, Cappello, Guido, Carpino, Sergio, Castorina, Amelia, Cataldi, Guido Angelo Cavaletti, Saverio, Cinti, Lucio Ildebrando Cocco, Ottavio, Cremona, Enrico, Crivellato, Antonio De Luca, Mirella, Falconi, Giuseppe, Familiari, Gianluca, Ferri, Francesco, Fornai, Marco, Gesi, Stefano, Geuna, Daniele Maria Gibelli, Antonio, Giordano, Pietro, Gobbi, Germano, Guerra, Massimo, Gulisano, Veronica, Macchi, Guido, Macchiarelli, Lucia, Manzoli, Fabrizio, Michetti, Sebastiano, Miscia, Stefania, Montagnani, Andrea, Costantino, Mario, Montella, Sergio, Morini, Paolo, Onori, Carla, Palumbo, Michele, Papa, Andrea, Porzionato, Daniela Elena Quacci, Mario, Raspanti, Rende, Mario, Rita, Rezzani, Domenico, Ribatti, Maurizio, Ripani, Luigi Fabrizio Rodella, Pellegrino, Rossi, Andrea, Sbarbati, Paola, Secchiero, Chiarella, Sforza, Carla, Stecco, Roberto, Toni, Alessandro, Vercelli, Marco, Vitale, Carlo, Zancanaro, Giorgio, Zauli, Sandra, Zecchi, Giuseppe Pio Anastasi, Eugenio, Gaudio, De Caro, R., Boscolo-Berto, R., Artico, M., Bertelli, E., Cannas, M., Cappello, F., Carpino, G., Castorina, S., Cataldi, A., Cavaletti, G. A., Cinti, S., Cocco, L. I., Cremona, O., Crivellato, E., De Luca, A., Falconi, M., Familiari, G., Ferri, G. L., Fornai, F., Gesi, M., Geuna, S., Gibelli, D. M., Giordano, A., Gobbi, P., Guerra, G., Gulisano, M., Macchi, V., Macchiarelli, G., Manzoli, L., Michetti, F., Miscia, S., Montagnani, S., Montella, A. C. M., Morini, S., Onori, P., Palumbo, C., Papa, M., Porzionato, A., Quacci, D. E., Raspanti, M., Rende, M., Rezzani, R., Ribatti, D., Ripani, M., Rodella, L. F., Rossi, P., Sbarbati, A., Secchiero, P., Sforza, C., Stecco, C., Toni, R., Vercelli, A., Vitale, M., Zancanaro, C., Zauli, G., Zecchi, S., Anastasi, G. P., Gaudio, E., De Caro, R, Boscolo-Berto, R, Artico, M, Bertelli, E, Cannas, M, Cappello, F, Carpino, G, Castorina, S, Cataldi, A, Cavaletti, G, Cinti, S, Cocco, L, Cremona, O, Crivellato, E, De Luca, A, Falconi, M, Familiari, G, Ferri, G, Fornai, F, Gesi, M, Geuna, S, Gibelli, D, Giordano, A, Gobbi, P, Guerra, G, Gulisano, M, Macchi, V, Macchiarelli, G, Manzoli, L, Michetti, F, Miscia, S, Montagnani, S, Montella, A, Morini, S, Onori, P, Palumbo, C, Papa, M, Porzionato, A, Quacci, D, Raspanti, M, Rende, M, Rezzani, R, Ribatti, D, Ripani, M, Rodella, L, Rossi, P, Sbarbati, A, Secchiero, P, Sforza, C, Stecco, C, Toni, R, Vercelli, A, Vitale, M, Zancanaro, C, Zauli, G, Zecchi, S, Anastasi, G, Gaudio, E, De Caro R., Boscolo-Berto R., Artico M., Bertelli E., Cannas M., Cappello F., Carpino G., Castorina S., Cataldi A., Cavaletti G.A., Cinti S., Cocco L.I., Cremona O., Crivellato E., De Luca A., Falconi M., Familiari G., Ferri G.L., Fornai F., Gesi M., Geuna S., Gibelli D.M., Giordano A., Gobbi P., Guerra G., Gulisano M., Macchi V., Macchiarelli G., Manzoli L., Michetti F., Miscia S., Montagnani S., Montella A.C.M., Morini S., Onori P., Palumbo C., Papa M., Porzionato A., Quacci D.E., Raspanti M., Rende M., Rezzani R., Ribatti D., Ripani M., Rodella L.F., Rossi P., Sbarbati A., Secchiero P., Sforza C., Stecco C., Toni R., Vercelli A., Vitale M., Zancanaro C., Zauli G., Zecchi S., Anastasi G.P., and Gaudio E.
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0301 basic medicine ,Students, Medical ,Tissue and Organ Procurement ,education ,Tissue Donor ,Legislation ,Body donation programme, Law Regulation, Cadaver lab, Education ,Cadaver lab ,NO ,Education ,03 medical and health sciences ,Body donation ,Anatomist ,Body donation programme Law Regulation Cadaver lab Education ,Political science ,Medical ,Cadaver ,Humans ,Anatomists ,Students ,Implementation ,Settore BIO/16 ,Body donation programme ,Settore BIO/16 - Anatomia Umana ,General Medicine ,Tissue Donors ,Transplantation ,Subordination (finance) ,030104 developmental biology ,Italy ,Law ,Donation ,Regulation ,Position paper ,030101 anatomy & morphology ,Anatomy ,Inefficiency ,Developmental Biology ,Human - Abstract
In Italy, recent legislation (Law No. 10/2020) has tuned regulations concerning the donation of one's postmortem body and tissues for study, training, and scientific research purposes. This study discusses several specific issues to optimise the applicability and effectiveness of such an important, novel regulatory setting. Some of these unsolved issues may involve the grantees of teaching and training activities, the role of academic anatomical institutes, the role of family members in the donation process, the universal time limit indicated for any donation, the handling of corpses, and the limited body donation and its subordination to the donation of organs and tissues. Critical issues arise concerning the learners, the type of training and teaching activities that can be planned, the position of academic anatomy institutes, the role of family members in the donation process, the time frame of the donation process, the eligibility of partial donation, or the simultaneous donation of organs and tissues to patients awaiting transplantation. In particular, a universal time limit for donations (i.e., one year) makes it impossible to plan the long-term use of specific body parts, which could be effectively preserved for the advanced teaching and training of medical students and surgeons. The abovementioned conditions lead to the limited use of corpses, thus resulting in the inefficiency of the whole system of body donation. Overall, the donors' scope for the donation of their body could be best honoured by a more flexible and tuneable approach that can be used on a case-by-case basis. Furthermore, it is deemed necessary to closely monitor the events scheduled for corpses in public nonacademic institutions or private enterprises. This paper presents useful insights from Italian anatomists with the hope of providing inspiration for drafting the regulations. In conclusion, this paper focuses on the critical issues derived from the recently introduced Italian law on the donation and use of the body after death and provides suggestions to lawmakers for future implementations.
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- 2021
14. Combinatorial control of Spo11 alternative splicing by modulation of RNA polymerase II dynamics and splicing factor recruitment during meiosis
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Donatella Farini, Maria Loiarro, Claudio Sette, Pamela Bielli, Eleonora Cesari, Marco Pieraccioli, Livia Pellegrini, Chiara Naro, and Vittoria Pagliarini
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Male ,Cancer Research ,Spo11 ,Immunology ,RNA polymerase II ,Article ,Chromosome segregation ,Mice ,Cellular and Molecular Neuroscience ,Exon ,Splicing factor ,Spermatocytes ,Animals ,Humans ,lcsh:QH573-671 ,Spermatogenesis ,Settore BIO/11 - BIOLOGIA MOLECOLARE ,Settore BIO/16 ,Endodeoxyribonucleases ,biology ,lcsh:Cytology ,fungi ,Alternative splicing ,rna splicing ,Cell Biology ,Cell biology ,Alternative Splicing ,Meiosis ,RNA splicing ,biology.protein ,RNA Polymerase II ,RNA Splicing Factors ,Homologous recombination - Abstract
Homologous recombination and chromosome segregation in meiosis rely on the timely expression of two splice variants of the endonuclease SPO11, named α and β, which respectively skip or include exon 2. However, in spite of its physiological importance, the mechanism underlying Spo11 alternative splicing in meiosis is still unknown. By screening the activity of factors that are predicted to bind the alternatively spliced region of Spo11, we identified hnRNPH as a key regulator of SPO11α splicing in mouse spermatocytes. Although hnRNPH was not upregulated in meiosis concomitantly with the switch in splicing, its recruitment to Spo11 pre-mRNA was favored by selective modulation of RNA polymerase II (RNAPII) phosphorylation and processivity in proximity of exon 2. The hnRNPH binding sites were localized near those of splicing factors that promote SPO11β splicing, suggesting that hnRNPH favors exon 2 skipping by competing out positive regulators. Indeed, hnRNPH binds proximal to a consensus motif for Sam68, a positive regulator of SPO11β splicing in vitro and in vivo, and it interferes with Sam68 binding to the Spo11 pre-mRNA. Thus, our work reveals that modulation of RNAPII dynamics in concert with hnRNPH recruitment exerts a combinatorial control of the timely regulated Spo11 splicing during meiosis.
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- 2020
15. Sempervirine inhibits RNA polymerase I transcription independently from p53 in tumor cells
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Susanna Dolci, Bruno Botta, Pamela Bielli, Deborah Quaglio, Eugenia Guida, Fabiola Moretti, Mattia Mori, Marco Barchi, Francesca Ghirga, Federica Todaro, Cinzia Caggiano, Paola Grimaldi, Emmanuele A. Jannini, and Pellegrino Rossi
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0301 basic medicine ,p53 ,Cancer Research ,Nucleolus ,Immunology ,tumor cells ,lcsh:RC254-282 ,Article ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Testicular cancer ,Downregulation and upregulation ,MDM2 ,Transcription (biology) ,Target identification ,Null cell ,RNA polymerase I ,E2F1 ,sempervirin ,lcsh:QH573-671 ,Settore BIO/16 ,lcsh:Cytology ,Cell growth ,Chemistry ,Sempervirine ,RNA polymerase ,Cell Biology ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Cell biology ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cancer cell - Abstract
In the search of small molecules that can target MDM2/p53 pathway in testicular germ cell tumors (TGCTs), we identified sempervirine (2,3,4,13-tetrahydro-1H-benz[g]indolo[2,3-a]quinolizin-6-ium), an alkaloid of Gelsemium sempervirens, that has been previously proposed as an inhibitor of MDM2 that targets p53-wildtype (wt) tumor cells. We found that sempervirine not only affects cell growth of p53-wt cancer cells, but it is also active in p53-mutated and p53-null cells by triggering p53-dependent and independent pathways without affecting non-transformed cells. To understand which mechanism/s could be activated both in p53-wt and -null cells, we found that sempervirine induced nucleolar remodeling and nucleolar stress by reducing protein stability of RPA194, the catalytic subunit of RNA polymerase I, that led to rRNA synthesis inhibition and to MDM2 block. As shown for other cancer cell models, MDM2 inhibition by nucleolar stress downregulated E2F1 protein levels both in p53-wt and p53-null TGCT cells with the concomitant upregulation of unphosphorylated pRb. Finally, we show that sempervirine is able to enter the nucleus and accumulates within the nucleolus where it binds rRNA without causing DNA damage. Our results identify semperivirine as a novel rRNA synthesis inhibitor and indicate this drug as a non-genotoxic anticancer small molecule.
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- 2020
16. Sam68 binds Alu-rich introns in SMN and promotes pre-mRNA circularization
- Author
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Claudio Sette, Pierre de la Grange, Vittoria Pagliarini, Valentina Di Rosa, Pamela Bielli, Ariane Jolly, Università cattolica del Sacro Cuore [Piacenza e Cremona] (Unicatt), Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Istituti di Ricovero e Cura a Carattere Scientifico (IRCCS), Centre d'investigation clinique Biothérapie [CHU Pitié-Salpêtrière] (CIC-BTi), Centre d'investigation clinique pluridisciplinaire [CHU Pitié Salpêtrière] (CIC-P 1421), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Fondazione Santa Lucia [IRCCS], Clinical and Behavioral Neurology [IRCCS Santa Lucia], Gestionnaire, Hal Sorbonne Université, Fondazione 'Policlinico Universitario A. Gemelli' [Rome], CIC Pitié BT, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)
- Subjects
animal diseases ,SAM68 ,SMN1 ,Biology ,Muscular Atrophy, Spinal ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Alu Elements ,Circular RNA ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,RNA Precursors ,Genetics ,Animals ,Humans ,Gene ,Adaptor Proteins, Signal Transducing ,030304 developmental biology ,Settore BIO/16 - ANATOMIA UMANA ,0303 health sciences ,Settore BIO/16 ,Binding Sites ,Gene regulation, Chromatin and Epigenetics ,Intron ,RNA-Binding Proteins ,RNA ,SMN Complex Proteins ,Exons ,RNA, Circular ,Survival of Motor Neuron 1 Protein ,Introns ,Cell biology ,nervous system diseases ,DNA-Binding Proteins ,Survival of Motor Neuron 2 Protein ,Alternative Splicing ,nervous system ,030220 oncology & carcinogenesis ,RNA splicing ,[SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Human genome ,Precursor mRNA - Abstract
The Spinal Muscular Atrophy (SMA) gene SMN wasrecently duplicated (SMN1 and SMN2) in higher primates.Furthermore, invasion of the locus by repetitiveelements almost doubled its size with respectto mouse Smn, in spite of an almost identicalprotein-coding sequence. Herein, we found that SMNranks among the human genes with highest densityof Alus, which are evolutionary conserved inprimates and often occur in inverted orientation. Invertedrepeat Alus (IRAlus) negatively regulate splicingof long introns within SMN, while promotingwidespread alternative circular RNA (circRNA) biogenesis.Bioinformatics analyses revealed the presenceof ultra-conserved Sam68 binding sites in SMNIRAlus. Cross-link-immunoprecipitation (CLIP), mutagenesisand silencing experiments showed thatSam68 binds in proximity of intronic Alus in theSMN pre-mRNA, thus favouring circRNA biogenesisin vitro and in vivo. These findings highlight a novellayer of regulation in SMN expression, uncover thecrucial impact exerted by IRAlus and reveal a role forSam68 in SMN circRNA biogenesis.
- Published
- 2020
17. Cannabinoid Receptors Signaling in the Development, Epigenetics, and Tumours of Male Germ Cells
- Author
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Marco Barchi, Paola Grimaldi, Teresa Giannattasio, Pellegrino Rossi, Elisa Innocenzi, and Susanna Dolci
- Subjects
embryonal carcinoma ,cannabis ,Male ,endocrine system ,Cannabinoid receptor ,Review ,Biology ,sperm ,Catalysis ,Epigenesis, Genetic ,Inorganic Chemistry ,Embryonal carcinoma ,Gonocyte ,Testicular Neoplasms ,medicine ,Animals ,Humans ,Epigenetics ,Physical and Theoretical Chemistry ,endocannabinoid system ,Receptor ,Receptors, Cannabinoid ,Molecular Biology ,Spectroscopy ,Settore BIO/16 ,male germ cells ,Reproduction ,Organic Chemistry ,General Medicine ,Neoplasms, Germ Cell and Embryonal ,medicine.disease ,Endocannabinoid system ,spermatogenesis ,Computer Science Applications ,Cell biology ,testicular tumors ,Gene Expression Regulation, Neoplastic ,medicine.anatomical_structure ,epigenetic ,intergenerational ,lipids (amino acids, peptides, and proteins) ,Disease Susceptibility ,Spermatogenesis ,Germ cell ,Biomarkers ,Signal Transduction - Abstract
Endocannabinoids are natural lipid molecules whose levels are regulated by specific biosynthetic and degradative enzymes. They bind to and activate two main cannabinoid receptors type 1 (CB1) and type 2 (CB2), and together with their metabolizing enzymes form the “endocannabinoid system” (ECS). In the last years, the relevance of endocannabinoids (eCBs) as critical modulators in various aspects of male reproduction has been pointed out. Mammalian male germ cells, from mitotic to haploid stage, have a complete ECS which is modulated during spermatogenesis. Compelling evidence indicate that in the testis an appropriate “eCBs tone”, associated to a balanced CB receptors signaling, is critical for spermatogenesis and for the formation of mature and fertilizing spermatozoa. Any alteration of this system negatively affects male reproduction, from germ cell differentiation to sperm functions, and might have also an impact on testicular tumours. Indeed, most of testicular tumours develop during early germ-cell development in which a maturation arrest is thought to be the first key event leading to malignant transformation. Considering the ever-growing number and complexity of the data on ECS, this review focuses on the role of cannabinoid receptors CB1 and CB2 signaling in male germ cells development from gonocyte up to mature spermatozoa and in the induction of epigenetic alterations in these cells which might be transmitted to the progeny. Furthermore, we present new evidence on their relevance in testicular cancer.
- Published
- 2019
18. Paternal activation of CB2 cannabinoid receptor impairs placental and embryonic growth via an epigenetic mechanism
- Author
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Michele Zampieri, Rosella Cicconi, Roberta Bernardini, Maurizio Mattei, Fabio Ciccarone, Paola Grimaldi, Gabriele Rossi, Emanuela De Domenico, and Elisa Innocenzi
- Subjects
Male ,Cannabinoid receptor ,Offspring ,medicine.medical_treatment ,Placenta ,DNA methylation ,DNA hydroxymethylation ,Cannabis sativa ,spermatogenesis, fertility ,lcsh:Medicine ,Embryonic Development ,Biology ,Article ,Receptor, Cannabinoid, CB2 ,Mice ,Pregnancy ,medicine ,Cannabinoid receptor type 2 ,Animals ,lcsh:Science ,Spermatogenesis ,Cannabis ,fertility ,Settore BIO/16 ,Multidisciplinary ,Sperm Count ,Cannabinoids ,lcsh:R ,Epigenome ,DNA Methylation ,Sperm ,Spermatozoa ,Placentation ,Spermatogonia ,Cell biology ,Risk factors ,Embryogenesis ,lcsh:Q ,lipids (amino acids, peptides, and proteins) ,Female ,Cannabinoid ,Genomic imprinting - Abstract
The cannabinoid receptor type 2 (CB2) is the peripheral receptor for cannabinoids, involved in the homeostatic control of several physiological functions. Male mitotic germ cells express a high level of CB2, whose activation promotes their differentiation in both in vitro and in vivo experiments, controlling the correct progression of spermatogenesis. However, it remains elusive if CB2 activation in spermatogonia could affect reproductive success in terms of fertility and healthy pregnancy outcomes. In this study, we explored the effects of male CB2 activation on sperm number and quality and its influence on next generation health. We show that exposure of male mice to JWH-133, a selective CB2 agonist, decreased sperm count, impaired placental development and reduced offspring growth. These defects were associated with altered DNA methylation/hydroxymethylation levels at imprinted genes in sperm and conserved in placenta. Our findings reveal that paternal selective activation of CB2 alters the sperm epigenome and compromises offspring growth. This study demonstrates, for the first time, a new role of CB2 signaling in male gametes in causing epigenetic alterations that can be transmitted to the next generation by sperm, highlighting potential risks induced by recreational cannabinoid exposure.
- Published
- 2019
19. Complete in vitro oogenesis: retrospects and prospects
- Author
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Wei Shen, Massimo De Felici, Francesca Gioia Klinger, Jun-Jie Wang, Jing-Cai Liu, Paul W. Dyce, and Wei Ge
- Subjects
0301 basic medicine ,Cell type ,In Vitro Oocyte Maturation Techniques ,Review ,Biology ,Oogenesis ,03 medical and health sciences ,medicine ,Animals ,Humans ,Molecular Biology ,Settore BIO/16 ,Settore BIO/17 ,Follicular atresia ,Settore BIO/13 ,Ovary ,Povidone ,Estrogens ,Cell Biology ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Immunology ,Female ,Folliculogenesis ,Stem cell ,Developmental biology ,Germ Layers ,Germ cell - Abstract
Precise control of mammalian oogenesis has been a traditional focus of reproductive and developmental biology research. Recently, new reports have introduced the possibility of obtaining functional gametes derived in vitro from stem cells. The potential to produce functional gametes from stem cells has exciting applications for regenerative medicine though still remains challenging. In mammalian females ovulation and fertilization is a privilege reserved for a small number of oocytes. In reality the vast majority of oocytes formed from primordial germ cells (PGCs) will undergo apoptosis, or other forms of cell death. Removal occurs during germ cell cyst breakdown and the establishment of the primordial follicle (PF) pool, during the long dormancy at the PF stage, or through follicular atresia prior to reaching the ovulatory stage. A way to solve this limitation could be to produce large numbers of oocytes, in vitro, from stem cells. However, to recapitulate mammalian oogenesis and produce fertilizable oocytes in vitro is a complex process involving several different cell types, precise follicular cell-oocyte reciprocal interactions, a variety of nutrients and combinations of cytokines, and precise growth factors and hormones depending on the developmental stage. In 2016, two papers published by Morohaku et al. and Hikabe et al. reported in vitro procedures that appear to reproduce efficiently these conditions allowing for the production, completely in a dish, of a relatively large number of oocytes that are fertilizable and capable of giving rise to viable offspring in the mouse. The present article offers a critical overview of these results as well as other previous work performed mainly in mouse attempting to reproduce oogenesis completely in vitro and considers some perspectives for the potential to adapt the methods to produce functional human oocytes.
- Published
- 2017
20. Regulation of PDE5 expression in normal prostate, benign prostatic hyperplasia, and adenocarcinoma
- Author
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Susanna Dolci, Francesco Pierconti, Maurizio Martini, Emmanuele A. Jannini, S. M. Di Stasi, Paola Grimaldi, Luigi Maria Larocca, G.L. Gravina, Pellegrino Rossi, and C. Bisegna
- Subjects
Male ,Pathology ,PDE5 inhibitor ,Endocrinology, Diabetes and Metabolism ,Prostatic Hyperplasia ,Cancer, Human, Immunohistochemistry, PDE5 inhibitor, Phosphodiesterase, prostate, Adenocarcinoma, Adult, Aged, Aged, 80 and over, Cyclic Nucleotide Phosphodiesterases, Type 5, Humans, Male, Middle Aged, Prostate, Prostatic Hyperplasia, Prostatic Neoplasms, Young Adult ,phosphodiesterase 5 ,urologic and male genital diseases ,Prostate cancer ,Settore MED/13 ,0302 clinical medicine ,Endocrinology ,Prostate ,80 and over ,Medicine ,Cancer ,Aged, 80 and over ,Settore BIO/16 ,030219 obstetrics & reproductive medicine ,Tissue microarray ,Hyperplasia ,Middle Aged ,Immunohistochemistry ,medicine.anatomical_structure ,Type 5 ,Adenocarcinoma ,Human ,Cyclic Nucleotide Phosphodiesterases ,Adult ,medicine.medical_specialty ,Stromal cell ,Urology ,prostatic cancer ,03 medical and health sciences ,Young Adult ,Lower urinary tract symptoms ,Phosphodiesterase PDE5 inhibitor ,Humans ,Phosphodiesterase ,Aged ,Cyclic Nucleotide Phosphodiesterases, Type 5 ,benign prostatic hyperplasia ,Settore MED/08 - ANATOMIA PATOLOGICA ,business.industry ,Prostatic Neoplasms ,medicine.disease ,Settore MED/24 ,Reproductive Medicine ,phosphodiesterase 5, prostate, benign prostatic hyperplasia, prostatic cancer ,business - Abstract
Background Type 5 phosphodiesterase (PDE5) expression in the normal and pathological prostate is controversial. Objectives This study aimed at identifying the cell type/s, if any, expressing PDE5 in human healthy or pathological prostate sections in order to further validate the rationale of PDE5 inhibitor (PDE5i) treatment of benign prostatic hyperplasia (BPH) and their safety in the treatment of erectile dysfunction following prostate cancer (PCa) surgery. Materials and methods By immunohistochemical analysis, we studied PDE5 expression in tissue microarrays containing sections obtained from healthy, BPH, and PCa samples. Results Our results showed that PDE5 is barely expressed in the epithelial or stromal compartment of normal human prostates, but it is highly expressed in the stromal compartment of BPH sections. We also found that a low but significant number of PCa samples (22%) expressed PDE5 in the epithelial cancer cells but not in stromal cells and that such expression was not correlated with the tumor aggressiveness, according to their Gleason score. Discussion and conclusion PDE5 overexpression in the stromal compartment of BPH samples supports the rationale of PDE5 as a target in lower urinary tract symptoms of BPH. PDE5 expression in a significant percentage of PCa samples but the lack of correlation with the Gleason score suggests that this enzyme is not correlated with tumor aggressiveness; however, a role of PDE5 in the minimal residual disease of PCa cannot be excluded.
- Published
- 2019
21. c-MYC empowers transcription and productive splicing of the oncogenic splicing factor Sam68 in cancer
- Author
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Pamela Bielli, Marco Pieraccioli, Claudio Sette, Valentina Panzeri, and Cinzia Caggiano
- Subjects
c-MYC empowers transcription ,Male ,Transcriptional Activation ,Transcription Elongation, Genetic ,alternative splicing ,cancer ,RNA binding proteins ,Biology ,Proto-Oncogene Proteins c-myc ,03 medical and health sciences ,Exon ,Splicing factor ,0302 clinical medicine ,Cell Line, Tumor ,Genetics ,Humans ,Promoter Regions, Genetic ,Transcription factor ,030304 developmental biology ,Adaptor Proteins, Signal Transducing ,Settore BIO/16 - ANATOMIA UMANA ,Regulation of gene expression ,0303 health sciences ,Settore BIO/16 ,Heterogeneous-Nuclear Ribonucleoprotein Group F-H ,Alternative splicing ,Gene regulation, Chromatin and Epigenetics ,Prostatic Neoplasms ,RNA-Binding Proteins ,Promoter ,Exons ,Cell biology ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Alternative Splicing ,HEK293 Cells ,030220 oncology & carcinogenesis ,RNA splicing ,RNA Polymerase II ,KHDRBS1 - Abstract
The splicing factor Sam68 is upregulated in many human cancers, including prostate cancer (PCa) where it promotes cell proliferation and survival. Nevertheless, in spite of its frequent upregulation in cancer, the mechanism(s) underlying its expression are largely unknown. Herein, bioinformatics analyses identified the promoter region of the Sam68 gene (KHDRBS1) and the proto-oncogenic transcription factor c-MYC as a key regulator of Sam68 expression. Upregulation of Sam68 and c-MYC correlate in PCa patients. c-MYC directly binds to and activates the Sam68 promoter. Furthermore, c-MYC affects productive splicing of the nascent Sam68 transcript by modulating the transcriptional elongation rate within the gene. Importantly, c-MYC-dependent expression of Sam68 is under the tight control of external cues, such as androgens and/or mitogens. These findings uncover an unexpected coordination of transcription and splicing of Sam68 by c-MYC, which may represent a key step in PCa tumorigenesis.
- Published
- 2019
22. Regulation of PDE5 expression in human aorta and thoracic aortic aneurysms
- Author
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Carmela Rita Balistreri, Emmanuele A. Jannini, G. Antonelli, Susanna Dolci, Calogera Pisano, Giovanni Ruvolo, Gabriele Rossi, Flavia Botti, Valeriana Cesarini, Cesarini V., Pisano C., Rossi G., Balistreri C.R., Botti F., Antonelli G., Ruvolo G., Jannini E.A., and Dolci S.
- Subjects
0301 basic medicine ,Male ,Cell ,lcsh:Medicine ,Stimulation ,Muscle, Smooth, Vascular ,Aortic aneurysm ,chemistry.chemical_compound ,0302 clinical medicine ,PDE5 expression, human aorta and thoracic aortic aneurysms ,Myocyte ,Thoracic aorta ,lcsh:Science ,Settore BIO/16 ,Multidisciplinary ,Transfection ,Middle Aged ,Isoenzymes ,medicine.anatomical_structure ,cardiovascular system ,Female ,Gene isoform ,Adult ,medicine.medical_specialty ,Myocytes, Smooth Muscle ,Article ,Gene Expression Regulation, Enzymologic ,Nitric oxide ,03 medical and health sciences ,medicine.artery ,Internal medicine ,medicine ,Humans ,Settore MED/05 - Patologia Clinica ,Aged ,Cyclic Nucleotide Phosphodiesterases, Type 5 ,Aortic Aneurysm, Thoracic ,business.industry ,lcsh:R ,medicine.disease ,030104 developmental biology ,Endocrinology ,chemistry ,Risk factors ,thoracic aortic aneurysms ,lcsh:Q ,Angiogenesis ,PDE5 ,business ,030217 neurology & neurosurgery - Abstract
Aneurysms and dissections affecting thoracic aorta are associated with smooth muscle cell (SMC) dysfunction. NO/cGMP signaling pathway in smooth muscle cells has been shown to be affected in sporadic thoracic aortic aneurysms. We analyzed the mRNA levels of PDE5, a cGMP-hydrolyzing enzyme highly expressed in aortic SMCs, that regulates arterious vascular tone by lowering cGMP levels. We found that aortic tissue obtained from Marfan, tricuspid and bicuspid thoracic aneurysms expressed lower levels of PDE5 mRNA compared to control aortas. In particular, we found that affected aortas showed lower levels of all the PDE5A isoforms, compared to control aortas. Transfection of vascular SMCs (VSMCs) with NOTCH3 activated domain (NICD3) induced the expression of PDE5A1 and A3 protein isoforms, but not that of the corresponding mRNAs. VSMC stimulation with GSNO, a nitric oxide analogue or with 8-br-cGMP, but not with 8-br-cAMP, up-regulated PDE5 and NOTCH-3 protein levels, indicating a negative feedback loop to protect the arterial wall from excessive relaxation. Finally, we found that PDE5 is expressed early during human aorta development, suggesting that if loss of function mutations of PDE5 occur, they might potentially affect aortic wall development.
- Published
- 2019
23. Splicing Dysregulation as Oncogenic Driver and Passenger Factor in Brain Tumors
- Author
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Marco Pieraccioli, Pamela Bielli, Claudio Sette, Cinzia Caggiano, and Vittoria Pagliarini
- Subjects
Carcinogenesis ,RNA Splicing ,Brain tumor ,Review ,Biology ,EGFR signaling ,PRMT5 ,alternative splicing ,brain tumors ,hippo signaling ,splicing factors ,tumor microenvironment ,Gene expression ,medicine ,Tumor Microenvironment ,Animals ,Humans ,Settore BIO/16 - ANATOMIA UMANA ,Settore BIO/16 ,Tumor microenvironment ,Brain Neoplasms ,Alternative splicing ,Cancer ,General Medicine ,Oncogenes ,medicine.disease ,Gene Expression Regulation, Neoplastic ,Alternative Splicing ,Hippo signaling ,RNA splicing ,Mutation ,Cancer research ,Disease Progression ,RNA Splicing Factors ,Reprogramming - Abstract
Brain tumors are a heterogeneous group of neoplasms ranging from almost benign to highly aggressive phenotypes. The malignancy of these tumors mostly relies on gene expression reprogramming, which is frequently accompanied by the aberrant regulation of RNA processing mechanisms. In brain tumors, defects in alternative splicing result either from the dysregulation of expression and activity of splicing factors, or from mutations in the genes encoding splicing machinery components. Aberrant splicing regulation can generate dysfunctional proteins that lead to modification of fundamental physiological cellular processes, thus contributing to the development or progression of brain tumors. Herein, we summarize the current knowledge on splicing abnormalities in brain tumors and how these alterations contribute to the disease by sustaining proliferative signaling, escaping growth suppressors, or establishing a tumor microenvironment that fosters angiogenesis and intercellular communications. Lastly, we review recent efforts aimed at developing novel splicing-targeted cancer therapies, which employ oligonucleotide-based approaches or chemical modulators of alternative splicing that elicit an impact on brain tumor biology.
- Published
- 2019
24. Regulation of kit expression in early mouse embryos and ES cells
- Author
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Emmanuele A. Jannini, Pellegrino Rossi, Susanna Dolci, Manuela Pellegrini, Lino Tessarollo, Florencia Barrios, Silvia Di Cesare, Federica Campolo, and Federica Todaro
- Subjects
0301 basic medicine ,proto-oncogene proteins c-kit ,mouse embryonic stem cells ,knockout ,RNA-binding proteins ,nanog homeobox protein ,0302 clinical medicine ,xenogeneic stem cell transplantation ,Inner cell mass ,reproductive and urinary physiology ,C-Kit ,Embryo ,Embryonic stem cells ,LIF ,Proliferation ,Transgenic mouse ,Xenogeneic stem cell transplantation ,Mice, Knockout ,Settore BIO/16 ,Gene Expression Regulation, Developmental ,gene expression regulation ,embryonic stem cells ,Cell biology ,animals ,medicine.anatomical_structure ,Regulatory sequence ,embryonic structures ,Molecular Medicine ,biological phenomena, cell phenomena, and immunity ,Stem cell ,SOXB1 transcription factors ,amino acid substitution ,Homeobox protein NANOG ,Genetically modified mouse ,missense ,mice ,proliferation ,Mutation, Missense ,embryo ,Biology ,Article ,03 medical and health sciences ,SOX2 ,transcription factors ,DNA-binding proteins ,medicine ,developmental ,Blastocyst ,c-kit ,transgenic mouse ,blastocyst ,mutation ,response elements ,urogenital system ,fungi ,Cell Biology ,Embryonic stem cell ,030104 developmental biology ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Kit is a growth factor receptor that regulates proliferation and/or survival of many embryonic and postnatal stem cell types. When mutated, it can induce malignant transformation of the host cells. To dissect the Kit role in the control of ESC pluripotency, we studied its expression during early mouse embryogenesis and during the process of ESC derivation from inner cell mass (ICM) cells. We followed the in vitro development of early mouse embryos obtained from transgenic mice carrying Kit promoter regions fused to EGFP (Kit-EGFP) and found that they initiate EGFP expression at morula stage. EGFP expression is then maintained in the blastocyst, within the ICM, and its levels increase when cultured in the presence of MAPK and GSK3β inhibitors (2i) plus LIF compared with the LIF-only condition. Kit-EGFP ESCs showed nonhomogeneous EGFP expression pattern when cultured in LIF condition, but they upregulated EGFP expression, as well as that of Sox2, Nanog, Prdm14, when shifted to 2i-LIF culture. Similarly, primordial germ cells (PGCs) in the process of embryonic germ cell (EGC) conversion showed enhanced EGFP expression in 2i-LIF. Kit expression was affected by manipulating Sox2 levels in ESCs. Chromatin immunoprecipitation experiments confirmed that Sox2 binds Kit regulatory regions containing Sox2 consensus sequences. Finally, Kit constitutive activation induced by the D814Y mutation increased ESC proliferation and cloning efficiency in vitro and in teratoma assays in vivo. Our results identify Kit as a pluripotency-responsive gene and suggest a role for Kit in the regulation of ESC proliferation. Stem Cells 2019;37:332–344
- Published
- 2019
25. Mouse ANKRD31 Regulates Spatiotemporal Patterning of Meiotic Recombination Initiation and Ensures Recombination between X and Y Sex Chromosomes
- Author
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Marcello Stanzione, Erika Testa, Ian R. Adams, Ji-Feng Fei, Alexander Schleiffer, Frantzeskos Papanikos, Anastasiia Bondarieva, Marco Barchi, Bernard de Massy, Diana Lustyk, Julie A. J. Clément, Petr Jansa, Corinne Grey, Jiri Forejt, Ihsan Dereli, Attila Tóth, Sarai Valerio-Cabrera, Ramya Ravindranathan, Technische Universität Dresden = Dresden University of Technology (TU Dresden), Institut de génétique humaine (IGH), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Università degli Studi di Roma Tor Vergata [Roma], Institute of Molecular Biotechnology (IMBA), Austrian Academy of Sciences (OeAW), Czech Academy of Sciences [Prague] (CAS), South China Normal University, and University of Edinburgh
- Subjects
Male ,X Chromosome ,Pseudoautosomal region ,[SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC] ,Biology ,[SDV.BDLR.RS]Life Sciences [q-bio]/Reproductive Biology/Sexual reproduction ,Chromosome segregation ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Meiosis ,Spermatocytes ,Chromosome Segregation ,Y Chromosome ,Homologous chromosome ,Animals ,meiosisgenome integrity in the germlinemammalian reproductionrecombination between psuedoautosomal regions of sex chromosomesPRDM9IHO1MEI4REC114hotspotsrecombinosome assembly on chromosome axis ,DNA Breaks, Double-Stranded ,Homologous Recombination ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,[SDV.BDD.GAM]Life Sciences [q-bio]/Development Biology/Gametogenesis ,PRDM9 ,030304 developmental biology ,Pseudoautosomal Regions ,0303 health sciences ,Settore BIO/16 ,Autosome ,fungi ,Cell Biology ,Cell biology ,[SDV.GEN.GA]Life Sciences [q-bio]/Genetics/Animal genetics ,Carrier Proteins ,Homologous recombination ,030217 neurology & neurosurgery ,Recombination - Abstract
Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution because of reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers, leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects co-occur with a genome-wide delay in assembling DSB-promoting proteins on autosome axes and loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins in wild type. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated DSB-promoting proteins.
- Published
- 2019
26. The emerging role of PTBP1 in human cancer: Novel prognostic factor in non-muscle invasive bladder cancer
- Author
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Claudio Sette, Savino M. Di Stasi, and Pamela Bielli
- Subjects
0301 basic medicine ,Settore BIO/16 - ANATOMIA UMANA ,Messenger RNA ,Spliceosome ,Settore BIO/16 ,Urology ,Alternative splicing ,Intron ,PTBP1 ,Computational biology ,Biology ,Genome ,03 medical and health sciences ,Exon ,030104 developmental biology ,Reproductive Medicine ,RNA splicing - Abstract
Alternative splicing is a sophisticated RNA processing mechanism that expands the coding potential of the genome and the proteomic complexity in higher eukaryotes. Splicing requires the recognition of the intron/exon boundaries by a large ribonucleoprotein complex, named the spliceosome, followed by intron excision and ligation of exons to yield the messenger RNA (mRNA) (1).
- Published
- 2018
27. Functional Interaction between U1snRNP and Sam68 Insures Proper 3' End Pre-mRNA Processing during Germ Cell Differentiation
- Author
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Pierre de la Grange, Eleonora Cesari, Ariane Jolly, Pamela Bielli, Claudio Sette, Chiara Naro, Donatella Farini, Livia Pellegrini, Fondazione Santa Lucia [IRCCS], Clinical and Behavioral Neurology [IRCCS Santa Lucia], Università degli Studi di Roma Tor Vergata [Roma], Institut du Cerveau et de la Moëlle Epinière = Brain and Spine Institute (ICM), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], and Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)
- Subjects
Genetics and Molecular Biology (all) ,0301 basic medicine ,Male ,Polyadenylation ,Somatic cell ,RNA Splicing ,Biology ,Biochemistry ,male fertility ,General Biochemistry, Genetics and Molecular Biology ,alternative splicing ,03 medical and health sciences ,Exon ,Mice ,0302 clinical medicine ,Meiosis ,medicine ,Animals ,Humans ,alternative polyadenylation ,germ cell differentiation ,Sam68 ,spermatogenesis ,U1snRNP ,Biochemistry, Genetics and Molecular Biology (all) ,Spermatogenesis ,lcsh:QH301-705.5 ,Gene ,3' Untranslated Regions ,[SDV.BDD.GAM]Life Sciences [q-bio]/Development Biology/Gametogenesis ,Adaptor Proteins, Signal Transducing ,Settore BIO/16 - ANATOMIA UMANA ,Settore BIO/16 ,Alternative splicing ,RNA-Binding Proteins ,Ribonucleoproteins, Small Nuclear ,Spermatogonia ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,HEK293 Cells ,lcsh:Biology (General) ,Transcription Termination, Genetic ,RNA splicing ,030217 neurology & neurosurgery ,Germ cell ,Protein Binding - Abstract
Summary: Male germ cells express the widest repertoire of transcript variants in mammalian tissues. Nevertheless, factors and mechanisms underlying such pronounced diversity are largely unknown. The splicing regulator Sam68 is highly expressed in meiotic cells, and its ablation results in defective spermatogenesis. Herein, we uncover an extensive splicing program operated by Sam68 across meiosis, primarily characterized by alternative last exon (ALE) regulation in genes of functional relevance for spermatogenesis. Lack of Sam68 preferentially causes premature transcript termination at internal polyadenylation sites, a feature observed also upon depletion of the spliceosomal U1snRNP in somatic cells. Notably, Sam68-regulated ALEs are characterized by proximity between U1snRNP and Sam68 binding motifs. We demonstrate a physical association between Sam68 and U1snRNP and show that U1snRNP recruitment to Sam68-regulated ALEs is impaired in Sam68−/− germ cells. Thus, our study reveals an unexpected cooperation between Sam68 and U1snRNP that insures proper processing of transcripts essential for male fertility. : Naro et al. show that Sam68 regulates an extensive splicing program during male germ cell differentiation. Sam68 primarily regulates alternative last exon selection in genes with functional relevance for spermatogenesis by preventing premature transcript termination and conferring tissue specificity to the U1snRNP telescripting activity. Keywords: germ cell differentiation, alternative polyadenylation, alternative splicing, U1snRNP, Sam68, spermatogenesis, male fertility
- Published
- 2018
28. Atm reactivation reverses ataxia telangiectasia phenotypes in vivo
- Author
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Manuela Pellegrini, Daniela Marazziti, Chiara Di Pietro, Roberto Gimmelli, André Nussenzweig, Federica Campolo, Andrea Lenzi, Susanna Dolci, and Sara Di Siena
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0301 basic medicine ,Premature aging ,Genetically modified mouse ,Male ,Cancer Research ,DNA damage ,Genetic enhancement ,Immunology ,Mice, Transgenic ,Ataxia Telangiectasia Mutated Proteins ,transgenic mice ,Transgenic ,Article ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Ataxia Telangiectasia ,Mice ,Radioresistance ,medicine ,Animals ,Humans ,DNA Damage ,Enzyme Activation ,Female ,Phenotype ,Signal Transduction ,Disease Models, Animal ,lcsh:QH573-671 ,Immunodeficiency ,Settore BIO/16 ,tamoxifen ,lcsh:Cytology ,Animal ,business.industry ,Cell Biology ,medicine.disease ,Transplantation ,030104 developmental biology ,ATM ,Disease Models ,Ataxia-telangiectasia ,Cancer research ,business ,animals ,ataxia telangiectasia ,ataxia telangiectasia mutated proteins ,enzyme activation ,female ,humans ,male ,mice ,transgenic ,phenotype ,signal transduction ,disease model ,animal - Abstract
Hereditary deficiencies in DNA damage signaling are invariably associated with cancer predisposition, immunodeficiency, radiation sensitivity, gonadal abnormalities, premature aging, and tissue degeneration. ATM kinase has been established as a central player in DNA double-strand break repair and its deficiency causes ataxia telangiectasia, a rare, multi-system disease with no cure. So ATM represents a highly attractive target for the development of novel types of gene therapy or transplantation strategies. Atm tamoxifen-inducible mouse models were generated to explore whether Atm reconstitution is able to restore Atm function in an Atm-deficient background. Body weight, immunodeficiency, spermatogenesis, and radioresistance were recovered in transgenic mice within 1 month from Atm induction. Notably, life span was doubled after Atm restoration, mice were protected from thymoma and no cerebellar defects were observed. Atm signaling was functional after DNA damage in vivo and in vitro. In summary, we propose a new Atm mouse model to investigate novel therapeutic strategies for ATM activation in ataxia telangiectasia disease.
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- 2018
29. DNAJC17 is localized in nuclear speckles and interacts with splicing machinery components
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Elena Amendola, Andrea Scaloni, G. Ferrandino, C. Sette, A. Pascarella, G De Vita, Sara Carmela Credendino, M De Felice, O. Spadaro, P. Bielli, Michele Ceccarelli, Chiara D'Ambrosio, B. Miranda, Carmen Moccia, Fulvio D'Angelo, Pascarella, A., Ferrandino, G., Credendino, S. C., Moccia, C., D'Angelo, F., Miranda, B., D'Ambrosio, C., Bielli, P., Spadaro, O., Ceccarelli, M., Scaloni, A., Sette, C., De Felice, M., De Vita, G., and Amendola, E.
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0301 basic medicine ,Proteomics ,lcsh:Medicine ,Biology ,Interactome ,Article ,Transcriptome ,03 medical and health sciences ,Protein Interaction Mapping ,Humans ,lcsh:Science ,Gene ,Cell Nucleus ,Gene knockdown ,Settore BIO/16 ,Multidisciplinary ,Alternative Splicing ,HSP40 Heat-Shock Proteins ,HeLa Cells ,Spliceosomes ,Alternative splicing ,lcsh:R ,Cell biology ,030104 developmental biology ,RNA splicing ,Knockout mouse ,lcsh:Q ,Minigene - Abstract
DNAJC17 is a heat shock protein (HSP40) family member, identified in mouse as susceptibility gene for congenital hypothyroidism. DNAJC17 knockout mouse embryos die prior to implantation. In humans, germline homozygous mutations in DNAJC17 have been found in syndromic retinal dystrophy patients, while heterozygous mutations represent candidate pathogenic events for myeloproliferative disorders. Despite widespread expression and involvement in human diseases, DNAJC17 function is still poorly understood. Herein, we have investigated its function through high-throughput transcriptomic and proteomic approaches. DNAJC17-depleted cells transcriptome highlighted genes involved in general functional categories, mainly related to gene expression. Conversely, DNAJC17 interactome can be classified in very specific functional networks, with the most enriched one including proteins involved in splicing. Furthermore, several splicing-related interactors, were independently validated by co-immunoprecipitation and in vivo co-localization. Accordingly, co-localization of DNAJC17 with SC35, a marker of nuclear speckles, further supported its interaction with spliceosomal components. Lastly, DNAJC17 up-regulation enhanced splicing efficiency of minigene reporter in live cells, while its knockdown induced perturbations of splicing efficiency at whole genome level, as demonstrated by specific analysis of RNAseq data. In conclusion, our study strongly suggests a role of DNAJC17 in splicing-related processes and provides support to its recognized essential function in early development.
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- 2018
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30. SOHLH1 and SOHLH2 directly down-regulate STIMULATED BY RETINOIC ACID 8 (STRA8) expression
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M De Felici, Maria Giovanna Desimio, Federica Campolo, Susanna Dolci, and Donatella Farini
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Male ,Cellular differentiation ,Molecular Sequence Data ,Retinoic acid ,Down-Regulation ,Gene Expression ,Biology ,Promoter Regions ,chemistry.chemical_compound ,Genetic ,Gene expression ,Basic Helix-Loop-Helix Transcription Factors ,medicine ,STRA8 ,Humans ,Gene silencing ,Gene Silencing ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Adaptor Proteins, Signal Transducing ,Settore BIO/16 ,Base Sequence ,SOHLH1 ,HEK 293 cells ,SOHLH2 ,Signal Transducing ,Adaptor Proteins ,Cell Differentiation ,Cell Biology ,Molecular biology ,Spermatogonia ,Meiosis ,HEK293 Cells ,medicine.anatomical_structure ,chemistry ,Cell culture ,Germ cell ,Reports ,Protein Binding ,Developmental Biology - Abstract
As the name implies, Stimulated by Retinoic Acid 8 is an early retinoic acid (RA) responsive gene pivotal for the beginning of meiosis in female and male germ cells. Its expression is strictly time-dependent and cell-specific (pre-meiotic germ cells) and likely requires a complex mechanism of regulation. In this study, we demonstrate a direct negative control of SOHLH1 and SOHLH2, 2 germ cell specific bHLH transcription factors, on Stra8 expression. We observed a negative correlation between STRA8 and SOHLH1 expression in prepuberal differentiating mouse KIT(+) spermatogonia and found that SOHLH1 and SOHLH2 were able to directly and cooperatively repress STRA8 expression in cell lines in vitro through binding to its promoter. We also identified 2 canonical E-Box motives in the Stra8 promoter that mediated the negative regulation of SOHLH1 and SOHLH2 on these gene both in the cell lines and KIT(+) spermatogonia. We hypothesize that this novel negative activity of SOHLH1 and SOHLH2 in male cooperates with that of other transcription factors to coordinate spermatogonia differentiation and the RA-induced meiosis and in female ensures STRA8 down-regulation at mid-end stages of meiotic prophase I.
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- 2015
31. Splicing Regulation: A Molecular Device to Enhance Cancer Cell Adaptation
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Chiara Naro, Claudio Sette, and Vittoria Pagliarini
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Genetics and Molecular Biology (all) ,Immunology and Microbiology (all) ,Physiological ,Cell Plasticity ,lcsh:Medicine ,Adaptation, Physiological ,Alternative Splicing ,Animals ,Gene Expression Regulation ,Humans ,Neoplasms ,Models, Genetic ,Biochemistry, Genetics and Molecular Biology (all) ,Review Article ,Biology ,Biochemistry ,General Biochemistry, Genetics and Molecular Biology ,Genetic ,Models ,Gene expression ,medicine ,Adaptation ,Settore BIO/16 - ANATOMIA UMANA ,Genetics ,Regulation of gene expression ,Settore BIO/16 ,General Immunology and Microbiology ,lcsh:R ,Alternative splicing ,Cancer ,General Medicine ,medicine.disease ,Cell biology ,Cancer cell ,RNA splicing ,Proteome ,Human genome - Abstract
Alternative splicing (AS) represents a major resource for eukaryotic cells to expand the coding potential of their genomes and to finely regulate gene expression in response to both intra- and extracellular cues. Cancer cells exploit the flexible nature of the mechanisms controlling AS in order to increase the functional diversity of their proteome. By altering the balance of splice isoforms encoded by human genes or by promoting the expression of aberrant oncogenic splice variants, cancer cells enhance their ability to adapt to the adverse growth conditions of the tumoral microenvironment. Herein, we will review the most relevant cancer-related splicing events and the underlying regulatory mechanisms allowing tumour cells to rapidly adapt to the harsh conditions they may face during the occurrence and development of cancer.
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- 2015
32. Overactive type 2 cannabinoid receptor induces meiosis in fetal gonads and impairs ovarian reserve
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Gabriele Rossi, Susanna Dolci, Federica Todaro, Raffaele Geremia, Emanuela De Domenico, Paola Grimaldi, and Pellegrino Rossi
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0301 basic medicine ,Male ,Cancer Research ,medicine.medical_specialty ,Cannabinoid receptor ,Offspring ,Immunology ,Stimulation ,Apoptosis ,Biology ,reproduction ,Histones ,Receptor, Cannabinoid, CB2 ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Gonocyte ,Fetus ,Meiosis ,Ovarian Follicle ,Pregnancy ,Internal medicine ,Testis ,medicine ,Humans ,DNA Breaks, Double-Stranded ,endocannabinoids ,Ovarian reserve ,Gonads ,Ovarian Reserve ,Spermatogenesis ,Settore BIO/16 ,Cannabinoids ,Cell Biology ,Oocyte ,030104 developmental biology ,medicine.anatomical_structure ,Endocrinology ,Germ Cells ,Oocytes ,Female ,Original Article ,030217 neurology & neurosurgery - Abstract
Type 2 cannabinoid receptor (CB2R) has been proposed to promote in vitro meiotic entry of postnatal male germ cells and to maintain the temporal progression of spermatogenesis in vivo. However, no information is presently available on the role played by CB2R in male and female fetal gonads. Here we show that in vitro pharmacological stimulation with JWH133, a CB2R agonist, induced activation of the meiotic program in both male and female fetal gonads. Upon stimulation, gonocytes initiated the meiotic program but became arrested at early stages of prophase I, while oocytes showed an increased rate of meiotic entry and progression toward more advanced stage of meiosis. Acceleration of meiosis in oocytes was accompanied by a strong increase in the percentage of γ-H2AX-positive pachytene and diplotene cells, paralleled by an increase of TUNEL-positive cells, suggesting that DNA double-strand breaks were not correctly repaired during meiosis, leading to oocyte apoptosis. Interestingly, in vivo pharmacological stimulation of CB2R in fetal germ cells through JWH133 administration to pregnant females caused a significant reduction of primordial and primary follicles in the ovaries of newborns with a consequent depletion of ovarian reserve and reduced fertility in adult life, while no alterations of spermatogenesis in the testis of the offspring were detected. Altogether our findings highlight a pro-meiotic role of CB2R in male and female germ cells and suggest that the use of cannabis in pregnant female might represent a risk for fertility and reproductive lifespan in female offspring.
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- 2017
33. An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation
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Sara Di Persio, Pamela Bielli, Pierre de la Grange, Raffaele Geremia, Claudio Sette, Antonio José Alberdi, Chiara Naro, Ariane Jolly, Niclas Setterblad, and Elena Vicini
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Male ,Proteomics ,0301 basic medicine ,RNA Stability ,Messenger ,Inbred C57BL ,intron retention ,Mice ,Genetics ,Settore BIO/16 ,Cell Differentiation ,Spermatozoa ,Meiosis ,medicine.anatomical_structure ,Gamete ,Transcription ,Reprogramming ,Germ cell ,alternative splicing ,germ cell differentiation ,mRNA stability ,spermatogenesis ,transcriptome profiling ,Alternative Splicing ,Animals ,Cell Nucleus ,Gene Ontology ,Introns ,Mice, Inbred C57BL ,RNA, Messenger ,Regulatory Sequences, Nucleic Acid ,Spermatogenesis ,Transcription, Genetic ,Transcriptome ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Developmental Biology ,03 medical and health sciences ,Genetic ,medicine ,Molecular Biology ,Gene ,Settore BIO/16 - ANATOMIA UMANA ,Nucleic Acid ,Alternative splicing ,Intron ,Cell Biology ,030104 developmental biology ,RNA ,Regulatory Sequences ,Protein Processing, Post-Translational - Abstract
Summary Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis., Highlights • Alternative splicing in meiotic spermatocytes favors intron retention • Intron retention stabilizes nuclear transcripts in meiotic spermatocytes • A high transcription rate outcompetes the spliceosome from weak introns in meiosis • Long-lived intron-retaining mRNAs encode key proteins for male gamete function, Transcription during mammalian spermatogenesis is discontinuous and ceases in late post-meiotic stages. Naro et al. show that regulated intron retention in male meiotic germ cells generates long-lived intron-retaining transcripts that are preserved for days after their synthesis and can be timely translated by transcriptionally inactive post-meiotic germ cells.
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- 2017
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34. Effect Of Microgravity On Aromatase Expression In Sertoli Cells
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Paola Grimaldi, Raffaele Geremia, Elisa Cirelli, Renato Massoud, Emanuela De Domenico, and Flavia Botti
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0301 basic medicine ,Male ,medicine.medical_specialty ,medicine.drug_class ,Science ,Article ,03 medical and health sciences ,Mice ,Aromatase ,Downregulation and upregulation ,Internal medicine ,medicine ,Glial cell line-derived neurotrophic factor ,Animals ,Viability assay ,Cells, Cultured ,Settore BIO/16 ,Multidisciplinary ,Sertoli Cells ,biology ,Weightlessness ,Cell cycle ,Sertoli cell ,Cell biology ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,Cell culture ,Estrogen ,biology.protein ,Medicine ,Biomarkers - Abstract
Cytochrome P450-aromatase catalyzes estrogen biosynthesis from C19 steroids. In the testis, Sertoli cells express P450-aromatase and represent the primary source of estrogen during prepuberal age. This study focused on the effect of simulated microgravity (SM) on aromatase expression in primary mouse Sertoli cells. When cultured in Rotary Cell Culture System (RCCS), Sertoli cells, formed multicellular three dimensional spheroids (3D). Biological properties were first analyzed in terms of viability, cell cycle, expression of cytoskeletal components and growth factors in comparison to Sertoli cells cultured in spheroids at unit gravity (G). SM did not affect cell viability and proliferation, nor expression of the main cytoskeleton proteins and of growth factors like Kit Ligand (KL) and glial derived neurotrophic factor (GDNF). On the other hand, SM caused a strong increase in P450 aromatase mRNA and protein expression. Interestingly, P450-aromatase was no more inducible by 8-Br-cAMP. The presence of a functional aromatase was confirmed by enrichment of 17β-estradiol released in the medium by androgen precursors. We concluded that SM causes a significant upregulation of aromatase gene expression in Sertoli cells, leading to a consequent increase in 17β-estradiol secretion. High level of 17β-estradiol in the testis could have potentially adverse effects on male fertility and testicular cancer.
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- 2017
35. Type 5 phosphodiesterase regulates glioblastoma multiforme aggressiveness and clinical outcome
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Silvia Di Agostino, Lucia Ricci Vitiani, Pellegrino Rossi, Maurizio Martini, Susanna Dolci, Luigi Maria Larocca, Quintino Giorgio D'Alessandris, Emmanuele A. Jannini, Giovanni Luca Gravina, Roberto Pallini, Grazia Graziani, and Valeriana Cesarini
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Male ,0301 basic medicine ,Settore MED/27 - NEUROCHIRURGIA ,Glioblastoma ,MMP2 ,MYPT ,PARP ,PDE5 ,Adult ,Aged ,Aged, 80 and over ,Blotting, Western ,Brain Neoplasms ,Cell Line, Tumor ,Cell Survival ,Cyclic GMP-Dependent Protein Kinases ,Cyclic Nucleotide Phosphodiesterases, Type 5 ,Female ,Humans ,Kaplan-Meier Estimate ,Matrix Metalloproteinase 2 ,Middle Aged ,Neoplasm Invasiveness ,Poly(ADP-ribose) Polymerases ,RNA Interference ,Retrospective Studies ,Reverse Transcriptase Polymerase Chain Reaction ,Treatment Outcome ,Gene Expression Regulation, Neoplastic ,Oncology ,Cell ,medicine.disease_cause ,80 and over ,Settore BIO/16 ,Tumor ,Hematology ,Blotting ,humanities ,medicine.anatomical_structure ,Type 5 ,Western ,Research Paper ,Cyclic Nucleotide Phosphodiesterases ,medicine.medical_specialty ,Cell Line ,03 medical and health sciences ,Internal medicine ,medicine ,Gene silencing ,Epigenetics ,Neoplastic ,business.industry ,glioblastoma ,Cancer ,medicine.disease ,Molecular medicine ,Surgery ,030104 developmental biology ,Gene Expression Regulation ,Cancer cell ,Cancer research ,business ,Carcinogenesis - Abstract
// Valeriana Cesarini 1, * , Maurizio Martini 2, * , Lucia Ricci Vitiani 3 , Giovanni Luca Gravina 4 , Silvia Di Agostino 5 , Grazia Graziani 6 , Quintino Giorgio D’Alessandris 7 , Roberto Pallini 7 , Luigi M. Larocca 2 , Pellegrino Rossi 1 , Emmanuele A. Jannini 6 , Susanna Dolci 1 1 Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy 2 Institute of Pathological Anatomy, Catholic University of Rome, Rome, Italy 3 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita (ISS), Rome, Italy 4 Department of Biotechnological and Applied Clinical Sciences, Laboratory of Radiobiology, University of L’Aquila, L’Aquila, Italy 5 Regina Elena National Cancer Institute-IFO, Oncogenomic and Epigenetic Unit, Rome, Italy 6 Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy 7 Department of Neurosurgery, Catholic University of Rome, Rome, Italy * These authors have contributed equally to this work Correspondence to: Susanna Dolci, email: dolci@uniroma2.it Keywords: PDE5, PARP, glioblastoma, MYPT, MMP2 Received: May 24, 2016 Accepted: December 12, 2016 Published: January 14, 2017 ABSTRACT Expression of type 5 phosphodiesterase (PDE5), a cGMP-specific hydrolytic enzyme, is frequently altered in human cancer, but its specific role in tumorigenesis remains controversial. Herein, by analyzing a cohort of 69 patients affected by glioblastoma multiforme (GBM) who underwent chemo- and radiotherapy after surgical resection of the tumor, we found that PDE5 was strongly expressed in cancer cells in about 50% of the patients. Retrospective analysis indicated that high PDE5 expression in GBM cells significantly correlated with longer overall survival of patients. Furthermore, silencing of endogenous PDE5 by short hairpin lentiviral transduction (sh-PDE5) in the T98G GBM cell line induced activation of an invasive phenotype. Similarly, pharmacological inhibition of PDE5 activity strongly enhanced cell motility and invasiveness in T98G cells. This invasive phenotype was accompanied by increased secretion of metallo-proteinase 2 (MMP-2) and activation of protein kinase G (PKG). Moreover, PDE5 silencing markedly enhanced DNA damage repair and cell survival following irradiation. The enhanced radio-resistance of sh-PDE5 GBM cells was mediated by an increase of poly(ADP-ribosyl)ation (PARylation) of cellular proteins and could be counteracted by poly(ADP-ribose) polymerase (PARP) inhibitors. Conversely, PDE5 overexpression in PDE5-negative U87G cells significantly reduced MMP-2 secretion, inhibited their invasive potential and interfered with DNA damage repair and cell survival following irradiation. These studies identify PDE5 as a favorable prognostic marker for GBM, which negatively affects cell invasiveness and survival to ionizing radiation. Moreover, our work highlights the therapeutic potential of targeting PKG and/or PARP activity in this currently incurable subset of brain cancers.
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- 2017
36. Sox2 is not required for melanomagenesis, melanoma growth and melanoma metastasis in vivo
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Emmanuele A. Jannini, Silvia K. Nicolis, Simona Caporali, Valeriana Cesarini, Antonio Costanzo, Federica Todaro, Susanna Dolci, Rebecca Favaro, Pellegrino Rossi, Elisabetta Botti, P M Lacal, Eugenia Guida, V Tassinari, S Di Agostino, Cesarini, V, Guida, E, Todaro, F, Di Agostino, S, Tassinari, V, Nicolis, S, Favaro, R, Caporali, S, Lacal, P, Botti, E, Costanzo, A, Rossi, P, Jannini, E, and Dolci, S
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Proto-Oncogene Proteins B-raf ,0301 basic medicine ,PTEN ,Cancer Research ,Indoles ,Nevi and melanomas ,Sox2 ,SOX2, MELANOMA, BRAFV600E, PTEN, VEMURAFENIB, CONDITIONAL GENE MODIFICATION ,Metastasis ,Malignant transformation ,Mice ,03 medical and health sciences ,Genetic ,stomatognathic system ,CONDITIONAL GENE MODIFICATION ,Cell Line, Tumor ,Genetics ,medicine ,Animals ,Humans ,melanocyte transformation ,metastasis ,mouse model ,Vemurafenib ,Molecular Biology ,neoplasms ,Melanoma ,Settore BIO/16 ,Sulfonamides ,biology ,SOXB1 Transcription Factors ,PTEN Phosphohydrolase ,Cancer ,medicine.disease ,030104 developmental biology ,Drug Resistance, Neoplasm ,embryonic structures ,biology.protein ,Cancer research ,BrafV600E ,sense organs ,Skin cancer ,medicine.drug - Abstract
Melanoma is a dangerous form of skin cancer derived from the malignant transformation of melanocytes. The transcription factor SOX2 is not expressed in melanocytes, however, it has been shown to be differentially expressed between benign nevi and malignant melanomas and to be essential for melanoma stem cell maintenance and expansion in vitro and in xenograft models. By using a mouse model in which BRafV600E mutation cooperates with Pten loss to induce the development of metastatic melanoma, we investigated if Sox2 is required during the process of melanomagenesis, melanoma growth and metastasis and in the acquisition of resistance to BRAF inhibitors (BRAFi) treatments. We found that deletion of Sox2 specifically in Pten null and BRafV600E-expressing melanocytes did not prevent tumor formation and did not modify the temporal kinetics of melanoma occurrence compared to Sox2 wt mice. In addition, tumor growth was similar between Sox2 wt and Sox2 deleted (del) melanomas. By querying publicly available databases, we did not find statistically significant differences in SOX2 expression levels between benign nevi and melanomas, and analysis on two melanoma patient cohorts confirmed that Sox2 levels did not significantly change between primary and metastatic melanomas. Melanoma cell lines derived from both Sox2 genotypes showed a similar sensitivity to vemurafenib treatment and the same ability to develop vemurafenib resistance in long-term cultures. Development of vemurafenib resistance was not dependent on SOX2 expression also in human melanoma cell lines in vitro. Our findings exclude an oncogenic function for Sox2 during melanoma development and do not support a role for this transcription factor in the acquisition of resistance to BRAFi treatments.Oncogene advance online publication, 3 April 2017; doi:10.1038/onc.2017.53
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- 2017
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37. Alternative polyadenylation of ZEB1 promotes its translation during genotoxic stress in pancreatic cancer cells
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Gabriele Capurso, Valentina Panzeri, Ilaria Passacantilli, Donatella Farini, Pamela Bielli, Claudio Sette, Emanuela Pilozzi, and Gianfranco Delle Fave
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0301 basic medicine ,Cancer Research ,endocrine system diseases ,Polyadenylation ,Immunology ,Genotoxic Stress ,Biology ,Deoxycytidine ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Downregulation and upregulation ,Cell Line, Tumor ,Pancreatic cancer ,Polysome ,microRNA ,medicine ,Humans ,3' Untranslated Regions ,Transcription factor ,Settore BIO/16 - ANATOMIA UMANA ,Settore BIO/16 ,Binding Sites ,Base Sequence ,Cell Biology ,Three prime untranslated region ,Zinc Finger E-box-Binding Homeobox 1 ,medicine.disease ,Gemcitabine ,digestive system diseases ,Up-Regulation ,Pancreatic Neoplasms ,MicroRNAs ,030104 developmental biology ,Ribonucleoproteins ,Polyribosomes ,Cancer research ,Original Article ,Carcinoma, Pancreatic Ductal - Abstract
Pancreatic ductal adenocarcinoma (PDAC) is characterized by extremely poor prognosis. The standard chemotherapeutic drug, gemcitabine, does not offer significant improvements for PDAC management due to the rapid acquisition of drug resistance by patients. Recent evidence indicates that epithelial-to-mesenchymal transition (EMT) of PDAC cells is strictly associated to early metastasization and resistance to chemotherapy. However, it is not exactly clear how EMT is related to drug resistance or how chemotherapy influences EMT. Herein, we found that ZEB1 is the only EMT-related transcription factor that clearly segregates mesenchymal and epithelial PDAC cell lines. Gemcitabine treatment caused upregulation of ZEB1 protein through post-transcriptional mechanisms in mesenchymal PDAC cells within a context of global inhibition of protein synthesis. The increase in ZEB1 protein correlates with alternative polyadenylation of the transcript, leading to shortening of the 3' untranslated region (UTR) and deletion of binding sites for repressive microRNAs. Polysome profiling indicated that shorter ZEB1 transcripts are specifically retained on the polysomes of PDAC cells during genotoxic stress, while most mRNAs, including longer ZEB1 transcripts, are depleted. Thus, our findings uncover a novel layer of ZEB1 regulation through 3'-end shortening of its transcript and selective association with polysomes under genotoxic stress, strongly suggesting that PDAC cells rely on upregulation of ZEB1 protein expression to withstand hostile environments.
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- 2017
38. Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method
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Pamela Bielli and Claudio Sette
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0301 basic medicine ,Immunoprecipitation ,Strategy and Management ,RNA-binding protein ,Industrial and Manufacturing Engineering ,03 medical and health sciences ,0302 clinical medicine ,Polypyrimidine tract-binding protein ,Settore BIO/16 - ANATOMIA UMANA ,Settore BIO/16 ,biology ,Chemistry ,Mechanical Engineering ,Metals and Alloys ,RNA ,CLIP ,PTBP1 ,Molecular biology ,Protein-RNA interaction ,Cell biology ,030104 developmental biology ,RNA processing ,030220 oncology & carcinogenesis ,Protein-RNA-immunoprecipitation ,RNA splicing ,biology.protein ,RIP-Chip ,ICLIP - Abstract
RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of a RBP to specific regions of its target RNAs in adherent human cell lines.
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- 2017
39. Sam68 promotes self-renewal and glycolytic metabolism in mouse neural progenitor cells by modulating Aldh1a3 pre-mRNA 3'-end processing
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Claudia Compagnucci, Stefano Farioli Vecchioli, Piergiorgio La Rosa, Eleonora Cesari, Elisabetta Volpe, Claudio Sette, and Pamela Bielli
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0301 basic medicine ,Mouse ,Transcription, Genetic ,Regulator ,ALDH1A3 ,Sam68 ,alternative splicing ,cell biology ,glycolytic metabolism ,mouse ,neural progenitor cells ,neuroscience ,pre-mRNA processing ,Neocortex ,Gene Knockout Techniques ,Mice ,Adaptor Proteins, Signal Transducing ,Animals ,Cell Differentiation ,Cell Proliferation ,Neural Stem Cells ,Protein Binding ,RNA Precursors ,RNA-Binding Proteins ,Retinal Dehydrogenase ,Stem Cells ,Glycolysis ,Biology (General) ,Settore BIO/16 ,General Neuroscience ,Neurogenesis ,Adaptor Proteins ,General Medicine ,Cell cycle ,Neural stem cell ,Cell biology ,medicine.anatomical_structure ,Biochemistry ,Medicine ,Precursor mRNA ,Transcription ,Research Article ,QH301-705.5 ,Science ,Biology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Genetic ,medicine ,otorhinolaryngologic diseases ,General Immunology and Microbiology ,Alternative splicing ,Signal Transducing ,Cell Biology ,Embryonic stem cell ,stomatognathic diseases ,030104 developmental biology ,Neuroscience - Abstract
The balance between self-renewal and differentiation of neural progenitor cells (NPCs) dictates neurogenesis and proper brain development. We found that the RNA- binding protein Sam68 (Khdrbs1) is strongly expressed in neurogenic areas of the neocortex and supports the self-renewing potential of mouse NPCs. Knockout of Khdrbs1 constricted the pool of proliferating NPCs by accelerating their cell cycle exit and differentiation into post-mitotic neurons. Sam68 function was linked to regulation of Aldh1a3 pre-mRNA 3'-end processing. Binding of Sam68 to an intronic polyadenylation site prevents its recognition and premature transcript termination, favoring expression of a functional enzyme. The lower ALDH1A3 expression and activity in Khdrbs1-/- NPCs results in reduced glycolysis and clonogenicity, thus depleting the embryonic NPC pool and limiting cortical expansion. Our study identifies Sam68 as a key regulator of NPC self-renewal and establishes a novel link between modulation of ALDH1A3 expression and maintenance of high glycolytic metabolism in the developing cortex. DOI: http://dx.doi.org/10.7554/eLife.20750.001, eLife digest Neurons develop from cells called neural progenitors. These cells can either divide to produce more progenitor cells or develop into specific types of neurons. These two activities – known as self-renewal and differentiation – must be balanced to produce the right number of specialized neurons, without depleting the pool of progenitor cells. The self-renewal and differentiation of progenitor cells is balanced by essentially regulating which genes are active, or expressed, within the cells. In the first step of gene expression, the genetic instructions are copied to form a molecule of pre-messenger RNA (or pre-mRNA for short). Each pre-mRNA molecule is then processed to produce a final product that can be translated into protein. Importantly, two copies of the same pre-mRNA may sometimes be processed in different ways, which allows multiple proteins to be produced from a single gene. RNA-binding proteins control pre-mRNA processing. The expression of one such protein, called Sam68, oscillates during the development of the nervous system, such that its expression peaks when there is intense production of new neurons and then declines. However, it was not known whether Sam68 actually helps neurons to develop. La Rosa et al. have now analysed the role of Sam68 in the developing brain of mice. The experiments confirmed that Sam68 is highly expressed in neural progenitor cells and showed that its levels dictate the cell’s fate: high expression encourages a cell to self-renew, while low expression triggers it to develop into a specialized neuron. Further investigation revealed that Sam68 works by promoting the expression of a metabolic enzyme called Aldehyde Dehydrogenase 1A3 or ALDH1A3. This enzyme promotes the release of energy from molecules of glucose via a process known as anaerobic glycolysis. La Rosa et al. found that cells that lack Sam68 make a truncated version of the pre-mRNA encoding ALDH1A3. This truncated pre-mRNA encodes a shortened version of the enzyme that is inactive. Further experiments confirmed that Sam68 normally prevents this from happening by binding to the pre-mRNA and processing it to produce the full-length, working version of the ALDH1A3 enzyme. Also, La Rosa et al. found that progenitor cells need working ALDH1A3 to keep them dividing, and to stop them from developing into specialized neurons too soon. Finally, because the processing of pre-RNA plays a major role in brain development, problems with this process often lead to intellectual disabilities and neurodegenerative diseases, such as autism spectrum disorder and amyotrophic lateral sclerosis. The next step following on from these new findings will be to investigate whether defects in Sam68 contribute to such conditions and, if so, to look for ways to counteract these defects. DOI: http://dx.doi.org/10.7554/eLife.20750.002
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- 2016
40. Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells
- Author
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Maria Loiarro, Roberto Furlan, Elisabetta Volpe, Chiara Maiorino, Vito Ruggiero, Claudio Sette, Luca Battistini, and Grazia Gallo
- Subjects
animal diseases ,medicine.disease_cause ,Biochemistry ,Monocytes ,Interleukin-1 Receptor-Associated Kinases ,Site-Directed ,Toll IL-1 Receptor (TIR) Domain ,Settore BIO/16 ,Mutation ,Tumor ,NF-kappa B ,Signal transducing adaptor protein ,hemic and immune systems ,Toll-like Receptor (TLR) ,Signal transduction ,Signal Transduction ,Protein Structure ,Mutation, Missense ,chemical and pharmacologic phenomena ,Biology ,Cell Line ,Cell Line, Tumor ,medicine ,Humans ,Molecular Biology ,Death domain ,HEK 293 cells ,Dendritic Cells ,HEK293 Cells ,Cellular Immune Response ,Toll-Like Receptor 2 ,Protein Multimerization ,Toll-Like Receptor 4 ,Mutagenesis ,MyD88 ,Mutagenesis, Site-Directed ,Myeloid Differentiation Factor 88 ,Protein Structure, Tertiary ,Amino Acid Substitution ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Molecular biology ,TLR2 ,bacteria ,Missense ,Tertiary - Abstract
Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu(183), Ser(244), and Arg(288) impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88151-189 and GFP-MyD88168-189), comprising the Glu(183) residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.
- Published
- 2013
41. ATM kinase enables the functional axis of YAP, PML and p53 to ameliorate loss of Werner protein-mediated oncogenic senescence
- Author
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Francesca Fausti, Sabrina Strano, Moshe Oren, Pamela Bielli, Giovanni Blandino, Pier Paolo Pandolfi, Marius Sudol, S Di Agostino, Mario Cioce, and Claudio Sette
- Subjects
Senescence ,Premature aging ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,Cell Cycle Proteins ,Ataxia Telangiectasia Mutated Proteins ,Promyelocytic Leukemia Protein ,Biology ,Transfection ,p38 Mitogen-Activated Protein Kinases ,Promyelocytic leukemia protein ,medicine ,cellular senescence ,Humans ,Nuclear protein ,education ,Molecular Biology ,Werner syndrome ,Yes-associated protein (YAP) ,ATM kinase ,Cellular Senescence ,Exodeoxyribonucleases ,HCT116 Cells ,HEK293 Cells ,MCF-7 Cells ,Nuclear Proteins ,RecQ Helicases ,Signal Transduction ,Transcription Factors ,Tumor Suppressor Protein p53 ,Tumor Suppressor Proteins ,Original Paper ,Settore BIO/16 ,education.field_of_study ,nutritional and metabolic diseases ,Cell Biology ,medicine.disease ,Cell biology ,Cancer research ,biology.protein ,Signal transduction ,Cell aging - Abstract
Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.
- Published
- 2013
42. Essential Role of Sox2 for the Establishment and Maintenance of the Germ Cell Line
- Author
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Pellegrino Rossi, Rebecca Favaro, Federica Campolo, Flavia Botti, Manuela Pellegrini, Manuele Gori, Emmanuele A. Jannini, Susanna Dolci, Silvia K. Nicolis, Campolo, F, Gori, M, Favaro, R, Nicolis, S, Pellegrini, M, Botti, F, Rossi, P, Jannini, E, and Dolci, S
- Subjects
Male ,Sox2 ,Gene Expression ,Transgenic ,Mice ,Primordial germ cells ,Specification ,Animals ,Cell Differentiation ,Cells, Cultured ,DNA-Binding Proteins ,Female ,Germ Cells ,Mice, Transgenic ,Oocytes ,Pluripotent Stem Cells ,SOXB1 Transcription Factors ,Signal Transduction ,Telomere-Binding Proteins ,Transcription Factors ,Settore BIO/16 ,Cultured ,Embryo ,Cell biology ,medicine.anatomical_structure ,Kit tyrosine kinase ,embryogenesis ,germ cell line ,transcription ,embryonic structures ,Molecular Medicine ,Germ line development ,Stem cell ,Germ cell ,endocrine system ,Cells ,Biology ,Sox2, germ cells, stem cells, conditional knockout ,SOX2 ,medicine ,Germ plasm ,urogenital system ,fungi ,Cell Biology ,Oocyte ,Embryonic stem cell ,Molecular biology ,Developmental Biology - Abstract
Sox2 is a pluripotency-conferring gene expressed in primordial germ cells (PGCs) and postnatal oocytes, but the role it plays during germ cell development and early embryogenesis is unknown. Since Sox2 ablation causes early embryonic lethality shortly after blastocyst implantation, we generated mice bearing Sox2-conditional deletion in germ cells at different stages of their development through the Cre/loxP recombination system. Embryos lacking Sox2 in PGCs show a dramatic decrease of germ cell numbers at the time of their specification. At later stages, we found that Sox2 is strictly required for PGC proliferation. On the contrary, Sox2 deletion in meiotic oocytes does not impair postnatal oogenesis and early embryogenesis, indicating that it is not essential for oocyte maturation or for zygotic development. We also show that Sox2 regulates Kit expression in PGCs and binds to discrete transcriptional regulatory sequences of this gene, which is known to be important for PGCs survival and proliferation. Sox2 also stimulates the expression of Zfp148, which is required for normal development of fetal germ cells, and Rif1, a potential regulator of PGC pluripotency.
- Published
- 2013
43. Numerical constraints and feedback control of double-strand breaks in mouse meiosis
- Author
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Scott Keeney, Marco Barchi, Liisa Kauppi, Frédéric Baudat, Julian Lange, and Maria Jasin
- Subjects
Male ,Spo11 ,Animals ,Chromosomes ,Meiosis ,Synaptonemal Complex ,Mice ,Endodeoxyribonucleases ,Feedback, Physiological ,DNA Breaks, Double-Stranded ,Physiological ,Meiocyte ,Feedback ,Double-Stranded ,Prophase ,Genetics ,Homologous chromosome ,X chromosome ,Settore BIO/16 ,biology ,DNA Breaks ,fungi ,Synapsis ,enzymes and coenzymes (carbohydrates) ,Synaptonemal complex ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Research Paper ,Developmental Biology - Abstract
Different organisms display widely different numbers of the programmed double-strand breaks (DSBs) that initiate meiotic recombination (e.g., hundreds per meiocyte in mice and humans vs. dozens in nematodes), but little is known about what drives these species-specific DSB set points or the regulatory pathways that control them. Here we examine male mice with a lowered dosage of SPO11, the meiotic DSB catalyst, to gain insight into the effect of reduced DSB numbers on mammalian chromosome dynamics. An approximately twofold DSB reduction was associated with the reduced ability of homologs to synapse along their lengths, provoking prophase arrest and, ultimately, sterility. In many spermatocytes, chromosome subsets displayed a mix of synaptic failure and synapsis with both homologous and nonhomologous partners (“chromosome tangles”). The X chromosome was nearly always involved in tangles, and small autosomes were involved more often than large ones. We conclude that homolog pairing requirements dictate DSB set points during meiosis. Importantly, our results reveal that karyotype is a key factor: Smaller autosomes and heteromorphic sex chromosomes become weak links when DSBs are reduced below a critical threshold. Unexpectedly, unsynapsed chromosome segments trapped in tangles displayed an elevated density of DSB markers later in meiotic prophase. The unsynapsed portion of the X chromosome in wild-type males also showed evidence that DSB numbers increased as prophase progressed. These findings point to the existence of a feedback mechanism that links DSB number and distribution with interhomolog interactions.
- Published
- 2013
44. Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury
- Author
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Andrea Lenzi, A. Isidori, Ma Venneri, Federica Barbagallo, Fabio Naro, Roberto Gimmelli, Manuela Pellegrini, S. Di Siena, Susanna Dolci, Stefania Lucia Nori, Teresa Mancuso, Eleonora Cianflone, Federica Campolo, Pellegrino Rossi, Daniele Torella, Daniele Gianfrilli, Elisa Giannetta, and Lionel Feigenbaum
- Subjects
0301 basic medicine ,Cancer Research ,Pathology ,Cellular differentiation ,030204 cardiovascular system & hematology ,0302 clinical medicine ,C-KIT RECEPTOR, CARDIAC STEM CELLS, HEART REGENERATION, MAPK AND AKT ACTIVATION ,Phosphorylation ,Receptor ,Settore BIO/16 ,Stem Cells ,cardiac stem cells ,Cell Differentiation ,Cell biology ,Proto-Oncogene Proteins c-kit ,cardiac repair ,Original Article ,MAPK AND AKT ACTIVATION ,Mitogen-Activated Protein Kinases ,Stem cell ,C-KIT RECEPTOR ,ERK and AKT signalling ,Heart regeneration ,Genetically modified mouse ,medicine.medical_specialty ,Transgene ,Immunology ,Mice, Transgenic ,Biology ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,stem-cell factor ,randomized phase-1 trial ,myocardial-infarction ,c-kit(+) cells ,ischemic cardiomyopathy ,in-vivo ,w-locus ,mutations ,expression ,kinase ,c-Kit ,medicine ,Animals ,Regeneration ,Progenitor cell ,Protein kinase B ,Wound Healing ,Myocardium ,c-kit receptor, cardiac repair, stem cells ,Endothelial Cells ,Cell Biology ,Survival Analysis ,Cell Compartmentation ,Hematopoiesis ,Enzyme Activation ,030104 developmental biology ,Amino Acid Substitution ,Mutation ,Proto-Oncogene Proteins c-akt - Abstract
The role of endogenous c-Kit receptor activation on cardiac cell homeostasis and repair remains largely unexplored. Transgenic mice carrying an activating point mutation (TgD814Y) in the kinase domain of the c-Kit gene were generated. c-KitTgD814Y receptor was expressed in the heart during embryonic development and postnatal life, in a similar timing and expression pattern to that of the endogenous gene, but not in the hematopoietic compartment allowing the study of a cardiac-specific phenotype. c-KitTgD814Y mutation produced a constitutive active c-Kit receptor in cardiac tissue and cells from transgenic mice as demonstrated by the increased phosphorylation of ERK1/2 and AKT, which are the main downstream molecular effectors of c-Kit receptor signaling. In adult transgenic hearts, cardiac morphology, size and total c-Kit+ cardiac cell number was not different compared with wt mice. However, when c-KitTgD814Y mice were subjected to transmural necrotic heart damage by cryoinjury (CI), all transgenic survived, compared with half of wt mice. In the sub-acute phase after CI, transgenic and wt mice showed similar heart damage. However, 9 days after CI, transgenic mice exhibited an increased number of c-Kit+CD31+ endothelial progenitor cells surrounding the necrotic area. At later follow-up, a consistent reduction of fibrotic area, increased capillary density and increased cardiomyocyte replenishment rate (as established by BrdU incorporation) were observed in transgenic compared with wt mice. Consistently, CD45−c-Kit+ cardiac stem cells isolated from transgenic c-KitTgD814Y mice showed an enhanced endothelial and cardiomyocyte differentiation potential compared with cells isolated from the wt. Constitutive activation of c-Kit receptor in mice is associated with an increased cardiac myogenic and vasculogenic reparative potential after injury, with a significant improvement of survival.
- Published
- 2016
45. Type 2 cannabinoid receptor contributes to the physiological regulation of spermatogenesis
- Author
-
Emanuela De Domenico, Daniele Di Giacomo, Raffaele Geremia, Paola Grimaldi, and Claudio Sette
- Subjects
0301 basic medicine ,Male ,Cannabinoid receptor ,Indoles ,Cellular differentiation ,Retinoic acid ,Gene Expression ,Biochemistry ,Receptor, Cannabinoid, CB2 ,Histones ,chemistry.chemical_compound ,Mice ,Histone methylation ,retinoic acid ,meiosis ,endocannabinoid system ,Promoter Regions, Genetic ,Cells, Cultured ,Microscopy ,Settore BIO/16 ,Cultured ,Blotting ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Differentiation ,Endocannabinoid system ,CB2 ,medicine.anatomical_structure ,Signal transduction ,histone methylation ,Animals ,Blotting, Western ,Cannabinoids ,Histone-Lysine N-Methyltransferase ,Lysine ,Meiosis ,Methylation ,Microscopy, Fluorescence ,Signal Transduction ,Spermatogenesis ,Spermatogonia ,Tretinoin ,Western ,Germ cell ,Biotechnology ,Receptor ,medicine.medical_specialty ,Cells ,Biology ,Fluorescence ,Promoter Regions ,03 medical and health sciences ,Genetic ,Internal medicine ,Genetics ,medicine ,Molecular Biology ,Cannabinoid ,030104 developmental biology ,Endocrinology ,chemistry ,H3K4me3 - Abstract
Type 2 cannabinoid receptor (CB2) has been proposed to play a pivotal role in meiotic entry of male germ cells, similar to retinoic acid (RA). In this study, we showed that activation of CB2with the specific agonist JWH133 [3-(1',1'-dimethylbutyl)-1-deoxy-8-THC] (IC5010(-6)M) mimics epigenetic events induced by RA (IC5010(-7)M) in spermatogonia. Both JWH133 and RA treatments stimulate the expression of the meiotic genes c-KitandStra8, by up-regulating H3K4me3 and down-regulating H3K9me2 levels in genomic regions flanking the transcription start site. Moreover, both agents increase the expression ofPrdm9, the gene encoding a meiosis-specific histone, H3K4me3 methyltransferase, which marks hotspots of recombination in prophase I, thus resulting in a global increase in H3K4me3. Notably, prolonged administration of JWH133 to immature 7 dpp CD-1 mice induced an acceleration of the onset of spermatogenesis, whereas the specific CB2antagonist delayed germ cell differentiation. Thus, both hyper- and hypostimulation of CB2disrupted the temporal dynamics of the spermatogenic cycle. These findings highlight the importance of proper CB2signaling for the maintenance of a correct temporal progression of spermatogenesis and suggest a possible adverse effect of cannabis in deregulating this process.-Di Giacomo, D., De Domenico, E., Sette, C., Geremia, R., Grimaldi, P. Type 2 cannabinoid receptor contributes to the physiological regulation of spermatogenesis.
- Published
- 2016
46. Targeted inactivation of nuclear interaction partner of ALK disrupts meiotic prophase
- Author
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Illert, A, L, Kawaguchi, H, Antinozzi, C, Bassermann, F, Quintanilla Martinez, L, von Klitzing, C, Hiwatari, M, Peschel, C, De Rooij, D, Morris, S, Barchi, M, Duyster, J, Other Research, and Center for Reproductive Medicine
- Subjects
Male ,NIPA (ZC3HC1) ,Immunoblotting ,CCNB1 ,Biology ,Mice ,Knockout mouse ,Prophase ,Meiosis ,Spermatocytes ,Animals ,Immunoprecipitation ,DNA Breaks, Double-Stranded ,Cyclin B1 ,RNA, Small Interfering ,Kinase activity ,Nuclear protein ,Molecular Biology ,Mitosis ,Research Articles ,Mice, Knockout ,Settore BIO/16 ,Checkpoint ,Cell Cycle ,Synapsis ,Nuclear Proteins ,Cell cycle ,Flow Cytometry ,Immunohistochemistry ,Molecular biology ,Mice, Mutant Strains ,Cell cycle, Knockout mouse, Meiosis, NIPA (ZC3HC1), CCNB1, Checkpoint ,Developmental Biology - Abstract
NIPA (nuclear interaction partner of ALK) is an F-box-like protein that monitors the timing of mitotic entry. Constitutively active NIPA delays mitotic entry by preventing accumulation of nuclear cyclin B1. Here, we have investigated the consequences of Nipa inactivation by using a conditional knockout strategy. Nipa-deficient animals are viable but show a lower birth rate and reduced body weight. Furthermore, Nipa-deficient males are sterile owing to a block of spermatogenesis during meiotic prophase. Whereas Nipa−/− mouse embryonic fibroblasts show no severe phenotype, Nipa−/− spermatocytes arrest during stage IV of the epithelial cycle with subsequent TUNEL-positive apoptosis resulting from improper synapsis, defects in the repair of DNA double-stranded breaks and synaptonemal complex formation. Moreover, we show nuclear accumulation of cyclin B1 with a subsequent premature increase in G2/M kinase activity in Nipa−/− spermatocytes. Together, these results reveal a novel role for NIPA in meiosis.
- Published
- 2012
47. RanBPM is essential for mouse spermatogenesis and oogenesis
- Author
-
Lino Tessarollo, Susanna Dolci, Vincenzo Coppola, Colleen Barrick, and Sandrine Puverel
- Subjects
Male ,Primary Ovarian Insufficiency ,Biology ,medicine.disease_cause ,Oogenesis ,Mice ,medicine ,Animals ,Meiotic Prophase I ,Gonads ,Spermatogenesis ,Molecular Biology ,Research Articles ,Gametogenesis ,Adaptor Proteins, Signal Transducing ,Settore BIO/16 ,Mutation ,Synapsis ,Nuclear Proteins ,Embryonic stem cell ,Molecular biology ,Cell biology ,Cytoskeletal Proteins ,medicine.anatomical_structure ,Female ,Stem cell ,Germ cell ,Developmental Biology - Abstract
RanBPM is a recently identified scaffold protein that links and modulates interactions between cell surface receptors and their intracellular signaling pathways. RanBPM has been shown to interact with a variety of functionally unrelated proteins; however, its function remains unclear. Here, we show that RanBPM is essential for normal gonad development as both male and female RanBPM−/− mice are sterile. In the mutant testis there was a marked decrease in spermatogonia proliferation during postnatal development. Strikingly, the first wave of spermatogenesis was totally compromised, as seminiferous tubules of homozygous mutant animals were devoid of post-meiotic germ cells. We determined that spermatogenesis was arrested around the late pachytene-diplotene stages of prophase I; surprisingly, without any obvious defect in chromosome synapsis. Interestingly, RanBPM deletion led to a remarkably quick disappearance of all germ cell types at around one month of age, suggesting that spermatogonia stem cells are also affected by the mutation. Moreover, in chimeric mice generated with RanBPM−/− embryonic stem cells all mutant germ cells disappeared by 3 weeks of age suggesting that RanBPM is acting in a cell-autonomous way in germ cells. RanBPM homozygous mutant females displayed a premature ovarian failure due to a depletion of the germ cell pool at the end of prophase I, as in males. Taken together, our results highlight a crucial role for RanBPM in mammalian gametogenesis in both genders.
- Published
- 2011
48. The RNA-binding protein Sam68 is a multifunctional player in human cancer
- Author
-
Claudio Sette, Roberta Busà, Maria Paola Paronetto, and Pamela Bielli
- Subjects
Cancer Research ,Endocrinology, Diabetes and Metabolism ,RNA-binding protein ,Biology ,medicine.disease_cause ,Endocrinology ,Neoplasms ,medicine ,Humans ,Neoplastic transformation ,RNA, Neoplasm ,Nuclear export signal ,Adaptor Proteins, Signal Transducing ,Settore BIO/16 ,Alternative splicing ,Signal Transducing ,Adaptor Proteins ,RNA-Binding Proteins ,Cell cycle ,KH domain ,Cell biology ,DNA-Binding Proteins ,Oncology ,Tumor progression ,RNA ,Neoplasm ,Carcinogenesis - Abstract
Src associated in mitosis, of 68 kDa (Sam68) is a KH domain RNA-binding protein that belongs to the signal transduction and activation of RNA family. Although ubiquitously expressed, Sam68 plays very specialized roles in different cellular environments. In most cells, Sam68 resides in the nucleus and is involved in several steps of mRNA processing, from transcription, to alternative splicing, to nuclear export. In addition, Sam68 translocates to the cytoplasm upon cell stimulation, cell cycle transitions or viral infections, where it takes part to signaling complexes and associates with the mRNA translation machinery. Recent evidence has linked Sam68 function to the onset and progression of endocrine tumors, such as prostate and breast carcinomas. Notably, all the biochemical activities reported for Sam68 seem to be implicated in carcinogenesis. Herein, we review the recent advancement in the knowledge of Sam68 function and regulation and discuss it in the frame of its participation to neoplastic transformation and tumor progression. Endocrine-Related Cancer (2011) 18 R91–R102
- Published
- 2011
49. Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells
- Author
-
Marco Barchi, Stéphane Richard, Valeria Messina, Maria Paola Paronetto, Claudio Sette, and Raffaele Geremia
- Subjects
Male ,Transcription, Genetic ,RNA-binding protein ,Spermatocyte ,Gene Regulation, Chromatin and Epigenetics ,Biology ,Mice ,Splicing factor ,Exon ,Spermatocytes ,Sarcoglycans ,Genetics ,medicine ,Animals ,Meiotic Prophase I ,Spermatogenesis ,Adaptor Proteins, Signal Transducing ,Mice, Knockout ,Transcriptionally active chromatin ,Settore BIO/16 ,Alternative splicing ,Intron ,RNA-Binding Proteins ,Spermatozoa ,Molecular biology ,Alternative Splicing ,medicine.anatomical_structure ,RNA splicing ,RNA Polymerase II - Abstract
Sam68 plays an essential role in mouse spermatogenesis and male fertility. Herein, we report an interaction between Sam68 and the phosphorylated forms of the RNA polymerase II (RNAPII) in meiotic spermatocytes. RNase treatment decreased but did not abolish the interaction, consistently with in vitro binding of RNAPII to the Sam68 carboxyl-terminal region. Sam68 retention in the spermatocyte nucleus was dependent on the integrity of cellular RNAs, suggesting that the protein is recruited to transcriptionally active chromatin. Mouse knockout models characterized by stage-specific arrest of spermatogenesis and staining with the phosphorylated form of RNAPII documented that Sam68 expression is confined to the transcriptionally active stages of spermatogenesis. Furthermore, Sam68 associates with splicing regulators in germ cells and we report that alternative splicing of Sgce exon 8 is regulated in a Sam68-dependent manner during spermatogenesis. RNA and chromatin crosslink immunoprecipitation experiments showed that Sam68 binds in vivo to sequences surrounding the intron 7/exon 8 boundary, thereby affecting the recruitment of the phosphorylated RNAPII and of the general splicing factor U2AF65. These results suggest that Sam68 regulates alternative splicing at transcriptionally active sites in differentiating germ cells and provide new insights into the regulation of Sam68 expression during spermatogenesis.
- Published
- 2011
50. Targeted JAM-C deletion in germ cells by Spo11-controlled Cre recombinase
- Author
-
Raffaele Geremia, Pellegrino Rossi, Giuseppina Claps, Gabriele Rossi, Bernd Arnold, Shyam K. Sharan, Lionel Feigenbaum, Susanna Dolci, Florencia Barrios, Valeria V. Orlova, Triantafyllos Chavakis, André Nussenzweig, and Manuela Pellegrini
- Subjects
Male ,Genetically modified mouse ,Spo11 ,Knockout ,Transgene ,Cre recombinase ,Mice, Transgenic ,Inbred C57BL ,Nbs1 ,Transgenic ,Promoter Regions ,Mice ,Genetic ,Conditional gene knockout ,Animals ,JAM-C ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Research Articles ,Gene knockout ,Mice, Knockout ,Settore BIO/16 ,Endodeoxyribonucleases ,SPO11 Gene ,Integrases ,biology ,fungi ,Gene targeting ,Cell Biology ,Miosis ,Molecular biology ,Cell biology ,Mice, Inbred C57BL ,Meiosis ,Germ Cells ,Gene Targeting ,Female ,Gene Deletion ,Knockout mouse ,biology.protein ,Developmental Biology - Abstract
Meiosis is a crucial process for the production of functional gametes. However, the biological significance of many genes expressed during the meiotic phase remains poorly understood, mainly because of the lethal phenotypes of the knockout mice. Functional analysis of such genes using the conditional knockout approach is hindered by the lack of suitable Cre transgenic lines. We describe here the generation of transgenic mice expressing Cre recombinase under the control of the meiotic Spo11 gene. Using LacZ-R26loxP and EYFP-R26loxP reporter mice, we show the specific expression and activity of Cre during meiosis in males and females. Spo11Cre mice were then crossed with floxed Nbs1 and JAM-C mice to produce conditional knockouts. A strong reduction of Nbs1 and JAM-C protein levels was found in the testis. Although Nbs1-deleted mice developed minor gonadal abnormalities, JAM-C-knockout mice showed a spermiogenetic arrest, as previously described for the null mice. These results provide strong evidence that Spo11Cre transgenic mice represent a powerful tool for deleting genes of interest specifically in meiotic and/or in postmeiotic germ cells.
- Published
- 2011
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