Carol IMBRIANO - personale UniMoRe

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Carol IMBRIANO

Professore Associato
Dipartimento di Scienze della Vita sede ex-Biologia

Pubblicazioni

2023 - BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting [Articolo su rivista]
Sorbi, C.; Belluti, S.; Atene, C. G.; Marocchi, F.; Linciano, P.; Roy, N.; Paradiso, E.; Casarini, L.; Ronsisvalle, S.; Zanocco-Marani, T.; Brasili, L.; Lanfrancone, L.; Imbriano, C.; Di Rocco, G.; Franchini, S.
abstract

: The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

2023 - Deciphering the transcriptional network of NF-Y in prostate cancer development and progression [Poster]
Belluti, Silvia; Cuoghi, L.; Mularoni, V.; Forcato, M.; Torricelli, F.; Ciarrocchi, A.; Imbriano, C.
abstract

2023 - Development of Stable Amino-Pyrimidine–Curcumin Analogs: Synthesis, Equilibria in Solution, and Potential Anti-Proliferative Activity [Articolo su rivista]
Mari, Matteo; Boniburini, Matteo; Tosato, Marianna; Rigamonti, Luca; Cuoghi, Laura; Belluti, Silvia; Imbriano, Carol; Avino, Giulia; Asti, Mattia; Ferrari, Erika
abstract

2023 - Discovery of potent pyrrolo-pyrimidine and purine HDAC inhibitors for the treatment of advanced prostate cancer [Articolo su rivista]
Moi, Davide; Bonanni, Davide; Belluti, Silvia; Linciano, Pasquale; Citarella, Andrea; Franchini, Silvia; Sorbi, Claudia; Imbriano, Carol; Pinzi, Luca; Rastelli, Giulio
abstract

2023 - Dysregulation of NF–Y splicing drives metabolic rewiring and aggressiveness in colon cancer [Poster]
Belluti, S.; Mularoni, V.; Campani, Virginia; Rigillo, G.; Cuoghi, L.; Ronzio, M.; Miserocchi, G.; Dolfini, D.; Righi, Valeria; Alessandrini, A.; Zappavigna, V.; Imbriano, C.
abstract

NF-Y is an evolutionarily conserved transcription factor that binds specifically to the CCAAT elements of eukaryotic genes, most of which frequently deregulated in cancer. NF-YA, the regulatory subunit of the NF-Y complex, has two isoforms generated by alternative splicing, NF-YAl and NF-YAs, which differ in the transactivation domain. Transcriptomic data from The Cancer Genome Atlas (TCGA) database highlighted a significant increase in the expression of NF-YAs at the expense of NF-YAl in colorectal cancer (CRC), compared to healthy tissues. Despite this, high NF-YAl levels predict lower patients’ survival and distinguish the mesenchymal molecular subtype CMS4, which is characterized by the worst prognosis. Through the analysis of 3D cellular models, we demonstrated that altered expression of genes related to extracellular matrix and epithelial-mesenchymal transition sustains enhanced migratory and invasive behavior of NF-YAl-transduced cells. Moreover, the integration of metabolomics, bioenergetics and transcriptional analyses demonstrated a direct role for NFYAl in metabolic flexibility of cancer cells that adjust their metabolism in response to environmental changes to potentiate migration. The zebrafish xenograft model confirmed the metastatic potential triggered by NF-YAl in CRC cells. Altogether, our data highlight the transcriptional role of NF-YAl in CRC aggressiveness and suggest splice-switching strategies to hinder NF-YAl-induced metastatic dissemination.

2023 - Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives [Articolo su rivista]
Imbriano, Carol; Moresi, Viviana; Belluti, Silvia; Renzini, Alessandra; Cavioli, Giorgia; Maretti, Eleonora; Molinari, Susanna
abstract

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.

2023 - Histone deacetylase functions and therapeutic implications for adult skeletal muscle metabolism [Articolo su rivista]
Molinari, Susanna; Imbriano, Carol; Moresi, Viviana; Renzini, Alessandra; Belluti, Silvia; Lozanoska-Ochser, Biliana; Gigli, Giuseppe; Cedola, Alessia
abstract

2023 - Nuclear Estrogen Receptors in Prostate Cancer: From Genes to Function [Articolo su rivista]
Belluti, Silvia; Imbriano, Carol; Casarini, Livio
abstract

: Estrogens are almost ubiquitous steroid hormones that are essential for development, metabolism, and reproduction. They exert both genomic and non-genomic action through two nuclear receptors (ERα and ERβ), which are transcription factors with disregulated functions and/or expression in pathological processes. In the 1990s, the discovery of an additional membrane estrogen G-protein-coupled receptor augmented the complexity of this picture. Increasing evidence elucidating the specific molecular mechanisms of action and opposing effects of ERα and Erβ was reported in the context of prostate cancer treatment, where these issues are increasingly investigated. Although new approaches improved the efficacy of clinical therapies thanks to the development of new molecules targeting specifically estrogen receptors and used in combination with immunotherapy, more efforts are needed to overcome the main drawbacks, and resistance events will be a challenge in the coming years. This review summarizes the state-of-the-art on ERα and ERβ mechanisms of action in prostate cancer and promising future therapies.

2023 - The NF-Y splicing signature controls hybrid EMT and ECM-related pathways to promote aggressiveness of colon cancer [Articolo su rivista]
Rigillo, Giovanna; Belluti, Silvia; Campani, Virginia; Ragazzini, Gregorio; Ronzio, Mirko; Miserocchi, Giacomo; Bighi, Beatrice; Cuoghi, Laura; Mularoni, Valentina; Zappavigna, Vincenzo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

: Aberrant splicing events are associated with colorectal cancer (CRC) and provide new opportunities for tumor diagnosis and treatment. The expression of the splice variants of NF-YA, the DNA binding subunit of the transcription factor NF-Y, is deregulated in multiple cancer types compared to healthy tissues. NF-YAs and NF-YAl isoforms differ in the transactivation domain, which may result in distinct transcriptional programs. In this study, we demonstrated that the NF-YAl transcript is higher in aggressive mesenchymal CRCs and predicts shorter patients' survival. In 2D and 3D conditions, CRC cells overexpressing NF-YAl (NF-YAlhigh) exhibit reduced cell proliferation, rapid single cell amoeboid-like migration, and form irregular spheroids with poor cell-to-cell adhesion. Compared to NF-YAshigh, NF-YAlhigh cells show changes in the transcription of genes involved in epithelial-mesenchymal transition, extracellular matrix and cell adhesion. NF-YAl and NF-YAs bind similarly to the promoter of the E-cadherin gene, but oppositely regulate its transcription. The increased metastatic potential of NF-YAlhigh cells in vivo was confirmed in zebrafish xenografts. These results suggest that the NF-YAl splice variant could be a new CRC prognostic factor and that splice-switching strategies may reduce metastatic CRC progression.

2023 - The NF-YA Splicing Signature Controls Aggressiveness of Colon Cancer by Regulating Cell Metabolism and Different Types of Cell Migration [Relazione in Atti di Convegno]
Belluti, Silvia; Mularoni, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-Y isoforms, NFYAs and NF-YAl, differentially control cell proliferation and differentiation. The analysis of patient’s transcriptome profiles from The Cancer Genome Atlas database highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA have a lower overall survival probability. We investigated the role of NF-YA in the metabolism of CRC cells, and the measurement of mitochondrial fuel usage in live cells shows that NF-YAl overexpression enhances the capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Moreover, we demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT, extracellular matrix and adhesion genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NFYAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patients’ stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities.

2022 - Alternative splicing of the transcription factor NF-Y promotes cell migration and invasion in colon cancer [Abstract in Atti di Convegno]
Rigillo, Giovanna; Belluti, Silvia; Campani, Virginia; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

The heterotrimeric transcription factor NF-Y directly controls the expression of genes involved in cellular pathways commonly altered in cancer cells, such as cell cycle, apoptosis and metabolism. Consistently, the binding site for NF-Y is highly enriched in the regulatory regions of genes overexpressed in tumors, and mRNA levels of NF-Y subunits are altered in cancer tissues and cells. In particular, the DNA binding subunit NF-YA is up-regulated in various tumors, among which gastric, lung, breast, ovarian, osteosarcoma and prostate cancers. Moreover, a switch between the two alternatively NF-YA spliced transcripts, NF-YAs and NF-YAl, occurs in tumor tissues compared to normal ones. Colorectal cancer (CRC) is the third most common malignancy worldwide. Four internationally approved consensus molecular subtypes (CMS) represent the best current description of CRC heterogeneity at the gene-expression level: the CMS1 group is characterized by the immune infiltration signature, CMS2 is the canonical epithelial subtype, CMS3 represents the metabolic group, and CMS4 is the mesenchymal one, associated with a worse prognosis and poor response to therapies compared to other subtypes. Here we show that increased levels of NF-YA characterize CRC versus healthy tissues. We identified a significant association between NF-YA isoforms and CRC subtypes: NF-YAs is up-regulated in all CMSs in opposition to NF-YAl, which is down-regulated in all subtypes with the exception of aggressive and metastatic CMS4 group. By using in vitro cell models, we confirmed that NF-YAs is the predominant isoform in CRC cell lines, while NF-YAl levels proportionally increase from epithelial to hybrid and mesenchymal cells. The modulation of NF-YA isoforms in CRC cells significantly affects cancer cell behavior by modulating differently, even oppositely, the transcription of genes associated to extracellular-matrix (ECM) and epithelial-to-mesenchymal transition (EMT). We described different modes of migration and invasion properties for NF-YAs and NF-YAl overexpressing cells by using 2D and 3D culture conditions, time-lapse imaging of CRC cells and intravascular distribution of NF-YAs/l transduced CRC cells in the embryonic zebrafish xenograft model. Altogether, our data highlight the direct role of the longer NF-YA isoform in CRC cell dissemination and suggest its possible use as biomarker for molecular stratification predictive of progressive disease in CRC patients.

2022 - Histone Marks-Dependent Effect on Alternative Splicing: New Perspectives for Targeted Splicing Modulation in Cancer? [Articolo su rivista]
Imbriano, Carol; Belluti, Silvia
abstract

2022 - Monitoring DNA Hybridization with Organic Electrochemical Transistors Functionalized with Polydopamine [Articolo su rivista]
Sensi, M.; Migatti, G.; Beni, V.; D'Alvise, T. M.; Weil, T.; Berto, M.; Greco, P.; Imbriano, C.; Biscarini, F.; Bortolotti, C. A.
abstract

Organic electrochemical transistors (OECTs) are finding widespread application in biosensing, thanks to their high sensitivity, broad dynamic range, and low limit of detection. An OECT biosensor requires the immobilization of a biorecognition probe on the gate, or else on the channel, through several, often lengthy, chemical steps. In this work, a fast and straightforward way to functionalize the carbon gate of a fully screen-printed OECT by means of a polydopamine (PDA) film is presented. By chemical immobilization of an amine-terminated single-stranded oligonucleotide, containing the HSP70 promoter CCAAT sequence, on the PDA film, the detection of the complementary DNA strand is demonstrated. Furthermore, the specificity of the developed genosensor is assessed by comparing its response to the fully complementary strand with the one to partially complementary and noncomplementary oligonucleotides. The developed sensor shows a theoretical limit of detection (LOD) of 100 × 10−15 m and a dynamic range over four orders of magnitude.

2022 - The interplay between NF-Y, AR and lipid metabolism regulates tumor aggressiveness in prostate cancer. [Poster]
Belluti, Silvia; Mularoni, Valentina; Imbriano, Carol.
abstract

Prostate cancer (PCa) is the second most frequent cancer in Western men. Computational analyses linked the transcription factor NF-Y to progression from benign to localized PCa, aggressive signatures, response to androgen deprivation therapy (ADT) and metastasis. The NF-YA gene encodes two alternatively spliced transcripts, NF-YAs and NF-YAl. We demonstrated that PCa samples are characterized by increased NF-YA levels, as well as higher NF-YAs/NF-YAl transcriptional ratio. In vitro and in vivo, NF-YA depletion affects PCa tumorigenicity and changes in NF-YA isoforms expression are associated with key clinical and molecular features of aggressive PCa. NF-YAs enhances tumor growth and metastasis, while NF-YAl increases cell motility. Stratification of patients based on NF-YAs expression is predictive of clinical outcome, although a significant decrease in the NF-YAs/NF-YAl ratio characterizes PCa circulating cells. PCa cells depend on male sex hormones for growth and survival, which is the basis of ADT. While ADT is initially effective, patients eventually relapse with castration-resistant prostate cancer (CRPC). One of the cellular processes most affected by androgens is lipid metabolism, whose rewiring contributes to PCa development, progression, metastasis and recurrence. Androgens stimulate de novo lipogenesis and lipid uptake by activating SREBPs, the master transcription factors of cholesterol and fatty acid biosynthesis. SREBPs interact with target promoters in cooperation with NF-Y, which controls de novo lipid biosynthesis pathway. A feedforward mechanism between SREBP and the Androgen Receptor (AR) was also described. We modulated NF-YA in androgen-sensitive healthy and cancer cell lines, performing cellular and molecular studies. RNA-seq analysis highlighted the alteration of lipid and cholesterol metabolic pathways and of the unfolded protein response (UPR) in NF-YAl-overexpressing cells. Bioenergetics metabolic profiling of NF-YA transduced cells confirmed the key role of NF-YAl in PCa cell metabolism. We found a reciprocal regulation of AR and NF-Y that relies in particular on NF-YAl. Our data suggest that NF-Y, and specifically the longer NF-YA isoform, can participate in ADT responsiveness and resistance mediated by metabolic alterations. The characterization of these pathways could be useful in the stratification of PCa patients into sub-categories with different levels of aggressiveness and ADT sensitivity.

2022 - The NF-YA splicing signature controls aggressiveness of colon cancer by regulating different modes of cell migration and cell metabolism. [Poster]
Belluti, Silvia; Rigillo, Giovanna; Mularoni, Valentina; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-YA isoforms, NF-YAs and NF-YAl, differentially control cell proliferation and differentiation. TCGA data highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA levels have a lower overall survival probability. We demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NF-YAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Since metabolic reprogramming is a hallmark of cancer and metastasis, we also investigated the role of NF-YAl in the metabolism of CRC cells. The measure of mitochondrial fuel usage in live cells showed that NF-YAl overexpression enhances the dependency and capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patient stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities.

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Poster]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; REGGIANI BONETTI, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; Reggiani Bonetti, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes.

2021 - Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) [Articolo su rivista]
Klionsky, D. J.; Abdel-Aziz, A. K.; Abdelfatah, S.; Abdellatif, M.; Abdoli, A.; Abel, S.; Abeliovich, H.; Abildgaard, M. H.; Abudu, Y. P.; Acevedo-Arozena, A.; Adamopoulos, I. E.; Adeli, K.; Adolph, T. E.; Adornetto, A.; Aflaki, E.; Agam, G.; Agarwal, A.; Aggarwal, B. B.; Agnello, M.; Agostinis, P.; Agrewala, J. N.; Agrotis, A.; Aguilar, P. V.; Ahmad, S. T.; Ahmed, Z. M.; Ahumada-Castro, U.; Aits, S.; Aizawa, S.; Akkoc, Y.; Akoumianaki, T.; Akpinar, H. A.; Al-Abd, A. M.; Al-Akra, L.; Al-Gharaibeh, A.; Alaoui-Jamali, M. A.; Alberti, S.; Alcocer-Gomez, E.; Alessandri, C.; Ali, M.; Alim Al-Bari, M. A.; Aliwaini, S.; Alizadeh, J.; Almacellas, E.; Almasan, A.; Alonso, A.; Alonso, G. D.; Altan-Bonnet, N.; Altieri, D. C.; Alvarez, E. M. C.; Alves, S.; Alves da Costa, C.; Alzaharna, M. M.; Amadio, M.; Amantini, C.; Amaral, C.; Ambrosio, S.; Amer, A. O.; Ammanathan, V.; An, Z.; Andersen, S. U.; Andrabi, S. A.; Andrade-Silva, M.; Andres, A. M.; Angelini, S.; Ann, D.; Anozie, U. C.; Ansari, M. Y.; Antas, P.; Antebi, A.; Anton, Z.; Anwar, T.; Apetoh, L.; Apostolova, N.; Araki, T.; Araki, Y.; Arasaki, K.; Araujo, W. L.; Araya, J.; Arden, C.; Arevalo, M. -A.; Arguelles, S.; Arias, E.; Arikkath, J.; Arimoto, H.; Ariosa, A. R.; Armstrong-James, D.; Arnaune-Pelloquin, L.; Aroca, A.; Arroyo, D. S.; Arsov, I.; Artero, R.; Asaro, D. M. L.; Aschner, M.; Ashrafizadeh, M.; Ashur-Fabian, O.; Atanasov, A. G.; Au, A. K.; Auberger, P.; Auner, H. W.; Aurelian, L.; Autelli, R.; Avagliano, L.; Avalos, Y.; Aveic, S.; Aveleira, C. A.; Avin-Wittenberg, T.; Aydin, Y.; Ayton, S.; Ayyadevara, S.; Azzopardi, M.; Baba, M.; Backer, J. M.; Backues, S. K.; Bae, D. -H.; Bae, O. -N.; Bae, S. H.; Baehrecke, E. H.; Baek, A.; Baek, S. -H.; Baek, S. H.; Bagetta, G.; Bagniewska-Zadworna, A.; Bai, H.; Bai, J.; Bai, X.; Bai, Y.; Bairagi, N.; Baksi, S.; Balbi, T.; Baldari, C. T.; Balduini, W.; Ballabio, A.; Ballester, M.; Balazadeh, S.; Balzan, R.; Bandopadhyay, R.; Banerjee, S.; Banerjee, S.; Banreti, A.; Bao, Y.; Baptista, M. S.; Baracca, A.; Barbati, C.; Bargiela, A.; Barila, D.; Barlow, P. G.; Barmada, S. J.; Barreiro, E.; Barreto, G. E.; Bartek, J.; Bartel, B.; Bartolome, A.; Barve, G. R.; Basagoudanavar, S. H.; Bassham, D. C.; Bast, R. C.; Basu, A.; Batoko, H.; Batten, I.; Baulieu, E. E.; Baumgarner, B. L.; Bayry, J.; Beale, R.; Beau, I.; Beaumatin, F.; Bechara, L. R. G.; Beck, G. R.; Beers, M. F.; Begun, J.; Behrends, C.; Behrens, G. M. N.; Bei, R.; Bejarano, E.; Bel, S.; Behl, C.; Belaid, A.; Belgareh-Touze, N.; Bellarosa, C.; Belleudi, F.; Bello Perez, M.; Bello-Morales, R.; Beltran, J. S. D. O.; Beltran, S.; Benbrook, D. M.; Bendorius, M.; Benitez, B. A.; Benito-Cuesta, I.; Bensalem, J.; Berchtold, M. W.; Berezowska, S.; Bergamaschi, D.; Bergami, M.; Bergmann, A.; Berliocchi, L.; Berlioz-Torrent, C.; Bernard, A.; Berthoux, L.; Besirli, C. G.; Besteiro, S.; Betin, V. M.; Beyaert, R.; Bezbradica, J. S.; Bhaskar, K.; Bhatia-Kissova, I.; Bhattacharya, R.; Bhattacharya, S.; Bhattacharyya, S.; Bhuiyan, M. S.; Bhutia, S. K.; Bi, L.; Bi, X.; Biden, T. J.; Bijian, K.; Billes, V. A.; Binart, N.; Bincoletto, C.; Birgisdottir, A. B.; Bjorkoy, G.; Blanco, G.; Blas-Garcia, A.; Blasiak, J.; Blomgran, R.; Blomgren, K.; Blum, J. S.; Boada-Romero, E.; Boban, M.; Boesze-Battaglia, K.; Boeuf, P.; Boland, B.; Bomont, P.; Bonaldo, P.; Bonam, S. R.; Bonfili, L.; Bonifacino, J. S.; Boone, B. A.; Bootman, M. D.; Bordi, M.; Borner, C.; Bornhauser, B. C.; Borthakur, G.; Bosch, J.; Bose, S.; Botana, L. M.; Botas, J.; Boulanger, C. M.; Boulton, M. E.; Bourdenx, M.; Bourgeois, B.; Bourke, N. M.; Bousquet, G.; Boya, P.; Bozhkov, P. V.; Bozi, L. H. M.; Bozkurt, T. O.; Brackney, D. E.; Brandts, C. H.; Braun, R. J.; Braus, G. H.; Bravo-Sagua, R.; Bravo-San Pedro, J. M.; Brest, P.; Bringer, M. -A.; Briones-Herrera, A.; Broaddus, V. C.; Brodersen, P.; Brodsky, J. L.; Brody, S. L.; Bronson, P. G.; Bronstein, J. M.; Brown, C. N.; Brown, R. E.; Brum, P. C.; Brumell, J. H.; Brunetti-Pierri, N.; Bruno, D.;
abstract

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

2021 - Inhibitors of histone deacetylase 6 based on a novel 3-hydroxy-isoxazole zinc binding group [Articolo su rivista]
Linciano, P.; Pinzi, L.; Belluti, S.; Chianese, U.; Benedetti, R.; Moi, D.; Altucci, L.; Franchini, S.; Imbriano, C.; Sorbi, C.; Rastelli, G.
abstract

Histone deacetylase 6 (HDAC6) is an established drug target for cancer treatment. Inhibitors of HDAC6 based on a hydroxamic acid zinc binding group (ZBG) are often associated with undesirable side effects. Herein, we describe the identification of HDAC6 inhibitors based on a completely new 3-hydroxy-isoxazole ZBG. A series of derivatives decorated with different aromatic or heteroaromatic linkers, and various cap groups were synthesised and biologically tested. In vitro tests demonstrated that some compounds are able to inhibit HDAC6 with good potency, the best candidate reaching an IC50 of 700 nM. Such good potency obtained with a completely new ZBG make these compounds particularly attractive. The effect of the most active inhibitors on the acetylation levels of histone H3 and α- tubulin and their anti-proliferative activity of DU145 cells were also investigated. Docking studies were performed to evaluate the binding mode of these new derivatives and discuss structure-activity relationships.

2021 - The transcription factor NF-Y is required for satellite stem cell myogenic progression and skeletal muscle tissue repair [Poster]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Imbriano, Carol
abstract

2021 - The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle [Articolo su rivista]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Ronzio, Mirko; Sauta, Elisabetta; Ciarrocchi, Alessia; Latella, Lucia; Saclier, Marielle; Molinari, Susanna; Vallarola, Antonio; Messina, Graziella; Mantovani, Roberto; Dolfini, Diletta; Imbriano, Carol
abstract

2020 - Transcription Factors in Cancer: When Alternative Splicing Determines Opposite Cell Fates [Articolo su rivista]
Belluti, Silvia; Rigillo, Giovanna; Imbriano, Carol
abstract

Alternative splicing (AS) is a finely regulated mechanism for transcriptome and proteome diversification in eukaryotic cells. Correct balance between AS isoforms takes part in molecular mechanisms that properly define spatiotemporal and tissue specific transcriptional programs in physiological conditions. However, several diseases are associated to or even caused by AS alterations. In particular, multiple AS changes occur in cancer cells and sustain the oncogenic transcriptional program. Transcription factors (TFs) represent a key class of proteins that control gene expression by direct binding to DNA regulatory elements. AS events can generate cancer-associated TF isoforms with altered activity, leading to sustained proliferative signaling, differentiation block and apoptosis resistance, all well-known hallmarks of cancer. In this review, we focus on how AS can produce TFs isoforms with opposite transcriptional activities or antagonistic functions that severely impact on cancer biology. This summary points the attention to the relevance of the analysis of TFs splice variants in cancer, which can allow patients stratification despite the presence of interindividual genetic heterogeneity. Recurrent TFs variants that give advantage to specific cancer types not only open the opportunity to use AS transcripts as clinical biomarkers but also guide the development of new anti-cancer strategies in personalized medicine.

2019 - Potent Anti-Cancer Properties of Phthalimide-Based Curcumin Derivatives on Prostate Tumor Cells [Articolo su rivista]
Belluti, Silvia; Orteca, Giulia; Semeghini, Valentina; Rigillo, Giovanna; Parenti, Francesca; Ferrari, Erika; Imbriano, Carol
abstract

Metastatic castration-resistant prostate cancer is commonly treated with chemotherapy, whose effect is less than satisfactory. This raised the need for novel agents for the treatment of prostate cancer. In the present study, five phthalimide-based curcumin derivatives were synthesized and completely characterized to assess improved stability, pharmacodynamics, and radical scavenging ability. To investigate the potential application in anti-cancer therapy, the anti-proliferative activity of the synthesized molecules was determined on aggressive prostate tumor cells. We demonstrated that the K3F21 derivative has increased potency compared to curcumin, in terms of GI50, anti-proliferative and anti-migrating activities. K3F21 inhibits anchorage-dependent and -independent growth of prostate cancer cells by altering the expression of key genes controlling cell proliferation, such as Cylins D1, B1 and B2, and apoptosis, among which Puma, Noxa, and Bcl-2 family members. Finally, the anti-cancer activity of K3F21 was demonstrated by the analysis of cancer-associated PI3K/AKT, ERK, and p38 signaling pathways.

2019 - Scouting Sigma Receptor Ligands As New Tools For The Treatment Of Neurodegenerative Diseases And Cancer [Relazione in Atti di Convegno]
Sorbi, C.; Linciano, P.; Tait, A.; Atene, C. G.; Guglielmo, L.; Di Rocco, G.; Ronsisvalle, S.; Denora, N.; Imbriano, C.; Rigillo, G.; Fossa, P.; Cichero, E.; Benassi, L.; Vaschieri, C.; Marocchi, F.; Lanfrancone, M. L.; Brasili, L.; Franchini, S.
abstract

Sigma receptors (Rs) are nowadays recognized as an unique class of membrane receptors divided into two subtypes, R and R. Rs regulate a number of physiological functions and their role has been evaluated in many disorders. Deficits in R are associated with neurodegeneration while their activation may represent a valuable strategy for the treatment of a number of neurodegenerative disorders. Moreover, R is overexpressed in a variety of cancer cells and selective R antagonists are reported to modulate cancer cell viability.1 R are also highly expressed proliferating tumors. R agonists are giving promising results in preclinical studies for the treatment of resistant or hardly treatable tumors and R ligands have been proposed as biomarkers for tumors proliferation.2 However, the identification of potent and selective ligands and the comprehension of the chemical features behind agonism/antagonism still remain a primary challenge in this field. With this aim, following a ligand-based approach, a library of over 120 ligands have been designed and synthesized over the years, by combining different substituted five-membered heterocyclic rings with appropriate R pharmacophoric amines. Compounds were tested for R and R affinity showing Ki values in the micromolar / sub-nanomolar range, with a selectivity mainly shifted toward the R. A detailed SAR, supported by molecular modelling, was drawn up. The intrinsic activity was determined in vivo for the most promising molecules. According to their profile, R agonists were tested for neuroprotection, whereas R antagonists / R agonists for anticancer activity. Preliminary results in SH-SY5Y neuroblastoma cells showed the ability of some compounds to protect neuronal cells from death induced by four toxicity models. Cell viability assays were performed on different cancer cell lines to assess the anti-proliferative potential of selected molecules. In particular, dose and time dependent treatments were done on prostate cancer cells, which express higher levels of both R and R compared to normal samples. Similarly, we assessed the effect of the compounds on melanoma cells: BS148, a potent and selective R agonist, showed anti-proliferative activity on immortalized and PDX (metastatic melanoma patient-derived xenografts) cell lines.3 Confocal microscopy studies with BS148 fluorescent probe revealed the internalization of BS148 within melanoma cells, with a cytoplasmatic localization, mostly in the perinuclear region, according to R distribution. Finally, to verify whether TMEM97 / R mediates BS148-antiproliferative activity, we stably overexpressed the TMEM97 gene in HeLa cells: TMEM97-Hela were more sensitive to BS148 anti-proliferative activity compared to control cells, which express endogenous R levels. Taken together, these results support the idea that R is an innovative target in cancer, paving the way for improved tools for cancer diagnosis, monitoring and therapy.

2019 - Switch of NF-YA splice variants in prostate cancer development and progression [Poster]
Belluti, Silvia; Semeghini, Valentina; Dolfini, Diletta; Rigillo, Giovanna; Imbriano, Carol
abstract

The pioneer transcription factor NF-Y is a heterotrimer composed by NF-YA, -YB and -YC subunits: NF-YA is the limiting subunit and harbors the DNA CCAAT-box binding domain. NF-Y is a master transcriptional regulator that ensures proper cell proliferation, controlling cell cycle, DNA replication, apoptosis and metabolism. Recent analyses pointed out that NF-Y binding site is over-represented in promoters of genes overexpressed during the progression from benign epithelium to Prostate Cancer (PC). Moreover, cell cycle regulation genes have emerged as a signature that can discriminate between metastatic and benign prostate tissues. The analysis of TCGA-RNAseq data highlighted a slight but significant increase of NF-YA levels in PC tumor vs normal tissues, particularly in high Gleason score specimens. Since the NF-YA gene encodes for two splice isoforms, NF-YAl (long) and NF-YAs (short), we generated untransformed and cancer prostate cell lines stably over-expressing NF-YA isoforms, to dissect the functional role of NF-YA in PC development and progression. Our data indicate that NF-YAl and NF-YAs could play different activities in cancer-associated processes, such as clonogenic ability, anchorage-independent growth, 3D cell growth, migration and invasion. In particular, in cancer cells NF-YAl improves invasion ability, while NF-YAs seems to enhance the proliferation of tumor spheroids. These results are consistent with increased NF-YAs/NF-YAl splice ratio observed in the progression from normal to malignant prostatectomy samples. Besides, normal prostate cell lines preferentially express the long NF-YA isoform, while an increase in NF-YAs/NF-YAl ratio can be observed in PC cell lines. These results prompt us to speculate that NF-YAl could participate to metastatization, while NF-YAs could have a major role in tumor growth and colonization. Disclosing this dualism could be crucial to better stratify PC patients and identify new specific anti-cancer treatments.

2019 - The transcription factor NF-Y is required for satellite stem cell proliferation and skeletal muscle tissue repair [Poster]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Imbriano, Carol
abstract

The transcription factor NF-Y, composed by NF-YA, NF-YB and NF-YC subunits, has an important role in the regulation of cellular proliferation and differentiation in different cell types, among which muscle cells. While NF-YA, the DNA binding subunit of NF-Y, is down-regulated in the adult muscle of WT mice, its expression is observed in the mdx mouse, model for Duchenne Muscular Dystrophy, in which a massive regeneration mediated by resident muscle stem cells, namely Satellite Cells (SCs), occurs. With the aim to investigate the role of NF-YA in the SCs proliferation and differentiation, we generated and characterized a conditional knock out mouse model in which NF-YA is deleted in Pax7+ SCs by Tamoxifen induction in adult NF-YAflox/flox:Pax7CreER mice (NF-YA cKO). Cellular and molecular analysis carried out on isolated myofibers and SCs from WT and NF-YA cKO mice highlighted that NF-Y activity is important for the maintenance of SCs homeostasis. NF-YA loss depletes Pax7+ SCs pool and impairs SCs proliferation. Moreover, SCs-mediated regeneration following muscle damage induced by cardiotoxin is delayed in NF-YA cKO. The effect of NF-YA abrogation was also explored in post-natal muscle growth. Immunohistological analysis showed defects in muscle morphology and a decrease in SCs number in 3 weeks aged NF-YA cKO mice, period of major increment of muscle mass  by SCs-mediated myonuclear accretion.  The molecular mechanism underlying the impairment of SCs activity following NF-YA loss was investigated by AdenoCre-induced NF-YA deletion in ex vivo cultured SCs.   Overall, our results highlight a role of NF-Y in muscle regeneration and in SCs fate, whose modulation could be useful to improve stem cell based therapies to treat muscular dystrophies.

2019 - The 1,10-phenanthroline ligand enhances the antiproliferative activity of dna-intercalating thiourea-pd(Ii) and-pt(ii) complexes against cisplatin-sensitive and-resistant human ovarian cancer cell lines [Articolo su rivista]
Marverti, G.; Gozzi, G.; Lauriola, A.; Ponterini, G.; Belluti, S.; Imbriano, C.; Costi, M. P.; D’Arca, D.
abstract

Ovarian cancer is the most lethal gynecological malignancy, often because of the frequent insurgence of chemoresistance to the drugs currently used. Thus, new therapeutical agents are needed. We tested the toxicity of 16 new DNA-intercalating agents to cisplatin (cDDP)-sensitive human ovarian carcinoma cell lines and their resistant counterparts. The compounds were the complexes of Pt(II) or Pd(II) with bipyridyl (bipy) and phenanthrolyl (phen) and with four dierent thiourea ancillary ligands. Within each of the four series of complexes characterized by the same thiourea ligand, the Pd(phen) drugs invariably showed the highest anti-proliferative ecacy. This paralleled both a higher intracellular drug accumulation and a more ecient DNA intercalation than all the other metal-bidentate ligand combinations. The consequent inhibition of topoisomerase II activity led to the greatest inhibition of DNA metabolism, evidenced by the inhibition of the expression of the folate cycle enzymes and a marked perturbation of cell-cycle distribution in both cell lines. These findings indicate that the particular interaction of Pd(II) with phenanthroline confers the best pharmacokinetic and pharmacodynamic properties that make this class of DNA intercalators remarkable inhibitors, even of the resistant cell growth.

2018 - Alternative splicing of transcription factors genes in muscle physiology and pathology [Articolo su rivista]
Imbriano, Carol; Molinari, Susanna
abstract

Skeletal muscle formation is a multi-step process that is governed by complex networks of transcription factors. The regulation of their functions is in turn multifaceted, including several mechanisms, among them alternative splicing (AS) plays a primary role. On the other hand, altered AS has a role in the pathogenesis of numerous muscular pathologies. Despite these premises, the causal role played by the altered splicing pattern of transcripts encoding myogenic transcription factors in neuromuscular diseases has been neglected so far. In this review, we systematically investigate what has been described about the AS patterns of transcription factors both in the physiology of the skeletal muscle formation process and in neuromuscular diseases, in the hope that this may be useful in re-evaluating the potential role of altered splicing of transcription factors in such diseases.

2018 - An autoregulatory loop controls the expression of the transcription factor NF-Y [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Basile, Valentina; Rigillo, Giovanna; Salsi, Valentina; Genovese, Filippo; Dolfini, Diletta; Imbriano, Carol
abstract

The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer. Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate how the stability of the complex is severely affected by the absence of one subunit. Our results identified for the first time one of the mechanisms responsible for NF-Y expression, which may be involved in the aberrant expression and activity observed in tumor cells and other pathological conditions.

2018 - Curcumin derivatives and Aβ-fibrillar aggregates: An interactions’ study for diagnostic/therapeutic purposes in neurodegenerative diseases [Articolo su rivista]
Orteca, Giulia; Tavanti, Francesco; Bednarikova, Zuzana; Gazova, Zuzana; Rigillo, Giovanna; Imbriano, Carol; Basile, Valentina; Asti, Mattia; Rigamonti, Luca; Saladini, Monica; Ferrari, Erika; Menziani, Maria Cristina
abstract

Several neurodegenerative diseases, like Alzheimer's (AD), are characterized by amyloid fibrillar deposition of misfolded proteins, and this feature can be exploited for both diagnosis and therapy design. In this paper, structural modifications of curcumin scaffold were examined in order to improve its bioavailability and stability in physiological conditions, as well as its ability to interfere with β-amyloid fibrils and aggregates. The acid-base behaviour of curcumin derivatives, their pharmacokinetic stability in physiological conditions, and in vitro ability to interfere with Aβ fibrils at different incubation time were investigated. The mechanisms governing these phenomena have been studied at atomic level by means of molecular docking and dynamic simulations. Finally, biological activity of selected curcuminoids has been investigated in vitro to evaluate their safety and efficiency in oxidative stress protection on hippocampal HT-22 mouse cells. Two aromatic rings, π-conjugated structure and H-donor/acceptor substituents on the aromatic rings showed to be the sine qua non structural features to provide interaction and disaggregation activity even at very low incubation time (2h). Computational simulations proved that upon binding the ligands modify the conformational dynamics and/or interact with the amyloidogenic region of the protofibril facilitating disaggregation. Significantly, in vitro results on hippocampal cells pointed out protection against glutamate toxicity and safety when administered at low concentrations (1 μM). On the overall, in view of its higher stability in physiological conditions with respect to curcumin, of his rapid binding to fibrillar aggregates and strong depolymerizing activity, phtalimmide derivative K2F21 appeared a good candidate for both AD diagnostic and therapeutic purposes.

2018 - New curcumin-derived ligands and their affinity towards Ga3+, Fe3+and Cu2+: Spectroscopic studies on complex formation and stability in solution [Articolo su rivista]
Rigamonti, Luca; Orteca, Giulia; Asti, Mattia; Basile, Valentina; Imbriano, Carol; Saladini, Monica; Ferrari, Erika
abstract

The metal complexing ability in solution of four substituted curcumin (CUR)-derived ligands K3T, originated by the insertion of the -CH2CH2COOtBu branch on the central atom of the diketonic moiety of CUR and related derivatives with variable meta and para substituents (OH, OMe, H, OCOCH3) on the peripheral aromatic rings, is examined. These molecules can act as new chelators with biological properties comparable to those of CUR but with improved stability. In fact, curcuminoids represent new perspectives for the development of novel therapeutic agents for several diseases including Alzheimer's disease. CUR showed neuroprotective properties, and a probable mechanism of its action is related to the complexation ability towards endogenous metal ions Fe3+and Cu2+. K3T derivatives retain the solvent-dependent diketo-ketoenol tautomerism, with the prevalence of the diketonic form in aqueous solution. They show enhanced stability in simulated physiological conditions (phosphate buffered solution at pH = 7.4) compared to CUR, together with similar or even higher anti-proliferative activity against human colon carcinoma cells HCT116. The addition of the metal ion causes dissociation of the enolic proton creating chelate complexes and shift of the tautomeric equilibrium toward the keto-enol species. The formation of metal complexes was followed and confirmed by both NMR (using Ga3+as a diamagnetic probe for Fe3+) and UV-visible spectroscopy. All the ligands showed high affinity for Fe3+and Ga3+, forming M:L 1:2 species. In view of therapeutic applications, notable is the good affinity of K3T31, i.e. the ligand bearing only OH groups in para positions of the aromatic rings, for Cu2+, and the ability of the Cu:K3T31 1:1 complex to bind to DNA.

2017 - Dynamic Phosphorylation of the Myocyte Enhancer Factor 2Cα1 Splice Variant Promotes Skeletal Muscle Regeneration and Hypertrophy [Articolo su rivista]
Baruffaldi, Fiorenza; Montarras, Didier; Basile, Valentina; De Feo, Luca; Badodi, Sara; Ganassi, Massimo; Battini, Renata; Nicoletti, Carmine; Imbriano, Carol; Musarò, Antonio; Molinari, Susanna
abstract

Abstract: The transcription factor MEF2C (Myocyte Enhancer Factor 2C) plays an established role in the early steps of myogenic differentiation. However, the involvement of MEF2C in adult myogenesis and in muscle regeneration has not yet been systematically investigated. Alternative splicing of mammalian MEF2C transcripts gives rise to two mutually exclusive protein variants: MEF2Cα2 which exerts a positive control of myogenic differentiation, and MEF2Cα1, in which the α1 domain acts as trans-repressor of the MEF2C pro-differentiation activity itself. However, MEF2Cα1 variants are persistently expressed in differentiating cultured myocytes, suggesting a role in adult myogenesis. We found that overexpression of both MEF2Cα1/α2 proteins in a mouse model of muscle injury promotes muscle regeneration and hypertrophy, with each isoform promoting different stages of myogenesis. Besides the ability of MEF2Cα2 to increase differentiation, we found that overexpressed MEF2Cα1 enhances both proliferation and differentiation of primary myoblasts, and activates the AKT/mTOR/S6K anabolic signaling pathway in newly formed myofibers. The multiple activities of MEF2Cα1 are modulated by phosphorylation of Ser98 and Ser110, two amino acid residues located in the α1 domain of MEF2Cα1. These specific phosphorylations allow the interaction of MEF2Cα1 with the peptidyl-prolyl isomerase PIN1, a regulator of MEF2C functions. Overall, in this study we established a novel regulatory mechanism in which the expression and the phosphorylation of MEF2Cα1 are critically required to sustain the adult myogenesis. The described molecular mechanism will represent a new potential target for the development of therapeutical strategies to treat muscle-wasting diseases.

2017 - In situ immunofluorescent staining of autophagy in muscle stem cells [Articolo su rivista]
Castagnetti, F.; Fiacco, E.; Imbriano, C.; Latella, L.
abstract

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and agerelated muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

2016 - Direct non transcriptional role of NF-Y in DNA replication [Articolo su rivista]
Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Ethienne; Mantovani, Roberto; Blow J., Julian; Imbriano, Carol
abstract

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication.

2016 - Direct non transcriptional role of the CCAAT-factor NF-Y in DNA replication [Poster]
Belluti, Silvia; Benatti, Paolo; Benoit, Miotto; Julia, Neusiedler; Diletta, Dolfini; Marjorie, Drac; Etienne, Schwob; Roberto, Mantovani; Julian Blow, J.; Imbriano, Carol
abstract

The heterotrimeric transcription factor NF-Y plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation. The expression levels of the sequence specific subunit NF-YA increase at the onset of S phase, and NF-YA loss in primary and tumor mammalian cells triggers defects in S-phase progression. Here, we show that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Using a Xenopus cell-free system, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication. Spontaneous DNA damage occurs in NF-YA-deficient cells, while NF-YA overexpression reduces the sensitivity to replication stress, suggesting a role for NF-Y in replication stress response and genome maintenance. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific transcription factors required for proper and efficient DNA replication.

2016 - Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356 [Articolo su rivista]
Klionsky, D. J.; Abdelmohsen, K.; Abe, A.; Abedin, M. J.; Abeliovich, H.; Arozena, A. A.; Adachi, H.; Adams, C. M.; Adams, P. D.; Adeli, K.; Adhihetty, P. J.; Adler, S. G.; Agam, G.; Agarwal, R.; Aghi, M. K.; Agnello, M.; Agostinis, P.; Aguilar, P. V.; Aguirre-Ghiso, J.; Airoldi, E. M.; Ait-Si-Ali, S.; Akematsu, T.; Akporiaye, E. T.; Al-Rubeai, M.; Albaiceta, G. M.; Albanese, C.; Albani, D.; Albert, M. L.; Aldudo, J.; Algul, H.; Alirezaei, M.; Alloza, I.; Almasan, A.; Almonte-Beceril, M.; Alnemri, E. S.; Alonso, C.; Altan-Bonnet, N.; Altieri, D. C.; Alvarez, S.; Alvarez-Erviti, L.; Alves, S.; Amadoro, G.; Amano, A.; Amantini, C.; Ambrosio, S.; Amelio, I.; Amer, A. O.; Amessou, M.; Amon, A.; An, Z.; Anania, F. A.; Andersen, S. U.; Andley, U. P.; Andreadi, C. K.; Andrieu-Abadie, N.; Anel, A.; Ann, D. K.; Anoopkumar-Dukie, S.; Antonioli, M.; Aoki, H.; Apostolova, N.; Aquila, S.; Aquilano, K.; Araki, K.; Arama, E.; Aranda, A.; Araya, J.; Arcaro, A.; Arias, E.; Arimoto, H.; Ariosa, A. R.; Armstrong, J. L.; Arnould, T.; Arsov, I.; Asanuma, K.; Askanas, V.; Asselin, E.; Atarashi, R.; Atherton, S. S.; Atkin, J. D.; Attardi, L. D.; Auberger, P.; Auburger, G.; Aurelian, L.; Autelli, R.; Avagliano, L.; Avantaggiati, M. L.; Avrahami, L.; Azad, N.; Awale, S.; Bachetti, T.; Backer, J. M.; Bae, D. -H.; Bae, J. -S.; Bae, O. -N.; Bae, S. H.; Baehrecke, E. H.; Baek, S. -H.; Baghdiguian, S.; Bagniewska-Zadworna, A.; Bai, H.; Bai, J.; Bai, X. -Y.; Bailly, Y.; Balaji, K. N.; Balduini, W.; Ballabio, A.; Balzan, R.; Banerjee, R.; Banhegyi, G.; Bao, H.; Barbeau, B.; Barrachina, M. D.; Barreiro, E.; Bartel, B.; Bartolome, A.; Bassham, D. C.; Bassi, M. T.; Bast, R. C.; Basu, A.; Batista, M. T.; Batoko, H.; Battino, M.; Bauckman, K.; Baumgarner, B. L.; Bayer, K. U.; Beale, R.; Beaulieu, J. -F.; Beck, G. R.; Becker, C.; Beckham, J. D.; Bedard, P. -A.; Bednarski, P. J.; Begley, T. J.; Behl, C.; Behrends, C.; Behrens, G. M. N.; Behrns, K. E.; Bejarano, E.; Belaid, A.; Belleudi, F.; Benard, G.; Berchem, G.; Bergamaschi, D.; Bergami, M.; Berkhout, B.; Berliocchi, L.; Bernard, A.; Bernard, M.; Bernassola, F.; Bertolotti, A.; Bess, A. S.; Besteiro, S.; Bettuzzi, S.; Bhalla, S.; Bhattacharyya, S.; Bhutia, S. K.; Biagosch, C.; Bianchi, M. W.; Biard-Piechaczyk, M.; Billes, V.; Bincoletto, C.; Bingol, B.; Bird, S. W.; Bitoun, M.; Bjedov, I.; Blackstone, C.; Blanc, L.; Blanco, G. A.; Blomhoff, H. K.; Boada-Romero, E.; Bockler, S.; Boes, M.; Boesze-Battaglia, K.; Boise, L. H.; Bolino, A.; Boman, A.; Bonaldo, P.; Bordi, M.; Bosch, J.; Botana, L. M.; Botti, J.; Bou, G.; Bouche, M.; Bouchecareilh, M.; Boucher, M. -J.; Boulton, M. E.; Bouret, S. G.; Boya, P.; Boyer-Guittaut, M.; Bozhkov, P. V.; Brady, N.; Braga, V. M. M.; Brancolini, C.; Braus, G. H.; Bravo-San-Pedro, J. M.; Brennan, L. A.; Bresnick, E. H.; Brest, P.; Bridges, D.; Bringer, M. -A.; Brini, M.; Brito, G. C.; Brodin, B.; Brookes, P. S.; Brown, E. J.; Brown, K.; Broxmeyer, H. E.; Bruhat, A.; Brum, P. C.; Brumell, J. H.; Brunetti-Pierri, N.; Bryson-Richardson, R. J.; Buch, S.; Buchan, A. M.; Budak, H.; Bulavin, D. V.; Bultman, S. J.; Bultynck, G.; Bumbasirevic, V.; Burelle, Y.; Burke, R. E.; Burmeister, M.; Butikofer, P.; Caberlotto, L.; Cadwell, K.; Cahova, M.; Cai, D.; Cai, J.; Cai, Q.; Calatayud, S.; Camougrand, N.; Campanella, M.; Campbell, G. R.; Campbell, M.; Campello, S.; Candau, R.; Caniggia, I.; Cantoni, L.; Cao, L.; Caplan, A. B.; Caraglia, M.; Cardinali, C.; Cardoso, S. M.; Carew, J. S.; Carleton, L. A.; Carlin, C. R.; Carloni, S.; Carlsson, S. R.; Carmona-Gutierrez, D.; Carneiro, L. A. M.; Carnevali, O.; Carra, S.; Carrier, A.; Carroll, B.; Casas, C.; Casas, J.; Cassinelli, G.; Castets, P.; Castro-Obregon, S.; Cavallini, G.; Ceccherini, I.; Cecconi, F.; Cederbaum, A. I.; Cena, V.; Cenci, S.; Cerella, C.; Cervia, D.; Cetrullo, S.; Chaachouay, H.; Chae, H. -J.; Chagin, A. S.; Chai, C. -Y.; Chakrabarti, G.; Chamilos, G.; Chan, E. Y. W.; Chan, M. T. V.; Chandra, D.; Chandra, P.; Chang, C. -P.; Chang,
abstract

2016 - Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) [Articolo su rivista]
Klionsky, Daniel J; Abdelmohsen, Kotb; Abe, Akihisa; Abedin, Md Joynal; Abeliovich, Hagai; Acevedo Arozena, Abraham; Adachi, Hiroaki; Adams, Christopher M; Adams, Peter D; Adeli, Khosrow; Adhihetty, Peter J; Adler, Sharon G; Agam, Galila; Agarwal, Rajesh; Aghi, Manish K; Agnello, Maria; Agostinis, Patrizia; Aguilar, Patricia V; Aguirre Ghiso, Julio; Airoldi, Edoardo M; Ait Si Ali, Slimane; Akematsu, Takahiko; Akporiaye, Emmanuel T; Al Rubeai, Mohamed; Albaiceta, Guillermo M; Albanese, Chris; Albani, Diego; Albert, Matthew L; Aldudo, Jesus; Algül, Hana; Alirezaei, Mehrdad; Alloza, Iraide; Almasan, Alexandru; Almonte Beceril, Maylin; Alnemri, Emad S; Alonso, Covadonga; Altan Bonnet, Nihal; Altieri, Dario C; Alvarez, Silvia; Alvarez Erviti, Lydia; Alves, Sandro; Amadoro, Giuseppina; Amano, Atsuo; Amantini, Consuelo; Ambrosio, Santiago; Amelio, Ivano; Amer, Amal O; Amessou, Mohamed; Amon, Angelika; An, Zhenyi; Anania, Frank A; Andersen, Stig U; Andley, Usha P; Andreadi, Catherine K; Andrieu Abadie, Nathalie; Anel, Alberto; Ann, David K; Anoopkumar Dukie, Shailendra; Antonioli, Manuela; Aoki, Hiroshi; Apostolova, Nadezda; Aquila, Saveria; Aquilano, Katia; Araki, Koichi; Arama, Eli; Aranda, Agustin; Araya, Jun; Arcaro, Alexandre; Arias, Esperanza; Arimoto, Hirokazu; Ariosa, Aileen R; Armstrong, Jane L; Arnould, Thierry; Arsov, Ivica; Asanuma, Katsuhiko; Askanas, Valerie; Asselin, Eric; Atarashi, Ryuichiro; Atherton, Sally S; Atkin, Julie D; Attardi, Laura D; Auberger, Patrick; Auburger, Georg; Aurelian, Laure; Autelli, Riccardo; Avagliano, Laura; Avantaggiati, Maria Laura; Avrahami, Limor; Awale, Suresh; Azad, Neelam; Bachetti, Tiziana; Backer, Jonathan M; Bae, Dong Hun; Bae, Jae Sung; Bae, Ok Nam; Bae, Soo Han; Baehrecke, Eric H; Baek, Seung Hoon; Baghdiguian, Stephen; Bagniewska Zadworna, Agnieszka; Bai, Hua; Bai, Jie; Bai, Xue Yuan; Bailly, Yannick; Balaji, Kithiganahalli Narayanaswamy; Balduini, Walter; Ballabio, Andrea; Balzan, Rena; Banerjee, Rajkumar; Bánhegyi, Gábor; Bao, Haijun; Barbeau, Benoit; Barrachina, Maria D; Barreiro, Esther; Bartel, Bonnie; Bartolomé, Alberto; Bassham, Diane C; Bassi, Maria Teresa; Bast, Robert C; Basu, Alakananda; Batista, Maria Teresa; Batoko, Henri; Battino, Maurizio; Bauckman, Kyle; Baumgarner, Bradley L; Bayer, K. Ulrich; Beale, Rupert; Beaulieu, Jean François; Beck, George R; Becker, Christoph; Beckham, J. David; Bédard, Pierre André; Bednarski, Patrick J; Begley, Thomas J; Behl, Christian; Behrends, Christian; Behrens, Georg Mn; Behrns, Kevin E; Bejarano, Eloy; Belaid, Amine; Belleudi, Francesca; Bénard, Giovanni; Berchem, Guy; Bergamaschi, Daniele; Bergami, Matteo; Berkhout, Ben; Berliocchi, Laura; Bernard, Amélie; Bernard, Monique; Bernassola, Francesca; Bertolotti, Anne; Bess, Amanda S; Besteiro, Sébastien; Bettuzzi, Saverio; Bhalla, Savita; Bhattacharyya, Shalmoli; Bhutia, Sujit K; Biagosch, Caroline; Bianchi, Michele Wolfe; Biard Piechaczyk, Martine; Billes, Viktor; Bincoletto, Claudia; Bingol, Baris; Bird, Sara W; Bitoun, Marc; Bjedov, Ivana; Blackstone, Craig; Blanc, Lionel; Blanco, Guillermo A; Blomhoff, Heidi Kiil; Boada Romero, Emilio; Böckler, Stefan; Boes, Marianne; Boesze Battaglia, Kathleen; Boise, Lawrence H; Bolino, Alessandra; Boman, Andrea; Bonaldo, Paolo; Bordi, Matteo; Bosch, Jürgen; Botana, Luis M; Botti, Joelle; Bou, German; Bouché, Marina; Bouchecareilh, Marion; Boucher, Marie Josée; Boulton, Michael E; Bouret, Sebastien G; Boya, Patricia; Boyer Guittaut, Michaël; Bozhkov, Peter V; Brady, Nathan; Braga, Vania Mm; Brancolini, Claudio; Braus, Gerhard H; Bravo San Pedro, José M; Brennan, Lisa A; Bresnick, Emery H; Brest, Patrick; Bridges, Dave; Bringer, Marie Agnès; Brini, Marisa; Brito, Glauber C; Brodin, Bertha; Brookes, Paul S; Brown, Eric J; Brown, Karen; Broxmeyer, Hal E; Bruhat, Alain; Brum, Patricia Chakur; Brumell, John H; Brunetti Pierri, Nicola; Bryson Richardson, Robert J; Buch, Shilpa; Buchan, Alastair M; Budak
abstract

Guidelines for studying autophagy

2016 - NF-Y activates genes of metabolic pathways altered in cancer cells [Articolo su rivista]
Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A.; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta
abstract

The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.

2016 - NF-Y loss triggers p53 stabilization and apoptosis in HPV18-positive cells by affecting E6 transcription [Articolo su rivista]
Benatti, Paolo; Basile, Valentina; Dolfini, Diletta; Belluti, Silvia; Tomei, Margherita; Imbriano, Carol
abstract

The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NFYA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response.

2016 - NF-YA splice variants have different roles on muscle differentiation [Articolo su rivista]
Basile, Valentina; Baruffaldi, Fiorenza; Dolfini, Diletta; Belluti, Silvia; Benatti, Paolo; Ricci, Laura; Artusi, Valentina; Tagliafico, Enrico; Mantovani, Roberto; Molinari, Susanna; Imbriano, Carol
abstract

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.

2016 - Role of the CCAAT-transcription factor NF-Y and its splice variants in myogenic differentiation and muscle regeneration [Poster]
Basile, Valentina; Baruffaldi, Fiorenza; Laura, Ricci; Molinari, Susanna; Imbriano, Carol
abstract

The expression of NF-YA, the DNA-binding subunit of the CCAAT-binding transcription factor NF-Y, is down-regulated in adult skeletal muscle to allow a complete terminal differentiation. The NF-YA gene encodes for two alternative splice variants, NF-YAs and NF-YAl: in human haematopoietic and mouse embryonic stem cells, the expression of NF-YAs or NF-YAl is crucial to promote proliferation or differentiation, respectively. The role of NF-YA isoforms in skeletal myogenesis and satellite cells fate has never been investigated. Here we show that both NF-YA isoforms are induced in mouse regenerating muscle and are expressed in satellite cells (SCs). The abrogation of NF-Y activity impairs both proliferation and differentiation of SCs. Forced expression of NF-YAs stimulates SCs proliferation, while NF-YAl enhances their differentiation. The same effects are observed in mouse C2C12 myoblasts, in which the two isoforms activate opposite transcriptional programs. NF-YAs upregulates genes annotated in growth factor binding, cell adhesion and extra cellular matrix Gene Onthology terms, while NF-YAl increases the transcription of genes belonging to sarcomere, muscle cell differentiation, development and muscle contraction categories. NF-YAl boosts myoblasts differentiation by up-regulating the transcription of novel NF-Y target genes, among which Mef2D, Six4 and Cdkn1C, which are known to be involved in the differentiation program. Overall, our results highlight the functional difference between NF-YA isoforms, whose modulation could be useful to improve stem cell based therapies to treat muscular dystrophy.

2016 - Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase [Articolo su rivista]
Cocchiarella, Fabienne; Latella, Maria Carmela; Basile, Valentina; Miselli, Francesca; Galla, Melanie; Imbriano, Carol; Recchia, Alessandra
abstract

The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2(S)-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer.

2015 - NF-Y splice variants differentially affect skeletal myogenesis [Esposizione]
Basile, Valentina; Baruffaldi, Fiorenza; Dolfini, Diletta; Ricci, Laura; Mantovani, Roberto; Molinari, Susanna; Imbriano, Carol
abstract

The mechanisms that regulate skeletal muscle development involve the coordinated activity of transcription factors (TFs) and the precise timing of gene expression patterns. NF-Y is a heterotrimeric TF with a pioneer role in the transcriptional and epigenetic regulation of promoters containing the CCAAT-box. NF-Y activates the expression of various genes related to the cell cycle, particularly genes of the G2/M phase. NF-YA, the regulatory DNA-binding subunit of the complex, is expressed in proliferating myoblasts and down-regulated during terminal differentiation. The NF-YA gene encodes for two alternatively spliced isoforms, namely NF-YAs and NF-YAl, which are not functionally identical. Using mouse C2C12 cells, we provide evidence of a different role for NF-YA variants in the myogenic program. While NF-YAs enhances myoblasts proliferation, NF-YAl boosts their differentiation by up-regulating the transcription of novel target genes, among which Mef2D, Sixs and Cdkn1C, which are known to be involved in the differentiation program. We further demonstrate that NF-YA is expressed in resident stem cells (SCs) and the two isoforms are transcribed at different levels during SCs activation and differentiation. The inhibition of NF-Y activity impairs both proliferation and differentiation of SCs and the overexpression of the two NF-YA isoforms differentially affects their fate.

2015 - Phosphorylation and alternative splicing of MEF2C, a dual switch function in muscle regeneration [Abstract in Rivista]
Baruffaldi, Fiorenza; Badodi, Sara; De Feo, Luca; Ganassi, Massimo; Battini, Renata; Imbriano, Carol; Nicoletti, Carmine; Musarò, Antonio; Buckingham, Margaret; Montarras, Didier; Molinari, Susanna
abstract

Muscle regeneration is a multistep process that is regulated by a restricted number of transcription factors whose activity is modulated at multiple levels. However, how different layers of regulation are coordinated to promote adult myogenesis is not yet understood. Here we show that the MEF2C transcription factor controls multiple steps of muscle regeneration, including myogenic progression of satellite cells and muscle maturation of newly generated myofibers, exhibiting multiple functions that depend on alternative splicing and post-translational modifications. Inclusion of the 1 exon in Mef2c transcripts is upregulated in proliferating mouse satellite cells and in the early phases of muscle regeneration. The encoded MEF2C1 isoform stimulates expansion of primary myoblasts ex vivo and in vivo. The pro-proliferative activity of MEF2C is mediated by phosphorylation of two phosphoserines located in exon 1. Subsequent terminal differentiation and growth of newly formed myofibers are promoted by dephosphorylated MEF2C1 and MEF2C2. Our results thus reveal an important role for regulatory interactions between alternative splicing and post translational modifications of a single transcription factor in the control of the multilayered regulatory programs required for adult myogenesis.

2014 - NF-Y LOSS REACTIVATES p53 AND TRIGGERS p53-DEPENDENT APOPTOSIS IN HPV-POSITIVE HUMAN CERVICAL CARCINOMA CELLS [Poster]
Benatti, Paolo; Mantovani, Roberto; Imbriano, Carol
abstract

The two early HPV genes, E6 and E7, play an essential role in cellular transformation and abnormal proliferation. The oncoprotein E6 suppresses the activation of p53-dependent pathways through proteasomal degradation of p53 and inhibition of its transactivating activity. The CCAAT-binding factor NF-Y regulates the transcription of various genes related to the cell cycle. A biochemical and genetic link has been described between p53 and NF-Y: i) a large overlap of targets exists between NF-Y and p53, particularly many genes transactivated by NF-Y are repressed following p53 activation; ii) both wt and mutp53 proteins interact with NF-Y; iii) loss of NF-Y, as well as unrestricted activity, triggers cycle defects and p53-dependent cell death. Specifically, the inactivation of NF-YA, the DNA binding subunit of the NF-Y complex, leads to a delay in S phase progression and to the activation of a DNA-damage response. Here we show a novel mechanism through which NF-Y and p53 are connected in HPV-positive cells. The knock-down of NF-YA in Hela cervical carcinoma cells leads to p53 re-expression and re-activation of p53-dependent apoptosis. The analysis of microarray profiles of NF-YA inactivated versus control cells points at “p53 pathway” and “apoptosis” as the most represented KEGG pathways up-regulated following NF-Y loss. Western blot, RT-PCR, transient transfection and ChIP data clearly suggest that NF-Y positively regulates E6 expression, driven by HPV-18 upstream regulatory region, which contains two NF-Y binding sites. Consequently, NF-Y down-regulation by shRNA delivery results in E6 transcriptional repression and in the activation of p53 and its target genes. Our results demonstrate that NF-Y inactivation might represent an interesting anti-tumor strategy to induce apoptosis in high-risk HPV-infected cancer cells, through the re-establishment of p53 expression.

2013 - A non transcriptional role for NF-Y in DNA replication [Poster]
Benatti, Paolo; Belluti, Silvia; Neusiedler, Julia; Basile, Valentina; Blow, J. Julian; Imbriano, Carol
abstract

The heterotrimeric transcription factor NF-Y, composed by NF-YA, NF-YB and NF-YC subunits, is a key transcriptional regulator of cell cycle progression. Using data generated by ENCODE, we identified a striking overlap between loci bound by NF-Y-and ORC2, hinting at a possible role of NF-Y in DNA replication. NF-YA knock-down leads to replication defects and the activation an intra-S checkpoint. We investigated the role of NF-Y in DNA replication by using the Xenopus cell-free system. NF-Y subunits were found to be recruited to chromatin during DNA replication. Both immunodepletion of NF-YA or NF-YB and overexpression of a dominant-negative NF-YA mutant lead to a clear decrease in DNA synthesis. In mammalian cells, NF-Y colocalizes and directly interacts with DNA replication proteins. Nascent strand abundance assay in NF-YA inactivated cells corroborates that NF-Y participates to the DNA replication process. Our data highlight that, in addition to its transcriptional activity in controlling cell proliferation, NF-Y plays a key role in the non transcriptional control of DNA replication.

2013 - A specific role for the splice variants of the transcription factor NF-Y in modulating the transcriptional activity of the myogenic program [Poster]
Basile, Valentina; Baruffaldi, Fiorenza; Belluti, Silvia; Benatti, Paolo; Mantovani, Roberto; Molinari, Susanna; Imbriano, Carol
abstract

Cell proliferation and differentiation programs are highly regulated transcriptional processes essential for myogenesis. The transcription factor NF-Y has been long considered a fundamental player of cell growth by supporting the basal transcription of various cell cycle genes. It is composed by the NF-YB/NF-YC heterodimer and NF-YA, which interacts with the other two subunits and confers the strict sequence specificity to the complex. The NF-YA gene encodes two alternatively splice transcripts (NF-YAs and NF-YAl), which differently regulate cell proliferation and differentiation, as shown in haematopoietic and mouse embryonic stem cells. NF-YAl expression is down-regulated in terminally differentiated muscle cells and in skeletal and cardiac muscle tissues. Its forced expression in muscle cells committed to differentiate impairs their exit from the cell cycle and indirectly interferes with the differentiation program. Here we show that the two NF-YA isoforms play a different role in the transcriptional activity of the myogenic program and may regulate the activity of muscle satellite cells.

2013 - bis-Dehydroxy-Curcumin Triggers Mitochondrial-Associated Cell Death in Human Colon Cancer Cells through ER-Stress Induced Autophagy. [Articolo su rivista]
Basile, Valentina; Belluti, Silvia; Ferrari, Erika; Gozzoli, C; Ganassi, Sonia; Quaglino, Daniela; Saladini, Monica; Imbriano, Carol
abstract

Background: The activation of autophagy has been extensively described as a pro-survival strategy, which helps to keep cells alive following deprivation of nutrients/growth factors and other stressful cellular conditions. In addition to cytoprotective effects, autophagy can accompany cell death. Autophagic vacuoles can be observed before or during cell death, but the role of autophagy in the death process is still controversial. A complex interplay between autophagy and apoptosis has come to light, taking into account that numerous genes, such as p53 and Bcl-2 family members, are shared between these two pathways. Methodology/Principal Findings: In this study we showed a potent and irreversible cytotoxic activity of the stable Curcumin derivative bis-DeHydroxyCurcumin (bDHC) on human colon cancer cells, but not on human normal cells. Autophagy is elicited by bDHC before cell death as demonstrated by increased autophagosome formation -measured by electron microscopy, fluorescent LC3 puncta and LC3 lipidation- and autophagic flux -measured by interfering LC3-II turnover. The accumulation of poly-ubiquitinated proteins and ER-stress occurred upstream of autophagy induction and resulted in cell death. Cell cycle and Western blot analyses highlighted the activation of a mitochondrial-dependent apoptosis, which involves caspase 7, 8, 9 and Cytochrome C release. Using pharmacological inhibitions and RNAi experiments, we showed that ER-stress induced autophagy has a major role in triggering bDHC-cell death. Conclusion/Significance: Our findings describe the mechanism through which bDHC promotes tumor selective inhibition of proliferation, providing unequivocal evidence of the role of autophagy in contrasting the proliferation of colon cancer cells.

2013 - Concurrent inhibition of enzymatic activity and NF-Y-mediated transcription of Topoisomerase-IIα by bis-DemethoxyCurcumin in cancer cells [Articolo su rivista]
Belluti, Silvia; Basile, Valentina; Benatti, Paolo; Ferrari, Erika; Marverti, Gaetano; Imbriano, Carol
abstract

Topoisomerase-IIa (TOP2A) enzyme is essential for cell viability due to its fundamental role in DNA metabolism and in chromatin organization during interphase and mitosis. TOP2A expression is finely regulated at the transcriptional level through the binding of the CCAAT-transcription factor NF-Y to its promoter. Overexpression and/or amplification of TOP2A have been observed in many types of cancers. For this reason, TOP2A is the target of the most widely successful drugs in cancer chemotherapy, such as TOP2A poisons, which stabilize TOP2A-DNA cleavage complexes and create DSBs, leading to chromosome damage and cell death. We previously reported that the Curcumin-derivative bis-DemethoxyCurcumin (bDMC) is an anti-proliferative agent that inhibits cell growth by concomitant G1/S and G2/M arrest. Here we showed that bDMC irreversibly induces DSBs in cancer cells, but not in normal cells, by targeting TOP2A activity and expression. TOP2A ablation by siRNA corroborates its contribution to apoptosis induced by bDMC. Short-term exposure to bDMC induces retention of TOP2A-DNA intermediates, while longer exposure inhibits TOP2A transcription by affecting expression and sub-cellular localization of NF-Y subunits. ChIP analysis highlighted reduced recruitment of NF-Y to TOP2A regulatory regions, concomitantly to histone deacetylation and decreased gene transcription. Our findings suggest that the dual activity of bDMC on TOP2A represents a novel therapeutic strategy to induce persistent apoptosis in cancer cells and identify NF-Y regulation as a promising approach in anti-cancer therapy.

2013 - NF-Y co-associates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors. [Articolo su rivista]
J. D., Fleming; G., Pavesi; BENATTI, Paolo; IMBRIANO, Carol; R., Mantovani; K., Struhl
abstract

NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB and NF-YC) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5,000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of the NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (non-modified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively co-localizes with FOS in all genomic contexts, and this often occurs in the absence of JUN and the AP-1 motif. NF-Y also co-associates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly-used, proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve co-association with FOS.

2013 - Transcriptionally regulated and non-toxic delivery of the hyperactive Sleeping Beauty Transposase [Abstract in Atti di Convegno]
Cocchiarella, Fabienne; Latella, Maria Carmela; Basile, Valentina; Galla, Melanie; Imbriano, Carol; Ivics, Zoltan; Izsvak, Zsuzsanna; Recchia, Alessandra
abstract

The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raised increasing interest for gene therapy application, genomic modification and, more recently, iPS reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by a gamma retroviral vector (iRV) has been circumvented by transduction of SB100X mRNA packaged into retrovirus particles (Galla et al. NAR 2011, Vol. 39, No. 16). In this study, we developed an alternative, safe and efficient transposase delivery system based on the tetracycline-ON regulatory system and on integrase defective lentiviral vectors (IDLV). The un-integrated and transcriptionally regulated expression of SB100X may become crucial in gene therapy and in iPS reprogramming, where permanent transposase expression could result in uncontrollable transposition. We generated two IDLV vectors, one carrying the rtTA2-M2 transactivator and the second bearing the tightly regulated, drug-inducible SB100X expression cassette.

2012 - Development of new metal-based phyto-radiopharmaceuticals: diagnostic agents derived from Cur(e)cumin [Relazione in Atti di Convegno]
Ferrari, Erika; M., Asti; Malavasi, Gianluca; Imbriano, Carol; Pignedoli, Francesca; M., Bonavia; D., Daquino; Saladini, Monica
abstract

Curcumin, a yellow pigment extracted from the Indian spice Curcuma longa, has been widely linked with suppression of angiogenesis, inflammation, cardiovascular diseases, tumorigenesis and A-binding activities in the context of therapies for Alzheimer’s disease [1,2]. Besides, Curcumin has shown interesting binding ability towards different metal ions such as Fe(III) and Ga(III) with the involvement of its keto-enolic moiety [3]. Metal complexation triggers an increased kinetic stability and bioavailability of Curcumin in physiological conditions, reducing one of its main drawbacks in therapeutic applications. Anyway biologically active curcuminoids have little been investigated as labelled radiopharmaceuticals for cancer diagnosis or therapy, and they represent a novelty in the field. In this presentation we show an overview of new classes of Curcumin-based ligands with bidentate and tridentate coordinating mode, we investigate their binding ability towards Rhenium and Gallium and report a complete thermodynamic and pharmaco-kinetic study on ligands and metal complexes with the aim of developing new radiopharmaceuticals.

2012 - Gain-of-function p53 mutants have widespread genomic locations partially overlapping with p63 [Articolo su rivista]
E., Martynova; S., Pozzi; Basile, Valentina; D., Dolfini; F., Zambelli; Imbriano, Carol; G., Pavesi; R., Mantovani
abstract

p53 and p63 are transcription factors -TFs- playing master roles in the DNA-damage response and in the development and maintenance of pluristratified epithelia, respectively. p53 mutations are common in epithelial tumors and HaCaT keratinocytes harbor two p53 alleles -H179Y and R282Q- with gain-of-function (GOF) activity. Indeed, functional inactivation of mutp53 affects the growth rate of HaCaT. We investigated the strategy of mutp53, by performing ChIP-Seq experiments of mutp53 and p63 and analyzed the transcriptome after mutp53 inactivation. Mutp53 bind to 7135 locations in vivo, with a robust overlap with p63. De novo motifs discovery recovered a p53/p63RE with high information content in sites bound by p63 and mutp53/p63, but not by mutp53 alone: these sites are rather enriched in elements of other TFs. The HaCaT p63 locations are only partially overlapping with those of normal keratinocytes; importantly, and enriched in mutp53 sites which delineate a functionally different group of target genes. Our data favour a model whereby mutp53 GOF mutants act both by tethering growth-controlling TFs and highjacking p63 to new locations.

2012 - The NF-Y/p53 liaison: Well beyond repression. [Articolo su rivista]
Imbriano, Carol; N., Gnesutta; R., Mantovani
abstract

NF-Y is a sequence-specific transcription factor - TF - targeting the common CCAAT promoter element. p53 is a master TF controlling the response to stress signals endangering genome integrity, often mutated in human cancers. The NF-Y/p53 - and p63, p73 - interaction results in transcriptional repression of a subset of genes within the vast NF-Y regulome under DNA-damage conditions. Recent data shows that NF-Y is also involved in pro-apoptotic activities, either directly, by mediating p53 transcriptional activation, or indirectly, by being targeted by a non coding RNA, PANDA. The picture is subverted in cells carrying Gain-of-function mutant p53, through interactions with TopBP1, a protein also involved in DNA repair and replication. In summary, the connection between p53 and NF-Y is crucial in determining cell survival or death.

2011 - An acetylation-mono-ubiquitination switch on lysine 120 of H2B. [Articolo su rivista]
R., Gatta; D., Dolfini; F., Zambelli; Imbriano, Carol; G., Pavesi; R., Mantovani
abstract

Post-translational modifications (PTMs) of histones are crucial for transcriptional control, defining positive and negative chromatin territories. A switch of opposing functional significance between acetylation and methylation occurs on many residues. Lysine 120 of H2B is modified by two PTMs: ubiquitination, which is required for further trans-tail H3 methylations and elongation, and acetylation, whose role is less clear. ChIP-Seq with MNase I-treated chromatin indicates that H2BK120ac is present on nucleosomes immediately surrounding the TSS of transcribed or poised units, but not in core promoters. In kinetic ChIP analysis of ER-stress inducible genes, H2BK120ac precedes activation and H2B-ub deposition. Using in vitro acetylation assays, pharmacologic inhibition and RNAi, we established that KAT3 is responsible for H2BK120ac. Interestingly, the global levels of H2B-ub decreased in KAT3-inactivated cells. However, RNF20 recruitment was not impaired by KAT3-inactivation. Our data point at acetylation of Lysine 120 of H2B as an early mark of poised or active state and establish a temporal sequence between acetylation and mono-ubiquitination of this H2B residue.

2011 - Newly synthesized curcumin derivatives: crosstalk between chemico-physical properties and biological activity. [Articolo su rivista]
Ferrari, Erika; Francesca, Pignedoli; Imbriano, Carol; Marverti, Gaetano; Valentina, Basile; Ettore, Venturi; Saladini, Monica
abstract

New curcumin analogues (ester and acid series) were synthesized with the aim to improve the chemical stability in physiological conditions and potential anticancer activity. Cytotoxicity against different tumorigenic cell lines (human ovarian carcinoma cells – 2008, A2780, C13* and A2780/CP - and human colon carcinoma cells - HCT116 and LoVo) was tested to evaluate cellular specificity and activity.Physico/chemical properties such as acidity, lipophilicity, kinetic stability and free radical scavenging activity were investigated to shed light on the structure-activity relationship and provide new attractive candidates for drug development. Most of ester derivatives show IC50 values lower than curcumin and exhibit selectivity against colon carcinoma cells. Especially they are extremely active after 24h exposure showing enhanced inhibitory effect on cell viability.The best performances of ester curcuminoids could be ascribed to their high lipophilicity that favors a greater and faster cellular uptake overcoming their apparently higher instability in physiological condition.

2011 - Specific inhibition of NF-Y subunits triggers different cell proliferation defects. [Articolo su rivista]
Benatti, Paolo; D., Dolfini; A., Viganò; M., Ravo; A., Weisz; Imbriano, Carol
abstract

Regulated gene expression is essential for a proper progression through the cell cycle. The transcription factor NF-Y has a fundamental function in transcriptional regulation of cell cycle genes, particularly of G2/M genes. In order to investigate common and distinct functions of NF-Y subunits in cell cycle regulation, NF-YA, NF-YB and NF-YC have been silenced by shRNAs in HCT116 cells. NF-YA loss led to a delay in S-phase progression, DNA damage and apoptosis: we showed the activation of the replication checkpoint, through the recruitment of Δp53 and of the replication proteins PCNA and Mcm7 to chromatin. Differently, NF-YB depletion impaired cells from exiting G2/M, but did not interfere with S-phase progression. Gene expression analysis of NF-YA and NF-YB inactivated cells highlighted a common set of hit genes, as well as a plethora of uncommon genes, unveiling a different effect of NF-Y subunits loss on NF-Y binding to its target genes. Chromatin extracts and ChIP analysis showed that NF-YA depletion was more effective than NF-YB in hitting NF-Y recruitment to CCAAT-promoters. Our data suggest a critical role of NF-Y expression, highlighting that the lack of the single subunits are differently perceived by the cells, which activate diverse cell cycle blocks and signaling pathways.

2011 - Structure-Based Design of Potent Aromatase Inhibitors by High-Throughput Docking [Articolo su rivista]
Caporuscio, Fabiana; Rastelli, Giulio; Imbriano, Carol; Del Rio, Alberto
abstract

Cytochrome P450 aromatase catalyzes the conversion of androgen substrates into estrogens. Aromatase inhibitors (AIs) have been used as first-line drugs in the treatment of estrogen-dependent breast cancer in postmenopausal women. However, the search for new, more potent, and selective AIs still remains necessary to avoid the risk of possible resistances and reduce toxicity and side effects of current available drugs. The publication of a high resolution X-ray structure of human aromatase has opened the way to structure-based virtual screening to identify new small-molecule inhibitors with structural motifs different from all known AIs. In this context, a high-throughput docking protocol was set up and led to the identification of nanomolar AIs with new core structures.

2010 - Probing solute–solvent hydrogen bonding with fluorescent water-soluble curcuminoids [Articolo su rivista]
Caselli, Monica; Ferrari, Erika; Imbriano, Carol; Pignedoli, Francesca; Saladini, Monica; Ponterini, Glauco
abstract

Glycosylated water-soluble curcuminoids (C1–C3, first scheme of this article) differing in the 3,3-ringsubstituents (–OH, –OCH3 and H) equilibrate between the di-keto and the keto–enol forms. The formerare well observable in the absorption spectra in water, but their emissions are always negligible. Theketo–enol forms of C1–C3 exhibit a broad range of fluorescence quantum yields in different solvents,organic and water: formation of solute–solvent hydrogen bonds through the 3,3-ring substituents maychange the radiationless S1-state decay constant by up to a factor 200. Such a fluorescence quenchingmechanism is extremely efficient in water and, for C1, in accepting organic media. On the contrary, noeffects traceable to intermolecular hydrogen bonds involving the central -dicarbonyl moiety have beenobserved. So, fluorescence of these curcuminoids may probe the hydrogen bonding ability, particularly asacceptor, of their microenvironments, including hydrophilic/hydrophobic domains in complex biologicalsystems. Interaction of C1 and C2 with bovine serum albumin results in emission enhancements inverseto the quantum yields of the curcuminoids in water. The observations support the idea that, although thecurcuminoid microenvironment within its complex with the protein is less polar and hydrogen bondingthan water itself, residual water/ligand hydrogen bonds are active in enhancing radiationless transitions.Finally, fluorescence confocal images of HCT116 cells treated with C1–C3 suggest the apparently smallstructural differences to affect, besides their fluorescence behaviour, their interactions and fate withinliving cells.

2009 - Curcumin derivatives: molecular basis of their anti-cancer activity. [Articolo su rivista]
Basile, Valentina; Ferrari, Erika; Lazzari, Sandra; Belluti, S.; Pignedoli, Francesca; Imbriano, Carol
abstract

Curcumin, a phenolic compound from the plant Curcuma longa L., has shown a wide-spectrum of chemopreventive, anti-oxidant and anti-tumor properties. Although its promising chemotherapeutic activity, preclinical and clinical studies highlight Curcumin limited therapeutic application due to its instability in physiological conditions. To improve its stability and activity, many derivatives have been synthesized and studied, among which bis-DemethoxyCurcumin (bDMC) and diAcetylCurcumin (DAC). In this report, we show that both bDMC and DAC are more stable than Curcumin in physiological medium. To explore the mechanism of their chemotherapeutic effect, we studied their role in proliferation in the HCT116 human colon cancer cells. We correlated kinetic stability and cellular uptake data to their biological effects. Both bDMC and DAC impair correct spindles formation and induce a p53- and p21(CIP1/WAF1)- independent mitotic arrest, which is more stable and long-lasting for bDMC. A subsequent p53/p21(CIP1/WAF1)- dependent inhibition of G1 to S transition is triggered by Curcumin and DAC as a consequence of the mitotic slippage, preventing postmitotic cells from re-entering the cell cycle. Conversely, the G1/S arrest induced by bDMC is a direct effect of the drug and concomitant to the mitotic block. Finally, we demonstrate that bDMC induces rapid DNA double-strand breaks, moving for its possible development in anti-cancer clinical applications.

2009 - Development of new metal-chelating multi target drus derived from Curcumin [Relazione in Atti di Convegno]
Ferrari, Erika; Pignedoli, Francesca; Lazzari, Sandra; Imbriano, Carol; Marverti, Gaetano; Saladini, Monica
abstract

2009 - Development of new therapeutic Metal(III)-chelating agents derived from Curcumin [Relazione in Atti di Convegno]
Ferrari, Erika; Pignedoli, Francesca; Imbriano, Carol; Saladini, Monica
abstract

With the aim to develop new anti-cancer approaches, which encompass therapies based on drug combinations, we are searching for innovative anti-tumor multi-targets treatments.1 In this research Curcumin represents our referring and starting point for the design of new derivatives. Curcumin, a natural occurring molecule, was shown to inhibit growth of several types of malignant cells and its biological activity was also related to its iron chelating ability.2,3 Recently curcumin proceeded onto clinical trials however its use is limited by a poor stability and bioavailability. In order to improve these features, we have synthesized new derivatives which potentially conjugate anti-proliferative and anti-oxidant effects with metal chelating capacity, especially towards Fe(III) and Ga(III), reducing free iron level and potentially delivering a chemotherapic metal ion such as Ga(III). We have performed a complete characterization of the chelating ability of new ligands in vitro by means of potentiometry, UV-Vis spectroscopy and NMR spectrometry. Stability and cellular uptake together with cytotoxicity towards different cell lines were also investigated for all ligands and their Ga(III) complexes in order to shed light on their potential anti-cancer effects especially against human colon cancer cells, unravelling the molecular basis of their biological activity.

2009 - NF-YC complexity is generated by dual promoters and alternative splicing [Articolo su rivista]
M., Ceribelli; Benatti, Paolo; Imbriano, Carol; R., Mantovani
abstract

The CCAAT box is a DNA element present in the majority of human promoters, bound by the trimeric NF-Y, composed of NF-YA, NF-YB and NF-YC subunits. We describe and characterize novel isoforms of one of the two histone-like subunit, NF-YC. The locus generates a minimum of four splicing products, mainly located within the Q-rich activation domain. The abundance of each isoform is cell dependent: only one major NF-YC isoform is present in a given cell type. The the 37 and 50 kDa isoforms are mutually exclusive and preferential pairings with NF-YA isoforms possess different transcriptional activities, with specific combinations being more active on selected promoters. The transcriptional regulation of the NF-YC locus is also complex, and mRNAs arise from two promoters: P1 and P2. Transient transfections, ChIPs and RT-PCRs indicate that P1 has a robust, housekeeping activity; P2 possesses a lower basal activity, but it is induced in response to DNA damage, in a p53 dependent way. Alternative promoter usage directly affects NF-YC splicing, with the 50 kDa transcript being excluded from P2. Specific functional inactivation of the 37 kDa isoform affects the basal levels of G1/S blocking and pro-apoptotic genes, but not G2/M promoters. In summary, our data highlight an unexpected degree of complexity and regulation of the NF-YC gene, demonstrating the existence of a discrete cohort of NF-Y trimer subtypes resulting from the functional diversification of Q-rich transactivating subunits and a specific role of the 37 kDa isoform in suppression of the DNA-damage response under growing conditions.

2009 - Understanding the molecular mechanisms of Curcumin derivatives chemotherapic activity [Relazione in Atti di Convegno]
Basile, Valentina; Ferrari, Erika; Lazzari, Sandra; Belluti, Silvia; Pignedoli, Francesca; Imbriano, Carol
abstract

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2008 - A balance between NF-Y and p53 governs the pro- and anti-apoptotic transcriptional response [Articolo su rivista]
Benatti, Paolo; Basile, Valentina; Merico, D; Fantoni, Luca Isaia; Tagliafico, Enrico; Imbriano, Carol
abstract

The transcription factor NF-Y is a trimer with histone-likesubunits that binds and activates CCAAT-containingpromoters. NF-Y controls the expressionof several key regulators of the cell cycle. In thisstudy, we examined the functional and moleculareffects of NF-YB knockdown. Cell cycle progressionis affected with a G2/M-specific depletion. This isdue to the inability of activation of G2/M-specificgenes, as evidenced by expression profiling, RT-PCRand ChIP data. Surprisingly, apoptosis is alsoobserved, with Caspase 3/7/8 cleavage. A role ofp53 and Bcl-2 family members is important. NF-YBinactivation is sufficient to functionally activate p53,in the absence of DNA damage. Failure to maintain aphysiologic level of CCAAT-dependent transcriptionof anti-apoptotic genes contributes to impairment ofBax/Bcl-2 and Bax/Bcl-XL ratios. Our data highlightthe importance of fine balancing the NF-Y-p53 duofor cell survival by (i) maintaining transcription ofanti-apoptotic genes and (ii) preventing p53 activationthat triggers the apoptotic cascade.

2008 - Role of the transcriprion factor NF-Y in cell cycle regulation [Poster]
Benatti, Paolo; Basile, Valentina; Merico, Daniele; Fantoni, Luca Isaia; Tagliafico, Enrico; Imbriano, Carol
abstract

The CCAAT-binding factor NF-Y plays an important role in controlling the transcription of cell cycle regulated genes. NF-Y binding sites belong to the regulatory module NF-Y-CDE-CHR, which controls cell cycle-dependent transcription of G2/M genes. NF-Y functions as an heterotrimer composed by NF-YA, NF-YB and NF-YC subunits. NF-YB knock-down impairs cell cycle progression by reducing G2/M cells and inducing p53-dependent apoptosis. Failure to maintain a physiological level of anti-apoptotic genes by NF-Y transcriptional activity, contributes to the triggering of the apoptotic cascade. Increasing the levels of NF-Y expression protects cells from entering a p53-mediated apoptosis: NF-Y reverts cytochrome c release into the cytoplasm following Adriamycin treatment, preventing p53 transcriptional activation. To investigate the role of the different NF-Y subunits in controlling cell cycle progression, we have separately knocked-down the three subunits by lentiviral shRNAs. NF-YA silencing shows a more severe impairment in cell cycle progression with respect to NF-YB knock-down. p53 is a common player of the cell cycle block observed following both NF-YA and NF-YB silencing. The identification of the signaling pathways through which p53 is activated will shed light on the molecular mechanism controlling the cross-talk between NF-Y and p53.

2006 - DNA damage promotes HDAC4 nuclear localization and G2/M promoters repression via p53 C-terminal lysisnes [Poster]
Basile, Valentina; Mantovani, Roberto; Imbriano, Carol
abstract

Repression of G2/M promoters after DNA-damage is an active mechanism that requires the p53 tumor suppressor. We have recently found that HDAC4 is recruited on NF-Y-dependent repressed promoters. In this report, we describe the relationship between p53 and HDAC4 recruitment following DNA-damage. (i) HDAC4 shuttles from the cytoplasm into the nucleus following DNA-damage, independently of the activation of p53. (ii) HDAC4 becomes associated to promoters through a p53-dependent mechanism. (iii) The C-terminal Lysines of p53, which are acetylated and methylated, are required for HDAC4 recruitment and transcriptional repression. (iv) TSA treatment, but not HDAC4 functional inactivation, relieves the Adriamycin-mediated repression of G2/M promoters. Our results indicate that HDAC4 is a component of the DNA-damage response, and that post-translational modifications of p53 are important for repression of G2/M genes.

2006 - DNA damage promotes histone deacetylase 4 nuclear localization and repression of G2/M promoters via p53 C-terminal lysines [Articolo su rivista]
Basile, Valentina; R., Mantovani; Imbriano, Carol
abstract

Repression of G2/M promoters after DNA damage is an active mechanism that requires the p53 tumor suppressor. We have recently found that histone deacetylase 4 (HDAC4) is recruited on NF-Y-dependent repressed promoters. In this report, we describe the relationship between p53 and HDAC4 recruitment following DNA damage using immunofluorescence, chromatin immunoprecipitation, and transfection experiments. HDAC4 shuttles from the cytoplasm into the nucleus, following DNA damage, independently of the activation of p53 and becomes associated with promoters through a p53-dependent mechanism. The C-terminal lysines of p53, which are acetylated and methylated, are required for HDAC4 recruitment and transcriptional repression. Trichostatin treatment, but not HDAC4 functional inactivation, relieves the adriamycin-mediated repression of G2/M promoters. Our results indicate that HDAC4 is a component of the DNA damage response and that post-translational modifications of p53 are important for repression of G2/M genes.

2006 - Dynamic recruitment of transcription factors and epigenetic changes on the ER stress response gene promoters [Articolo su rivista]
G., Donati; Imbriano, Carol; R., Mantovani
abstract

Response to stresses that alter the function of the endoplasmic reticulum is an important cellular function, which relies on the activation of specific genes. Several transcription factors (TFs) are known to affect this pathway. Using RT-PCR and ChIP assays, we studied the recruitment of promoterspecific TFs, general TFs and epigenetic marks in activated promoters. H3-K4 di- and tri-methylation and H3-K79 di-methylation are present before induction. H3 acetylation is generally high before induction, and H4 acetylation shows a promoter-specific increase. Interestingly, there is a depletion of histone H3 under maximal induction, explaining an apparent decrease of H3-K4 tri-methylation and H3-K79 di-methylation. Poll 11 is found enriched on some promoters under basal conditions, unlike TBP and p300, which are recruited selectively. Most genes are bound by XBP-1 after induction, some before induction, presumably by the inactive isoform. ATF6 and CHOP associate to largely different set of genes. C/EBPP is selective and binding to the CHOP promoter precedes that of XBP-1, ATF6 and CHOP. Finally, one of the ER-stress inducible genes analyzed, HRD1, is not bound by any of these factors. Among the constitutive TFs, NF-Y, but not Sp1, is found on all genes before induction. Intriguingly, siRNA interference of the NF-YB subunit indicates transcriptional impairment of some, but not all genes. These data highlight a previously unappreciated complexity of TFs binding and epigenetic changes, pointing to different TFs-specific pathways within this broad response.

2005 - Direct p53 transcriptional repression: In vivo analysis of CCAAT-containing G2/M promoters [Articolo su rivista]
Imbriano, Carol; A., GURTNER A; Cocchiarella, Fabienne; S., DI AGOSTINO; Basile, Valentina; M., Gostissa; M., Dobbelstein; G., DEL SAL; G., Piaggio; R., Mantovani
abstract

In response to DNA damage, p53 activates G1/S blocking and apoptotic genes through sequence-specific binding. p53 also represses genes with no target site, such as those for Cdc2 and cyclin B, key regulators of the G2/M transition. Like most G2/M promoters, they rely on multiple CCAAT boxes activated by NF-Y, whose binding to DNA is temporally regulated during the cell cycle. NF-Y associates with p53 in vitro and in vivo through the alpha C helix of NF-YC (a subunit of NF-Y) and a region close to the tetramerization domain of p53. Chromatin immunoprecipitation experiments indicated that p53 is associated with cyclin B2, CDC25C, and Cdc2 promoters in vivo before and after DNA damage, requiring DNA-bound NF-Y. Following DNA damage, p53 is rapidly acetylated at K320 and K373 to K382, histories are deacetylated, and the release of PCAF and p300 correlates with the recruitment of histone deacetylases (HDACs)-HDAC1 before HDAC4 and HDAC5 and promoter repression. HDAC recruitment requires intact NF-Y binding sites. In transfection assays, PCAF represses cyclin B2, and a nonacetylated p53 mutant shows a complete loss of repression potential, despite its abilities to bind NF-Y and to be recruited on G2/M promoters. These data (i) detail a strategy of direct p53 repression through associations with multiple NF-Y trimers that is independent of sequence-specific binding of p53 and that requires C-terminal acetylation, (ii) suggest that p53 is a DNA damage sentinel of the G2/M transition, anti (iii) delineate a new role for PCAF in cell cycle control.

2005 - Expression of the CCAAT-binding factor NF-Y in Caenorhabditis elegans [Articolo su rivista]
Franchini, Antonella; Imbriano, Carol; Peruzzi, Elisa; R., Mantovani; Ottaviani, Enzo
abstract

NF-Y is a conserved trimer with histone-like subunits that binds and activates the common CCAAT promoter element. C. elegans NF-Y genes present two CeNF-YAs, a unique feature in kingdoms other than plants, one CeNF-YB and one CeNF-YC. The expression of both CeNF-YAs is restricted to the gonads and developing embryos, whereas the histone-like CeNF-YB- and CeNF-YC are also present in the pharyngeal bulb, in the neurons of ganglia surrounding the pharynx and in sensory organs of the head. Moreover, in infertile, 12-day-old worms, expression of the three subunits falls dramatically in the gonads. Our data indicate that NF-Y is not ubiquitously expressed.

2004 - Cell cycle regulation of NF-YC nuclear localization [Articolo su rivista]
Frontini, M.; Imbriano, C.; Manni, I.; Mantovani, R.
abstract

: Anti-cancer properties of palm oil have been attributed to the presence of tocotrienols and carotenoids. Studies from various laboratories have shown that tocotrienol-rich fraction (TRF) of palm oil inhibits cell growth and induces apoptosis in both preneoplastic and neoplastic cells. However, the mechanism by which TRF induces apoptosis remains largely unknown. Since several chemopreventive agents have been shown to utilize p53 pathway in negative regulation of cell growth, using human colon carcinoma RKO cells which express wild type p53, we investigated the effect of TRF on components of p53 signaling network. Treatment of cells with TRF resulted in a dose- and time- dependent inhibition of growth and colony formation. Further, TRF treatment of RKO cells resulted in the induction of WAF1/p21 which appears to be independent of cell cycle regulation and is transcriptionally upregulated in p53 dependent fashion. These results were further confirmed by using cells that express luciferase from a p53 responsive promoter where TRF treatment leads to activation of p53 reporter activity. TRF treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1. This altered expression of Bcl2 family members triggered the activation of initiator caspase-9 followed by activation of effector caspase-3. These signaling cascades lead to condensed chromatin, DNA fragmentation and shrinkage of cell membrane resulting into apoptosis. Our data suggest that TRF-induced apoptosis in colon carcinoma cells is mediated by p53 signaling network which appears to be independent of cell cycle association.

2004 - Cell cycle regulation of NF-YC nuclear localization [Articolo su rivista]
M., Frontini; Imbriano, Carol; I., Manni; Mantovani, Roberto
abstract

NF-Y is a trimeric activator with histone fold - HFM - subunits that binds to the CCAAT-box and is required for a majority of cell cycle promoters, often in conjuction with E2Fs. In vivo binding of NF-Y is dynamic during the cell cycle and correlates with gene activation. We performed immunofluorescence studies on endogenous, GFP- and Flag-tagged overexpressed NF-Y subunits. NF-YA, NF-YB are nuclear proteins. Unexpectedly, NF-YC localizes both in cytoplamatic and nuclear compartments and its nuclear localization is determined by the interaction with its heterodimerization partner NF-YB. Most importantly, compartmentalization is regulated during the cell cycle of serum restimulated NIH3T3 cells, accumulating in the nucleus at the onset of S phase. These data point to the control of HFM heterodimerization as an important layer of NF-Y regulation during cell cycle progression.

2003 - Dynamic recruitment of NF-Y and histone acetyltransferases on cell-cycle promoters [Articolo su rivista]
Caretti, G; Salsi, Valentina; Vecchi, Chiara; Imbriano, Carol; Mantovani, Roberto
abstract

Regulation of transcription during the cell-cycle is under the control of E2 factors (E2Fs), often in cooperation with nuclear factor Y (NF-Y), a histone-like CCAAT-binding trimer. NF-Y is paradigmatic of a constitutive, ubiquitous factor that pre-sets the promoter architecture for other regulatory proteins to access it. We analyzed the recruitment of NF-Y, E2F1/4/6, histone acetyltransferases, and histone deacetylase (HDAC) 1/3/4 to several cell-cycle promoters by chromatin immunoprecipitation assays in serum-starved and restimulated NIH3T3 cells. NF-Y binding is not constitutive but timely regulated in all promoters tested, being displaced when promoters are repressed. p300 association correlates with activation, and it is never found in the absence of NF-Y, whereas PCAF/hGCN5 is often found before NF-Y association. E2F4 and E2F6, together with HDACs, are bound to repressed promoters, including the G(2)/M Cyclin B2. As expected, an inverse relationship between HDACs association and histones H3/H4 acetylation is observed. Blocking cells in G(1) with the cyclin-dependent kinase 2 inhibitor R-roscovitine confirms that NF-Y is bound to G(1)/S but not to G(2)/M promoters in G(1). These data indicate that following the release of E2Fs/HDACs, a hierarchy of PCAF-NF-Y-p300 interactions and H3-H4 acetylations are required for activation of cell-cycle promoters.

2003 - Links between tumor suppressors: p53 is required for TGF-beta gene responses by cooperating with Smads [Articolo su rivista]
M., Cordenonsi; S., Dupont; S., Maretto; A., Insinga; Imbriano, Carol; S., Piccolo
abstract

The p53 tumor suppressor belongs to a family of proteins that sense multiple cellular inputs to regulate Cell proliferation, apoptosis, and differentiation. Whether and how these functions of p53 intersect with the activity of extracellular growth factors is not understood. Here, we report that key cellular responses to TGF-beta signals rely on p53 family members. During Xenopus embryonic development, p53 promotes the activation of multiple TGF-beta target genes. Moreover, mesoderm differentiation is inhibited in p53-depleted embryos. In mammalian cells, the full transcriptional activation of the CDK inhibitor p21(WAF1) by TGF-beta requires p53. p53-deficient cells display an impaired cytostatic response to TGF-beta signals. Smad and p53 protein complexes converge on separate cis binding elements on a target promoter and synergistically activate TGF-beta induced transcription. p53 can physically interact in vivo with Smad2 in a TGF-beta-dependent fashion. The results unveil a previously unrecognized link between two primary tumor suppressor pathways in vertebrates.

2003 - Transcriptional activation of the cyclin A gene by the architectural transcription factor HMGA2 [Articolo su rivista]
Tessari, Ma; Gostissa, M; Altamura, S; Sgarra, R; Rustighi, A; Salvagno, C; Caretti, G; Imbriano, Carol; Mantovani, Roberto; Del Sal, G; Giancotti, V; Manfioletti, G.
abstract

The HMGA2 protein belongs to the HMGA family of architectural transcription factors, which play an important role in chromatin organization. HMGA proteins are overexpressed in several experimental and human tumors and have been implicated in the process of neoplastic transformation. Hmga2 knockout results in the pygmy phenotype in mice and in a decreased growth rate of embryonic fibroblasts, thus indicating a role for HMGA2 in cell proliferation. Here we show that HMGA2 associates with the E1A-regulated transcriptional repressor p120(E4F), interfering with p120(E4F) binding to the cyclin A promoter. Ectopic expression of HMGA2 results in the activation of the cyclin A promoter and induction of the endogenous cyclin A gene. In addition, chromatin immunoprecipitation experiments show that HMGA2 associates with the cyclin A promoter only when the gene is transcriptionally activated. These data identify the cyclin A gene as a cellular target for HMGA2 and, for the first time, suggest a mechanism for HMGA2-dependent cell cycle regulation.

2002 - NF-Y recruitment of TFIID, multiple interactions with histone fold TAF(II)s [Articolo su rivista]
Frontini, Mattia; Imbriano, Carol; A., Disilvio; B., Bell; A., Bogni; C., Romier; D., Moras; L., Tora; I., Davidson; Mantovani, Roberto
abstract

The nuclear factor y (NF-Y) trimer and TFIID contain histone fold subunits, and their binding to the CCAAT and Initiator elements of the major histocompatibility complex class II Ea promoter is required for transcriptional activation. Using agarose-electrophoretic mobility shift assay we found that NF-Y increases the affinity of holo-TFIID for Ea in a CCAAT- and Inr-dependent manner. We began to dissect the interplay between NF-Y- and TBP-associated factors PO1II (TAF(II)s)-containing histone fold domains in protein-protein interactions and transfections. hTAF(II)20, hTAF(II)28, and hTAF(II)18-hTAF(II)28 bind to the NF-Y B-NF-YC histone fold dimer; hTAF(II)80 and hTAF(II)31-hTAF(II)80 interact with the trimer but not with the NF-YB-NF-YC dimer. The histone fold alpha2 helix of hTAF(II)80 is not required for NF-Y association, as determined by interactions with the naturally occurring splice variant hTAF(II)80delta. Expression of hTAF(II)28 and hTAF(II)18 in mouse cells significantly and specifically reduced NF-Y activation in GAL4-based experiments, whereas hTAF,120 and hTAF(II)135 increased it. These results indicate that NF-Y (i) recruits purified holo-TFIID in vitro and (ii) can associate multiple TAF(II)s, potentially accommodating different core promoter architectures.

2002 - Ternary complex formation between MADS-box transcription factors and the histone fold protein NF-YB [Articolo su rivista]
S., Masiero; Imbriano, Carol; F., Ravasio; R., Favaro; N., Pelucchi; Ms, Gorla; Mantovani, Roberto; L., Colombo; Mm, Kater
abstract

MADS-box proteins are transcription factors present in different eukaryotic kingdoms. In contrast to plants, for mammalian and yeast MADS-box proteins ternary complex formation with unrelated transcription factors was reported. We show here the first identification of such ternary interaction in plants. A rice seed-specific NF-YB was identified as partner of OsMADS18 by two-hybrid screening. NF-YB contains a histone fold motif, HFM,(1) and is part of the trimeric CCAAT-binding NF-Y complex. OsMADS18, alone or in combination with a natural partner, interacts with OsNF-YB1 through the MADS and I regions. The mouse NF-YB also associates with OsMADS18 in vivo and in vitro as a NF-YB-NF-YC dimer. Other rice MADS-box proteins do not interact in these assays, indicating specificity for the interaction. OsNF-YB1 is capable of heterodimerizing with NF-YC, but not trimerizing with NF-YA, thus precluding CCAAT binding. Mutation of the variant Asp at position 99 of the HFM alpha2-helix into a conserved serine recovers the capacity to interact with NF-YA, but not with DNA. This is the first indication that members of the NF-YB family work through mechanisms independent of the CCAAT box.

2001 - HSP-CBF is an NF-Y-dependent coactivator of the heat shock promoters CCAAT boxes [Articolo su rivista]
Imbriano, Carol; F., Bolognese; A., Gurtner; G., Piaggio; Mantovani, Roberto
abstract

The cellular response to toxic stimuli is elicited through the expression of heat shock proteins, a transcriptional process that relies upon conserved DNA elements in the promoters: the Heat Shock Elements, activated by the heat shock factors, and the CCAAT boxes. The identity of the CCAAT activator(s) is unclear because two distinct entities, NF-Y and HSP-CBF, have been implicated in the HSP70 system, The former is a conserved ubiquitous trimer containing histone-like subunits, the latter a 110-kDa protein with an acidic N-terminal, We analyzed two CCAAT-containing promoters, HSP70 and HSP40, with recombinant NF-Y and HSP-CBF using electrophoretic mobility shift assay, protein-protein interactions, transfections and chromatin immunoprecipitation assays (ChIP) assays. Both recognize a common DNA-binding protein in nuclear extracts, identified in vitro and in vivo as NF-Y, Both CCAAT boxes show high affinity for recombinant NF-Y but not for HSP-CBF, However, HSP-CBF does activate HSP70 and HSP40 transcription under basal and heat shocked conditions; for doing so, it requires an intact NF-Y trimer as judged by cotransfections with a diagnostic NF-YA dominant negative vector. HSP-CBF interacts in solution and on DNA with the NF-Y trimer through an evolutionary conserved region. In yeast two-hybrid assays HSP-CBF interacts with NF-YB, These data implicate HSP-CBF as a non-DNA binding coactivator of heat shock genes that act on a DNA-bound NF-Y.

2000 - Cloning and characterization of the histone-fold proteins YBL1 and YCL1 [Articolo su rivista]
F., Bolognese; Imbriano, Carol; G., Caretti; Mantovani, Roberto
abstract

Histones are among the most conserved proteins in evolution, sharing a histone fold motif, A number of additional histonic proteins exist and are involved in the process of transcriptional regulation. We describe here the identification, cloning and characterization of two small members of the H2A-H2B sub-family (YBL1 and YCL1) related to the NF-YB and NF-YC subunits of the CCAAT-binding activator NF-Y and to the TATA-binding protein (TBP) binding repressor NC2, Unlike the latters, YBL1 and YCL1 have no intrinsic CCAAT or TATA-binding capacity. In nucleosome reconstitution assays, they can form complexes with histones in solution and on DNA and they are part of relatively large complexes, as determined by glycerol gradient experiments. Our data support the idea that YBL1 and YCL1 are divergent with respect to NF-YB and NF-YC for specific functions, but have coevolved the capacity to interact with nucleosomal structures.

1999 - Dissection of the NF-Y transcriptional activation potential [Articolo su rivista]
DI SILVIO, Alberto; Imbriano, Carol; Mantovani, Roberto
abstract

NF-Y is a trimeric CCAAT-binding factor with histone fold subunits (NF-YB/NF-YC) and bipartite activation domains located on NF-YA and NF-YC, We reconstituted the NF-Y activation potential in vivo with GAL4 DBD fusions, In the GAL4-YA configuration, activation requires co-expression of the three subunits; with GAL4-YB and GAL4-YC, transfections of the histone fold partners are sufficient, provided that the Q-rich domain of NF-YC is present, Combinations of mutants indicate that the Q-rich domains of NF-YA and NF-YC are redundant in the trimeric complex, Glutamines 101 and 102 of NF-YA are required for activity, We assayed NF-Y on different promoter targets, containing single or multiple GAL4 sites: whereas on a single site NF-Y is nearly as powerful as VP16, on multiple sites neither synergistic nor additive effects are observed, NF-Y activates TATA and Inr core elements and the overall potency is in the same range as other Q-rich and Pro-rich activation domains. These results represent the first in vivo evidence of subunit interactions studies and further support the hypothesis that NF-Y is a general promoter organizer rather than a brute activator.

Università degli studi di Modena e Reggio Emilia

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