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Valentina SALSI

Ricercatore t.d. art. 24 c. 3 lett. A
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche
Docente a contratto
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Neuroscienze

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2024 - Physical activity practiced at a young age is associated with a less severe subsequent clinical presentation in facioscapulohumeral muscular dystrophy [Articolo su rivista]
Bettio, C.; Banchelli, F.; Salsi, V.; Vicini, R.; Crisafulli, O.; Ruggiero, L.; Ricci, G.; Bucci, E.; Angelini, C.; Berardinelli, A.; Bonanno, S.; D’Angelo, M. G.; Di Muzio, A.; Filosto, M.; Frezza, E.; Maggi, L.; Mongini, T.; Pegoraro, E.; Rodolico, C.; Scarlato, M.; Vattemi, G.; Velardo, D.; Tomelleri, G.; D’Amico, R.; D’Antona, G.; Tupler, R.

Background: In facioscapulohumeral muscular dystrophy (FSHD), it is not known whether physical activity (PA) practiced at young age is associated with the clinical presentation of disease. To assess this issue, we performed a retrospective cohort study concerning the previous practice of sports and, among them, those with medium-high cardiovascular commitment in clinically categorized carriers of a D4Z4 reduced allele (DRA). Methods: People aged between 18 and 60 were recruited as being DRA carriers. Subcategory (classical phenotype, A; incomplete phenotype, B; asymptomatic carriers, C; complex phenotype, D) and FSHD score, which measures muscle functional impairment, were assessed for all participants. Information on PAs was retrieved by using an online survey dealing with the practice of sports at a young age. Results: 368 participants were included in the study, average age 36.6 years (SD = 9.4), 47.6% male. The FSHD subcategory A was observed in 157 (42.7%) participants with average (± SD) FSHD score of 5.8 ± 3.0; the incomplete phenotype (category B) in 46 (12.5%) participants (average score 2.2 ± 1.7) and the D phenotype in 61 (16.6%, average score 6.5 ± 3.8). Asymptomatic carriers were 104 (subcategory C, 28.3%, score 0.0 ± 0.2). Time from symptoms onset was higher for patients with A (15.8 ± 11.1 years) and D phenotype (13.3 ± 11.9) than for patients with B phenotype (7.3 ± 9.0). The practice of sports was associated with lower FSHD score (-17%) in participants with A phenotype (MR = 0.83, 95% CI = 0.73-0.95, p = 0.007) and by 33% in participants with D phenotype (MR = 0.67, 95% CI = 0.51-0.89, p = 0.006). Conversely, no improvement was observed in participants with incomplete phenotype with mild severity (B). Conclusions: PAs at a young age are associated with a lower clinical score in the adult A and D FSHD subcategories. These results corroborate the need to consider PAs at the young age as a fundamental indicator for the correct clinical stratification of the disease and its possible evolution.

2023 - De novo variants and recombination at 4q35: hints for preimplantation genetic testing in facioscapulohumeral muscular dystrophy [Articolo su rivista]
Pini, Sara; Napoli, Floriana Maria; Tagliafico, Enrico; Marca, Antonio La; Bertucci, Emma; Salsi, Valentina; Tupler, Rossella

2023 - The FSHD jigsaw: are we placing the tiles in the right position? [Articolo su rivista]
Salsi, Valentina; Vattemi, Gaetano Nicola Alfio; Tupler, Rossella

Purpose of review: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common myopathies, involving over 870,000 people worldwide and over 20 FSHD national registries. Our purpose was to summarize the main objectives of the scientific community on this topic and the moving trajectories of research from the past to the present. Recent findings: To date, research is mainly oriented toward deciphering the molecular and pathogenetic basis of the disease by investigating DUX4-mediated muscle alterations. Accordingly, FSHD drug development has been escalating in the last years in an attempt to silence DUX4 or to block its downstream effectors. Breakthroughs in the field include the awareness that new biomarkers and outcome measures are required for tracking disease progression and patient stratification. The need to develop personalized therapeutic strategies is also crucial according to the phenotypic variability observed in FSHD subjects. Summary: We analysed 121 literature reports published between 2021 and 2023 to assess the most recent advances in FSHD clinical and molecular research.

2022 - Counseling and prenatal diagnosis in facioscapulohumeral muscular dystrophy: A retrospective study on a 13‐year multidisciplinary approach [Articolo su rivista]
Di Feo, Maria Francesca; Bettio, Cinzia; Salsi, Valentina; Bertucci, Emma; Tupler, Rossella

2021 - The Italian National Registry for FSHD: an enhanced data integration and an analytics framework towards Smart Health Care and Precision Medicine for a rare disease [Articolo su rivista]
Bettio, C.; Salsi, V.; Orsini, M.; Calanchi, E.; Magnotta, L.; Gagliardelli, L.; Kinoshita, J.; Bergamaschi, S.; Tupler, R.

Background: The Italian Clinical network for FSHD (ICNF) has established the Italian National Registry for FSHD (INRF), collecting data from patients affected by Facioscapulohumeral dystrophy (FSHD) and their relatives. The INRF has gathered data from molecular analysis, clinical evaluation, anamnestic information, and family history from more than 3500 participants. Methods: A data management framework, called Mediator Environment for Multiple Information Sources (MOMIS) FSHD Web Platform, has been developed to provide charts, maps and search tools customized for specific needs. Patients’ samples and their clinical information derives from the Italian Clinical network for FSHD (ICNF), a consortium consisting of fourteen neuromuscular clinics distributed across Italy. The tools used to collect, integrate, and visualize clinical, molecular and natural history information about patients affected by FSHD and their relatives are described. Results: The INRF collected the molecular data regarding FSHD diagnosis conducted on 7197 subjects and identified 3362 individuals carrying a D4Z4 Reduced Allele (DRA): 1634 were unrelated index cases. In 1032 cases the molecular testing has been extended to 3747 relatives, 1728 carrying a DRA. Since 2009 molecular analysis has been accompanied by clinical evaluation based standardized evaluation protocols. In the period 2009–2020, 3577 clinical forms have been collected, 2059 follow the Comprehensive Clinical Evaluation form (CCEF). The integration of standardized clinical information and molecular data has made possible to demonstrate the wide phenotypic variability of FSHD. The MOMIS (Mediator Environment for Multiple Information Sources) data integration framework allowed performing genotype–phenotype correlation studies, and generated information of medical importance either for clinical practice or genetic counseling. Conclusion: The platform implemented for the FSHD Registry data collection based on OpenClinica meets the requirement to integrate patient/disease information, as well as the need to adapt dynamically to security and privacy concerns. Our results indicate that the quality of data collection in a multi-integrated approach is fundamental for clinical and epidemiological research in a rare disease and may have great value in allowing us to redefine diagnostic criteria and disease markers for FSHD. By extending the use of the MOMIS data integration framework to other countries and the longitudinal systematic collection of standardized clinical data will facilitate the understanding of disease natural history and offer valuable inputs towards trial readiness. This approach is of high significance to FSHD medical community and also to rare disease research in general.

2020 - Does DNA Methylation Matter in FSHD? [Articolo su rivista]
Salsi, Valentina; Magdinier, Frédérique; Tupler, Rossella

Facioscapulohumeral muscular dystrophy (FSHD) has been associated with the genetic and epigenetic molecular features of the CpG-rich D4Z4 repeat tandem array at 4q35. Reduced DNA methylation of D4Z4 repeats is considered part of the FSHD mechanism and has been proposed as a reliable marker in the FSHD diagnostic procedure. We considered the assessment of D4Z4 DNA methylation status conducted on distinct cohorts using different methodologies. On the basis of the reported results we conclude that the percentage of DNA methylation detected at D4Z4 does not correlate with the disease status. Overall, data suggest that in the case of FSHD1, D4Z4 hypomethylation is a consequence of the chromatin structure present in the contracted allele, rather than a proxy of its function. Besides, CpG methylation at D4Z4 DNA is reduced in patients presenting diseases unrelated to muscle progressive wasting, like Bosma Arhinia and Microphthalmia syndrome, a developmental disorder, as well as ICF syndrome. Consistent with these observations, the analysis of epigenetic reprogramming at the D4Z4 locus in human embryonic and induced pluripotent stem cells indicate that other mechanisms, independent from the repeat number, are involved in the control of the epigenetic structure at D4Z4.

2020 - Interpretation of the epigenetic signature of facioscapulohumeral muscular dystrophy in light of genotype-phenotype studies [Articolo su rivista]
Nikolic, A.; Jones, T. I.; Govi, M.; Mele, F.; Maranda, L.; Sera, F.; Ricci, G.; Ruggiero, L.; Vercelli, L.; Portaro, S.; Villa, L.; Fiorillo, C.; Maggi, L.; Santoro, L.; Antonini, G.; Filosto, M.; Moggio, M.; Angelini, C.; Pegoraro, E.; Berardinelli, A.; Maioli, M. A.; D'Angelo, G.; Di Muzio, A.; Siciliano, G.; Tomelleri, G.; D'Esposito, M.; Ragione, F. D.; Brancaccio, A.; Piras, R.; Rodolico, C.; Mongini, T.; Magdinier, F.; Salsi, V.; Jones, P. L.; Tupler, R.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.

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

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 - HOX cluster-embedded micro-RNAs and cancer [Articolo su rivista]
Fantini, S.; Salsi, V.; Zappavigna, V.

HOX cluster-embedded micro-RNAs and cancer.

2017 - NUP98-fusion transcripts characterize different biological entities within acute myeloid leukemia: A report from the AIEOP-AML group [Articolo su rivista]
Bisio, V.; Zampini, M.; Tregnago, C.; Manara, E.; Salsi, Valentina; Di Meglio, A.; Masetti, R.; Togni, M.; Di Giacomo, D.; Minuzzo, S.; Leszl, A.; Zappavigna, Vincenzo; Rondelli, R.; Mecucci, C.; Pession, A.; Locatelli, F.; Basso, G.; Pigazzi, M.

In the last years, collaborative studies have joined to link the degree of genetic heterogeneity of acute myeloid leukemia (AML) to clinical outcome,1, 2 allowing risk stratification before therapy and guiding post-induction treatment of children with AML. So far, still half of these patients, whose disease is usually characterized by a grim prognosis, lack a known biomarker offering opportunities of targeted treatment.

2017 - The miR-196b miRNA inhibits the GATA6 intestinal transcription factor and is upregulated in colon cancer patients [Articolo su rivista]
Fantini, Sebastian; Salsi, Valentina; Reggiani, Luca; Maiorana, Antonino; Zappavigna, Vincenzo

Objective: To explore the possible misexpression of the microRNA miR-196b in colorectal cancer (CRC) and its role in controlling the expression of GATA6, a putative target gene crucial to intestinal cell homeostasis and tumorigenesis. Design: The expression of miR-196b was analysed by qRT-PCR in surgical resection samples from a cohort of sporadic colon cancer patients. Manipulations of miR-196b expression were performed to demonstrate its inhibition of GATA6 protein levels. Results: We found that miR-196b is significantly upregulated in pre-treatment surgical resection samples from a cohort of sporadic colon cancer patients. The upregulation of miR-196b correlates with less severe clinicopathological characteristics, such as early tumor stage and absence of lymph node metastases. We show that in CRC cells, miR-196b targets the mRNA of GATA6, a transcription factor involved in the homeostasis and differentiation of intestinal epithelial cells, and a positive regulator of the Wnt/β-catenin pathway. We moreover found that the increase of miR-196b correlates with a reduced GATA6 protein expression in colon cancer patients. Conclusion: Our results establish miR-196b as a post-transcriptional inhibitor of GATA6 in CRC cells, implicating miR-196b function in gene regulatory pathways crucial to intestinal cell homeostasis and tumorigenesis. Our results furthermore suggest a role of miR-196b expression in CRC, as an antagonist of GATA6 function in tumor cells, thus providing the basis for a potential targeting strategy for the treatment of CRC.

2016 - NUP98 fusion oncoproteins interact with the APC/CCdc20 as a pseudosubstrate and prevent mitotic checkpoint complex binding [Articolo su rivista]
Salsi, Valentina; Fantini, Sebastian; Zappavigna, Vincenzo

NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/CCdc20 in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/CCdc20. NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/CCdc20. We found that NUP98 wild-type is a substrate of APC/CCdc20 prior to mitotic entry, and that its binding to APC/CCdc20 is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/CCdc20. We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/CCdc20 during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/CCdc20 and to interfere with its function.

2015 - MicroRNA-196b is transcribed from an autonomous promoter and is directly regulated by Cdx2 and by posterior Hox proteins during embryogenesis [Articolo su rivista]
Fantini, Sebastian; Salsi, Valentina; Vitobello, Antonio; Rijli, Filippo M.; Zappavigna, Vincenzo

The miR-196 miRNA gene family located within the Hox gene clusters has been shown to function during embryogenesis and to be aberrantly expressed in various malignancies, including leukaemia, melanoma, and colorectal cancer. Despite its involvement in numerous biological processes, the control of miR-196 expression is still poorly defined. We identified the miR-196b promoter and found that the mature miR-196b originates from a large, non-coding primary transcript, which starts within an autonomous TATA box promoter and is not in physical continuity with either the Hoxa10 or Hoxa9 main primary transcripts. A ~680bp genomic fragment, spanning the pri-miR-196b transcription start site, is sufficient to recapitulate the neural tube expression pattern of miR-196 during embryogenesis. This region contains potential binding sites for Cdx and 5'Hox transcription factors. Two of these sites revealed to be necessary for neural tube expression and were bound in vivo by Cdx2 and Hoxd13. We show that Cdx2 is required for miR-196 expression and that both Cdx2 and 5'Hox, but not 3'Hox, are able to activate the miR-196b promoter. The possible role of Cdx2- and 5'Hox-mediated regulation of miR-196 expression in vertebrate anterior-posterior (AP) axis formation during embryogenesis is discussed.

2014 - MafB is a downstream target of the IL-10/STAT3 signaling pathway, involved in the regulation of macrophage de-activation [Articolo su rivista]
Gemelli, C.; Zanocco Marani, T.; Bicciato, S.; Mazza, E. M. C.; Boraschi, D.; Salsi, V.; Zappavigna, V.; Parenti, S.; Selmi, T.; Tagliafico, E.; Ferrari, S.; Grande, A.

In spite of the numerous reports implicating MafB transcription factor in the molecular control of monocyte-macrophage differentiation, the precise genetic program underlying this activity has been, to date, poorly understood. To clarify this issue, we planned a number of experiments that were mainly conducted on human primary macrophages. In this regard, a preliminary gene function study, based on MafB inactivation and over-expression, indicated MMP9 and IL-. 7R genes as possible targets of the investigated transcription factor. Bioinformatics analysis of their promoter regions disclosed the presence of several putative MARE elements and a combined approach of EMSA and luciferase assay subsequently demonstrated that expression of both genes is indeed activated by MafB through a direct transcription mechanism. Additional investigation, performed with similar procedures to elucidate the biological relevance of our observation, revealed that MafB is a downstream target of the IL-10/STAT3 signaling pathway, normally inducing the macrophage de-activation process. Taken together our data support the existence of a signaling cascade by which stimulation of macrophages with the IL-10 cytokine determines a sequential activation of STAT3 and MafB transcription factors, in turn leading to an up-regulated expression of MMP9 and IL-. 7R genes. © 2014 Elsevier B.V.

2014 - NUP98 fusion oncoproteins promote aneuploidy by attenuating the mitotic spindle checkpoint [Articolo su rivista]
Salsi, V.; Ferrari, S.; Gorello, P.; Fantini, S.; Chiavolelli, F.; Mecucci, C.; Zappavigna, V.

NUP98 is a recurrent fusion partner in chromosome translocations that cause acute myelogenous leukemia. NUP98, a nucleoporin, and its interaction partner Rae1, have been implicated in the control of chromosome segregation, but their mechanistic contributions to tumorigenesis have been unclear. Here, we show that expression of NUP98 fusion oncoproteins causes mitotic spindle defects and chromosome missegregation, correlating with the capability of NUP98 fusions to cause premature securin degradation and slippage from an unsatisfied spindle assembly checkpoint (SAC). NUP98 fusions, unlike wild-type NUP98, were found to physically interact with the anaphase promoting complex/cyclosome (APC/C)(Cdc20) and to displace the BubR1 SAC component, suggesting a possible mechanistic basis for their interference with SAC function. In addition, NUP98 oncoproteins displayed a prolonged half-life in cells. We found that NUP98 stability is controlled by a PEST sequence, absent in NUP98 oncoproteins, whose deletion reproduced the aberrant SAC-interfering activity of NUP98 oncoproteins. Together, our findings suggest that NUP98 oncoproteins predispose myeloid cells to oncogenic transformation or malignant progression by promoting whole chromosome instability.

2013 - The Orosomucoid1 protein is involved in the vitamin D – mediated macrophage de-activation process [Articolo su rivista]
Gemelli, Claudia; Martello, Andrea; Montanari, Monica; ZANOCCO MARANI, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 - VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

2009 - HOXD13 binds DNA replication origins to promote origin licensing and is inhibited by geminin. [Articolo su rivista]
Salsi, Valentina; Ferrari, Silvia; Ferraresi, Roberta; Cossarizza, Andrea; Grande, Alexis; Zappavigna, Vincenzo

HOX DNA-binding proteins control patterning during development by regulating processes such as cell aggregation and proliferation. Recently, a possible involvement of HOX proteins in replication origin activity was suggested by results showing that a number of HOX proteins interact with the DNA replication licensing regulator geminin and bind a characterized human origin of replication. The functional significance of these observations, however, remained unclear. We show that HOXD13, HOXD11, and HOXA13 bind in vivo all characterized human replication origins tested. We furthermore show that HOXD13 interacts with the CDC6 loading factor, promotes pre-replication complex (pre-RC) proteins assembly at origins, and stimulates DNA synthesis in an in vivo replication assay. HOXD13 expression in cultured cells accelerates DNA synthesis initiation in correlation with the earlier pre-RC recruitment onto origins during G(1) phase. Geminin, which interacts with HOXD13 as well, blocks HOXD13-mediated assembly of pre-RC proteins and inhibits HOXD13-induced DNA replication. Our results uncover a function for Hox proteins in the regulation of replication origin activity and reveal an unforeseen role for the inhibition of HOX protein activity by geminin in the context of replication origin licensing.

2008 - Hoxd13 binds in vivo and regulates the expression of genes acting in key pathways for early limb and skeletal patterning [Articolo su rivista]
Salsi, Valentina; Vigano, M. A.; Cocchiarella, Fabienne; Mantovani, R.; Zappavigna, Vincenzo

5' HoxD genes are required for the correct formation of limb skeletal elements. Hoxd13, the most 5'-located HoxD gene, is important for patterning the most distal limb region, and its mutation causes human limb malformation syndromes. The mechanisms underlying the control of developmental processes by Hoxd13, and by Hox genes in general, are still elusive, due to the limited knowledge on their direct downstream target genes. We identified by ChIP-on-chip 248 known gene loci bound invivo by Hoxd13. Genes relevant to limb patterning and skeletogenesis were further analysed. We found that Hoxd13 binds invivo, in developing limbs, the loci of Hand2, a gene crucial to limb AP axis patterning, of Meis1 and Meis2, involved in PD patterning, of the Sfrp1, Barx1, and Fbn1 genes, involved in skeletogenesis, and of the Dach1, Bmp2, Bmp4, andEmx2 genes. We show that Hoxd13 misexpression in developing chick limbs alters the expression of the majority of these genes, supporting the conclusion that Hoxd13 directly regulates their transcription. Our results indicate that 5' Hox proteins regulate directly both key genes for early limb AP and PD axis patterning and genes involved, at later stages, in skeletal patterning.

2006 - Hoxd13 and Hoxa13 directly control the expression of the EphA7 ephrin tyrosine kinase receptor in developing limbs [Articolo su rivista]
Salsi, Valentina; Zappavigna, Vincenzo

Hoxa and Hoxd genes, related to the Drosophila Abd-B gene, display regionally restricted expression patterns and are necessary for the formation of the limb skeletal elements. Hox genes encode transcription factors, which are supposed to control the expression of a series of downstream target genes, whose nature has remained largely elusive. Several genes were identified that are differentially expressed in relation to Hox gene activity; few studies, however, explored their direct regulation by Hox proteins. Ephrin tyrosine kinase receptors and ephrins have been proposed as Hox targets, and recently, evidence was gained for their role in limb development. The expression of the EphA7 gene in developing limbs was shown to correlate with the expression of Hoxa13 and Hoxd13; however, its direct regulation by these genes has never been assessed. We have characterized the EphA7 promoter region and show that it contains multiple binding sites for paralog group 13 Hox proteins. We found that one of these sites is bound in vivo by HOXA13 and HOXD13 and by endogenous Hoxd13 in developing mouse limbs. Moreover, we show that HOXD13 and HOXA13 activate transcription from the EphA7 promoter and that a mutation of the HOXA13/HOXD13 binding site was sufficient to abolish activation. Conversely, the HOXD13(147L) mutation, identified in patients displaying a novel brachydactyly-polydactyly syndrome, does not bind to in vivo, and fails to transactivate the EphA7 promoter. These results establish that EphA7 is a direct downstream target of Hoxd13 and Hoxa13 during limb development, thus providing further insight into the regulatory networks that control limb patterning.

2006 - Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression [Articolo su rivista]
Capellini, Td; Di Giacomo, G; Salsi, Valentina; Brendolan, A; Ferretti, E; Srivastava, D; Zappavigna, Vincenzo; Selleri, L.

Vertebrate limb development occurs along three cardinal axes-proximodistal, anteroposterior and dorsoventral-that are established via the organization of signaling centers, such as the zone of polarizing activity ( ZPA). Distal limb development, in turn, requires a molecular feedback loop between the ZPA expression of sonic hedgehog ( Shh) and the apical ectodermal ridge. The TALE homeoprotein Pbx1 has been shown to be essential for proximal limb development. In this study, we first uncover that Pbx1 and Pbx2 are co-expressed in the lateral plate and early limb field mesoderm. Later, Pbx2 is expressed throughout the limb, unlike Pbx1, which is expressed only in the proximal bud. By exploiting a Pbx1/Pbx2 loss-of-function mouse model, we demonstrate that, despite the lack of limb abnormalities in Pbx2-deficient ( Pbx2(-/-)) embryos, compound Pbx1(-/-); Pbx2(+/-) mutants, in addition to their exacerbated proximal limb defects, exhibit novel and severe distal abnormalities. Additionally, we reveal that Pbx1(-/-); Pbx2(-/-) embryos lack limbs altogether. Furthermore, we establish that, unlike in flies, where the leg develops independently of Hox and where the Pbx ortholog Exd is required for specification of proximal ( but not distal) limbs, in vertebrates, distal limb patterning is Pbx1/Pbx2 dependent. Indeed, we demonstrate that Pbx genetic requirement is mediated, at least in part, through their hierarchical control of Hox spatial distribution and Shh expression. Overall, we establish that, by controlling the spatial expression of Hox genes in the posterior limb and regulating ZPA function, Pbx1/Pbx2 exert a primary hierarchical function on Hox genes, rather than behaving merely as Hox ancillary factors.

2005 - A Pbx1-dependent genetic and transcriptional network regulates spleen ontogeny [Articolo su rivista]
Brendolan, A.; Ferretti, E.; Salsi, V.; Moses, K.; Quaggin, S.; Blasi, F.; Cleary, M. L.; Selleri, L.

The genetic control of cell fate specification, morphogenesis and expansion of the spleen, a crucial lymphoid organ, is poorly understood. Recent studies of mutant mice implicate various transcription factors in spleen development, but the hierarchical relationships between these factors have not been explored. In this report, we establish a genetic network that regulates spleen ontogeny, by analyzing asplenic mice mutant for the transcription factors Pbx1, Hox11 (Tlx1), Nkx3.2 (Bapx1) and Pod1 (capsulin, Tcf21). We show that Hox11 and Nkx2.5, among the earliest known markers for splenic progenitor cells, are absent in the splenic anlage of Pbx1 homozygous mutant (-/-) embryos, implicating the TALE homeoprotein Pbx1 in splenic cell specification. Pbx1 and Hox11 genetically interact in spleen formation and loss of either is associated with a similar reduction of progenitor cell proliferation and failed expansion of the splenic anlage. Chromatin immunoprecipitation assays show that Pbx1 binds to the Hox11 promoter in spleen mesenchymal cells, which co-express Pbx1 and Hox11. Furthermore, Hox11 binds its own promoter in vivo and acts synergistically with TALE proteins to activate transcription, supporting its role in an auto-regulatory circuit. These studies establish a Pbx1-Hox11-dependent genetic and transcriptional pathway in spleen ontogeny. Additionally, we demonstrate that while Nkx3.2 and Pod1 control spleen development via separate pathways, Pbx1 genetically regulates key players in both pathways, and thus emerges as a central hierarchical co-regulator in spleen genesis.

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

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 - Interactions between p300 and multiple NF-Y trimers govern cyclin B2 promoter function [Articolo su rivista]
Salsi, V.; Caretti, G.; Wasner, M.; Reinhard, W.; Haugwitz, U.; Engeland, K.; Mantovani, R.

The CCAAT box is one of the most common elements in eukaryotic promoters and is activated by NF-Y, a conserved trimeric transcription factor with histone-like subunits. Usually one CCAAT element is present in promoters at positions between -60 and -100, but an emerging class of promoters harbor multiple NF-Y sites. In the triple CCAAT-containing cyclin B2 cell-cycle promoter, all CCAAT boxes, independently from their NF-Y affinities, are important for function. We investigated the relationships between NF-Y and p300. Chromatin immunoprecipitation analysis found that NF-Y and p300 are bound to the cyclin B2 promoter in vivo and that their binding is regulated during the cell cycle, positively correlating with promoter function. Cotransfection experiments determined that the coactivator acts on all CCAAT boxes and requires a precise spacing between the three elements. We established the order of in vitro binding of the three NF-Y complexes and find decreasing affinities from the most distal Y1 to the proximal Y3 site. Binding of two or three NF-Y trimers with or without p300 is not cooperative, but association with the Y1 and Y2 sites is extremely stable. p300 favors the binding of NF-Y to the weak Y3 proximal site, provided that a correct distance between the three CCAAT is respected. Our data indicate that the precise spacing of multiple CCAAT boxes is crucial for coactivator function. Transient association to a weak site might be a point of regulation during the cell cycle and a general theme of multiple CCAAT box promoters.