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Professore Ordinario
Dipartimento di Scienze della Vita sede ex-Biologia

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2019 - ‘Building a perfect body’: Control of vertebrate organogenesis by PBX-dependent regulatory networks [Articolo su rivista]
Selleri, L.; Zappavigna, V.; Ferretti, E.

Pbx genes encode transcription factors that belong to the TALE (three-amino-acid loop extension) superclass of homeodomain proteins. We have witnessed a surge in information about the roles of this gene family as leading actors in the transcriptional control of development. PBX proteins represent a clear example of how transcription factors can regulate developmental processes by combinatorial properties, acting within multimeric complexes to implement activation or repression of transcription depending on their interaction partners. Here, we revisit long-emphasized functions of PBX transcription factors as cofactors for HOX proteins, major architects of the body plan. We further discuss new knowledge on roles of PBX proteins in different developmental contexts as upstream regulators of Hox genes—as factors that interact with non-HOX proteins and can work independently of HOX—as well as potential pioneer factors. Committed to building a perfect body, PBX proteins govern regulatory networks that direct essential morphogenetic processes and organogenesis in vertebrate development. Perturbations of PBX-dependent networks can cause human congenital disease and cancer.

2019 - Corrigendum: ‘Building a perfect body’: Control of vertebrate organogenesis by PBX-dependent regulatory networks (Genes & Development 33: 258–275 (2019) [Articolo su rivista]
Selleri, L.; Zappavigna, V.; Ferretti, E.

In the above-mentioned article, the funding source NNF17CC0027852 to Elisabetta Ferretti was missing from Acknowledgments and has been added as follows: “This work was supported by The Novo Nordisk Foundation Center for Stem Cell Biology (NNF17CC0027852 to E.F.).”.

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 - Special issue on HOX genes in development [Articolo su rivista]
Zappavigna, V.

This Special Issue of Journal of Developmental Biology (JDB) covers an indeed very "special" (at least to me) family of highly evolutionarily conserved genes, the Hox genes.[...].

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, Valentina; Ferrari, Silvia; Gorello, Paolo; Fantini, Sebastian; Chiavolelli, Francesca; Mecucci, Cristina; Zappavigna, Vincenzo

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.

2012 - An N-terminal G11A mutation in HOXD13 causes synpolydactyly and interferes with Gli3R function during limb pre-patterning. [Articolo su rivista]
Brison, N; Debeer, P; Fantini, Sebastian; Oley, C; Zappavigna, Vincenzo; Luyten, Fp; Tylzanowski, P.

Synpolydactyly (SPD) is a distal limb anomaly characterized by incomplete digit separation and the presence of supernumerary digits in the syndactylous web. This phenotype has been associated with mutations in the homeodomain or polyalanine tract of the HOXD13 gene. We identified a novel mutation (G11A) in HOXD13 that is located outside the previously known domains and affects the intracellular half life of the protein. Misexpression of HOXD13(G11A) in the developing chick limb phenocopied the human SPD phenotype. Finally, we demonstrated through in vitro studies that this mutation has a destabilizing effect on GLI3R uncovering an unappreciated mechanism by which HOXD13 determines the patterning of the limb.

2012 - Cellule e segnali [Monografia/Trattato scientifico]
Marigo, Valeria; Zappavigna, Vincenzo

È un libro di testo indirizzato agli studenti delle lauree triennali e magistrali in Scienze Biologiche e Biotecnologie per lo studio dei principali meccanismi di comunicazione tra cellule che controllano la proliferazione cellulare, il differenziamento e la morfogenesi. Il testo tratta le principali vie di segnalazione quali: Vie mediate dai recettori accoppiati a proteine G e recettori tirosina chinasi, Famiglia di proteine TGFb, Wnt, Hedgehog, Recettori nucleari per ormoni, Notch, Efrine. Si propone altresì di discutere i meccanismi molecolari attivati da queste vie di segnalazione e la loro importanza nello sviluppo embrionale e nella proliferazione cellulare. Inoltre illustra il coinvolgimento di queste vie di trasduzione del segnale in patologie come il cancro e le malattie ereditarie.

2012 - Emx2 is a dose-dependent negative regulator of Sox2 telencephalic enhancers. [Articolo su rivista]
Mariani, J; Favaro, R; Lancini, C; Vaccari, G; Ferri, Al; Bertolini, J; Tonoli, D; Latorre, E; Caccia, R; Ronchi, A; Ottolenghi, S; Miyagi, S; Okuda, A; Zappavigna, Vincenzo; Nicolis, Sk

The transcription factor Sox2 is essential for neural stem cells (NSC) maintenance in the hippocampus and in vitro. The transcription factor Emx2 is also critical for hippocampal development and NSC self-renewal. Searching for 'modifier' genes affecting the Sox2 deficiency phenotype in mouse, we observed that loss of one Emx2 allele substantially increased the telencephalic β-geo (LacZ) expression of a transgene driven by the 5' or 3' Sox2 enhancer. Reciprocally, Emx2 overexpression in NSC cultures inhibited the activity of the same transgene. In vivo, loss of one Emx2 allele increased Sox2 levels in the medial telencephalic wall, including the hippocampal primordium. In hypomorphic Sox2 mutants, retaining a single 'weak' Sox2 allele, Emx2 deficiency substantially rescued hippocampal radial glia stem cells and neurogenesis, indicating that Emx2 functionally interacts with Sox2 at the stem cell level. Electrophoresis mobility shift assays and transfection indicated that Emx2 represses the activities of both Sox2 enhancers. Emx2 bound to overlapping Emx2/POU-binding sites, preventing binding of the POU transcriptional activator Brn2. Additionally, Emx2 directly interacted with Brn2 without binding to DNA. These data imply that Emx2 may perform part of its functions by negatively modulating Sox2 in specific brain areas, thus controlling important aspects of NSC function in development.

2011 - Control of pelvic girdle development by genes of the Pbx family and Emx2. [Articolo su rivista]
Capellini, Td; Handschuh, K; Quintana, L; Ferretti, E; Di Giacomo, G; Fantini, Sebastian; Vaccari, G; Clarke, Sl; Wenger, Am; Bejerano, G; Sharpe, J; Zappavigna, Vincenzo; Selleri, L.

Genes expressed in the somatopleuric mesoderm, the embryonic domain giving rise to the vertebrate pelvis, appear important for pelvic girdle formation. Among such genes, Pbx family members and Emx2 were found to genetically interact in hindlimb and pectoral girdle formation. Here, we generated compound mutant embryos carrying combinations of mutated alleles for Pbx1, Pbx2, and Pbx3, as well as Pbx1 and Emx2, to examine potential genetic interactions during pelvic development. Indeed, Pbx genes share overlapping functions and Pbx1 and Emx2 genetically interact in pelvic formation. We show that, in compound Pbx1;Pbx2 and Pbx1;Emx2 mutants, pelvic mesenchymal condensation is markedly perturbed, indicative of an upstream control by these homeoproteins. We establish that expression of Tbx15, Prrx1, and Pax1, among other genes involved in the specification and development of select pelvic structures, is altered in our compound mutants. Lastly, we identify potential Pbx1-Emx2-regulated enhancers for Tbx15, Prrx1, and Pax1, using bioinformatics analyses.

2011 - Pbx homeodomain proteins: TALEnted regulators of limb patterning and outgrowth. [Articolo su rivista]
Capellini, Td; Zappavigna, Vincenzo; Selleri, L.

Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5' HoxA/D gene expression.

2010 - Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1 [Articolo su rivista]
T. D., Capellini; G., Vaccari; E., Ferretti; Fantini, Sebastian; M., He; Pellegrini, Massimo; L., Quintana; G., Di Giacomo; J., Sharpe; L., Selleri; Zappavigna, Vincenzo

The genetic pathways underlying shoulder blade development are largely unknown, as gene networks controlling limb morphogenesis have limited influence on scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has suggested their potential roles in girdle development. In this study, by generating compound mutant mice, we examined the genetic control of scapula development by Pbx genes and their functional relationship with Emx2. Analyses of Pbx and Pbx1;Emx2 compound mutants revealed that Pbx genes share overlapping functions in shoulder development and that Pbx1 genetically interacts with Emx2 in this process. Here, we provide a biochemical basis for Pbx1;Emx2 genetic interaction by showing that Pbx1 and Emx2 can bind specific DNA sequences as heterodimers. Moreover, the expression of genes crucial for scapula development is altered in these mutants, indicating that Pbx genes act upstream of essential pathways for scapula formation. In particular, expression of Alx1, an effector of scapula blade patterning, is absent in all compound mutants. We demonstrate that Pbx1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and cooperatively activate its transcription via this potential regulatory element. Our results establish an essential role for Pbx1 in genetic interactions with its family members and with Emx2 and delineate novel regulatory networks in shoulder girdle development.

2009 - A G220V substitution within the N-terminal transcription regulating domain of HOXD13 causes a variant synpolydactyly phenotype. [Articolo su rivista]
Fantini, Sebastian; Vaccari, Giulia; Brison, N; Debeer, P; Tylzanowski, P; Zappavigna, Vincenzo

The 5' members of the HoxD gene cluster (paralogous groups 9-13) are crucial for correct vertebrate limb patterning. Mutations in the HOXD13 gene have been found to cause synpolydactyly (SPD) and other limb malformations in human. We report the identification in a Greek family of a variant form of SPD caused by a novel missense mutation that substitutes glycine for valine in position 220 (G220V) of HOXD13. This mutation represents the first substitution of an amino acid located outside of the HOXD13 homeodomain that causes autopodal limb malformations. We have characterized this mutation at the molecular level and found that the G220V substitution causes a significant impairment of the capacity of HOXD13 to bind DNA and regulate transcription. HOXD13(G220V) was found to be deficient in both activating and repressing transcription through HOXD13-responsive regulatory elements. In accordance with these results, a comparison of the activities of HOXD13 and HOXD13(G220V) in vivo, using retrovirus-mediated misexpression in developing chick limbs, showed that the G220V mutation impairs the capacity of HOXD13 to perturb the development of proximal limb skeletal elements and to ectopically activate the transcription of the Hand2 target gene. We moreover show that the G220V mutation compromises the stability of the HOXD13 protein within cells and causes its partial accumulation in the cytosol in the form of subtle aggregates. Taken together, our results establish that the G220V substitution does not produce a dominant-negative effect or a gain-of-function, but represents a dominant loss-of-function mutation revealing haploinsufficiency of HOXD13 in human.

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 - Developed to cull: How a master control gene of development turned into a regulator of innate immune homeostasis [Articolo su rivista]
Zappavigna, V.

A striking novel role for the Caudal "master control" gene of development in the regulation of innate immune functions in insects has emerged. A recent study now adds further insight into the function of this homeobox gene in the maintenance of the immune homeostasis that is required to preserve the normal commensal community within the Drosophila gut. These results point to a possible more widespread co-option of developmental regulatory genes during evolution to add tissue- and/or organ-specific regulatory plasticity to innate immune systems.

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 cup full of functions [Articolo su rivista]
Piccioni, F.; Zappavigna, Vincenzo; Verrotti, A.

Data from different laboratories have recently indicated that Cup is a multi-functional protein acting both during Drosophila ovary development and early embryogenesis. Cup directly and/or indirectly affects the activity of different mRNAs and proteins to achieve a broad range of biological functions: (1) Cup interacts with Nanos to promote maintenance and survival of the female germ-line stem cells; (2) it binds eIF4E and 3'-UTR-bound factors to repress translation of oskar and nanos mRNAs; (3) it interacts genetically with eIF4E to control translation initiation during ovary development and growth; (4) it may play a role in the control of the phosphorylation status of eIF4E within the developing ovary; finally, (5) it possesses nucleo-cytoplasmic shuttling properties thus pointing to a still uncharacterized function in the cell nucleus. A multi-disciplinary approach, ranging from genetics to proteomics, will be required to shed light on the diverse molecular mechanisms involving Cup and the growing family of its specific interactors.

2005 - Identification of a conserved 125 base-pair Hb9 enhancer that specifies gene expression to spinal motor neurons [Articolo su rivista]
Nakano, T; Windrem, M; Zappavigna, Vincenzo; Goldman, Sa

The homeobox gene Hb9 is expressed selectively by motor neurons (MNs) in the developing CNS. Previous studies have identified a 9-kb 5´ fragment of the mouse Hb9 gene that is sufficient to direct gene expression to spinal MNs in vivo. Here, we sought to identify more discrete MN-specifying elements, using homology searches between genomic sequences of evolutionarily distant species. Based on homology screening of the mouse and human Hb9 promoters, we identified a 3.6-kb Hb9 enhancer that proved sufficient to drive MN-specific lacZ expression. We then compared mouse, human, and pufferfish (Fugu rubripes) genomic sequences, and identified a conserved 438-bp sequence, consisting of noncontiguous 313-bp and 125-bp fragments, residing within the 3.6-kb Hb9 enhancer. The zebrafish (Danio rerio) Hb9 genomic region was then found to have two identical copies of the 125-bp sequence, but no counterpart for the 313-bp sequence. Transgenic analysis showed that the 125-bp alone was both necessary and sufficient to direct spinal MN-specific lacZ expression, whereas the 313-bp sequence had no such enhancer activity. Moreover, the 125-bp Hb9 enhancer was found to harbor two Hox/Pbx consensus-binding sequences, mutations of which completely disrupted thoracolumbar Hb9 expression. These data suggest that Hox/Pbx plays a critical role in the segmental specification of spinal MNs. Together, these results indicate that the molecular pathways regulating Hb9 expression are evolutionarily conserved, and that MN-specific gene expression may be directed and achieved using a small 125-bp 5´ enhancer.

2005 - Translational regulation during oogenesis and early development: the cap-poly(A) tail relationship [Articolo su rivista]
F., Piccioni; Zappavigna, Vincenzo; A. C., Verrotti

Metazoans rely on the regulated translation of select maternal mRNAs to control oocyte maturation and the initial stages of embryogenesis. These transcripts usually remain silent until their translation is temporally and spatially required during early development. Different translational regulatory mechanisms, varying front cytoplasmic polyadenylation to localization of maternal mRNAs, have evolved to assure coordinated initiation of development. A common feature of these mechanisms is that they share a few key trans-acting factors. Increasing evidence suggest that ubiquitous conserved mRNA-binding factors, including the eukaryotic translation initiation factor 4E (eIF4E) and the cytoplasmic polyadenylation element binding protein (CPEB), interact with cell-specific molecules to accomplish the correct level of translational activity necessary for normal development. Here we review how capping and polyadenylation of mRNAs modulate interaction with multiple regulatory factors, thus controlling translation during oogenesis and early development. To cite this article: E Piccioni et al., C. R. Biologies 328 (2005).

2004 - Cup is a nucleocytoplasmic shuttling protein that interacts with the eukaryotic translation initiation factor 4E to modulate Drosophila ovary development [Articolo su rivista]
Zappavigna, Vincenzo; F., Piccioni; Jc, Villaescusa; Ac, Verrotti

In Drosophila, the product of the is (2)cup gene (Cup) is known to be crucial for diverse aspects of female germ-line development. Its functions at the molecular level, however, have remained mainly unexplored. Cup was found to directly associate with eukaryotic translation initiation factor 4E (eIF4E). In this report, we show that Cup is a nucleocytoplasmic shuttling protein and that the interaction with eIF4E promotes retention of the Cup protein in the cytoplasm. Cup is required for the correct accumulation and localization of eIF4E within the posterior cytoplasm of developing oocytes. We furthermore show that cup and eIF4E interact genetically, because a reduction in the level of eIF4E activity deteriorates the development and growth of ovaries bearing homozygous cup mutant alleles. Our results reveal a crucial role for the Cup-eIF4E complex in ovary-specific developmental programs.

2004 - Integration of anteroposterior and dorsoventral regulation of Phox2b transcription in cranial motoneuron progenitors by homeodomain proteins [Articolo su rivista]
Oa, Samad; Mj, Geisen; G., Caronia; I., Variet; Zappavigna, Vincenzo; J., Ericson; C., Goridis; Fm, Rijli

Little is known about the molecular mechanisms that integrate anteroposterior (AP) and dorsoventral (DV) positional information in neural progenitors that specify distinct neuronal types within the vertebrate neural tube. We have previously shown that in ventral rhombomere (r)4 of Hoxb1 and Hoxb2 mutant mouse embryos, Phox2b expression is not properly maintained in the visceral motoneuron progenitor domain (pMNv), resulting in a switch to serotonergic fate. Here, we show that Phox2b is a direct target of Hoxb1 and Hoxb2. We found a highly conserved Phox2b proximal enhancer that mediates rhombomere-restricted expression and contains separate Pbx-Hox (PH) and Prep/Meis (P/M) binding sites. We further show that both the PH and P/M sites are essential for Hox-Pbx-Prep ternary complex formation and regulation of the Phox2b enhancer activity in ventral r4. Moreover, the DV factor Nkx2.2 enhances Hox-mediated transactivation via a derepression. mechanism. Finally, we show that induction of ectopic Phox2b-expressing visceral motoneurons in the chick hindbrain requires the combined activities of Hox and Nkx2 homeodomain proteins. This study takes an important first step to understand how activators and repressors, induced along the AP and DV axes in response to signaling pathways, interact to regulate specific target gene promoters, leading to neuronal fate specification in the appropriate developmental context.

2004 - MAB21L2, a vertebrate member of the Male-abnormal 21 family, modulates BMP signaling and interacts with SMAD1. [Articolo su rivista]
Baldessari, D; Badaloni, A; Longhi, R; Zappavigna, Vincenzo; Consalez, G. G.

Through in vivo loss-of-function studies, vertebrate members of the Male abnormal 21 (mab-21) gene family have been implicated in gastrulation, neural tube formation and eye morphogenesis. Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs. RESULTS: Through a gain-of-function approach, we analyze the interaction of Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4) overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor. CONCLUSIONS: Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances.

2003 - An 147L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function [Articolo su rivista]
G., Caronia; F. R., Goodman; C. M. E., Mckeown; P. J., Scambler; Zappavigna, Vincenzo

The 5' members of the Hoxa and Hoxd gene clusters play major roles in vertebrate limb development. One such gene, HOXD]3, is mutated in the human limb malformation syndrome synpolydactyly. Both polyalanine tract expansions and frameshifting deletions in HOXD13 cause similar forms of this condition, but it remains unclear whether other kinds of HOXD13 mutations could produce different phenotypes. We describe a six-generation family in which a novel combination of brachydactyly and central polydactyly co-segregates with a missense mutation that substitutes leucine for isoleucine at position 47 of the HOXD13 homeodomain. We compared the HOXD13(I47L) mutant protein both in vitro and in vivo to the wild-type protein and to an artificial HOXD13 mutant, HOXD13(IQN), which is completely unable to bind DNA. We found that the mutation causes neither a dominant-negative effect nor a gain of function, but instead impairs DNA binding at some sites bound by wild-type HOXD13. Using retrovirus-mediated misexpression in developing chick limbs, we showed that wild-type HOXD13 could upregulate chick EphA7 in the autopod, but that HOXD13(I47L) could not. In the zeugopod, however, HOXD13(I47L) produced striking changes in tibial morphology and ectopic cartilages, which were never produced by HOXD13(IQN), consistent with a selective rather than generalised loss of function. Thus, a mutant HOX protein that recognises only a subset of sites recognised by the wild-type protein causes a novel human malformation, pointing to a hitherto undescribed mechanism by which missense mutations in transcription factors can generate unexpected phenotypes. Intriguingly, both HOXD13(I47L) and HOXD13(IQN) produced more severe shortening in proximal limb regions than did wildtype HOXD13, suggesting that functional suppression of anterior Hox genes by more posterior ones does not require DNA binding and is mediated by protein:protein interactions.

2003 - Erratum: An I47L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function (Development (2003) vol. 130 (1701-1712)) [Articolo su rivista]
Caronia, G.; Goodman, F. R.; McKeown, C. M. E.; Scambler, P. J.; Zappavigna, V.

2003 - PBX1 nuclear export is regulated independently of PBX-MEINOX interaction by PKA phosphorylation of the PBC-B domain. [Articolo su rivista]
Kilstrup Nielsen, C.; Alessio, M.; Zappavigna, Vincenzo

The regulation of PBC protein function through subcellular distribution is a crucial evolutionarily conserved mechanism for appendage patterning. We investigated the processes controlling PBX1 nuclear export. Here we show that in the absence of MEINOX proteins nuclear export is not a default pathway for PBX1 subcellular localization. In different cell backgrounds, PBX1 can be imported or exported from the nucleus independently of its capacity to interact with MEINOX proteins. The cell context-specific balance between nuclear export and import of PBX1 is controlled by the PBC-B domain, which contains several conserved serine residues corresponding to phosphorylation sites for Ser/Thr kinases. PBX1 subcellular localization correlates with the phosphorylation state of these residues whose dephosphorylation induces nuclear export. Protein kinase A (PKA) specifically phosphorylates PBX1 at these serines, and stimulation of endogenous PKA activity in vivo blocks PBX1 nuclear export in distal limb mesenchymal cells. Our results reveal a novel mechanism for the control of PBX1 nuclear export in addition to the absence of MEINOX protein, which involves the inhibition of PKA-mediated phosphorylation at specific sites within the PBC-B domain.

2002 - Characterization of PREP2, a paralog of PREP1, which defines a novel sub-family of the MEINOX TALE homeodomain transcription factors [Articolo su rivista]
Fognani, C; KILSTRUP NIELSEN, C; Berthelsen, J; Ferretti, E; Zappavigna, Vincenzo; Blasi, F.

TALE (three amino acid loop extension) homeodomain proteins include the PBC and the MEINOX sub-families. MEINOX proteins form heterodimer complexes with PBC proteins. Heterodimerization is crucial to DNA binding and for nuclear localization. PBC-MEINOX heterodimers bind DNA also in combination with HOX proteins, thereby modulating their DNA-binding specificity. TALE proteins therefore play crucial roles in multiple developmental and differentiation pathways in vivo. We report the identification and characterization of a novel human gene homologous to PREP1, called PREP2. Sequence comparisons indicate that PREP1 and PREP2 define a novel sub-family of MEINOX proteins, distinct from the MEIS sub-family. PREP2 is expressed in a variety of human adult tissues and displays a more restricted expression pattern than PREP1. PREP2 is capable of heterodimerizing with PBC proteins. Heterodimerization with PBX1 appears to be essential for nuclear localization of both PREP2 and PBX1. A comparison between the functional properties of PREP1 and PREP2 reveals that PREP2-PBX display a faster DNA-dissociation rate than PREP1-PBX heterodimers, suggesting different roles in controlling gene expression. Like PREP1, PREP2-PBX heterodimers are capable of forming ternary complexes with HOXB1. The analysis of some PREP2 in vitro properties suggests a functional diversification among PREP and between PREP and MEIS MEINOX proteins.

2001 - The recruitment of SOX/OCT complexes and the differential activity of HOXA1 and HOXB1 modulate the Hoxb1 auto-regulatory enhancer function [Articolo su rivista]
G., Di Rocco; A., Gavalas; H., Popperl; R., Krumlauf; Mavilio, Fulvio; Zappavigna, Vincenzo

Regionally restricted expression patterns of Hox genes in developing embryos rely on auto-, cross-, and para-regulatory transcriptional elements. One example is the Hoxb1 auto-regulatory element (b1-ARE), which drives expression of Hoxb1 in the fourth rhombomere of the hindbrain, We previously showed that HOXB1 and PBX1 activate transcription from the b1-ARE: by binding to sequences required for the expression of a reporter gene in rhombomere 4 in vivo, We now report: that in embryonal carcinoma cells, which retain characteristics of primitive neuroectodermal cells, the b1-ARE displays higher basal and HOX/PBX-induced activities than in other cell backgrounds. We have identified a bipartite-binding site for SOX/OCT heterodimers within the b1-ARE that accounts for its cell context-specific activity and is required for maximal transcriptional activity of HOX/PBX complexes in embryonal carcinoma cells. Furthermore, we found that in an embryonal carcinoma cell background, HOXB1 has a significantly higher transcriptional activity than its paralog HOXA1. We map the determinants for this differential activity within the HOXB1 N-terminal transcriptional activation domain. By using analysis in transgenic and HOXA1 mutant mice, we extended these findings on the differential activities of HOXA1 and HOXB1 in vivo, and we demonstrated that they are important for regulating aspects of HOXB1 expression in the hindbrain. We found that mutation of the SOX/OCT site and targeted inactivation of Hoxa1 both impair the response of the b1-ARE to retinoic acid in transgenic mice. Our results show that Hoxa1 is the primary mediator of the response of b1-ARE to retinoic acid in vivo and that this function is dependent on the binding of SOX/OCT heterodimers to the b1-ARE. These results uncover novel functional differences between Hox paralogs and their modulators.

1999 - Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin [Articolo su rivista]
Capdevila, J.; Tsukui, T.; Esteban, C. R.; Zappavigna, V.; Belmonte, J. C. I.

The mechanisms controlling growth and patterning along the proximal-distal axis of the vertebrate limb are yet to be understood. We show that restriction of expression of the homeobox gene Meis2 to proximal regions of the limb bud is essential for limb development, since ectopic Meis2 severely disrupts limb outgrowth. We also uncover an antagonistic relationship between the secreted factors Gremlin and BMPs required to maintain the Shh/FGF loop that regulates distal outgrowth. These proximal and distal factors have coordinated activities: Meis2 can repress distal genes, and Bmps and Hoxd genes restrict Meis2 expression to the proximal limb bud. Moreover, combinations of BMPs and AER factors are sufficient to distalize proximal limb cells. Our results unveil a novel set of proximal-distal regulatory interactions that establish and maintain outgrowth of the vertebrate limb.

1999 - The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH [Articolo su rivista]
Berthelsen, J.; Kilstrup-Nielsen, C.; Blasi, F.; Mavilio, F.; Zappavigna, V.

Nuclear localization of the Extradenticle (EXD) and PBX1 proteins is regionally restricted during Drosophila and mammalian development. We studied the subcellular localization of EXD, PBX, and their partners Homothorax (HTH) and PREP1, in different cell contexts. HTH and PREP1 are cytoplasmic and require association with EXD/PBX for nuclear localization. EXD and PBX1 are nuclear in murine fibroblasts but not in Drosophila Schneider cells, in which they are actively exported to the cytoplasm. Coexpression of EXD/PBX with HTH/PREP1 causes nuclear localization of their heterodimers in both cell contexts. We propose that heterodimerization with HTH/PREP induces nuclear translocation of EXD and PBX1 in specific cell contexts by blocking their nuclear export.

1999 - Two murine and human homologs of mab-21, a cell fate determination gene involved in Caenorhabditis elegans neural development [Articolo su rivista]
Mariani, M.; Baldessari, D.; Francisconi, S.; Viggiano, L.; Rocchi, M.; Zappavigna, V.; Malgaretti, N.; Consalez, G. G.

We report the cloning and genetic characterization of one human and two murine homologs of the mab-21 cell fate specification gene. mab-21 participates in the formation of sensory organs in the male nematode tail, and is essential for other developmental functions elsewhere in the Caenorhabditis elegans embryo. The expanding mab-21 gene family, which is strikingly conserved in evolution, includes two putative Drosophila members. The two mammalian genes, encoding 41 kDa nuclear basic proteins, are expressed in partially overlapping territories in the embryonic brain, eye and limbs, as well as in neural crest derivatives. Recent genetic data implicating mab-21 as a downstream target of TGF-β signaling, together with the distribution of mab-21 transcripts in the mouse embryo, propose these novel genes as relevant factors in various aspects of vertebrate neural development.

1998 - Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-Binding context [Articolo su rivista]
Ma, Vigano'; G., DI ROCCO; Zappavigna, Vincenzo; Mavilio, Fulvio

Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

1998 - Prep1, a novel functional partner of Pbx proteins [Articolo su rivista]
Berthelsen, J.; Zappavigna, V.; Mavilio, F.; Blasi, F.

The human transcription factor, UEF3, is important in regulating the activity of the urokinase plasminogen activator (uPA) gene enhancer. The UEF3 DNA target site is a regulatory element in the promoters of several growth factor and protease genes. We reported previously that purified UEF3 is a complex of several subunits. In this paper we report the cloning of the cDNA of one of the subunits which encodes for a novel human homeodomain protein, which we have termed Prep1. The Prep1 homeodomain belongs to the TALE class of homeodomains, is most closely related to those of the TGIF and Meis1 proteins, and like these, recognizes a TGACAG motif. We further identify the other UEF3 subunit as a member of the Pbx protein family. Unlike other proteins known to interact with Pbx, Prep1 forms a stable complex with Pbx independent of DNA binding. Heterodimerization of Prep1 and Pbx results in a strong DNA binding affinity towards the TGACAG target site of the uPA promoter. Overall, these data indicate that Prep1 is a stable intracellular partner of Pbx in vivo.

1998 - The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity [Articolo su rivista]
Berthelsen, J.; Zappavigna, V.; Ferretti, E.; Mavilio, F.; Blasi, F.

The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity, Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

1997 - A novel homeo-domain protein, prep 1, forms a regulatory complex with pbx proteins in vivo [Articolo su rivista]
Berthelsen, J.; Zappavigna, V.; Mavilio, F.; Blasi, F.

Expression of the human urokinase (uPA) gene is regulated by an enhancer containing a combined PEA3/AP-1 site and an AP-1 site, spaced 72bp apart. These two sites have been shown to synergistically cooperate in both basal level and PMA induced activity of the enhancer, dependent on an element, the co-operation mediator (COM) element, positioned between the two sites, and which bind several different nuclear factors. uPA Enhancer Factor 3 (UEF3) has been identified in various cell lines binding specifically to a site within the COM element and in addition, binds to similar regulating motifs found in various other promoters, showing similar organization and activity as the uPA enhancer. We have purified UEF3 from HeLa cell nuclei by standard chromatography. Purification data show that UEF3 is a complex formed by three polypeptides, p64, p50 and p40. Overall, the data further suggest that UEF3 is a heterodimer consiting of p64 complexed with either p50 orp40. We have cloned the cDNA p64, which encodes for a novel protein, termed Prepl, containing a homeodomain similar to those found in mammalian PBX and Drosophila Extradenticle (exd) homeo domain proteins and in yeast Mat2a protein. We find that Prepl exist as a stable complex with Pbx proteins in vivo and that this complex can be made in vitro reconstituting UEF3 activity. Pbx are proteins known to functionally interact with Hox proteins during development. Both its structure and its way of interacting with Pbx, show that Prepl is different from Hox proteins, and our data point to a novel regulatory role for Pbx/Prepl complexes proteins independent on Hox.

1997 - Functional dissection of a transcriptionally active, target specific Hox/Pbx complex. [Articolo su rivista]
G., DI ROCCO; Mavilio, Fulvio; Zappavigna, Vincenzo

breve descrizione

1996 - HMG1 interacts with HOX proteins and enhances their DNA binding and transcriptional activation [Articolo su rivista]
Zappavigna, V.; Falciola, L.; Citterich, M. H.; Mavilio, F.; Bianchi, M. E.

High mobility group protein 1 (HMG1) is a nonhistone, chromatin-associated nuclear protein with a proposed role in the regulation of eukaryotic gene expression. We show that HMG1 interacts with proteins encoded by the HOX gene family by establishing protein-protein contacts between the HMG box domains and the HOX homeodomain. The functional role of these interactions was studied using the transcriptional activity of the human HOXD9 protein as a model, HMG1 enhances, in a dose-dependent fashion, the sequence-specific DNA binding activity in vitro, and the transcriptional activation in a co-transfection assay in vivo, of the HOXD9 protein. Functional interaction between HMG1 and HOXD9 is dependent on the DNA binding activity of the homeodomain, and requires the HOXD9 transcriptional activation domain, HMG1 enhances activation by HOXD9, but not by HOXD8, of the HOXD9-controlled element. Specific target recognition and functional interaction with HMG1 can be transferred to HOXD8 by homeodomain swapping. We propose that HMG1-like proteins might be general co-factors in HOX-mediated transcriptional activation, which facilitate access of HOX proteins to specific DNA targets, and/or introduce architectural constraints in the assembly of HOX-containing transcriptional complexes.

1994 - Inhibition of proliferation and induction of differentiation of pluripotent human embryonal carcinoma cells by osteogenic protein-1 (or bone morphogenetic protein-7) [Articolo su rivista]
Andrews, P. W.; Damjanov, I.; Berends, J.; Kumpf, S.; Zappavigna, V.; Mavilio, F.; Sampath, K.

BACKGROUND: Osteogenic protein-1 (OP-1) is a member of the transforming growth factor-β super family closely related to the bone morphogenetic proteins and also known as bone morphogenetic protein-7. Other members of this family of growth factors influence cell differentiation as well as cell growth in a number of systems. The Drosophila homolog encoded by the decapentaplegic locus is involved in dorsal-ventral pattern formation during embryogenesis, whereas the expression of several bone morphogenetic proteins including OP-1 is developmentally regulated in mammalian embryos. EXPERIMENTAL DESIGN: The effect of recombinant human OP-1 on the proliferation and differentiation of an established pluripotent human embryonal carcinoma (EC) cell line, NTERA2, and three nullipotent human EC cell lines, 2102Ep, 833KE and TERA-1, was investigated. These cells were grown under reduced serum conditions, and differentiation was monitored by morphology and expression of marker antigens. RESULTS: OP-1 inhibited proliferation of NTERA2 and induced their differentiation, marked by changes in cellular morphology, the loss of EC cell antigens (SSEA3, SSEA4, the liver isozyme of alkaline phosphatase), and the appearance of new antigens, notably SSEA1 and class 1 major histocompatibility complex antigens. These changes were irreversible and did not involve significant cell degeneration or cell death. The OP-1-induced differentiation of NTERA2 appeared distinct from that induced by either retinoic acid or hexamethylene bisacetamide. Nevertheless, OP-1 did induce the homeobox gene, HOXA1. By contrast, OP-1 elicited only a limited and partial response from the nullipotent EC cell lines. CONCLUSIONS: Our results suggest that pluripotent human EC cells differentiate in response to OP-1 and that this factor can modulate the differentiation induced by retinoic acid. Like other members of the transforming growth factor-β super family, OP-1 might play an inductive role in the early embryo. The results also suggest a possible therapeutic value for OP-1 in the treatment of some germ cell tumors.

1994 - Inhibition of retinoic acid-induced activation of 3' human HOXB genes by antisense oligonucleotides affects sequential activation of genes located upstream in the four HOX clusters [Articolo su rivista]
Faiella, A.; Zappavigna, V.; Mavilio, F.; Boncinelli, E.

Most homeobox genes belonging to the Hox family are sequentially activated in embryonal carcinoma cells upon treatment with retinoic acid. Genes located at the 3' end of each one of the four Hox clusters are activated progressively later. This activation has been extensively studied for human HOX genes in the NT2/D1 cell line and shown to take place at the transcriptional level. To understand the molecular mechanisms of sequential HOX gene activation in these cells, we tried to modulate the expression of 3' HOX genes through the use of antisense oligonucleotides added to the culture medium. We chose the HOXB locus. A 5- to 15-fold reduction of the expression of HOXB1 and HOXB3 was sufficient to produce a significant inhibition of the activation of the upstream HOXB genes, as well as of their paralogs in the HOXA, HOXC, and HOXD clusters. Conversely, no effect was detectable on downstream HOX genes. The extent of this inhibition increased for progressively more-5' genes. The stability of the corresponding mRNAs appeared to be unaffected, supporting the idea that the observed effect might be mediated at the transcriptional level. These data suggest a cascade model of progressive activation of Hox genes, with a 3'-to-5' polarity.

1994 - Specificity of HOX protein function depends on DNA-protein and protein- protein interactions, both mediated by the homeo domain [Articolo su rivista]
Zappavigna, V.; Sartori, D.; Mavilio, F.

Transcription of human HOX gene promoters in cultured cells is positively and negatively regulated by HOX proteins interacting with specific target sequences. The human HOXD9 protein activates transcription of the HOXD9 promoter by interacting with the HCR sequence and is antagonized by the HOXD8 protein. HOXD8 is not intrinsically a repressor, since it can activate transcription on different targets. Complete or partial HOXD8/HOXD9 homeo domain swapping indicates that the ability to recognize, and activate transcription from, the HCR target in vivo depends on the amino terminus and helix 1 of the homeo domain. The inhibitory activity of HOXD8 is not affected by deletion of the homeo domain helix 2/3 region, whereas it requires the amino terminus/helix 1 region and an additional, effector domain located at the protein amino-terminal end. This activity is therefore DNA-binding independent, and possibly mediated by protein-protein interactions. Affinity chromatography experiments show that the homeo domain amino terminus/helix 1 region is able to mediate direct interactions between HOX proteins in solution. These data indicate that specificity of HOX protein function in vivo depends on both DNA-protein and protein-protein interactions, mediated by the same sub region of the homeo domain.

1992 - Comparison of mouse and human HOX-4 complexes defines conserved sequences involved in the regulation of Hox-4.4 [Articolo su rivista]
Renucci, A.; Zappavigna, V.; Zakany, J.; Izpisua-Belmonte, J. -C.; Burki, K.; Duboule, D.

We have cloned and sequenced, in both mouse and human, regions of the HOX-4 complex which contain two Abd-B like genes, Hox-4.4 and Hox-4.5 (HOX4C and HOX4D in human, respectively). The high degree of conservation between the homeoprotein sequences extends to non-coding areas, which suggests that the mechanisms of regulation have been conserved. We show that the Hox-4.5/Hox-4.4 intergenic region can be broadly subdivided into three domains based on DNA conservation between rodents and primates. The presence of all these domains in association with sequences located 3' to the transcription termination site are required to mimick the spatial regulation of Hox-4.4 in transgenic mouse embryos. Several highly conserved short sequences located in this region were studied in gel retardation assays for their binding to potential regulatory factors. One such factor is detected in embryonal carcinoma cells but absent from other differentiated cell lines. This specific binding activity is down regulated upon retinoic acid treatment.

1992 - The upstream region of the human homeobox gene HOX3D is a target for regulation by retinoic acid and HOX homeoproteins [Articolo su rivista]
Arcioni, L.; Simeone, A.; Guazzi, S.; Zappavigna, V.; Boncinelli, E.; Mavilio, F.

We studied the structure, regulation and expression of HOX3D, a human homeobox gene located in the HOX3 cluster on chromosome 12. HOX3D is developmentally regulated during embryogenesis and is activated by retinoic acid (RA) in cultured embryonal carcinoma (EC) cells. Transfection of HOX3D upstream genomic sequences linked to a reporter gene allowed the functional definition of its promoter, containing a canonical TATA element. This promoter directs the expression of the reporter gene in EC cells after induction with RA, and binds RA-induced nuclear factor(s) through a conserved palindromic sequence located ~ 100 bp upstream of the transcription start site. The HOX3D promoter is transactivated in both human and murine cells when cotransfected with vectors expressing the protein product of the upstream gene HOX3C and the paralogs of further upstream genes in the HOX4 cluster (i.e. HOX4D, HOX4C and the murine HOX 4.3). The HOX3D protein, and those encoded by the downstream gene HOX3E and its paralog HOX4B are instead inactive. HOX4C and HOX4D proteins synthesized in bacteria bind to the same conserved sequence located around position -120, as well as to the TATA box and immediately upstream and downstream nucleotides. These data provide evidence that cross-regulatory interactions between mammalian homeogenes take place in cultured cells, thus raising the possibility that a regulatory network may exist in vivo. The sequences on the HOX3D promoter involved in cross-regulation are different from those binding nuclear factors induced by RA.

1991 - HOX4 genes encode transcription factors with potential auto- and cross-regulatory capacities [Articolo su rivista]
Zappavigna, V.; Renucci, A.; Izpisua-Belmonte, J. -C.; Urier, G.; Peschle, C.; Duboule, D.

We have looked for the binding of several HOX4 complex homeoproteins in the genomic region containing the HOX4C promoter, between the human HOX4C and HOX4D genes. The HOX4C, HOX4D and Hox-4.3 homeoproteins bind to a phylogenetically highly conserved DNA fragment, which is located in the proximal part of this intergenic region and contains multiple binding sites for these HOX4 proteins. Using cotransfection experiments, we show that this endogenous DNA sequence can mediate transactivation by the HOX4D and HOX4C proteins and that this effect requires the presence of TAAT-related binding sites. The Hox-4.3 protein, in contrast, is unable to activate and can repress the activation observed with the two other proteins. These results show that the HOX4D and HOX4C genes are genuine sequence-specific transcription factors and suggest that, as in Drosophila, cross-regulatory interactions between these genes might be essential for their proper expression.

1990 - Molecular mechanisms underlying the expression of the human HOX-5.1 gene [Articolo su rivista]
Cianetti, L.; Cristofaro, A. D.; Zappavigna, V.; Bottero, L.; Boccoli, G.; Testa, U.; Russo, G.; Boncinelli, E.; Peschle, C.

The complex mechanisms underlying homeobox genes expression involve regulation at transcriptional, posttranscriptional and translational levels. The multiple transcripts of the human HOX-5.1 gene are expressed differentially in tissue- and stage-specific patterns during embryogenesis, and differentially induced by retinoic acid (RA) in human embryonal carcinoma (EC) NT2/D1 cells. We have sequenced 6.3 Kb of the genomic region containing the HOX-5.1 gene and analyzed its mechanisms of expression. Two alternative promoters underlie the transcription of two classes of HOX-5.1-specific mRNAs. These classes differ in tissue and subcellular distribution, induction by RA, structure of the 5'-UT region and mRNA stability: these features are compatible with a differential function of the two classes of transcripts in embryogenesis. © 1990 Oxford University Press.

1990 - Primary structure and embryonic expression pattern of the mouse Hox-4,3 homeobox gene [Articolo su rivista]
Izpisua-Belmonte, J. -C.; Dolle, P.; Renucci, A.; Zappavigna, V.; Falkenstein, H.; Duboule, D.

We report the cloning, genomic localization, primary structure and developmental expression pattern of the novel mouse Hox-4.3 gene. This gene is located within the HOX-4(5) complex, at a position which classifies it as a member of the Hox-3.1 and -2.4 subfamily, the DNA and predicted protein sequences further confirmed this classification. Hox-4.3 has a primary structure characteristic of a Hox gene but, in addition, contains several monotonic stretches of amino acids, one of the 'paired'-like type. As expected from its presence and position within the complex, Hox-4.3 is developmentally expressed in structures of either mesodermal or neurectodermal origin located or derived from below a precise craniocaudal level. However, a very important offset between anteroposterior boundaries within neuroectoderm versus mesoderm derivatives is observed. Like other genes of the HOX-4(5) complex, Hox-4.3 is expressed in developing limbs and gonads, suggesting that 'cluster specificity' could be a feature of the HOX network.

1989 - Differential expression of human HOX-2 genes along the anterior-posterior axis in embryonic central nervous system [Articolo su rivista]
Giampaolo, A.; Acampora, D.; Zappavigna, V.; Pannese, M.; D'Esposito, M.; Care, A.; Feiella, A.; Stornaiuolo, A.; Russo, G.; Simeone, A.; Boncinelli, E.; Peschle, C.

We have investigated the structure of the human HOX-2 locus, which encompasses a 90-kb region on chromosome 17q21. Five new human HOX-2 homeoboxes, termed HOX-2.5, 2.4, 2.6, 2.7 and 2.8, Have been identified, and their nucleotide sequences are reported. They have the same 5′–3′transcriptional korientation and are clustered with three previously described HOX-2 homeoboxes (5′–2.5–2.4–2.3–2.2-2.1–2.6–2.7–2.8-3′). We have also investigated the region-specific expression of HOX-2 genes in human embryonic-fetal life by Northern-blot analysis. All genes are expressed in whole embrvos and fetuses at 5–9 weeks from conception. Their major site of expression lies within the central nervous system (CNS), althought they are transcribed at a lower level in body structures other than the CNS. Their relatively abundant experession in CNS has been analyzed along the anterior-posterior axis by dissecting the brain, the Medulla oblongata and the spinal cord proper. HOX-2.5, 2.4 and 2.3 transcripts are markedly more abundant in spinal cord than in medulla, whereas 2.2, 2.2, 2.6 and 2.7 mRNAs are progressively more abundant in the medull. Additionall, expression in brain was detected, although at lower level, for HOX-2.1, 2.7, 2.8. Thus, the relative position of HOX-2 homeobox genes along the chromosome in the 5′–3′direction appears to correlate with CNS longitudinal axis in the caudal-cephalic direction. © 1989, International Society of Differentiation. All rights reserved.

1987 - Human acute myelogenous and lymphoid leukemias: pattern of expression of cellular oncogenes [Articolo su rivista]
Sposi, N. M.; Mavilio, F.; Petrini, M.; Bottero, L.; Zappavigna, V.; Mastroberardino, G.; De Rossi, G.; Amadori, S.; Mandelli, F.; Peschle, C.

1987 - Two human homeobox genes, c1 and c8: Structure analysis and expression in embryonic development [Articolo su rivista]
Simeone, A.; Mavilio, F.; Acampora, D.; Giampaolo, A.; Faiella, A.; Zappavigna, V.; D'Esposito, M.; Pannese, M.; Russo, G.; Boncinelli, E.

Two human cDNA clones (HHO.c1.95 and HHO.c8.5111) containing a homeobox region have been characterized, and the respective genomic regions have been partially analyzed. Expression of the corresponding genes, termed c1 and c8, was evaluated in different organs and body parts during human embryonic/fetal development. HHO.c1.95 apparently encodes a 217-amino acid protein containing a class I homeodomain that shares 60 out of 61 amino acid residues with the Antennapedia homeodomain of Drosophila melanogaster. HHO.c8.5111 encodes a 153-amino acid protein containing a homeodomain identical to that of the frog AC1 gene. Clones HHO.c1 and HHO.c8 detect by blot-hybridization one and two specific polyadenylylated transcripts, respectively. These are differentially expressed in spinal cord, backbone rudiments, limb buds (or limbs), heart, and skin of human embros and early fetuses in the 5- to 9-week postfertilization period, thus suggesting that the c1 and c8 genes play a key role in a variety of developmental processes. Together, the results of the embryonic/fetal expression of c1 and c8 and those of two previously analyzed genes (c10 and c13) indicate a coherent pattern of expression of these genes in early human ontogeny.

1986 - Differential and stage-related expression in embryonic tissues of a new human homoeobox gene [Articolo su rivista]
Mavilio, F.; Simeone, A.; Giampaolo, A.; Faiella, A.; Zappavigna, V.; Acampora, D.; Poiana, G.; Russo, G.; Peschle, C.; Boncinelli, E.

The homoeobox is a 183 base-pair (bp) DNA sequence1 conserved in several Drosophila genes controlling segmentation and segment identity 2-4. Homoeobox sequences have been detected in the genome of species ranging from insects and anellids to vertebrates5,6 and homoeobox containing genes have been cloned from Xenopus7-9, mouse 10-22 and man23. We recently isolated human homoeobox containing complementary DNA clones, that represent transcripts from four different human genes24. One clone (HHO.c10) is selectively expressed in a 2.1 kilobase (kb) polyadenylated transcript in the spinal cord of human embryos and fetuses 5-10 weeks after fertilization25. We report the characterization of a second cDNA clone, termed HHO.c13, that represents a new homoeobox gene. This clone encodes a protein of 255 amino-acid residues, which includes a pentapeptide, upstream of the homoeo domain, conserved in other Drosophila, Xenopus, murine and human homoeobox genes. By Northern analysis HHO.c13 detects multiple embryonic transcripts, which are differentially expressed in spinal cord, brain, backbone rudiments, limb buds and heart in 5-9-week-old human embryos and fetuses, in a striking organ- and stage-specific pattern. These observations suggest that in early mammalian development homoeobox genes may exert a wide spectrum of control functions in a variety of organs and body parts, in addition to the spinal cord. © 1986 Nature Publishing Group.

1986 - Translocation of c-myc into the immunoglobulin heavy-chain locus in human acute B-cell leukemia. A molecular analysis [Articolo su rivista]
Care, A.; Cianetti, L.; Giampaolo, A.; Sposi, N. M.; Zappavigna, V.; Mavilio, F.; Alimena, G.; Amadori, S.; Mandelli, F.; Peschle, C.

We report the molecular analysis of primary cells from four cases of human B-cell malignancies each with an 8;14 chromosomal translocation involving the c-myc proto-oncogene and the immunoglobulin (Ig) gene cluster. In two cases of B-cell acute lymphocytic leukemia (B-ALL) the c-myc is truncated, rearranged into the Ig C alpha 1 locus and over-expressed in two abnormal mRNAs of approximately 2.0 and 2.8 kb. Conversely, in two cases of B-cell lymphoma progressed into leukemia the c-myc locus was translocated intact in its coding and 5'-flanking region into an Ig region different from C alpha 1, and over-expressed in two normal mRNA species. Cloning and sequencing of the breakpoint region on chromosome 14q+ from one of the two B-ALL cases showed that the myc gene is truncated 1077 nucleotides upstream from the translation start site, and rearranged in the opposite transcriptional orientation into an Ig class-switch segment approximately 4.8 kb upstream from the C alpha 1 gene. The c-myc anti-sense strand contains two class-switch recombination consensus sequences in the immediate boundaries of the breakpoint on chromosome 8: this allows us to postulate that an erroneous, class-switch-like recombination between Ig and myc sequences gave rise to the chromosomal translocation. Furthermore, we report 13 point mutations clustered in a region spanning from the first intron to the second exon of the translocated c-myc gene, five of which cause amino acid changes leading to an abnormal myc protein. This is the first evidence of mutations in a translocated c-myc in primary tumor cells.