PED/PEA-15 induces autophagy and mediates TGF-beta1 effect on muscle cell differentiation.
S. Iovino1,4, F. Oriente1,4, G. Botta1, S. Cabaro1, V. Iovane2, O. Paciello2, D. Viggiano3, G. Perruolo1, P. Formisano1 and F. Beguinot*,1

TGF-beta1 has been shown to induce autophagy in certain cells but whether and how this action is exerted in muscle and whether this activity relates to TGF-beta1 control of muscle cell differentiation remains unknown. Here, we show that expression of the autophagy-promoting protein phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) progressively declines during L6 and C2C12 skeletal muscle cell differentiation. PED/PEA-15 underwent rapid induction upon TGF-beta1 exposure of L6 and C2C12 myoblasts, accompanied by impaired differentiation into mature myotubes. TGF-beta1 also induced autophagy in the L6 and C2C12 cells through a PP2A/FoxO1-mediated mechanism. Both the TGF-beta1 effect on differentiation and that on autophagy were blocked by specific PED/PEA-15 ShRNAs. Myoblasts stably overexpressing PED/PEA-15 did not differentiate and showed markedly enhanced autophagy. In these same cells, the autophagy inhibitor 3-methyladenine rescued TGF-beta1 effect on both autophagy and myogenesis, indicating that PED/PEA-15 mediates TGF-beta1 effects in muscle. Muscles from transgenic mice overexpressing PED/PEA-15 featured a significant number of atrophic fibers, accompanied by increased light chain 3 (LC3)II to LC3I ratio and reduced PP2A/FoxO1 phosphorylation. Interestingly, these mice showed significantly impaired locomotor activity compared with their non-transgenic littermates. TGF-beta1 causes transcriptional upregulation of the autophagy-promoting gene PED/PEA-15, which in turn is capable to induce atrophic responses in skeletal muscle in vivo.

[PMID: 22281705 ] [see .PDF]

1Department of Cellular and Molecular Biology and Pathology & Institute of Sperimental Oncology and Endocrinology, University of Naples Federico II, via Sergio Pansini 5, Naples 80131, Italy;2Department of Pathology and Animal Health, University of Naples Federico II, via Delpino, 1, Naples 80137, Italy and 3Department ofHealth Sciences, University of Molise, Campobasso 86100, Italy;3Department ofHealth Sciences, University of Molise, Campobasso 86100, Italy;*Corresponding author: F Beguinot, Department of Cellular and Molecular Biology and Pathology, ‘Federico II’ University of Naples, Via Pansini 5, 80131 Naples, Italy.44These authors contributed equally to this work.
Homeodomain transcription factor and tumor suppressor Prep1 is required to maintain genomic stability.
Giorgio Iotti1, Elena Longobardi1, Silvia Masella, Leila Dardaei, Francesca De Santis, Nicola Micali, and Francesco Blasi2

Prep1 is a homeodomain transcription factor that is essential in embryonic development and functions in the adult as a tumor suppressor. We show here that Prep1 is involved in maintaining genomic stability and preventing neoplastic transformation. Hypomorphic homozygous Prep1(i/i) fetal liver cells and mouse embryonic fibroblasts (MEFs) exhibit increased basal DNA damage and normal DNA damage response after γ-irradiation compared with WT. Cytogenetic analysis shows the presence of numerous chromosomal aberrations and aneuploidy in very early-passage Prep1(i/i) MEFs. In human fibroblasts, acute Prep1 down-regulation by siRNA induces DNA damage response, like in Prep1(i/i) MEFs, together with an increase in heterochromatin-associated modifications: rapid increase of histone methylation and decreased transcription of satellite DNA. Ectopic expression of Prep1 rescues DNA damage and heterochromatin methylation. Inhibition of Suv39 activity blocks the chromatin but not the DNA damage phenotype. Finally, Prep1 deficiency facilitates cell immortalization, escape from oncogene-induced senescence, and H-Ras(V12)-dependent transformation. Importantly, the latter can be partially rescued by restoration of Prep1 level. The results show that the tumor suppressor role of Prep1 is associated with the maintenance of genomic stability.

[PMID: 21715654 ] [see .PDF]

1IFOM Istituto FIRC (Fondazione Italiana per la Ricerca sul Cancro) di Oncologia Molecolare, 20139 Milan, Italy
Prep1 Controls Insulin Glucoregulatory Function in Liver by Transcriptional Targeting of SHP1 Tyrosine Phosphatase
Francesco Oriente1 Salvatore Iovino1 Serena Cabaro1 Angela Cassese1 Elena Longobardi2,3 Claudia Miele1 Paola Ungaro1 Pietro Formisano1 Francesco Blasi2,3 and Francesco Beguinot1

OBJECTIVE: We investigated the function of the Prep1 gene in insulin-dependent glucose homeostasis in liver. RESEARCH DESIGN AND METHODS: Prep1 action on insulin glucoregulatory function has been analyzed in liver of Prep1-hypomorphic mice (Prep1(i/i)), which express 2-3% of Prep1 mRNA. RESULTS: Based on euglycemic hyperinsulinemic clamp studies and measurement of glycogen content, livers from Prep1(i/i) mice feature increased sensitivity to insulin. Tyrosine phosphorylation of both insulin receptor (IR) and insulin receptor substrate (IRS)1/2 was significantly enhanced in Prep1(i/i) livers accompanied by a specific downregulation of the SYP and SHP1 tyrosine phosphatases. Prep1 overexpression in HepG2 liver cells upregulated SYP and SHP1 and inhibited insulin-induced IR and IRS1/2 phosphorylation and was accompanied by reduced glycogen content. Consistently, overexpression of the Prep1 partner Pbx1, but not of p160MBP, mimicked Prep1 effects on tyrosine phosphorylations, glycogen content, and on SYP and SHP1 expression. In Prep1 overexpressing cells, antisense silencing of SHP1, but not that of SYP, rescued insulin-dependent IR phosphorylation and glycogen accumulation. Both Prep1 and Pbx1 bind SHP1 promoter at a site located between nucleotides -2,113 and -1,778. This fragment features enhancer activity and induces luciferase function by 7-, 6-, and 30-fold, respectively, in response to Prep1, Pbx1, or both. CONCLUSIONS: SHP1, a known silencer of insulin signal, is a transcriptional target of Prep1. In liver, transcriptional activation of SHP1 gene by Prep1 attenuates insulin signal transduction and reduces glucose storage.

[PMID: 20864515 ] [see .PDF]

1Dipartimento di Biologia e Patologia Cellulare e Molecolare and Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Universita` degli Studi di Napoli Federico II, Naples, Italy; 2Istituto FIRC di Oncologia Molecolare (Fondazione Italiana per la Ricerca sul Cancro Institute of Molecular Oncology), Milano, Italy; 3Universita` Vita Salute San Raffaele, Milano, Italy.
Characterization of the regulatory region of the zebrafish Prep1.1 gene: analogies to the promoter of the human PREP1.
Elisa Bernardi1, Gianluca Deflorian1, Federica Pezzinenti1, Victor M. Diaz2, Marina Mione1, Francesco Blasi1,2,*

Prep1 is a developmentally essential TALE class homeodomain transcription factor. In zebrafish and mouse, Prep1 is already ubiquitously expressed at the earliest stages of development, with important tissue-specific peculiarities. The Prep1 gene in mouse is developmentally essential and has haploinsufficient tumor suppressor activity [1]. We have determined the human Prep1 transcription start site (TSS) by primer extension analysis and identified, within 20 bp, the transcription start region (TSR) of the zebrafish Prep1.1 promoter. The functions of the zebrafish 5' upstream sequences were analyzed both by transient transfections in Hela Cells and by injection in zebrafish embryos. This analysis revealed a complex promoter with regulatory sequences extending up to -1.8, possibly -5.0 Kb, responsible for tissue specific expression. Moreover, the first intron contains a conserved tissue-specific enhancer both in zebrafish and in human cells. Finally, a two nucleotides mutation of an EGR-1 site, conserved in all species including human and zebrafish and located at a short distance from the TSS, destroyed the promoter activity of the -5.0 Kb promoter. A transgenic fish expressing GFP under the -1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development. In the adult fish, GFP was expressed in hematopoietic regions like the kidney, in agreement with the essential function of Prep1 in mouse hematopoiesis. Sequence comparison showed conservation from man to fish of the sequences around the TSS, within the first intron enhancer. Moreover, about 40% of the sequences spread throughout the 5 Kbof the zebrafish promoter are concentrated in the -3 to -5 Kb of the human upstream region.

[PMID: 21203543 ] [see .PDF]

1IFOM-FIRC Institute of Molecular Oncology Foundation, Milan, Italy,2Universita` Vita Salute San Raffaele, Milan, Italy.
The absence of Prep1 causes p53-dependent apoptosis of mouse pluripotent epiblast cells.
Luis C. Fernandez-Diaz1, Audrey Laurent1, Sara Girasoli1, Margherita Turco2, Elena Longobardi1, Giorgio Iotti1, Nancy A. Jenkins3, Maria Teresa Fiorenza4, Neal G. Copeland3 and Francesco Blasi1,5,*

Disruption of mouse Prep1, which codes for a homeodomain transcription factor, leads to embryonic lethality during post-implantation stages. Prep1(-/-) embryos stop developing after implantation and before anterior visceral endoderm (AVE) formation. In Prep1(-/-) embryos at E6.5 (onset of gastrulation), the AVE is absent and the proliferating extra-embryonic ectoderm and epiblast, marked by Bmp4 and Oct4, respectively, are reduced in size. At E.7.5, Prep1(-/-) embryos are small and very delayed, showing no evidence of primitive streak or of differentiated embryonic lineages. Bmp4 is expressed residually, while the reduced number of Oct4-positive cells is constant up to E8.5. At E6.5, Prep1(-/-) embryos retain a normal mitotic index but show a major increase in cleaved caspase 3 and TUNEL staining, indicating apoptosis. Therefore, the mouse embryo requires Prep1 when undergoing maximal expansion in cell number. Indeed, the phenotype is partially rescued in a p53(-/-), but not in a p16(-/-), background. Apoptosis is probably due to DNA damage as Atm downregulation exacerbates the phenotype. Despite this early lethal phenotype, Prep1 is not essential for ES cell establishment. A differential embryonic expression pattern underscores the unique function of Prep1 within the Meis-Prep family.

[PMID: 20826531 ] [see .PDF]

1IFOM, FIRC Institute of Molecular Oncology Foundation,2Department ofExperimental Oncology, European Institute of Oncology (IEO),IFOM-IEO Campus,via Adamello 16, 20130 Milan, Italy;3Institute of Molecular and Cell biology, 61 Biopolis Drive, Proteos, 138673, Singapore;4Pasteur Institute, Cenci Bolognetti Foundation and Department of Psychology, Section of Neuroscience, University ‘Sapienza’ of Rome, 00185 Rome, Italy;5Università Vita Salute San Raffaele andIstituto Scientifico San Raffaele, via Olgettina 60, 20132 Milan, Italy.*Author for correspondence.
Down syndrome fibroblasts and mouse Prep1-overexpressing cells display increased sensitivity to genotoxic stress.
Nicola Micali1, Elena Longobardi2, Giorgio Iotti2, Carmelo Ferrai3, Laura Castagnaro3, Mario Ricciardi3, Francesco Blasi2,3,4 and Massimo P. Crippa1,*

PREP1 (PKNOX1) maps in the Down syndrome (DS) critical region of chromosome 21, is overexpressed in some DS tissues and might be involved in the DS phenotype. By using fibroblasts from DS patients and by overexpressing Prep1 in F9 teratocarcinoma and Prep1(i/i) MEF to single out the role of the protein, we report that excess Prep1 increases the sensitivity of cells to genotoxic stress and the extent of the apoptosis directly correlates with the level of Prep1. The apoptotic response of Prep1-overexpressing cells is mediated by the pro-apoptotic p53 protein that we show is a direct target of Prep1, as its depletion reverts the apoptotic phenotype. The induction of p53 overcomes the anti-apoptotic role of Bcl-X(L), previously shown to be also a Prep1 target, the levels of which are increased in Prep1-overexpressing cells as well. Our results provide a rationale for the involvement of PREP1 in the apoptotic phenotype of DS tissues and indicate that differences in Prep1 level can have drastic effects.

[PMID: 20110257 ] [see .PDF]

1Laboratory of Molecular Dynamics of the Nucleus, Division of Genetics and Cell Biology, S. Raffaele Scientific Institute, via Olgettina 60, Milan 20132, 2IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), via Adamello 16, Milan 20139,3Laboratory of Molecular Genetics, Division of Genetics and Cell Biology, S. Raffaele Scientific Institute, via Olgettina 60, Milan 20132 and 4 Universita` Vita e Salute S. Raffaele, via Olgettina 58, Milan 20132, Italy.
Prep1 (pKnox1)-deficiency leads to spontaneous tumor development in mice and accelerates EmuMyc lymphomagenesis: a tumor suppressor role for Prep1.
E. Longobardia,b,1, G. Iottib,1, P. Di Rosaa, S. Mejettab, F. Bianchib, L.C. Fernandez-Diazb, N. Micalib, P. Nuciforob, E. Lentia, M. Ponzonic, C. Doglionic, M. Caniatti d, P.P. Di Fioreb,e,f, F. Blasia,b,*

The Prep1 homeodomain transcription factor is essential for embryonic development. 25% of hypomorphic Prep1(i/i) embryos, expressing the gene at 2% of the normal levels, survive pregnancy and live a normal-length life. Later in life, however, these mice develop spontaneous pre-tumoral lesions or solid tumors (lymphomas and carcinomas). In addition, transplantation of E14.5 fetal liver (FL) Prep1(i/i) cells into lethally irradiated mice induces lymphomas. In agreement with the above data, haploinsufficiency of a different Prep1-deficient (null) allele accelerates EmuMyc lymphoma growth. Therefore Prep1 has a tumor suppressor function in mice. Immunohistochemistry on tissue micrroarrays (TMA) generated from three distinct human cohorts comprising a total of some 1000 human tumors revealed that 70% of the tumors express no or extremely low levels of Prep1, unlike normal tissues. Our data in mice are thus potentially relevant to human cancer.

[PMID: 20106730 ] [see .PDF]

a Universita` Vita Salute San Raffaele, via Olgettina 60, Milano 20132, Italy;bIFOM (Fondazione Istituto FIRC di Oncologia Molecolare), IFOM-IEO-Campus, via Adamello 16, 20139 Milano, Italyc Servizio di Anatomia Patologica, Istituto Scientifico S. Raffaele, via Olgettina 60, 20132 Milano, Italy;d Dipartimento di Patologia, Igiene e Sanita` Pubblica Veterinaria, Sezione di Anatomia Patologica Veterinaria e Patologia Aviaria, Universita`degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy; e Department of Experimental Oncology, Istituto Europeo di Oncologia, IFOM-IEO-Campus, via Adamello 16, 20139 Milan, Italy; f Dipartimento di Medicina, Chirurgia ed Odontoiatria, Universita` degli Studi di Milano, Via di Rudinı` 8, 20122 Milan, Italy.
Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1.
Hesse D, Hommel A, Jaschke A, Moser M, Bernhardt U, Zahn C, Kluge R, Wittschen P, Gruber AD, Al-Hasani H, Joost HG, Schürmann A.1

The GTPase ADP-ribosylation factor related protein 1 (ARFRP1) controls the recruitment of proteins such as golgin-245 to the trans-Golgi. ARFRP1 is highly expressed in adipose tissues in which the insulin-sensitive glucose transporter GLUT4 is processed through the Golgi to a specialized endosomal compartment, the insulin-responsive storage compartment from which it is translocated to the plasma membrane in response to a stimulation of cells by insulin. In order to examine the role of ARFRP1 for GLUT4 targeting, subcellular distribution of GLUT4 was investigated in adipose tissue specific Arfrp1 knockout (Arfrp1(ad)(-/-)) mice. Immunohistochemical and ultrastructural studies of brown adipocytes demonstrated an abnormal trans-Golgi in Arfrp1(ad)(-/-) adipocytes. In addition, in Arfrp1(ad)(-/-) adipocytes GLUT4 protein accumulated at the plasma membrane rather than being sequestered in an intracellular compartment. A similar missorting of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which was associated with significantly elevated uptake of deoxyglucose under basal conditions. Thus, Arfrp1 appears to be involved in sorting of GLUT4.

[PMID: 20230794] [see .PDF]

1Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany.
The ARF-like GTPase ARFRP1 is essential for lipid droplet growth and is involved in the regulation of lipolysis.
Hommel A, Hesse D, Völker W, Jaschke A, Moser M, Engel T, Blüher M, Zahn C, Chadt A, Ruschke K, Vogel H, Kluge R, Robenek H, Joost HG, Schürmann A.1

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a GTPase regulating protein trafficking between intracellular organelles. Here we show that mice lacking Arfrp1 in adipocytes (Arfrp1(ad-/-)) are lipodystrophic due to a defective lipid droplet formation in adipose cells. Ratios of mono-, di-, and triacylglycerol, as well as the fatty acid composition of triglycerides, were unaltered. Lipid droplets of brown adipocytes of Arfrp1(ad-/-) mice were considerably smaller and exhibited ultrastructural alterations, such as a disturbed interaction of small lipid-loaded particles with the larger droplets, suggesting that ARFRP1 mediates the transfer of newly formed small lipid particles to the large storage droplets. SNAP23 (synaptosomal-associated protein of 23 kDa) associated with small lipid droplets of control adipocytes but was located predominantly in the cytosol of Arfrp1(ad-/-) adipocytes, suggesting that lipid droplet growth is defective in Arfrp1(ad-/-) mice. In addition, levels of phosphorylated hormone-sensitive lipase (HSL) were elevated, and association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue from Arfrp1(ad-/-) mice. Accordingly, basal lipolysis was increased after knockdown of Arfrp1 in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of lipolysis.

[PMID: 20038528] [see .PDF]

1Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany.
Prep1 directly regulates the intrinsic apoptotic pathway by controlling Bcl-XL levels.
Nicola Micali1, Carmelo Ferrai1, Luis C. Fernandez-Diaz2,Francesco Blasi1,2,3,* and Massimo P. Crippa1,*

The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1(i/i)) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1(i/i) mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1(i/i) MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-X(L) mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1(i/i) MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression.

[PMID: 19103748 ] [see .PDF]

1Laboratory of Molecular Genetics, S. Raffaele Scientific Institute, Milan, Italy;2 IFOM (FIRC Institute of Molecular Oncology), Milan, Italy; 3School of Medicine, Universita` Vita-Salute S. Raffaele, Milan, Italy.
p66Shc-generated Oxidative Signal Promotes Fat Accumulation.
Ina Berniakovich1,2, Mirella Trinei3, Massimo Stendardo1,2, Enrica Migliaccio1,2, Saverio Minucci1,3, Paolo Bernardi4, Pier Giuseppe Pelicci1,2,**, and Marco Giorgio1,2

Reactive oxygen species (ROS) and insulin signaling in the adipose tissue are critical determinants of aging and age-associated diseases. It is not clear, however, if they represent independent factors or they are mechanistically linked. We investigated the effects of ROS on insulin signaling using as model system the p66(Shc)-null mice. p66(Shc) is a redox enzyme that generates mitochondrial ROS and promotes aging in mammals. We report that insulin activates the redox enzyme activity of p66(Shc) specifically in adipocytes and that p66(Shc)-generated ROS regulate insulin signaling through multiple mechanisms, including AKT phosphorylation, Foxo localization, and regulation of selected insulin target genes. Deletion of p66(Shc) resulted in increased mitochondrial uncoupling and reduced triglyceride accumulation in adipocytes and in vivo increased metabolic rate and decreased fat mass and resistance to diet-induced obesity. In addition, p66(Shc-/-) mice showed impaired thermo-insulation. These findings demonstrate that p66(Shc)-generated ROS regulate the effect of insulin on the energetic metabolism in mice and suggest that intracellular oxidative stress might accelerate aging by favoring fat deposition and fat-related disorders.

[PMID: 18838380] [see .PDF]

From the 1European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy, 2Firc Institute for Molecular Oncology and 3Genextra SpA, Via Adamello 16, 20139 Milan, Italy, 4Dipartimento di Scienze Biochimiche, University of Padova, 35128 Padova, Italy, and **Dipartimento di Medicina, Chirurgia e Odontoiatria, University of Milano, 20142 Milan, Italy
Prep1 deficiency induces protection from diabetes and increased insulin sensitivity through a p160-mediated mechanism.
Francesco Oriente1, Luis Cesar Fernandez Diaz3, Claudia Miele1, Salvatore Iovino1, Silvia Mori3, Victor Manuel Diaz4, Giancarlo Troncone2, Angela Cassese1, Pietro Formisano1, Francesco Blasi3,4, and Francesco Beguinot1,*

We have examined glucose homeostasis in mice hypomorphic for the homeotic transcription factor gene Prep1. Prep1-hypomorphic (Prep1(i/i)) mice exhibit an absolute reduction in circulating insulin levels but normal glucose tolerance. In addition, these mice exhibit protection from streptozotocin-induced diabetes and enhanced insulin sensitivity with improved glucose uptake and insulin-dependent glucose disposal by skeletal muscle. This muscle phenotype does not depend on reduced expression of the known Prep1 transcription partner, Pbx1. Instead, in Prep1(i/i) muscle, we find normal Pbx1 but reduced levels of the recently identified novel Prep1 interactor p160. Consistent with this reduction, we find a muscle-selective increase in mRNA and protein levels of PGC-1alpha, accompanied by enhanced expression of the GLUT4 transporter, responsible for insulin-stimulated glucose uptake in muscle. Indeed, using L6 skeletal muscle cells, we induced the opposite effects by overexpressing Prep1 or p160, but not Pbx1. In vivo skeletal muscle delivery of p160 cDNA in Prep1(i/i) mice also reverses the molecular phenotype. Finally, we show that Prep1 controls the stability of the p160 protein. We conclude that Prep1 controls insulin sensitivity through the p160-GLUT4 pathway.

[PMID: 18644868 ] [see .PDF]

1Dipartimento di Biologia e Patologia Cellulare e Molecolare & Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Universita` degli Studi di Napoli Federico II, Naples, Italy;2Dipartimento di Scienze Biomorfologiche e Funzionali, Universita` degli Studi di Napoli Federico II, Naples, Italy;1IFOM (FIRC Institute of Molecular Oncology), via Adamello 16, 20134 Milan, Italy;4Universita` Vita Salute San Raffaele, via Olgettina 60, 20132 Milan, Italy.
p160 Myb-binding protein interacts with Prep1 and inhibits its transcriptional activity.
Victor M. Diaz, Silvia Mori1, Elena Longobardi1,4, Guillermo Menendez,1‡ Carmelo Ferrai 1 Rebecca A. Keough2 Angela Bachi3 and Francesco Blasi1,4,*

Prep1 is known to interact in vivo with Pbx1 to regulate development and organogenesis. We have identified a novel Prep1-interacting protein, p160 c-Myb binding protein (p160). p160 and Pbx1 compete for Prep1 in vitro, and p160 inhibits Prep1-dependent HoxB2 expression in retinoic acid-treated NT2-D1 cells. The N-terminal physiologically truncated form of p160, p67, binds the sequence 63LFPLL67 in the HR1 domain of Prep1. Mutation of both L63 and L66 impairs the binding of Prep1 to both p160/p67 and Pbx1. The sequences required to bind Prep1 are mainly located in residues 51 to 151. Immunofluorescence colocalization and coimmunoprecipitation of endogenous p160 and Prep1 are induced by ActD, which translocates p160 from the nucleolus to the nucleoplasm. These data therefore show that p160 is a novel regulator of Prep1-Pbx1 transcriptional activity.

[PMID: 17875935 ] [see .PDF]

1Molecular Genetics Unit, Department of Molecular Biology and Functional Genomics, Universita` Vita Salute San Raffaele and DIBIT, H San Raffaele, via Olgettina 58, 20132 Milan, Italy; 2School of Molecular and Biomedical Sciences (Biochemistry), University of Adelaide, Adelaide, SA 5005, Australia; 3Mass Spectrometry Unit, San Raffaele Scientific Institute, via Olgettina 60, 20132 Milan, Italy; 4IFOM (FIRC Institute of Molecular Oncology), via Adamello 16, 20139 Milan, Italy.