Publications (50) View all
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Article: UtroUp is a novel six zinc finger artificial transcription factor that recognises 18 base pairs of the utrophin promoter and efficiently drives utrophin upregulation.
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ABSTRACT: BACKGROUND: Duchenne muscular dystrophy (DMD) is the most common X-linked muscle degenerative disease and it is due to the absence of the cytoskeletal protein dystrophin. Currently there is no effective treatment for DMD. Among the different strategies for achieving a functional recovery of the dystrophic muscle, the upregulation of the dystrophin-related gene utrophin is becoming more and more feasible. RESULTS: We have previously shown that the zinc finger-based artificial transcriptional factor "Jazz" corrects the dystrophic pathology in mdx mice by upregulating utrophin gene expression. Here we describe a novel artificial transcription factor, named "UtroUp", engineered to further improve the DNA-binding specificity. UtroUp has been designed to recognise an extended DNA target sequence on both the human and mouse utrophin gene promoters. The UtroUp DNA-binding domain contains six zinc finger motifs in tandem, which is able to recognise an 18-base-pair DNA target sequence that statistically is present only once in the human genome. To achieve a higher transcriptional activation, we coupled the UtroUp DNA-binding domain with the innovative transcriptional activation domain, which was derived from the multivalent adaptor protein Che-1/AATF. We show that the artificial transcription factor UtroUp, due to its six zinc finger tandem motif, possesses a low dissociation constant that is consistent with a strong affinity/specificity toward its DNA-binding site. When expressed in mammalian cell lines, UtroUp promotes utrophin transcription and efficiently accesses active chromatin promoting accumulation of the acetylated form of histone H3 in the utrophin promoter locus. CONCLUSIONS: This novel artificial molecule may represent an improved platform for the development of future applications in DMD treatment.BMC Molecular Biology 01/2013; 14(1):3. · 2.86 Impact Factor -
Article: Hormonal regulation of β myosin heavy chain expression in the mouse left ventricle.
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ABSTRACT: We investigated the influence of sex-hormones on the expression of a- and b-cardiac myosin heavy chain isoforms (a-MHC and b-MHC) in C57bl/6 mice of both sexes, under physiological and pathological conditions. In the left ventricles of fertile female mice, b-MHC expression were 10 fold higher compared to the age-matched males, whereas no differences were found in the a-MHC expression. These differences disappeared after ovariectomy or in sexually immature mice. We also found a sex-related difference in expression of b1-adrenoceptors (b1-AR), since mRNA levels of this gene were 40% lower in fertile females compared to males of the same age, but did not differ in prepuberal or ovariectomized animals. Interestingly, the deletion of both b1- and b2-AR abolished sex difference in the b-MHC expression , since mRNA levels in knockout males were increased and reached values comparable to those of knockout females. Moreover, the b1-AR antagonist metoprolol induced about three-fold increase of b-MHC expression in male mice. The capability of gender to regulate b-MHC expression was also evaluated in the presence of hemodynamic load. Thoracic aortic coarctation (TAC) produced cardiac hypertrophy along with a 12 fold increase of b-MHC and a 50% decrease of b1AR expression in males but not in females, thus abolishing the gender difference observed in sham animals for such genes. In conclusion our results show that the expression of b-MHC and b1-AR in the left ventricles undergo gender-related and correlated changes under both physiological and pathological conditions, and suggest a role of b1-adrenoceptor-mediated signalling.Journal of Endocrinology 11/2012; · 3.55 Impact Factor -
Article: PC4/Tis7/IFRD1 stimulates skeletal muscle regeneration and is involved in myoblast differentiation as a regulator of MyoD and NF-kappaB.
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ABSTRACT: In skeletal muscle cells, the PC4 (Tis7/Ifrd1) protein is known to function as a coactivator of MyoD by promoting the transcriptional activity of myocyte enhancer factor 2C (MEF2C). In this study, we show that up-regulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions as a negative regulator of NF-κB, known to inhibit MyoD expression post-transcriptionally. In fact, up-regulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-κB p65, in concomitance with induction of MyoD expression. On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We also observe that PC4 potentiates the inhibition of NF-κB transcriptional activity mediated by histone deacetylases and that PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis.Journal of Biological Chemistry 02/2011; 286(7):5691-707. · 4.77 Impact Factor -
Article: PC4/Tis7/IFRD1 stimulates skeletal muscle regeneration and is involved in myoblast differentiation as a regulator of MyoD and NF-kB
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ABSTRACT: In skeletal muscle cells the PC4 (Tis7/IFRD1) protein is known to function as a coactivator of MyoD by promoting the transcriptional activity of myocyte enhancer factor 2C (MEF2C). In this study we show that upregulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that PC4-silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and show that such effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions as a negative regulator of NF-kB, known to inhibit MyoD expression post-transcriptionally. In fact, upregulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-kB p65, in concomitance with induction of MyoD expression. On the contrary, PC4-silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We also observe that PC4 potentiates the inhibition of NF-kB transcriptional activity mediated by histone deacetylases (HDACs) and that PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such positively regulates regenerative myogenesis.Journal of Biological Chemistry 12/2010; · 4.77 Impact Factor -
Article: The eEF1γ subunit contacts RNA polymerase II and binds vimentin promoter region.
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ABSTRACT: Here, we show that the eukaryotic translation elongation factor 1 gamma (eEF1γ) physically interacts with the RNA polymerase II (pol II) core subunit 3 (RPB3), both in isolation and in the context of the holo-enzyme. Importantly, eEF1γ has been recently shown to bind Vimentin mRNA. By chromatin immunoprecipitation experiments, we demonstrate, for the first time, that eEF1γ is also physically present on the genomic locus corresponding to the promoter region of human Vimentin gene. The eEF1γ depletion causes the Vimentin protein to be incorrectly compartmentalised and to severely compromise cellular shape and mitochondria localisation. We demonstrate that eEF1γ partially colocalises with the mitochondrial marker Tom20 and that eEF1γ depletion increases mitochondrial superoxide generation as well as the total levels of carbonylated proteins. Finally, we hypothesise that eEF1γ, in addition to its role in translation elongation complex, is involved in regulating Vimentin gene by contacting both pol II and the Vimentin promoter region and then shuttling/nursing the Vimentin mRNA from its gene locus to its appropriate cellular compartment for translation.PLoS ONE 01/2010; 5(12):e14481. · 4.09 Impact Factor
