Sp1 and Sp3 physically interact and co-operate with GABP for the activation of the utrophin promoter (original) (raw)
Related papers
Journal of molecular biology, 2001
The utrophin gene codes for a large cytoskeletal protein closely related to dystrophin which, in the absence of dystrophin, can functionally substitute it. Utrophin is transcribed by two independently regulated promoters about 50 kb apart. The upstream promoter is TATA-less and contains a functional GABP binding site which, in muscle, restricts the promoter activity to post-synaptic nuclei. Transient transfections analysis of mutant promoters in rhabdomyosarcoma cells showed that the upstream promoter contains three functional GC elements that are recognised by Sp1 and Sp3 factors in vitro. Co-transfections of the promoter with Sp1, Sp3 and GABP factors in Drosophila SL2 Schneider cells, which lack of endogenous Sp factors, demonstrated that both Sp1 and Sp3 are positive regulators of the utrophin promoter and that they activate transcription synergistically with GABP. Consistent with this result, we observed physical interaction of both Sp factors with the GABPa subunit in vitro. Functional domain interaction analysis of Sp1 and Sp3 revealed that both factors interact with GABPa through their DNA binding zinc ®nger domain. The modulation and correct interaction between Sp1, Sp3 and GABP in muscle cells may be critical for the regulation of the utrophin promoter, and provide new targets for therapies of Duchenne muscular dystrophy.
Utrophin Up-Regulation by an Artificial Transcription Factor in Transgenic Mice
PLoS ONE, 2007
Duchenne Muscular Dystrophy (DMD) is a severe muscle degenerative disease, due to absence of dystrophin. There is currently no effective treatment for DMD. Our aim is to up-regulate the expression level of the dystrophin related gene utrophin in DMD, complementing in this way the lack of dystrophin functions. To this end we designed and engineered several synthetic zinc finger based transcription factors. In particular, we have previously shown that the artificial three zinc finger protein named Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from the utrophin promoter ''A''. Here we report on the characterization of Vp16-Jazz-transgenic mice that specifically over-express the utrophin gene at the muscular level. A Chromatin Immunoprecipitation assay (ChIP) demonstrated the effective access/binding of the Jazz protein to active chromatin in mouse muscle and Vp16-Jazz was shown to be able to up-regulate endogenous utrophin gene expression by immunohistochemistry, western blot analyses and real-time PCR. To our knowledge, this is the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger based transcription factor. The achievement of Vp16-Jazz transgenic mice validates the strategy of transcriptional targeting of endogenous genes and could represent an exclusive animal model for use in drug discovery and therapeutics. Citation: Mattei E, Corbi N, Di Certo MG, Strimpakos G, Severini C, et al (2007) Utrophin Up-Regulation by an Artificial Transcription Factor in Transgenic Mice. PLoS ONE 2(8): e774.
Molecular Biology of the Cell, 1999
Utrophin/dystrophin-related protein is the autosomal homologue of the chromosome X-encoded dystrophin protein. In adult skeletal muscle, utrophin is highly enriched at the neuromuscular junction. However, the molecular mechanisms underlying regulation of utrophin gene expression are yet to be defined. Here we demonstrate that the growth factor heregulin increases de novo utrophin transcription in muscle cell cultures. Using mutant reporter constructs of the utrophin promoter, we define the N-box region of the promoter as critical for heregulin-mediated activation. Using this region of the utrophin promoter for DNA affinity purification, immunoblots, in vitro kinase assays, electrophoretic mobility shift assays, and in vitro expression in cultured muscle cells, we demonstrate thatets-related GA-binding protein α/β transcription factors are activators of the utrophin promoter. Taken together, these results suggest that the GA-binding protein α/β complex of transcription factors binds ...
Journal of Biological Chemistry, 1998
Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349 -353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997) J. Biol. ). In the present study, we have therefore focused on the effect of agrin on utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the utrophin gene, we transfected muscle cells with a 1.3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24 -48 h. Under these conditions, both muscle-and nerve-derived agrin increased the activity of -galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the utro-
S.Scaramuzza et al.,J.Control.Rel. 2012
We describe the preparation and characterization of a new monoPEGylated derivate of a recombinant form of filgrastim (methionyl human granulocite colony stimulating factor, rh-Met-G-CSF), BK0026, prepared by enzymatic site-specific 20kDa PEG conjugation to glutamine 135 residue by microbial transglutaminase catalyzed reaction. BK0026 was purified to a clinical grade by a single cation exchange chromatography step and characterized by using a panel of physicochemical analyses. NH 2 -terminal sequence and peptide mapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim. The circular dichroism and fluorescence spectroscopy showed the preservation of high order protein structure. The single conjugation site on glutamine 135 was identified by endoproteinase Glu-C peptide mapping combined with mass spectrometry analysis and NH 2 -terminal sequence of the PEGylated peptides. BK0026 purity as well as product-and process-related contaminants was determined by several analytical methods, which showed that BK0026 is stable for more than 2years when stored at 4-8°C. The advantages of enzymatic PEGylation of filgrastim are the absolute specificity of glutamine 135 conjugation combined with high PEGylation yields under very mild reaction conditions. The new site specific monoPEGylated filgrastim is a promising candidate for preclinical and clinical studies aimed at developing a long-lasting treatment of neutropenia in oncological patients under chemotherapy treatments.
BMC Molecular Biology, 2013
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.
The Journal of Gene Medicine, 2005
Background Duchenne muscular dystrophy (DMD) is caused by the absence of the muscle cytoskeletal protein dystrophin. Utrophin is an autosomal homologue of dystrophin, and overexpression of the protein is expected to compensate for the defect of dystrophin. The utrophin gene has two promoters, A and B, and promoter A of the utrophin gene is a possible target of pharmacological interventions for DMD because A-utrophin is up-regulated in dystrophin-deficient mdx skeletal and cardiac muscles. To investigate the utrophin promoter A activity in vivo, we generated nuclear localization signaltagged LacZ transgenic mice, where the LacZ gene was driven by the 5-kb flanking region of the A-utrophin gene. Methods Four transgenic lines were established by mating four independent founders with C57BL/6J mice. The levels of mRNA for β-galactosidase in several tissues were examined by RT-PCR. Cryosections from several tissues were stained with hematoxylin and eosin (H&E) and with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). Results The 5-kb upstream region of the A-utrophin gene showed high transcriptional activity in liver, testis, colon, submandibular gland, and small intestine, consistent with the endogenous expression of utrophin protein. Surprisingly, the levels of both β-gal protein and mRNA for the transgene in cardiac and skeletal muscles were extremely low, even in nuclei near the neuromuscular junctions. These results indicate that the regulation of the utrophin gene in striated muscle is different from that in non-muscle tissues.
Chemistry & Biology, 2011
Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter-and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of magnitude in both the catalytic turnover (k cat ) and ubiquitin (Ub) binding (K M ) between USPs. Further activity modulation by intramolecular domains affects both the k cat and K M , whereas the intermolecular activators UAF1 and GMPS mainly increase the k cat . Also, we provide the first comprehensive analysis comparing Ub chain preference. USPs can hydrolyze all linkages and show modest Ub-chain preferences, although some show a lack of activity toward linear di-Ub. This comprehensive kinetic analysis highlights the variability within the USP family.