Search

Up-regulation of the dystrophin-related gene utrophin represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy (DMD). In order to re-program the utrophin expression level in muscle, we engineered artificial zinc finger transcription factors (ZF-ATFs) that target the utrophin 'A' promoter. We have previously shown that the ZF-ATF "Jazz", either by transgenic manipulation or by systemic adeno-associated viral delivery, induces significant rescue of muscle function in dystrophic "mdx" mice. We present the full characterization of an upgraded version of Jazz gene named "JZif1" designed to minimize any possible host immune response. JZif1 was engineered on the Zif268 gene-backbone using selective amino acid substitutions to address JZif1 to the utrophin 'A' promoter. Here, we show that JZif1 induces remarkable amelioration of the pathological phenotype in mdx mice. To investigate the molecular mechanisms underlying Jazz and JZif1 induced muscle functional rescue, we focused on utrophin related pathways. Coherently with utrophin subcellular localization and role in neuromuscular junction (NMJ) plasticity, we found that our ZF-ATFs positively impact the NMJ. We report on ZF-ATF effects on post-synaptic membranes in myogenic cell line, as well as in wild type and mdx mice. These results candidate our ZF-ATFs as novel therapeutic molecules for DMD treatment., (Copyright © 2018. Published by Elsevier B.V.)

Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD., (© 2014 Wiley Periodicals, Inc.)

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 absence of the cytoskeletal protein dystrophin results in Duchenne muscular dystrophy (DMD). The utrophin protein is the best candidate for dystrophin replacement in DMD patients. To obtain therapeutic levels of utrophin expression in dystrophic muscle, we developed an alternative strategy based on the use of artificial zinc finger transcription factors (ZF ATFs). The ZF ATF 'Jazz' was recently engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment we generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. Here, we show that the artificial Jazz protein restores sarcolemmal integrity and prevents the development of the dystrophic disease in mdx mice. This exclusive animal model establishes the notion that utrophin-based therapy for DMD can be efficiently developed using ZF ATF technology and candidates Jazz as a novel therapeutic molecule for DMD therapy.

Zinc finger (ZF) proteins belonging to the Cys2-His2 class provide a simple and versatile framework to design novel artificial transcription factors (ATFs) targeted to the desired genes. Our work is based on ZF ATFs engineered to up-regulate the expression level of the dystrophin-related gene utrophin in Duchenne muscular dystrophy (DMD). In particular, on the basis of the "recognition code" that defines specific rules between zinc finger primary structure and potential DNA-binding sites we engineered and selected a new family of artificial transcription factors, whose DNA-binding domain consists in a three zinc finger peptide called "Jazz." Jazz protein binds specifically the 9 bp DNA sequence (5(')-GCT-GCT-GCG-3(')) present in the promoter region of both the human and mouse utrophin gene. We generated a transgenic mouse expressing Jazz protein fused to the strong transcriptional activation domain VP16 and under the control of the muscle specific promoter of the myosin light chain gene. Vp16-Jazz mice display a strong up-regulation of the utrophin at both mRNA and protein levels. To our knowledge, this represents the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger-based transcription factor.

Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

Che-1 is a nuclear protein involved in the regulation of gene transcription and cell proliferation. It has also been shown to localize to the cytoplasm of postmitotic neuronal cells, where it is able to interact with the microtubule-associated protein tau. Cyclin-dependent kinase 5 (Cdk5) is a postmitotic proline-directed serine/threonine kinase that hyperphosphorylates tau under pathological conditions. We observed that Che-1 overexpression induces Cdk5 expression both at the mRNA and protein levels. Furthermore, we show that Che-1 directly interacts with Cdk5 protein in vivo. Cdk5/Che-1 complex formation does not compete with Cdk5/p35 interaction, thus Che-1 is able to bind the active kinase complex. Finally, we demonstrated that Che-1 is itself a Cdk5 substrate.

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.

Che-1/AATF (Che-1) was originally characterized as an interacting protein for RNA polymerase II. In addition to transcriptional regulation, the evidence suggests that Che-1 has a viral factor-like S phase promoting role in counteracting Rb repression to facilitate E2F-dependent transactivation during G1-S transition. Recently, Che-1 was found to play an important role in the DNA damage response and cell-cycle checkpoint control. Genetic studies in mice revealed that Che-1 is essential for preimplantation development and the establishment of embryonic gene expression. Importantly, several findings showed that Che-1 participates in inhibiting apoptotic process. Thus, Che-1 emerges as an important adaptor that connects transcriptional regulation, cell-cycle progression, checkpoint control, and apoptosis.

We have previously demonstrated that DNA damage leads to stabilization and accumulation of Che-1, an RNA polymerase II-binding protein that plays an important role in transcriptional activation of p53 and in maintenance of the G(2)/M checkpoint. Here we show that Che-1 is down-regulated during the apoptotic process. We found that the E3 ligase HMD2 physically and functionally interacts with Che-1 and promotes its degradation via the ubiquitin-dependent proteasomal system. Furthermore, we found that in response to apoptotic stimuli Che-1 interacts with the peptidyl-prolyl isomerase Pin1 and that conformational changes generated by Pin1 are required for Che-1/HDM2 interaction. Notably, a Che-1 mutant lacking the capacity to bind Pin1 exhibits an increased half-life and this correlates with a diminished apoptosis in response to genotoxic stress. Our results establish Che-1 as a new Pin1 and HDM2 target and confirm its important role in the cellular response to DNA damage.

Neurotrophin receptor-interacting MAGE homolog (NRAGE) has been recently identified as a cell-death inducer, involved in molecular events driving cells through apoptotic networks during neuronal development. Recently, we have focused on the functional role of Che-1, also known as apoptosis-antagonizing transcription factor (AATF), a protein involved in cell cycle control and gene transcription. Increasing evidence suggests that Che-1 is involved in apoptotic signalling in neural tissues. In cortical neurons Che-1 exhibits an anti-apoptotic activity, protecting cells from neuronal damage induced by amyloid beta-peptide. Here, we report that Che-1 interacts with NRAGE and that an EGFP-NRAGE fusion protein inhibits nuclear localization of Che-1, by sequestering it within the cytoplasmic compartment. Furthermore, NRAGE overexpression downregulates endogenous Che-1 by targeting it for proteasome-dependent degradation. Finally, we propose that Che-1 is a functional antagonist of NRAGE, because its overexpression completely reverts NRAGE-induced cell-death.

Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage.

We recently identified and cloned a novel human gene, Che-1, whose product interacts with both RNA polymerase II and the retinoblastoma gene product (Rb). Furthermore, we found that Che-1 overexpression counteracts the growth inhibitory effects of Rb, regulating in such way both transcription and cell proliferation. In this paper, we describe the genomic organization of the mouse orthologous Che-1 gene and its promoter region. The promoter is TATA less and presents several potential transcription factor-binding motifs. Importantly, we showed that Che-1 expression is regulated by a negative feedback mechanism, in which this protein is present on its own promoter repressing transcription.

RPB3 is a core subunit of RNA polymerase II (pol II) that, together with the RPB11 subunit, forms the heterodimer considered as a functional counterpart of the bacterial alpha subunit homodimer involved in promoter recognition. We previously employed the yeast two-hybrid system and identified an interaction between RPB3 and the myogenic transcription factor myogenin, demonstrating an involvement of this subunit in muscle differentiation. In this paper we report the interaction between RPB3 and another known transcription factor, ATF4. We found that the intensity of the interaction between RPB3 and ATF4 is similar to the one between RPB3 and myogenin. This interaction involves an RPB3 specific region not homologous to the prokaryotic alpha subunit. We demonstrated that RBP3 is able to enhance ATF4 transactivation, whereas the region of RPB3 (Sud) that contacts ATF4, when used as a dominant negative, markedly inhibits ATF4 transactivation activity. Interestingly, ATF4 protein level, as reported for its partner RPB3, increases during C2C7 cell line muscle differentiation.

Memory consolidation requires gene expression regulation by transcription factors, which eventually may induce chromatin modifica-tions as histone acetylation. This mechanism is regulated by histone acetylases and deacetylases. It is not yet clear whether memory consolidation always recruits histone acetylation or it is only engaged in more persistent memories. To address this question, we used different strength of training for novel object recognition task in mice. Only strong training induced a long-lasting memory and an increase in hippocampal histone H3 acetylation. Histone acetylase inhibition in the hippocampus during consolidation impaired mem-ory persistence, whereas histone deacetylase inhibition caused weak memory to persist. Nuclear factor kB (NF-kB) transcription factor inhibition impaired memory persistence and, concomitantly, reduced the general level of H3 acetylation. Accordingly, we found an important increase in H3 acetylation at a specific NF-KB-regulated promoter region of the Camk2d gene, which was reversed by NF-kB inhibition. These results show for the first time that histone acetylation is a specific molecular signature of enduring memories. [ABSTRACT FROM AUTHOR]

Background: Aberrant hypermethylation of CpG islands in housekeeping gene promoters and widespread genome hypomethylation are typical events occurring in cancer cells. The molecular mechanisms behind these cancer-related changes in DNA methylation patterns are not well understood. Two questions are particularly important: (i) how are CpG islands protected from methylation in normal cells, and how is this protection compromised in cancer cells, and (ii) how does the genome-wide demethylation in cancer cells occur. The latter question is especially intriguing since so far no DNA demethylase enzyme has been found. Methodology/Principal Findings: Our data show that the absence of ADP-ribose polymers (PARs), caused by ectopic overexpression of poly(ADP-ribose) glycohydrolase (PARG) in L929 mouse fibroblast cells leads to aberrant methylation of the CpG island in the promoter of the Dnmt1 gene, which in turn shuts down its transcription. The transcriptional silencing of Dnmt1 may be responsible for the widespread passive hypomethylation of genomic DNA which we detect on the example of pericentromeric repeat sequences. Chromatin immunoprecipitation results show that in normal cells the Dnmt1 promoter is occupied by poly(ADP-ribosyl)ated Parp1, suggesting that PARylated Parp1 plays a role in protecting the promoter from methylation. Conclusions/Significance: In conclusion, the genome methylation pattern following PARG over-expression mirrors the pattern characteristic of cancer cells, supporting our idea that the right balance between Parp/Parg activities maintains the DNA methylation patterns in normal cells. The finding that in normal cells Parp1 and ADP-ribose polymers localize on the Dnmt1 promoter raises the possibility that PARylated Parp1 marks those sequences in the genome that must remain unmethylated and protects them from methylation, thus playing a role in the epigenetic regulation of gene expression. [ABSTRACT FROM AUTHOR]

Copyright of Biochemistry & Cell Biology is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Here, we show that the sub cellular localization of α-like RNA polymerase II core subunit 3 (RPB3) is regulated during muscle differentiation. We have recently demonstrated that the expression of RPB3 is regulated during muscle differentiation and that, inside RNA polymerase II (RNAP II), it is directly involved in contacting regulatory proteins such as the myogenic transcription factor Myogenin and activating transcription factor ATF4. We show for the first time, that RPB3, in addition to its presence and role inside the RNAP II core enzyme, accumulates in the cytoplasm of cycling myogenic cells and migrates to the nucleus upon induction of the differentiation program. Furthermore, using human RPB3 as bait in a yeast two-hybrid system, we have isolated a novel RPB3 cytoplasmic interacting protein, HCR. HCR, previously identified as a-helix coiled-coil rod homologue, is one of the psoriasis vulgaris (PV) candidate genes. In cycling myogenic C2C7 cells, we show that the RPB3 protein directly interacts with HCR within the cytoplasm. Finally, knocking down HCR expression by RNA interference, we demonstrate that HCR acts as cytoplasmic docking site for RPB3. [ABSTRACT FROM AUTHOR]

Copyright of Biochemistry & Cell Biology is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)