UtroUp is a novel six zinc finger artificial transcription factor that recognises 18 base pairs of the utrophin promoter and efficiently drives utrophin upregulation

Onori, Annalisa; Pisani, Cinzia; Strimpakos, Georgios; Monaco, Lucía; Mattei, Elisabetta; Passananti, Claudio; Corbi, Nicoletta

doi:10.1186/1471-2199-14-3

Cited by 16 publications

(12 citation statements)

References 38 publications

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“…AZF-based DNA-binding platforms are currently being developed for therapeutic purposes. Throughout the short history of the development of the artificial DNA-binding platform, the field has moved from the early use of three ZF proteins based on the natural ZF protein ZNF268 ( 15 , 22 ) to adopting six ZF proteins that were expected to be more specific in targeting sites within the whole human genome ( 37 , 38 ).…”

Section: Discussionmentioning

confidence: 99%

Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain

et al. 2015

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Transcription factors are often regarded as having two separable components: a DNA-binding domain (DBD) and a functional domain (FD), with the DBD thought to determine target gene recognition. While this holds true for DNA binding in vitro, it appears that in vivo FDs can also influence genomic targeting. We fused the FD from the well-characterized transcription factor Krüppel-like Factor 3 (KLF3) to an artificial zinc finger (AZF) protein originally designed to target the Vascular Endothelial Growth Factor-A (VEGF-A) gene promoter. We compared genome-wide occupancy of the KLF3FD-AZF fusion to that observed with AZF. AZF bound to the VEGF-A promoter as predicted, but was also found to occupy approximately 25 000 other sites, a large number of which contained the expected AZF recognition sequence, GCTGGGGGC. Interestingly, addition of the KLF3 FD re-distributes the fusion protein to new sites, with total DNA occupancy detected at around 50 000 sites. A portion of these sites correspond to known KLF3-bound regions, while others contained sequences similar but not identical to the expected AZF recognition sequence. These results show that FDs can influence and may be useful in directing AZF DNA-binding proteins to specific targets and provide insights into how natural transcription factors operate.

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Section: Discussionmentioning

confidence: 99%

Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain

et al. 2015

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“…A chromatin immunoprecipitation assay was performed as previously described [ 41 ]. Equal amounts of chromatin from each sample were immunoprecipitated overnight with anti-eEF1Bγ rabbit polyclonal antibodies.…”

Section: Methodsmentioning

confidence: 99%

eEF1Bγ binds the Che-1 and TP53 gene promoters and their transcripts

Pisani

Onori

Gabanella

et al. 2016

J Exp Clin Cancer Res

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BackgroundWe have previously shown that the eukaryotic elongation factor subunit 1B gamma (eEF1Bγ) interacts with the RNA polymerase II (pol II) alpha-like subunit “C” (POLR2C), alone or complexed, in the pol II enzyme. Moreover, we demonstrated that eEF1Bγ binds the promoter region and the 3’ UTR mRNA of the vimentin gene. These events contribute to localize the vimentin transcript and consequentially its translation, promoting a proper mitochondrial network.MethodsWith the intent of identifying additional transcripts that complex with the eEF1Bγ protein, we performed a series of ribonucleoprotein immunoprecipitation (RIP) assays using a mitochondria-enriched heavy membrane (HM) fraction.ResultsAmong the eEF1Bγ complexed transcripts, we found the mRNA encoding the Che-1/AATF multifunctional protein. As reported by other research groups, we found the tumor suppressor p53 transcript complexed with the eEF1Bγ protein. Here, we show for the first time that eEF1Bγ binds not only Che-1 and p53 transcripts but also their promoters. Remarkably, we demonstrate that both the Che-1 transcript and its translated product localize also to the mitochondria and that eEF1Bγ depletion strongly perturbs the mitochondrial network and the correct localization of Che-1. In a doxorubicin (Dox)-induced DNA damage assay we show that eEF1Bγ depletion significantly decreases p53 protein accumulation and slightly impacts on Che-1 accumulation. Importantly, Che-1 and p53 proteins are components of the DNA damage response machinery that maintains genome integrity and prevents tumorigenesis.ConclusionsOur data support the notion that eEF1Bγ, besides its canonical role in translation, is an RNA-binding protein and a key player in cellular stress responses. We suggest for eEF1Bγ a role as primordial transcription/translation factor that links fundamental steps from transcription control to local translation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0424-x) contains supplementary material, which is available to authorized users.

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“…DMD myoblasts repair by NHEJ (resulting in micro-insertions or microdeletions, INDELs) elicited by meganucleases and ZFN targeting either introns or exons in the dystrophin gene was also reported [34,35]. ZFNs were also designed to target the utrophin promoter region, which has resulted in increased promoter activity, utrophin up-regulation and strong amelioration of the dystrophic phenotype in mdx mice [36]. Although CRISPR systems have shown promise in genome engineering with easily designed target recognition, they exhibit high off-target cleavage when used with single-guide RNAs [37,38].…”

Section: Gene Editingmentioning

confidence: 96%