p53 Gene Repair with Zinc Finger Nucleases Optimised by Yeast 1-Hybrid and Validated by Solexa Sequencing

The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation ‘hotspots’. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci

Control of neurone-specific gene expression: Transcriptional regulation of the M1 muscarinic acetylcholine receptor gene

The establishment of differentiated neuronal phenotype remains an outstanding problem of molecular neurobiology. One of the clearest manifestations of this molecular diversity is provided by the G-protein coupled receptor (GPCR) family. Each of the more than 1,000 members of this gene family has a unique expression profile and thereby offer an ideal model to examine the transcriptional mechanisms that underwrite this molecular diversity. Muscarinic acetylcholine receptors (M1- M5) make up one of the subfamilies of GPCR genes. The M1 gene is the most abundant of the muscarinic receptor genes and is mainly expressed in telencephalic regions and autonomic ganglia. I have now investigated the regions of this gene that are capable of driving expression of a reporter gene in an M1 specific manner. One of these regions is a polypyrimidine/polypurine (PPY/PPU) sequence capable of forming single stranded DNA, and the other (found downstream of the PPY/PPU tract) is conserved across species, has no recognisable motifs and is not able to form single stranded DNA by itself although it shows sensitivity to specific single stranded nucleases when next to the PPY/PPU tract. Both the PPY/PPU tract and the conserved region are bound by nucleolin, a multifunctional phosphoprotein, and act as cis-enhancing elements. A second region important for expression of the M1 gene has been identified to be bound by SHARP-1, a basic helix loop helix protein of unknown function expressed in the adult nervous system. Gal4 fusion experiments have shown that SHARP-1 functions as a repressor of both basal and activated transcription driven either by a TATA-containing or a TATA-less promoter in a position independent manner. Furthermore, SHARP-1 contains two independent repression domains, one at the C- terminus, which acts by a mechanism sensitive to the histone deacetylase inhibitor Trichostatin A (TSA), and the other at the bHLH domain, whichworks through a TSA insensitive mechanism. Co-transfection assays showed that SHARP-1 downregulates expression of the M1 gene in M1 expressing cells. Data presented here shows that the trancriptional mechanisms that control expression of the M1 gene are different to those that control expression of the other members of the same family. These results provideinsight into the molecules and mechanisms employed in the stablishment of aspects of differentiated neuronal phenotype

Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice

BACKGROUND: Synthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene therapy. We recently developed a ZF transcription repressor (ZF-KOX1) able to bind to expanded DNA CAG-repeats in the huntingtin (HTT) gene, which are found in Huntington's disease (HD). This ZF acutely repressed mutant HTT expression in a mouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In the present work, we sought to develop a long-term single-injection gene therapy approach in the brain. METHOD: Since non-self proteins can elicit immune and inflammatory responses, we designed a host-matched analogue of ZF-KOX1 (called mZF-KRAB), to treat mice more safely in combination with rAAV vector delivery. We also tested a neuron-specific enolase promoter (pNSE), which has been reported as enabling long-term transgene expression, to see whether HTT repression could be observed for up to 6 months after AAV injection in the brain. RESULTS: After rAAV vector delivery, we found that non-self proteins induce significant inflammatory responses in the brain, in agreement with previous studies. Specifically, microglial cells were activated at 4 and 6 weeks after treatment with non-host-matched ZF-KOX1 or GFP, respectively, and this was accompanied by a moderate neuronal loss. In contrast, the host-matched mZF-KRAB did not provoke these effects. Nonetheless, we found that using a pCAG promoter (CMV early enhancer element and the chicken β-actin promoter) led to a strong reduction in ZF expression by 6 weeks after injection. We therefore tested a new non-viral promoter to see whether the host-adapted ZF expression could be sustained for a longer time. Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat) resulted in up to 77 % repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 10(10) virions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that longer term effects are possible. CONCLUSION: Host-adapted ZF-AAV constructs displayed a reduced toxicity and a non-viral pNSE promoter improved long-term ZF protein expression and target gene repression. The optimized constructs presented here have potential for treating HD.Authors were funded by the European Research Council grants: FP7 ERC 201249 ZINC-HUBS and H2020 - ERC-2014-PoC 641232 - Fingers4Cure

Neuronal expression of the rat M1 muscarinic acetylcholine receptor gene is regulated by elements in the first exon

Muscarinic acetylcholine receptor genes are members of the G-protein coupled receptor superfamily. Each member of this family studied to date appears to have a distinct expression profile, however the mechanisms determining these expression patterns remain largely unknown. We have previously isolated a genomic clone containing the M1 muscarinic receptor gene and determined its gene structure [Pepitoni, Wood and Buckley (1997) J. Biol. Chem. 272, 17112-17117]. We have now identified DNA elements responsible for driving cell specific expression in transient transfection assays of immortalized cell lines. A region of the gene spanning 974 nucleotides and containing 602 nucleotides of the first exon is sufficient to drive specific expression in cell lines. Like the M4 and M2 gene promoters, the M1 promoter contains an Sp1 motif which can recruit transcription factor Sp1 and at least one other protein, although this site does not appear to be functionally important for M1 expression in our assay. We have identified a region within the first exon of the M1 gene that regulates expression in cell lines, contains several positive and negative acting elements and is able to drive expression of a heterologous promoter. A polypyrimidine/polypurine tract and a sequence conserved between M1 genes of various species act in concert to enhance M1 transcription and are able to activate a heterologous promoter. We show that DNA binding proteins interact in vitro with single-stranded DNA derived from these regions and suggest that topology of the DNA is important for regulation of M1 expression

Additional file 3: of Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice

Primers used in qRT-PCR analyses. (DOCX 100 kb

rec-Y3H screening allows the detection of simultaneous RNA-protein interface mutations

In Press.Understanding which proteins and RNAs directly interact is crucial for revealing cellular mechanisms of gene regulation. Efficient methods allowing to detect RNA-protein interactions and dissect the underlying molecular origin for RNA-binding protein (RBP) specificity are in high demand. The recently developed recombination-Y3H screening (rec-Y3H) enabled many-by-many detection of interactions between pools of proteins and RNA fragments for the first time. Here, we test different conditions for protein-RNA interaction selection during rec-Y3H screening and provide information on the screen performance in several selection media. We further show that rec-Y3H can detect the nucleotide and amino acid sequence determinants of protein-RNA interactions by mutating residues of interacting proteins and RNAs simultaneously. We envision that systematic RNA-protein interface mutation screening will be useful to understand the molecular origin of RBP selectivity and to engineer RBPs with targeted specificities in the future.This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2017-85361-P] and the National Research Foundation of Korea (NRF) funded by the Ministry of Education [2018R1A6A3A11045727]. We further acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the ‘Centro de Excelencia Severo Ochoa’ [SEV-2012-0208] and the CERCA Programme/Generalitat de Catalunya.Peer reviewe

Additional file 2: of Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice

Summary of mice injected with ZF-KOX1, mZF-KRAB, GFP or PBS. (DOCX 48 kb

Additional file 4: of Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice

Identification of reference genes for qPCR from whole brain of the R6/1 and WT mice. (DOCX 51 kb

rec-YnH enables simultaneous many-by-many detection of direct protein-protein and protein-RNA interactions

Knowing which proteins and RNAs directly interact is essential for understanding cellular mechanisms. Unfortunately, discovering such interactions is costly and often unreliable. To overcome these limitations, we developed rec-YnH, a new yeast two and three-hybrid-based screening pipeline capable of detecting interactions within protein libraries or between protein libraries and RNA fragment pools. rec-YnH combines batch cloning and transformation with intracellular homologous recombination to generate bait–prey fusion libraries. By developing interaction selection in liquid–gels and using an ORF sequence-based readout of interactions via next-generation sequencing, we eliminate laborious plating and barcoding steps required by existing methods. We use rec-Y2H to simultaneously map interactions of protein domains and reveal novel putative interactors of PAR proteins. We further employ rec-Y2H to predict the architecture of published coprecipitated complexes. Finally, we use rec-Y3H to map interactions between multiple RNA-binding proteins and RNAs—the first time interactions between protein and RNA pools are simultaneously detected.We also acknowledge the support from the Spanish Ministry of Economy and Competitiveness (MINECO) for Juan de la Cierva-Incorporación Programme (IJCI‐2014‐22070) to J.-S.Y., L.S. (BFU2015-63571-P), and S.M. (BFU2014-54278-P and BFU2015-62550-ERC). We further acknowledge support of the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013-2017”, SEV-2012-0208 and the CERCA Programme/Generalitat de Catalunya. This work was funded by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) reference MINECO PE 2013-2016 PN FEDER and the European Regional Development Fund (ERDF). All sequencing was done in the CRG Genomics Core Facility