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An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes.

Barbon E, Pignani S, Branchini A, Bernardi F, Pinotti M, Bovolenta M - Sci Rep (2016)

Bottom Line: We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model.Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene.These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.

ABSTRACT
Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.

No MeSH data available.


Related in: MedlinePlus

Evaluation of TF4 specificity and off-targets.(a) Trans-activation effects of pTF4 on the expression of pBasic (lacking the promoter) pSlug (carrying the unrelated ubiquitous Slug promoter) or F7 promoter variants with the mutated (5 out of 22 bp) TF4 target sequence (pFVII-lucwtΔ5, pFVII-luc−61Δ5) in HepG2 cells. (b,c) Results from conventional RT-PCR and RT followed by qPCR analysis of F10 mRNA isolated from HepG2 (b) or HEK293 (c) cells transfected with TF4 (+) or untransfected cells (−). The amplicons were separated on 2% agarose gel. (d) Relative transcript quantification of the predicted off-targets genes by qPCR. The data analyses were performed with Student’s t-test comparing TF4-treated cells with untreated cells. The results are expressed as the mean ± standard deviation from at least three independent experiments.
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f4: Evaluation of TF4 specificity and off-targets.(a) Trans-activation effects of pTF4 on the expression of pBasic (lacking the promoter) pSlug (carrying the unrelated ubiquitous Slug promoter) or F7 promoter variants with the mutated (5 out of 22 bp) TF4 target sequence (pFVII-lucwtΔ5, pFVII-luc−61Δ5) in HepG2 cells. (b,c) Results from conventional RT-PCR and RT followed by qPCR analysis of F10 mRNA isolated from HepG2 (b) or HEK293 (c) cells transfected with TF4 (+) or untransfected cells (−). The amplicons were separated on 2% agarose gel. (d) Relative transcript quantification of the predicted off-targets genes by qPCR. The data analyses were performed with Student’s t-test comparing TF4-treated cells with untreated cells. The results are expressed as the mean ± standard deviation from at least three independent experiments.

Mentions: Target specificity is a crucial issue when proposing a new therapeutic approach. Thus, we assessed our designed TALE-TFs in multiple control experiments for off-target effects. The key role of the TALE-DNA binding domain was to guarantee specific targeting. This specificity was demonstrated in control experiments where the VP64 activation domain was expressed alone. Negligible effects on the expression of the pHD plasmid were observed in these experiments (Fig. 1c). In complementary experiments, TF4 was unable to increase the transcription of neither the pBasic construct, which does not have a promoter, or the pSlug vector, which possesses a ubiquitous and F7-unrelated Slug promoter30 (Fig. 4a). Most importantly, the TF4 efficacy was abolished upon the partial mutagenesis (five out of the 22 nucleotides) of the target sequence (Fig. 4a, inset), either in the wild-type (pFVII-lucwtΔ5) or mutated (pFVII-luc−61Δ5) contexts, a finding that supports high specificity and a reduced probability of off-target effects (Fig. 4a).


An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes.

Barbon E, Pignani S, Branchini A, Bernardi F, Pinotti M, Bovolenta M - Sci Rep (2016)

Evaluation of TF4 specificity and off-targets.(a) Trans-activation effects of pTF4 on the expression of pBasic (lacking the promoter) pSlug (carrying the unrelated ubiquitous Slug promoter) or F7 promoter variants with the mutated (5 out of 22 bp) TF4 target sequence (pFVII-lucwtΔ5, pFVII-luc−61Δ5) in HepG2 cells. (b,c) Results from conventional RT-PCR and RT followed by qPCR analysis of F10 mRNA isolated from HepG2 (b) or HEK293 (c) cells transfected with TF4 (+) or untransfected cells (−). The amplicons were separated on 2% agarose gel. (d) Relative transcript quantification of the predicted off-targets genes by qPCR. The data analyses were performed with Student’s t-test comparing TF4-treated cells with untreated cells. The results are expressed as the mean ± standard deviation from at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920032&req=5

f4: Evaluation of TF4 specificity and off-targets.(a) Trans-activation effects of pTF4 on the expression of pBasic (lacking the promoter) pSlug (carrying the unrelated ubiquitous Slug promoter) or F7 promoter variants with the mutated (5 out of 22 bp) TF4 target sequence (pFVII-lucwtΔ5, pFVII-luc−61Δ5) in HepG2 cells. (b,c) Results from conventional RT-PCR and RT followed by qPCR analysis of F10 mRNA isolated from HepG2 (b) or HEK293 (c) cells transfected with TF4 (+) or untransfected cells (−). The amplicons were separated on 2% agarose gel. (d) Relative transcript quantification of the predicted off-targets genes by qPCR. The data analyses were performed with Student’s t-test comparing TF4-treated cells with untreated cells. The results are expressed as the mean ± standard deviation from at least three independent experiments.
Mentions: Target specificity is a crucial issue when proposing a new therapeutic approach. Thus, we assessed our designed TALE-TFs in multiple control experiments for off-target effects. The key role of the TALE-DNA binding domain was to guarantee specific targeting. This specificity was demonstrated in control experiments where the VP64 activation domain was expressed alone. Negligible effects on the expression of the pHD plasmid were observed in these experiments (Fig. 1c). In complementary experiments, TF4 was unable to increase the transcription of neither the pBasic construct, which does not have a promoter, or the pSlug vector, which possesses a ubiquitous and F7-unrelated Slug promoter30 (Fig. 4a). Most importantly, the TF4 efficacy was abolished upon the partial mutagenesis (five out of the 22 nucleotides) of the target sequence (Fig. 4a, inset), either in the wild-type (pFVII-lucwtΔ5) or mutated (pFVII-luc−61Δ5) contexts, a finding that supports high specificity and a reduced probability of off-target effects (Fig. 4a).

Bottom Line: We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model.Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene.These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Italy.

ABSTRACT
Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.

No MeSH data available.


Related in: MedlinePlus