Utrophin Up-Regulation by an Artificial Transcription Factor in Transgenic Mice

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 sustains hypoxic response of colorectal cancer cells by affecting Hif-1α stabilization

BACKGROUND: Solid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia. METHODS: The human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses. RESULTS: Here we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism. CONCLUSIONS: We show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13046-017-0497-1) contains supplementary material, which is available to authorized users

Additional file 1: Figure S1. of Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1Îą stabilization

Che-1 is involved in the metabolic switch in response to hypoxia. A- and B- HT29 (A) and A549 (B) cells were transiently transfected with stealth siRNA negative control (siControl) or siRNA Che-1 (siChe-1) and exposed to hypoxia for 16 h where indicated and pH was measured. C- and D- Score plots indicating metabolic differences between hypoxic and normoxic samples from HT29 (C) and A549 (D) cells transiently transfected as in A. E- HT29 cells were transfected as in A and the medium lactate content was evaluated. (TIF 5841 kb

Additional file 2: Figure S2. of Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1α stabilization

Che-1 regulates genes transcription in response to hypoxia. A- Quantitative RT–PCR (qRT–PCR) for metabolic genes expression was performed in HT29 cells transiently transfected with Stealth siRNA negative control (siControl) or siRNA Che-1 (siChe-1) and exposed to hypoxia for 4 h. Values were normalized to RPL19 mRNA expression. Error bars represent the standard error of three different experiments. *P = 0,0010, **P ≤ 0,0003, ***P ≤ 0,004, n.s., not significant. (TIF 1835 kb

Muscle specific expression of Vp16-Jazz in transgenic mice.

<p>A: Western Blot analysis of total proteins extracted from the skeletal muscle of wild type (wt) and transgenic mice derived from two different founders (tg9 and tg41). The expression of Vp16-Jazz transgene was monitored by the anti-myc monoclonal 9E10 antibody. Detection of α−tubulin was used to normalize the amount of proteins. B: Western Blot analysis of total proteins extracted from the skeletal muscle, heart and brain of transgenic mice from families tg9 and tg41.</p

Vp16-Jazz and its DNA target sequence.

<p>A: Schematic representation of the Vp16-Jazz gene in the pMex-vector, used to generate transgenic mice. The 9 base pair long Vp16-Jazz DNA target sequence is indicated. B: The nucleotide sequence of the mouse utrophin promoter A. The Vp16-Jazz DNA target sequence is indicated in bold characters and underlined. The main transcription factor binding sites present in this promoter region are indicated.</p

Vp16-Jazz and utrophin up-regulation.

<p>A: Vp16-Jazz chromatin immunoprecipitation, performed in skeletal muscle derived from wt mice and transgenic mice (family tg9) using myc monoclonal antibody/protein G-agarose beads or protein G-agarose beads as a control (no-Ab). Immunoprecipitates from each sample were analyzed for the presence of utrophin promoter by PCR. A sample representing linear amplification of the total input chromatin (input) was included (lane 1). As control, samples from transgenic mice were also tested for the presence of dystrophin promoter sequence. B: Real-time PCR analysis of the utrophin gene expression rate in Vp16-Jazz transgenic mice (tg9 and tg41) and control wt mice. The gene expression ratio between utrophin and β-glucoronidase (GUS) and β2-microglobulin (β2M) is shown as means±S.D. from three independent experiments performed in triplicate. C: Western blot of total protein extracts derived from skeletal muscle and heart from wt mice and Vp16-Jazz transgenic mice (tg9 and tg41) incubated with monoclonal antibody against utrophin. The same membrane was incubated with anti-α-tubulin monoclonal antibody for loading normalization. D: Relative utrophin expression of wt and transgenic mice (tg9 and tg41) was determined by densitometric analysis. E: Total protein extracts from skeletal muscle of wt and transgenic mice (tg9 and tg41) were subjected to immunoblotting to detect the expression levels of the dystrophin and α-sarcoglycan proteins. The anti-α-tubulin and anti-myc antibodies were used to normalize the protein content and to test the Vp16-Jazz transgene expression respectively.</p

Effects of Vp16-Jazz in transgenic mice.

<p>Immunohistochemistry of Tibialis Anterior (TA) muscle derived from wt (panel A) and transgenic mice tg9 (panel B) stained with anti-utrophin antibody. Nuclei are counterstained with Hoechst 33258. C: TA from wt and transgenic mice tg9 were co-stained with anti-utrophin antibody and the α-bungarotoxin-Alexa Fluor to visualize the acetylcholine receptor (AChR) at the neuromuscular junctions. The anti-utrophin monoclonal antibody reveals an extra-synaptic distribution of utrophin only in transgenic mice. D: Relationship between contractile response (g) and stimulation frequency (Volts) in diaphragm and EDL muscle preparations obtained from wt, tg9 and tg41 mice. E: Scatter plots of the DEG with natural log transformed expression values averaged over the 4 replicates. The x–axis reports the mean of the replicates of transgenic mice, while the y-axis is the mean across the replicates of the control wt mice. Expression values are colour coded with red representing up-regulated genes in tg9/tg41 and green down-regulated genes compared to wild-type and lines on the graph set at 2-folds differential expression.</p