Wnt signalling mediates miR-133a nuclear re-localization for the transcriptional control of Dnmt3b in cardiac cells

MiR-133a is a muscle-enriched miRNA, which plays a key role for proper skeletal and cardiac muscle function via regulation of transduction cascades, including the Wnt signalling. MiR-133a modulates its targets via canonical mRNA repression, a process that has been largely demonstrated to occur within the cytoplasm. However, recent evidence has shown that miRNAs play additional roles in other sub-cellular compartments, such as nuclei. Here, we show that miR-133a translocates to the nucleus of cardiac cells following inactivation of the canonical Wnt pathway. The nuclear miR-133a/AGO2 complex binds to a complementary miR-133a target site within the promoter of the de novo DNA methyltransferase 3B (Dnmt3b) gene, leading to its transcriptional repression, which is mediated by DNMT3B itself. Altogether, these data show an unconventional role of miR-133a that upon its relocalization to the nucleus is responsible for epigenetic repression of its target gene Dnmt3b via a DNMT3B self-regulatory negative feedback loop.

Universal RT microRNA PCR Polyadenylation and cDNA synthesis kit (Exiqon). Quantitative real time polymerase chain reaction (qRT-PCR) was performed with microRNA LNA™ PCR primers (Exiqon) using the GoTaq Probe qPCR Master Mix (Promega). Relative expression was calculated using the ΔΔ(Ct) method. MiRNA expression in cells or nuclei was normalized to U6 snRNA, while mRNA expression in cells was normalized to GAPDH. Sequences of primers used for qRT-PCR are reported in Supplementary Table 1 and Supplementary Table 2.

DNA constructs
For the BRET assay, Ago2 and Ago1 cDNAs were cloned into the pNLF1-N vector, while DNMT3B and IPO8 cDNA were cloned into the HaloTag-pFN21A vector. All vectors for Nano-Luciferase and BRET assays were obtained from Promega. All cloning steps were performed using the In-fusion HD Cloning Plus kit (Clontech).

Nano-BRET assay
The NanoBRET assay was performed as described by the manufacturer (Promega) [60]. Briefly, for proteinprotein interaction assays, HL-1 transfected cells were treated with 100 nM NanoBRET 618 Ligand (Promega) and signals were detected 6 hours after treatment. Signals were detected using a Synergy H4 instrument (BioTek) and results were analysed using Prism 6.0 software (GraphPad Software, CA).

Chromatin Imunoprecipitation (ChIP) assay
For chromatin immunoprecipitation, HL-1 cells were seeded at a density of 1 x 106 on gelatin/fibronectin plates and treated with IWR-1 for 48 hours as previously reported. On the day of harvest, cells were crosslinked with 1% of formaldehyde for 10 min. The cross-linking reaction was stopped with 0.125 M Glycine for 5 min after which cells were washed with cold PBS and collected into IP Buffer (2 volumes of SDS Buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.1, 5 mM EDTA pH 8.0, 0.5% SDS), and 1 volume of Triton Dilution Buffer (100 mM NaCl, 100 mM Tris-HCl pH 8.1, 5 mM EDTA, 5% Triton X-100)). Samples were sonicated using a Bioruptor® Plus sonication device, clarified by centrifugation, and subsequently checked on a 0.8% agarose gel following DNA purification. 20% of clarified lysates was saved as input, before addition of 20 µl of Dynabeads® Protein G (Thermo Fisher Scientific) and specific antibodies (AGO2; DNMT3B; H3K4me3; H3K27me3) and incubation overnight at 4°C. The day after, samples were washed 3 times with Mixed Micelle Buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA pH 8.0, 1% Triton X-100, 0.2% SDS), 2 times with Buffer 500 (50 mM HEPES, 0.1% Deoxycholic Acid, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA pH 8.0); and 1 time with LiCl detergent buffer (10 mM Tris-HCl pH 8.0; 0.5% Deoxycholic Acid, 0.5% NP40, 250 mM LiCl). Subsequently, elution was performed in elution buffer (1% SDS and 100 mM NaHCO3) after which de-cross-linking was performed by addition of 200 mM NaCl and incubation at 65°C overnight. The following day, samples were incubated with 20 ug/ml of Proteinase K at 45°C for 2 hours, where after DNA was extracted by ethanol precipitation following treatment with UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (Thermo Fisher Scientific). qRT-PCR was subsequently performed using the primers reported in Supplementary Table 3. Evaluation of methylation enrichment The methylation enrichment (%) of CpG nucleotides was determined using the MethylCollector™Ultra Kit (ActiveMotif), according to the manufacturer's instruction. The resulting methylated DNA was analysed through qRT-PCR using specific primers to amplify the locus of miR-133a putative binding site.

AGO2 nuclear pull-down
HL-1 cells were seeded at a density of 1 x 106 on gelatin/fibronectin plates and treated with IWR-1 for 48 hours as previously described. Cells were collected and processed for nuclear extracts using the Nuclear Complex Co-IP Kit, according to the manufacturer's protocol. The nuclear protein lysate was then incubated with antibodies against AGO2 or IgG, and 20 µl of Dynabeads® Protein G (Thermo Fisher Scientific) overnight at 4 °C with rotation. For protein analysis, samples were subjected to Western blot assays, while for miRNA analysis, the RNA derived from AGO2 or IgG pull-down assay was isolated and subsequently analysed by qRT-PCR.

Fluorescent in situ hybridization (FISH) and Duo-Link assay
HL-1 cells were seeded at 5.0 X 104 cells/well on VWR Micro cover slips coated with gelatin/fibronectin in complete Claycomb medium, and incubated overnight at 37°C with 5% CO2. The following day, HL-1 cells were treated with 5 µM IWR-1 for 24-48 hours. Cultured cells were fixed for 5 min in 4% paraformaldehyde at room temperature, after which cells were permeabilized with PBS containing 0.2% Triton X-100 for 5 min. MiRCURY LNA™ Detection probe was used to stain for miR-133a-3p or scramble, and tyramide signal amplification (TSA) system (Life technologies) was used to detect miRNA or scramble signal. After three washes with PBS, nuclei were counterstained with 4', 6-diamidino-2-phenylindole, dihydrochloride (Life Technologies) for 5 min at room temperature. Fluorescent images were taken with a laser scanning confocal microscope (Olympus, FV1000/SIMS). The AGO2-DNMT3B interaction, the Duo-Link assay (Sigma Aldrich) was used following the manufacturer's instructions.

FACS Sorting
FITC positive and negative PSCs-derived cardiomyocytes were sorted using a FACS Aria III instrument (Becton Dickinson, Franklin Lakes, CA, USA). Photomultiplier voltages and laser time delay were checked on a daily basis to ensure the maximum reproducibility of results. Cell sorting was performed at room temperature using a 100 µm nozzle, 20 psi pressure, 3500 plate voltage and low speed to reduce cell stress to the minimum. Doublet discrimination was performed by plotting events in a FSC-A vs. FSC-H dot plot.

Statistical Analysis
Data are presented as mean ± SD. The normality of data was assessed using the Kolmogorov-Smirnov test. Statistical comparison was performed in at least 3 independent experiments with the Mann-Whitney test, and comparisons between groups were analysed by ANOVA repeated-measures in combination with the Tukey multicomparison. Prism 6.0 software (GraphPad Software) was used to verify the normality of the data and for statistical calculation. A value of P < 0.05 was considered statistically significant.