Phenylbutazone induces expression of MBNL1 and suppresses formation of MBNL1-CUG RNA foci in a mouse model of myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is caused by abnormal expansion of CTG repeats in the 3′ untranslated region of the DMPK gene. Expanded CTG repeats are transcribed into RNA and make an aggregate with a splicing regulator, MBNL1, in the nucleus, which is called the nuclear foci. The nuclear foci sequestrates and downregulates availability of MBNL1. Symptomatic treatments are available for DM1, but no rational therapy is available. In this study, we found that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone (PBZ), upregulated the expression of MBNL1 in C2C12 myoblasts as well as in the HSALR mouse model for DM1. In the DM1 mice model, PBZ ameliorated aberrant splicing of Clcn1, Nfix, and Rpn2. PBZ increased expression of skeletal muscle chloride channel, decreased abnormal central nuclei of muscle fibers, and improved wheel-running activity in HSALR mice. We found that the effect of PBZ was conferred by two distinct mechanisms. First, PBZ suppressed methylation of an enhancer region in Mbnl1 intron 1, and enhanced transcription of Mbnl1 mRNA. Second, PBZ attenuated binding of MBNL1 to abnormally expanded CUG repeats in cellulo and in vitro. Our studies suggest that PBZ is a potent therapeutic agent for DM1 that upregulates availability of MBNL1.

C2C12 cells were lysed with PLC lysis buffer with protease inhibitors, and whole cell lysates were extracted as described above. The lysates were incubated for 30 min on ice, then cleared after centrifugation for 30 min at 15,400 × g at 4˚C. The cleared lysates were resolved with SDS/PAGE and blotted on to a PVDF membrane, as previously described 1 . The membrane was stained with anti-MBNL1 mouse monoclonal antibody (1:200, 4A8, sc-136165, Santa Cruz) and anti-CELF1 mouse monoclonal antibody (1:200, 3B1, sc-20003, Santa Cruz), and re-blotted with anti-GAPDH rabbit polyclonal antibody (1:5000, G9545, Sigma-Aldrich).

Expression and purification of recombinant proteins.
The bacterial expression vector, GST-MBNL1, carrying GST cDNA fused to MBNL1 cDNA (amino acids 1-260) was kindly provided by Dr. Andrew Berglund at University of Oregon 2 . Another bacterial expression vector, GST-PTBP1, carrying GST cDNA fused to PTBP1 cDNA was constructed by inserting a previously cloned PTBP1 cDNA (BC013684) 3 into the GST-fusion vector pGEX-6P-1 (GE Healthcare Life Sciences) at EcoRI and XhoI sites. Protein expression was induced using BL21 Star (DE3)pLysS One Shot Chemically Competent E. coli (Thermo Fisher Scientific) with 0.25 mM IPTG (Sigma) at an OD600 ≈ 0.5-1.0, for 4 h at 37˚C. Cells that express MBNL1 were lysed in 10 ml of buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.5, 1 mM EDTA, and 1 mM DTT).
The cell extracts were centrifuged for 15 min at 21,160 × g, and the supernatants were collected. GST-fusion protein was bound to Glutathione-Sepharose 4B beads (GE Healthcare Life sciences) for 45 min at 4˚C, and the beads were washed 3 times with cleavage buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, and 1 mM DTT). GST-MBNL1 was cleaved from the affinity tag with PreScission Protease (GE Healthcare Life Sciences) according to the manufacturer's instructions. GST-PTBP1 was eluted in elusion buffer (50 mM Tris-HCl pH 8.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA and 30 mM Glutathione Reduced Form). The supernatants containing recombinant proteins were collected from the beads, and stored at -80˚C.

DNA methylation-inhibition assay.
C2C12 cells were seeded in a 60-mm culture dish. After confluency, the culture medium was replaced with the differentiation medium containing 2% horse serum. Cells were treated with 10 µM 5-AC (A2385, Sigma) on differentiation day 0. RNA was extracted 72 h after treatment. Expressions of Mbnl1 and Gapdh mRNAs were quantified by real-time RT-PCR with primers shown in Supplementary Table S3.

DNA extraction and CpG methylation analysis by bisulfite-sequencing PCR.
Genomic DNA was extracted from C2C12 cells using QIAamp DNA Mini Kit (Qiagen). Non-methylated cytidines in DNA (2.5 µg) were converted to uridines with MethylEasy TM Xceed Rapid DNA Bisulfite modification kit (Cat # ME002, Human Genetic Signatures) according to the manufacturer's instructions. Pairs of bisulfite-sequencing PCR (BSP) primers were used to amplify the methylated MeR1 and MeR2 regions of Mbnl1 ( Supplementary Fig. S3). Primer sequences are shown in Supplementary Table S4 and S5. The PCR products were run on an agarose gel followed by purification with the Wizard SV Gel and PCR Clean-Up System (Promega), and subcloned into the TA cloning vector (pGEM-T Easy Vector, Promega). To identify the methylated CpG dinucleotides, we sequenced 14 independent clones for each sample with the CEQ 2000 DNA Analysis System (Beckman Coulter). The methylation pattern was analyzed with the QUMA (Quantitative Tool for Methylation Analysis) software (http://quma.cdb.riken.jp/) using the default parameters 4 .

Quantitative RT-PCR.
To quantify Mbnl1 mRNAs initiated at different TSSs (1a, 1b, 2, and 1' in Supplementary Fig. S5a), we performed real-time RT-PCR using primers shown in Supplementary Table S6. Mbnl1 transcripts 1a and 1b were quantified using a primer pair E/1F-E/2R and E1/F-E3/R, respectively. Whole Mbnl1 transcripts were quantified with a primer pair E3/F-E4/R. Then, the expression level of residual Mbnl1 transcripts (2+1') was estimated by subtracting expression level of 1a and 1b from that of whole Mbnl1 transcripts.
These parameters were all analyzed at Oriental Yeast Co., Ltd. (Japan).

Legends for Supplementary Figures
Supplementary Figure S1 performed using the primers shown in (a). Expression level of each transcript, which is calculated as in Methods, is normalized to that of Gapdh, and the relative mRNA expression levels are further normalized to that of transcript (1a) of WT FVB/N mice. The mean and SD of five mice in each group are indicated. The data were analyzed by two-way ANOVA followed by Bonferroni post-hoc test. ***p < 0.001.