Thomas et al. 2017

Supplemental Materials and Methods

Disrupted prenatal RNA processing and myogenesis in congenital

James D. Thomas1, Łukasz J. Sznajder1, Olgert Bardhi1, Faaiq N. Aslam1, Zacharias P. Anastasiadis1, Marina M. Scotti1, Ichizo Nishino2, Masayuki Nakamori3, Eric T. Wang1 and Maurice S. Swanson1*

1Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, University of Florida, College of Medicine, Gainesville, Florida, USA 2Department of Neuromuscular Research, National Center of Neurology and Psychiatry, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan 3Department of Neurology, Osaka University Graduate School of Medicine, Osaka 565- 0871, Japan

Content Figures S1 to S6 Tables S1 to S6 Videos S1 to S2 Supplemental Materials & Methods Supplemental References

*Correspondence: [email protected]

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A MAPT PDLIM5 RPKM MAPT - CE NCOR2 - A5SS PDLIM5 - A3SS DOM3Z - RI 1.0 1.0 18 30 600 60 0.8 0.8 0.6 0.6 ** 18 30 600 60 Ψ 0.4 Ψ 0.4 0.2 *** 0.2 18 30 600 60 0.0 0.0 Ctrl. CDM Ctrl. CDM 18 30 600 60 NCOR2 DOM3Z 1.0 1.0 *** 18 30 600 60 0.8 0.8 0.6 0.6 18 30 600 60 Ψ 0.4 Ψ 0.4 0.2 0.2 *** e6 e46 e47 e4 e5 e5 92 e4 0.0 0.0 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ Ctrl. CDM Ctrl. CDM

B CEs excluded in DM1 CEs included in DM1 C MAPT LDB3 Ctrl. Ctrl. DM1 CDM Ctrl. DM1 CDM CDM 1.0 1.0 1.0

0.8 0.8 0.8

0.6 0.6 0.6 Ψ Ψ Ψ 0.4 0.4 0.4 0.2 0.2 0.2 0.0 0.0 0.0 NFIX INSR LDB3 RYR1 MAPT [MBNL] [MBNL] BIN1.2 BIN1.1 inferred inferred CAPZB COPZ2 MBNL2 MBNL1 LDB3.1 MEF2C MAPT.1 PDLIM3 ATP2A1 CLASP1 SORBS1 CAMK2B MBNL2.1 CAMK2G PDLIM3.2 CACNA1S CACNA2D1 ARHGEF10L Supplemental Figure S1. RNA mis-processing in CDM . (A) RNA-seq read coverage across alternative MAPT exon 6, NCOR2 exon 46, PDLIM5 exon 5, and DOM3Z intron 4. Quantification of MISO Ψ for each alternative event is shown to the right with 95% confidence intervals shown as gray lines (** and ***, monotonicity Z-score > 1.5 and 2.0, respectively). (B) Individual control and CDM patient Ψ for events mis-regulated in adult-onset DM1 (Wagner et al. 2016) or CDM patient-derived cells (Fugier et al. 2011). (C) Individual adult control (Ctrl.), adult-onset DM1, and CDM patient Ψ for MAPT and

LDB3 alternative exons according to ranked [MBNL]inferred levels with fitted curves shown in black. [MBNL]inferred levels were not calculated for CDM so these points are plotted outside the fitted curve.

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A Mapping C Align to hg19 mutually exclusive exons retained introns Keep primary, unique alignments 350 851 622 696 600 355 815 575 Quality control (as in Fig. S2B) 300 500 Splicing Analysis 250 400 Quantify MISO Ψ 200 300 (MISO annotations v2.0) 150 200 Identify myogenic RNA isoforms: 100 Bayes-factor ≥ 5 50 100 number of events monotonicity Z-score ≥ 1.5 0 number of events 0 −1.0 0.0 1.0 |ΔΨ| ≥ 0.10 −1.0 0.0 1.0 ΔΨ ΔΨ B body coverage D ITGA7 1.0 -24 Ψ = 0.01

1.0 24 Ψ = 0.08

0.6 48 Ψ = 0.30 “in utero” MPC (hr) ΔΨ ≥ 0.74 0.4 72 Coverage Ψ = 0.30 0.2 fetal muscle Ψ = 0.75 day 127 0.0 0 20 40 60 80 100 e25 Gene body percentile (5’ to 3’) 3’ 5’ E CORO6 NEB F TPM1 ALE 1.0 1.0 -24 0.8 0.8

0.6 0.6 24 Ψ Ψ

0.4 0.4 RNA-seq

0.2 0.2 48

0.0 0.0 72 MEF2D MAPT 1.0 1.0 127 0.8 0.8 PolyA-seq 0.6 0.6 Ctrl. Ψ Ψ 0.4 0.4 CDM 0.2 0.2

0.0 0.0 5’ 3’

G representative CEs affected in CDM 1.0 1.0 1.0 Gradual Late No change 0.8 0.8 0.8

0.6 0.6 0.6 Ψ Ψ Ψ 0.4 0.4 0.4

0.2 0.2 0.2

0.0 0.0 0.0

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H ChIP-seq: C2C12 differentiation 0 hr

24 hr

MYOD 60 hr

7 d

0 hr

24 hr

MYOG 60 hr

7 d

Dmwd Dmpk Six5 Supplemental Figure S2. Prenatal RNA isoform transitions in development and disease. (A) Schematic of RNA-seq pipeline used in this study. (B) Gene body coverage for all myogenesis datasets used in this study. (C) Histograms depicting the number of MXE and RI events with consistently increasing (red) or decreasing (blue) Ψ from -24 hr of myogenic differentiation in vitro to day 127 fetal muscle. (D) RNA-seq read coverage across ITGA7 exon 25 with quantification of MISO Ψ and 95% confidence interval shown to the right. Prenatal ΔΨ is ≥ 0.74 for this exon. (E) Additional examples of CEs mis- spliced in CDM that undergo prenatal alternative splicing transitions. Plots are as in Fig. 2D except the average CDM Ψ, rather than individual points, is plotted as a horizontal red line. The vertical dashed line demarcates pre- and postnatal datasets (i.e., the transition from day 127 fetal muscle to 3 month infant muscle). (F) Representative alternative last exon (ALE) use identified in CDM PolyA-seq with read coverage across the TPM1 ALE depicting the normal RNA processing (upper, gray) and mis-processing in CDM (lower, red). (G) Distinct exon splicing dynamics during prenatal myogenesis including gradual, late, and no change in Ψ during human muscle development. Gray solid lines represent individual CE splicing transitions. Solid black lines represent averages based on total plotted CEs. The dashed, vertical black line represents the border between pre- (day 127 fetal muscle) and postnatal (3 month infant muscle) datasets. All exons shown are mis- spliced in CDM muscle compared to controls. (H) MYOD and MYOG ChIP-seq (GSM915186, GSM915183, GSM915185, GSM915165, GSM915166, GSM915159, GSM915163, GSM915164) read coverage across the Dmpk locus during C2C12 cell differentiation in vitro.

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A P0 distal hindlimb H&E C myofiber csa 1.0 ****

n **

o 0.5 i

TA t c a

r soleus f

0.0

e 0 125 250 v i 1.0 t

a **** l u

m 0.5 u C Sol. TA 0.0 0 100 200 300 µm2 B P0 skeletal muscle WT 1KO DKO TA soleus

Wheat germ agglutinin DAPI

Supplemental Figure S3. Analysis of newborn Mbnl KO mice. (A) H&E stained cross- section of P0 hind-limb with soleus (Sol.) and tibialis anterior (TA) muscles indicated. (B) Immunofluorescence image of AF488 labelled, wheat germ agglutinin (green) and DAPI (blue) stained cross-sections of TA and soleus muscles from WT, 1KO, and DKOs. Myofiber disorganization is noted (white arrowheads) as regions of discontinuity in WGA staining. (C) Distribution of myofiber cross-sectional area (csa) in P0 WT (gray), 1KO (yellow), and DKO (orange) soleus (upper) and TA (lower) muscle. ** and ****, p < 0.01 and 0.0001, respectively, Kruskal-Wallis test with Dunn’s post-hoc.

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Supplemental Figure S4. Congenital spliceopathy in mouse DKO muscle. (A) Representative RT-PCR validations of CE mis-splicing in P0 muscle from WT, 2KO, 1KO, and DKOs. For Atp2a1, exon 22 loss is responsive to both single and compound loss of MBNL activity. (B) Dynamics of representative CEs undergoing prenatal splicing transitions during mouse myogenesis. (C) Quantification of Sorbs1 exon 15 Ψ during mouse muscle maturation. The Ψ value for DKO muscle is indicated (orange circle). (D) Venn diagram representing overlap of homologous with mis-splicing (CE, MXE, A5SS, A3SS, and/or RI) in human CDM and mouse DKO muscle. (E) Quantification of Cacna2d1 exon 19 Ψ during mouse muscle maturation. The Ψ value for DKO mouse is indicated (orange circle). (F) RNA-seq read coverage across Cacna2d1 exon 19 during mouse myogenesis in vitro (Trapnell et al. 2010) and in P0 WT and DKO muscle.

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Supplemental Figure S5. Analysis of 3KO myoblasts. (A) Schematic of targeting strategy used to generate the Mbnl3 conditional and KO alleles. The targeting construct contains puromycin (P) positive selection and thymidine kinase (TK) negative selection cassettes, and Cre-mediated excision resulted in a modified Mbnl3 allele lacking exons 2-7c. (B) Southern blot confirming the presence of mutant Mbnl3 alleles in targeted mouse embryonic stem cells (mESCs). (C) RT-PCR analysis of Mbnl3 WT and KO myoblasts using forward primers in exons 2 or 3 and a reverse primer in exon 8. (D) Immunoblot of MBNL3 in WT and 3KO thymus. (E) RNA-seq read coverage across the Mbnl3 locus in WT and 3KO myoblasts. The region with the expected deletion is outlined with a gray box as in panel A. (F) Phase contrast image of WT and 3KO myoblasts grown in proliferation

7 Thomas et al. 2017 media at high density. For WT (left panel), myoblast- (green dotted circle) and myocyte- like (black dotted oval) morphologies are indicated. Quantification of cell spreading (i.e., myocyte-like morphology) based on multiple WT and 3KO myoblast isolates is shown (right) (***, p < 0.001, Student’s t-test). (G) Scatter plot of mis-spliced CEs in WT and 3KO myoblasts with significant events indicated (green circles). (H) RNA-seq read coverage across Fermt2 tandem CEs in WT and 3KO myoblasts. (I) Scatter plot of changes in mRNA abundance (all mRNAs ≥ 10 TPM) in WT (gray) and 3KO (green) myoblasts 0 and 6 hours after induction of differentiation in vitro. Few changes occur in WT myoblasts within 6 hours (R = 0.94), but 3KO myoblasts show rapid changes in steady state RNA levels for many transcripts (R = 0.86). (J) Quantification of representative muscle-specific mRNAs showing increased abundance in 3KO compared to WT myoblasts within 6 hours of differentiation. (K) RNA-seq read coverage across the Dystrophin (Dmd) locus in WT and 3KO myoblasts 0 (upper) and 6 (lower) hr post-differentiation.

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Supplemental Figure S6. Analysis of Mbnl TKO mice. (A) Adult TKO mouse presenting with kyphosis, skeletal muscle wasting, and respiratory distress (Supplemental Video S2). (B) Average read depth for DKO and TKO RNA-seq. (C) Counts of the total number of MXE, A5SS, A3SS, and RI mis-splicing events in DKO and TKOs. (D) Quantification of Ap2m1 and Clip1 CE mis-splicing in TKO compared to WT and DKO muscle (***, monotonicity Z-score > 2.0). (E) Venn diagram depicting the overlap in biological pathways identified in CDM and TKO muscle. (F) Network analysis of biological pathways identified in CDM and TKO muscle with the 10 most enriched pathways highlighted.

Numbers in parenthesis represent log10P values for CDM, TKO, and DKO muscle, respectively (n/a is used when a pathway did not reach statistical significance). (G) Labelling of individual AChR clusters in WT, DKO, and TKO P0 diaphragms. (H) Distribution of individual AChR cluster size in WT (gray), DKO (orange), and TKO (red) P0 diaphragms (***, p < 0.001, Kruskal-Wallis test with Dunn’s post-hoc).

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Supplementary Table S1. RNA-seq sample descriptions.

Supplementary Table S2. CDM RNA mis-processing events.

Supplementary Table S3. RNA alternative splicing changes during normal human myogenesis.

Supplementary Table S4. DKO and TKO RNA mis-splicing events, and RNA alternative splicing changes during normal mouse myogenesis.

Supplementary Table S5. Orthologous mis-spliced cassette exons in mouse DKO and human CDM muscle.

Supplementary Table S6. Differential gene expression and enriched biological pathways in mouse DKO and TKO and human CDM muscle.

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Supplemental Video 1. Respiratory distress in moribund P0 DKO mice.

Supplemental Video 2. Adult TKO mouse.

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Supplemental Materials and Methods

Mbnl3 conditional knockout mice To generate the Mbnl3 conditional whole locus (condWL) allele, an ~7.5 kb fragment containing Mbnl3 intron 7 was retrieved from a C57/BL6 BAC into a high copy plasmid backbone containing HSV-TK. A puromycin resistance cassette with a single downstream LoxP sequence was inserted into the intron 7 sequence creating a 3.3 kb 5’ arm of homology (AOH) and 4.2 kb 3’ AOH within the Mbnl3condWL targeting construct. The Mbnl3condWL targeting construct was linearized with NotI, and electroporated into CMTI-2 BL/6 ESCs previously targeted for the Mbnl3 conditional large isoform knockout (Mbnl3condE2/Y) (Poulos et al. 2013). Clones were subjected to positive (neomycin/puromycin) and negative (HSV-TK) selection and screened by genomic blot analysis using 5’ probes (forward primer: GGT GAC AAG ATG ATG TGA GTG AGT GGA C reverse primer: GGA ACA GCT AAT CCT TCA GTA ACA GG) or 3’ probes (forward primer: TAC CCT CTA CAA TCC ACA GTT CAA reverse primer: ATC AAT GTC GAG CTT GTT CTA CTG) by digesting with either SacI or BmtI, respectively. The 5’ WT allele fragment size was 9.4 kb and the mutant allele 6.6 kb and the 3’ WT allele fragment size was 9.6 kb and mutant allele 7.2 kb. Positive euploid Mbnl3condWL mESCs were injected into B6(Cg)-Tyrc-2J/J blastocysts and transferred to pseudo-pregnant C57BL/6 females. Germline transmission of the Mbnl3condWL allele was obtained by mating chimeric males to B6(Cg)-Tyrc-2J/J females. To create the Mbnl3 WLKO (3KO), Mbnl3+/condWL females were crossed to male B6.C-Tg(CMV-Cre)1Cgn/J (JAX, X-linked) to create F2 Mbnl3+/-; Cre+ females which were subsequently crossed to male Mbnl3condWL to generate F3 Mbnl3 KO males. Mbnl3 KO females were generated in the F4 generation by crossing Mbnl3 KO males with female Mbnl3+/condWL. Myog-Cre (Li et al. 2005) induced Mbnl1; Mbnl2; Mbnl3 compound knockout mice were generated by crossing Mbnl1+/ΔE3; Mbnl2C/C; Mbnl3condWL/Y; Myog-Cre+/- males to Mbnl1+/ΔE3; Mbnl2C/C; Mbnl3condWL/ condWL; Myog-Cre-/- females and gDNA was isolated from tail biopsies of P21 mice for genotyping.

Immunoblotting

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Dissected tissues or cell pellets were homogenized in lysis buffer (20 mM HEPES-KOH, pH 8.0, 100 mM KCl, 0.1% Igepal CA-630 (Sigma), and protease inhibitors) on ice. Crude lysates were sonicated, centrifuged (16,000 g, 15 min, 4°C), and the supernatant was collected and used for immunoblotting. were detected using a rabbit polyclonal antibody (rpAb) to MBNL3 (Poulos et al. 2013), mouse monoclonal antibody (mAb) to

GAPDH (mAb 6C5, Abcam), and HRP-conjugated anti-mouse, or anti-rabbit, secondary antibody followed by ECL (GE Healthcare).

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Supplemental References

Fugier C, Klein AF, Hammer C, Vassilopoulos S, Ivarsson Y, Toussaint A, Tosch V, Vignaud A, Ferry A, Messaddeq N et al. 2011. Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy. Nat Med 17: 720- 725. Li S, Czubryt MP, McAnally J, Bassel-Duby R, Richardson JA, Wiebel FF, Nordheim A, Olson EN. 2005. Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice. Proc Natl Acad Sci USA 102: 1082-1087. Poulos MG, Batra R, Li M, Yuan Y, Zhang C, Darnell RB, Swanson MS. 2013. Progressive impairment of muscle regeneration in muscleblind-like 3 isoform knockout mice. Hum Mol Genet 22: 3547-3558. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. 2010. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28: 511-515. Wagner SD, Struck AJ, Gupta R, Farnsworth DR, Mahady AE, Eichinger K, Thornton CA, Wang ET, Berglund AJ. 2016. Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy. PLoS Genet 12.

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