Figure S1, eyer et al. Pax7 Myogenin si sitrl Hoechst T = 72h 14 1.8.6.4.2 12 1 8 6 4 2 24h 48h 96h diff. sitrl siset1 212 72h diff. b1 td r t Se km MyH Vinculin Myogenin β-ctin Vinculin MW b1 ka td r t Se 17 45 18 115 5 4 115 Gel 1 ** % apoptotic cells signal (U) Gel 2 Figure S1: is required for accurate skeletal muscle stem cell proliferation. () depletion inhibits MuS proliferation. EL single myofibres were cultured for 72 h following transfection with control (sitrl) or sir (si). For detection of proliferating and differentiating MuSs indirect IF was performed to detect Pax7 (green) and Myogenin (red), respectively. was labelled with Hoechst (blue). Representative myogenic cell clusters are shown. Scale bar = 5 µm. This figure supplements Figure 1E. () protein decreases with progressive differentiation. Quantification of W for as described in Figure 1G. The signal intensity was measured with ImageJ software. Every signal was normalized to the corresponding loading control. () Proliferating 212 myoblasts were transfected and differentiated for 72 h as described in Figure 1H. TUEL assay was performed to detect apoptotic cells, as in Figure F. ata are presented as mean +/- SEM of three independent experiments. For significance Student paired t-test was applied. The difference between the two conditions was not statistically significant. () Overexpression of inhibits expression of muscle differentiation markers. W analysis of, MyH, km and Myogenin was performed in whole cell extracts from 212 cells stably overexpressing or an empty expression vector (tr). ells were proliferating () or differentiated (diff.) for 72 h. Vinculin and β-ctin; loading controls. ote that the shown lanes were cut from the same gels (1 or 2) and have strictly the same exposure time. For and : Images are representative of a minimum of three independent experiments. For and : ata are presented as mean +/- SEM of three independent experiments. For significance Student paired t-test was applied. **: p-values less than.1 and are considered significant.
Figure S2, eyer et al. 12 +/- 12 -/- 12 hip-seq tf7ip2 5 kb 5 kb hip-seq 9 +/- 9 -/- 9 nat 5 4 Log2 enrichment 2 1-1 -2 - pg-island Promoter Exon TTS Intergenic Intron SIE LTR LIE HK9me >5. regions 82 71 Read count Per Million mapped reads.1.2..4.5.6 SET1- HK9me HK9me_212_sort.bam.bam.1.2..4.5.6 SET1- HK27me HK27me_M_SRX1886 HK27me_M_SRX1469.1.2..4.5.6 SET1- HK6me/ K79me HK6me_M_SRX62112 HK6me_M_SRX14618 HK79me_M_SRX14611 2 1 enter 1 2 2 1 enter 1 2 2 1 enter 1 2 Read count Per Million mapped reads.1.2..4.5.6 SET1- HK4me1/ HK4me1_M_SRX6214 HK4me_M_SRX14616.1.2..4.5.6 SET1- HKac Hac_M_SRX1462.1.2..4.5.6 SET1- HK9/18/27ac HK9ac_M_SRX62116 HK18ac_M_SRX62118 HK27ac_M_SRR4965 HK27ac_M_SRX144166 212 2 1 enter 1 2 2 1 enter 1 2 2 1 enter 1 2
Figure S2: omparison of genomic distribution of and histone marks in proliferating myoblasts and hip-seq specificity controls () Genome rowser presentation of binding at the promoter of tf7ip2 and nat, analysed by hip-seq in mouse embryonic stem cells (mes) heterozygous (+/-) or knockout (-/-) for. () Presentation of genomic elements bound by in 212. Enrichments were analysed by hipseq. This figure supplements Figure 2. () omparison of enrichments between various histone modifications and. Presented are hipseq analyses in proliferating myoblasts (M). /HK9me enrichment and input were analysed in proliferating 212 myoblasts. HK27me data were re-analysed from 1 and HK27ac data from 2. Remaining histone modifications were re-analysed from. The scale is +/- 2kb from peak summits and the enrichment corresponds to the normalised number of reads. () Venn iagram showing 82 genes commonly enriched by and HK9me in proliferating 212 myoblasts. ata were analysed by hip-seq: for HK9me enrichment, 2 peak callers were used and merged (MS1.4, p-value <.1; and SIER v..1, e-value <.1). References 1 Mousavi K, Zare H, Wang H, Sartorelli V. Polycomb protein Ezh1 promotes R polymerase II elongation. Molecular cell 212; 45:255-262. 2 lum R, Vethantham V, owman, Rudnicki M, ynlacht. Genome-wide identification of enhancers in skeletal muscle: the role of Myo1. Genes ev 212; 26:276-2779. sp P, lum R, Vethantham V et al. Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proceedings of the ational cademy of Sciences of the United States of merica 211; 18:E149-158.
Figure S, eyer et al. HK9me 2 Relative mr expression 5 kb 2 txn1 2.5 2. * 1.5 1..5 24h 48h 72h diff. 27 kb M HK9me M HK4me1 HK4me2 HK4me HK27ac M MT M MT M MT M MT M R polii MT promoter IgG % input HK9ac.4..2.1.25 1..8.6.4.2 24h 48h diff. 2.5 2. 1.5 1..5 8 7 HK9me 6 5 4 2 1 24h diff..15.1.5 txn1 % input normalized to IgG txn1.2 24h 48h diff. HK27ac 1.8.6.4.2 24h diff. HK4me1.4.5..25.2.15.1.5 24h diff. 24h diff. H G sitrl 12 si % apoptotic cells % input.5 % input normalized to IgG IgG 1.4.6. HK9me 1.6.7 F E % input.8 enhancer 1 8 6 4 2 sitrl si 48h diff.
Figure S: subset of target genes are bound differently during terminal differentiation in myoblasts. () Genome rowser presentation of and HK9me binding profiles at txn1 promoter in proliferating 212 myoblasts analysed by hip-seq. () Relative mr expression analysis of increases during differentiation in primary myoblasts. ells were proliferating () or differentiated (diff.) for the indicated time (24, 48 or 72 h). This figure supplements Figure 2H. () hip-seq representation of and HK9me bindings (both red) at the gene in proliferating 212 myoblasts. inding profiles are compared with published data for HK4me1, HK4me2, HK4me, and R pol II by sp P et al., 211 1. HK27ac binding data are published by lum R. et al., 212 2. HK9me binding data are from. inding was analysed in myoblasts (M) in green and myotubes (MT) in blue. () (left) and HK9me (right) enrichments at the enhancer. 212 cells were proliferating () or differentiating (diff.) for 24 h or 48 h. inding was analysed by hip-qpr. Results are presented as immunoprecipitated compared to input (% input). IgG served as a negative control. Presented data are mean +/- SEM of a minimum of three independent experiments. This figure supplements Figure 2I, J. (E) HK9ac, HK27ac and HK4me1 binding was analysed by hip-qpr at the enhancer. 212 cells were proliferating () or differentiating for 24 h (24h diff.). Results are presented as immunoprecipitated compared to input (% input). Presented data are mean +/- SEM of a minimum of three independent experiments. (F) (left) and HK9me (right) enrichments at the txn1 promoter were analysed by hip and qpr. 212 myoblasts were proliferating () or differentiating (diff.) for 24 h or 48 h. Results are presented as % input and normalised to IgG (negative control). Results are from one experiment as representative of a minimum of three independent experiments. (G) Phase contrast images of 212 myoblasts after knockdown. 8-9% confluent cells were transfected with control sir (sitrl) or sir (si). ifferentiation was started simultaneously for 72 h. Scale bar = 1 µm. (H) Proliferating 212 myoblasts were transfected and differentiated as described in Fig S1. poptotic cells were stained by performing the TdT-mediated dutp-biotin nick end labeling (TUEL) reaction. ells containing a signal inside the nucleus were considered as apoptotic. minimum of 4 cells was counted. References 1 sp P, lum R, Vethantham V et al. Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proceedings of the ational cademy of Sciences of the United States of merica 211; 18:E149-158. 2 lum R, Vethantham V, owman, Rudnicki M, ynlacht. Genome-wide identification of enhancers in skeletal muscle: the role of Myo1. Genes ev 212; 26:276-2779. Mousavi K, Zare H, Wang H, Sartorelli V. Polycomb protein Ezh1 promotes R polymerase II elongation. Molecular cell 212; 45:255-262.
Figure S4, eyer et al. sitrl si Second b 212 PI Merge ontrol 212 212- + LM PI Merge E HeLa PI Merge Homogenous ytoplasmic LM TRL uclear = ytoplasm; = ucleus F Vinculin LM (ng/ml) 4 1 MW ka 17 115 PI Merge G PI Rel Merge ESs MR5 MEFs HeLa 24h diff. + LM 24h diff. H diff. MW ka 17 α-tubulin 5 lanes are from the same gel, same exposure time
Figure S4: uclear and cytoplasmic localisation of. () Sepcificity if the immunofluorescence (IF) signal. ellular (green) was detected by indirect IF in 212 myoblasts either transfected with a control sir (sitrl) or sir (si) (2 left upper panels); or in 212 cells stably overexpressing (212-) or an empty expression vector (control 212) (lower panels). The signal obtained with the secondary antibody is shown (Second b, upper right). () emonstration of different subcellular localisations. Enlargement of 212 myoblasts after indirect IF of (green). was classified as cytoplasmic, homogenous or nuclear if cells had the represented phenotypes. ucleus and cytoplasm are marked as and respectively. was stained with PI (red). nalysis was done by confocal microscopy. Scale bar = 5 µm. () localisation in HeLa, MEFs, MR5 and proliferating ESs. (green) was stained by indirect IF. was stained with PI (red). Scale bar =1 µm. () Leptomycin (LM) changes localisation in proliferating 212 myoblasts. Indirect IF of (green) in cells, non-treated () or treated with LM for 18 h ( + LM). was stained with PI (red). nalysis was done by confocal microscopy. Scale bar = 1 µm. (E) localisation is changed after LM treatment in HeLa cells. Indirect IF of (green) in HeLa cells, non-treated (TRL) or treated with LM for 1 h (LM). was stained with PI (red). Scale bar = 1 µm. (F) global protein levels are not affected by LM. W analysis of in whole cell extracts from 212 myoblasts after 24 h of differentiation. ells were in parallel treated with the indicated concentrations of LM for the last 18 h. Vinculin; loading control. (G) uclear export of Rel is diminished by LM. Indirect IF of Rel (green) in 212 myoblasts after 24 h of differentiation (24 h diff.) and in parallel treated with LM for the last 18 h (24 h diff. + LM). was co-stained with api (red). nalysis was done by confocal microscopy. Scale bar 1 µm. (H) nalysis of in nuclear () and cytoplasmic () fractions by W in the nuclear () versus cytoplasmic () fractions of proliferating 212 myoblasts () and after 24 h of differentiation (diff.). typical experiment is shown. a-tubulin, specific control for the cytoplasmic fraction. ote that the shown lanes were cut from the same gel. ll results are representative of a minimum of three independent experiments.
Figure S5, eyer et al. Hoechst Phalloidin Merge Merge Hoechst Merge -at. active Vinculin Myog. Vinculin tr 212 myoblasts 24h diff. Wnta IWP2 tr Wnta IWP2 MW ka 17 8 115 25 115 -at. active -Tub. MSO Primary myoblasts Wnta IWP2 24h diff. 6h 24h MW ka 17 8 5 45 4 5 25 2 15 1 5 xin 2 tr IWP2 tr IWP2 Wnta Wnta 24h diff. =1 1.4 1..8.6.4.2 tr IWP2 tr IWP2 Wnta Wnta 24h diff. F PI HeLa cells Merge G PI HeLa cells Merge Wnta IWP2 TRL TRL + Wnta 24h diff. + IWP2 24h diff. E relative mr expression cnd1 H Hoechst Merge 24h diff.
Figure S5: cellular localisation is dependent on Wnta signalling. () localisation changes with increased Wnta signalling in proliferating primary mouse myoblasts. ells were non-treated () or stimulated with Wnta protein for 24 h ( + Wnta). Indirect IF of (green) and confocal analysis was performed. Phalloidin (red) was used to co-stain for actin filaments and Hoechst to visualize (blue). Scale bar = 1 µm. () Inhibition of Wnt signalling restricts delocalisation in primary myoblasts. ells were differentiated for 24 h and simultaneously treated with IWP2. IF was performed as described in (). (), active b-atenin and Myogenin protein levels were analysed in whole cell extracts of proliferating () and 24 h differentiating (24 h diff.) 212 myoblasts. ells were treated in parallel with Wnta or IWP2 for 24 h. Vinculin; loading control. () and active b-atenin protein levels were analysed in whole cell extracts from proliferating () or 24 h differentiating (24h diff.) MuS-derived primary mouse myoblasts. ells were treated simultaneously with MSO, Wnta or IWP2 for 24 h. α-tubulin served as loading control. (E) xin2 and cnd1 relative mr expression analysis in 212 myoblasts. ells were treated and cultured as described in. ata are represented as fold change relative to proliferation values and normalised to yclo and TP. Presented data are mean +/- SEM of a minimum of three independent experiments. (F) localisation changes with increased Wnta signalling in HeLa cells. ells were non-treated (TRL) or treated with Wnta for 24 h (Wnta). Indirect IF of (green) was performed. was stained with PI (red). Scale bar = 1 µm. (G) delocalisation is restricted when Wnt signalling is inhibited in HeLa cells. Indirect IF of (green) in cells, non-treated (TRL) or treated with IWP2 for 24 h (IWP2). was stained with PI (red). Scale bar = 1 µm. (H) b-atenin translocates to the nucleus during differentiation of primary myoblasts. ells were proliferating () or differentiating for 24 h. Indirect IF of b-atenin (green) was performed. was visualized by Hoechst. Scale bar = 2 µm. For -, F - H: Images are representatives of a minimum of three independent experiments.
Figure S6, eyer et al. hip 2 ontrol 2 Wnta txn1 5 kb Primary myoblasts 212 myoblasts Relative mr expression (%) 2 1 * TRL Wnta relative mr expression 1.8 1.6 1.4 1..8.6.4.2 * TRL Wnta IWP2 1.6 Ttn 6 MMP1 Relative mr expression 1.4 1..8.6.4.2 Relative mr expression 5 4 2 1 sitrl si sitrl si Figure S6: Wnta changes occupancy of at certain target genes. () Genome rowser presentation of binding profile at txn1 promoter in proliferating 212 myoblasts non-treated (ontrol) or treated with Wnta for 24 h (Wnta). () mr increases in differentiating mouse primary myoblasts additionally treated with Wnta. ells were differentiated for 24 h and simultaneously treated with Wnta. () mr changes in differentiating 212 myoblasts when Wnt signalling is additionally stimulated or inhibited. ells were differentiated and simultaneously treated with Wnta or IWP2 for 24 h. () knockdown in proliferating myoblasts decreases Ttn and increases MMP1 mr level. Proliferating 212 myoblasts were transfected with control sir (sitrl) or sir (si), as described for the R-seq assay. Relative mr expression levels of MMP1 and Ttn quantified by the normalized number of read counts for their transcripts in the R-seq data. For and : ata are represented as fold change relative to proliferation values and normalised to yclo. Presented data are mean +/- SEM of a minimum of three independent experiments. *: p-values less than.5 and are considered significant.