bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Transcriptome and epigenome diversity and plasticity of muscle stem cells following

2 transplantation

3

1,2* 3* 3* 1,2 4 Authors: Brendan Evano , Diljeet Gill , Irene Hernando-Herraez , Glenda Comai , Thomas M.

3,4 5 3 # 1,2 # 5 Stubbs , Pierre-Henri Commere , Wolf Reik , Shahragim Tajbakhsh

6

7 Affiliations:

1 8 Stem Cells & Development, Department of Developmental & Stem Cell Biology, Institut Pasteur, 25

9 rue du Dr. Roux, 75015, Paris, France.

2 10 CNRS UMR 3738, Institut Pasteur, Paris 75015, France.

3 11 Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, United Kingdom.

4 12 Current address: Chronomics Limited, 1 St James Court, Norwich NR3 1RU.

5 13 Cytometry and Biomarkers, Center for Technological Resources and Research, Institut Pasteur, 28

14 rue du Dr. Roux, 75015, Paris, France.

15

* 16 These authors contributed equally.

# 17 These authors contributed equally.

18

19 Correspondence to [email protected] and [email protected]

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20 ABSTRACT

21 Adult skeletal muscles are maintained during homeostasis and regenerated upon injury by muscle stem

22 cells (MuSCs). A heterogeneity in self-renewal, differentiation and regeneration properties has been

23 reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular

24 muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the

25 molecular determinants that govern these differences remain undefined. Here we show that EOM and

26 limb MuSCs have distinct DNA methylation signatures associated with enhancers of location-specific

27 genes, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle

28 environment. Notably, EOM MuSCs expressed host-site specific positional Hox codes after engraftment

29 and self-renewal within the host muscle. However, about 10% of EOM-specific genes showed

30 engraftment-resistant expression, pointing to cell-intrinsic molecular determinants of the higher

31 engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC

32 populations and molecularly define their plasticity in response to microenvironmental cues. These

33 findings provide insights into strategies designed to improve the functional capacity of MuSCs in the

34 context of regenerative medicine.

35

36 INTRODUCTION

37 Skeletal muscles are essential for physiological functions such as locomotion, breathing, and

38 metabolism, and they represent up to 40% of the human body mass. Tissue-specific muscle stem cells

1–3 39 (MuSCs) ensure skeletal muscle homeostasis and regeneration . MuSCs have been implicated in the

4,5 6–11 40 etiology of some muscular dystrophies as well as age-associated impaired muscle regeneration

12–15 41 leading to an incapacitating decrease of muscle mass and strength . Stem-cell therapies have

42 proven to be challenging for muscular dystrophies, as they require delivering enough functional MuSCs

43 to the right muscle groups and ex vivo amplification of healthy MuSCs results in a major decline in their

16 44 regenerative capacity and stemness properties .

45

46 Most of the knowledge about MuSC biology arises from the study of limb muscles, whereas MuSCs

47 from other muscle groups remain less well characterized. Extraocular muscles (EOMs) are responsible

17 48 for eye movements, with the basic pattern of 6 muscles conserved in most vertebrate species . Limb

18–20 49 muscles derive from the somitic mesoderm and rely in part on Pax3 expression and function . In

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50 contrast, EOMs derive from cranial mesoderm and rely initially on Mesp1 and Pitx2 for their emergence,

21,22 51 yet unlike the majority of other cranial-derived muscles, their founder stem cell population arises

17,23–29 52 independently of Tbx1 function . After the distinct specification of cranial and trunk progenitors, the

53 core myogenic regulatory factors Myf5, Mrf4, Myod, and Myogenin regulate myogenic cell commitment

30 54 and differentiation . In adult homeostatic muscles, MuSCs are mostly quiescent. They are activated

55 upon muscle injury, proliferate and differentiate to contribute to new muscle fibers, or self-renew to

31–34 56 reconstitute the stem cell pool . This process is accompanied by a temporal expression of cell fate

57 markers, such as the transcription factors Pax7 (stem), Myod (commitment) and Myogenin

58 (differentiation). Several reports indicate that EOMs are functionally different from their hindlimb

35–38 59 counterparts since they are preferentially spared in ageing and several muscular dystrophies .

60 Interestingly, adult EOM-derived MuSCs cells have superior in vitro proliferation, self-renewal and

61 differentiation capacities, as well as a superior in vivo engraftment potential, compared to limb-derived

39 39 62 MuSCs . These properties were maintained by EOM-derived MuSCs from dystrophic or aged mice .

63 However, the unique functional properties of adult EOM MuSCs remain undefined at the molecular level,

64 as well as whether their specificity is instructed by cell-intrinsic factors or through interactions with their

65 microenvironment (niche).

66

67 DNA methylation is a critical epigenetic mechanism involved in establishing and maintaining cellular

68 identity. Dynamic changes in expression of DNA (de)methylation enzymes were reported in MuSCs

40–42 69 during muscle regeneration , indicating a potential increase of DNA methylation from quiescent to

70 activated MuSCs. Several studies investigated the DNA methylation signatures of proliferating and

43–45 71 differentiating cultures of MuSCs and reported some changes in DNA methylation patterns during

72 late myogenic differentiation. Further, aged MuSCs show increased DNA methylation heterogeneity at

46 73 promoters, associated with a degradation of coherent transcriptional networks . However, whether

74 quiescent MuSCs from homeostatic muscles at different anatomical locations display similar or different

75 DNA methylation patterns remains unknown.

76

77 Here we performed parallel DNA methylation and transcriptome sequencing to characterize the specific

78 identity of adult mouse EOM and limb MuSCs at the molecular level. We used heterotopic

79 transplantation of EOM MuSCs into limb muscles to challenge their fate and assess their plasticity. We

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80 show that their specific identity is mostly niche-driven as they adopt a limb-like molecular signature.

81 Nevertheless, we also identify EOM-specific genes that resist reprogramming by the microenvironment,

82 indicating potential candidates to manipulate in limb-derived MuSCs for improving their regenerative

83 capacity in the context of cellular therapies.

84

85 RESULTS

86 To investigate the molecular differences between EOM and TA MuSCs in vivo, MuSCs from both

17 87 locations were isolated by FACS (Fluorescence-Activated Cell Sorting) from 10 adult Tg:Pax7-nGFP

47,48 88 mice and processed for RNA-sequencing and BS-sequencing as in (Figure 1A). Unsupervised

89 Principal Component Analysis (PCA) on the transcriptomes revealed clear discrimination between TA

90 and EOM MuSCs samples, indicating location-specific transcriptional identities (Figure 1B). To further

91 investigate these transcriptional signatures, we performed differential expression analysis and found

92 261 genes significantly upregulated in EOM MuSCs (EOM Differentially Expressed Genes, DEGs) and

93 339 genes significantly upregulated in TA MuSCs (TA DEGs) (DE-seq, P <0.05, FC >2) (Figure 1C).

94 The muscle stem cell marker Pax7 and the myogenic factors Myf5 and Myod showed slightly higher

95 expression levels in TA compared to EOM MuSCs, while the transcription factor Pitx2 showed higher

49 96 expression levels in EOM MuSCs (Figure 1D). As reported , Pax3 was expressed in TA MuSCs and

97 not detected in EOM MuSCs. Other genes specifically up-regulated in TA MuSCs included the

98 developmental factors Lbx1 and Tbx1. Lbx1 is a homeobox transcription factor required for the migration

99 of myogenic progenitor cells to the limbs, and Tbx1 is a T-box containing transcription factor necessary

50–52 100 for the development of craniofacial muscles . Examples of EOM up-regulated genes included the

30,53,54 55 101 homeobox transcription factors Lmx1a and Alx4 as well as Mobp (involved in myelination )

102 (Figure 1D). Further, genes of the Hox gene family were upregulated in TA samples (Figure 1E),

56 103 consistent with their anteroposterior expression pattern in vertebrates . Overall, 13 Hox genes were

104 upregulated, representing 64% of the HoxA cluster and 75% of the HoxC cluster. Notably, these Hox

57 105 genes are expressed more posteriorly along the body axis . Overall, differentially expressed genes

106 were enriched for developmental processes suggesting that location-specific patterns reflect their

107 different origins during embryogenesis (Supplementary Figure 1).

108 Overall, global DNA methylation levels were similar between EOM and TA MuSCs, with EOM MuSCs

109 showing slightly higher levels of DNA methylation relative to TA MuSCs. On average genome-wide DNA

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110 methylation levels were around 50%, promoters and enhancers were hypomethylated (25% and 30%

111 respectively) and repeat elements were hypermethylated (85%) (Figure 2A). We restricted our more

58 112 detailed analysis to enhancers (regions marked by H3K27Ac in MuSCs that did not overlap promoters)

113 and promoters (-2000bp to 500bp of the TSS of Ensembl genes) and calculated DNA methylation levels

114 over each genomic element. PCA analysis for promoter regions showed no clear differences between

115 TA and EOM MuSCs (Figure 2B). In contrast, a clear separation by anatomical location was observed

116 when considering enhancer regions (Figure 2C). This clustering was also observed when restricting the

117 analysis to 544 enhancers associated with TA and EOM DEGs (enhancer regions within 1 Mb to the

118 closest transcription start site, Supplementary Figure 2A). Furthermore, enhancers associated with

119 genes specifically upregulated in TA MuSCs were less methylated in TA MuSCs compared to EOM

120 MuSCs. Similarly, enhancers associated with genes upregulated in EOM MuSCs were less methylated

121 in EOM MuSCs compared to TA MuSCs (Figure 2D). To examine which enhancers were responsible

122 for shifts in the distributions, we determined which enhancers were significantly differentially methylated

123 using a rolling Z-score. Not all of the enhancers associated with TA or EOM DEGs were classified as

124 differentially methylated, suggesting only a subset of these genes was regulated by enhancer

125 methylation (Figure 2E). We also identified 29 Differentially Methylated Regions (DMRs) with more than

126 5 consecutive CpG methylation sites and 10% methylation differences. The majority of the DMRs

127 overlapped enhancer or promoter regions such as the DMR overlapping the Tbx1 promoter which is

128 hypomethylated in TA MuSCs (Mean methylation levels in TA: 13%; mean methylation levels in EOM:

129 80%). These results suggest that DNA methylation patterns at regulatory regions contribute to the

130 location-specific transcriptional profile of MuSCs.

131

132 To investigate MuSC plasticity and the influence of the cellular microenvironment on the observed

133 location-specific signatures, we performed heterotopic transplantations of EOM MuSCs into TA

134 muscles. Specifically, EOM MuSCs from Tg:Pax7-nGFP mice were transplanted into pre-injured TA

-/- -/- 59 135 muscles of immunodeficient Rag2 ;gC mice. As a control, and for each donor mouse, TA MuSCs

136 were transplanted into the same recipient mouse, in the contralateral pre-injured TA muscle. After 28

137 days when muscle regeneration is complete and self-renewal of MuSCs can be evaluated, engrafted

138 EOM and TA MuSCs were re-isolated by FACS based on GFP positivity (post-graft samples).

139 Additionally, a fraction of the EOM and TA MuSCs before grafting was kept as a control (pre-graft

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140 samples). Pre-graft and post-graft EOM and TA MuSCs were then processed for RNA-sequencing and

141 BS-sequencing (Figure 3A). A total of 10 donor mice were analysed (Supplementary Table 1). We first

142 observed important transcriptome differences between pre and post-graft MuSCs (Supplementary

143 Figure 3A). Importantly, although muscle regeneration is generally considered to be largely achieved

60 144 within 28 days post-injury , transcriptome analysis showed substantial differences between pre-graft

145 and post-graft TA MuSCs (Supplementary Figure 3B), indicating that our transplantation of MuSCs per

146 se had a direct effect on gene expression. Genes upregulated after transplantation of TA MuSCs were

147 enriched for immunological processes (Supplementary Figure 3C), including genes encoding interferon-

148 induced proteins and complement proteins. However, as expected for this time point, the myogenic

149 differentiation marker Myogenin was downregulated in the post-graft cells, indicating that cells at the

150 time of re-isolation were not undergoing active myogenic commitment or differentiation. We also

151 observed a slight global increase in the DNA methylation levels after transplantation (mean TA pre-graft:

152 56.6%, mean TA post-graft: 59.2%; Supplementary Figure 3D). Whether these observations reflect

153 molecular changes with slow kinetics on MuSCs during muscle regeneration or are the result of the

154 adaptation of engrafted MuSCs to an immunodeficient environment remains to be explored. The latter

155 is favoured as recent RNA-seq analyses of MuSCs during regeneration showed that MuSCs re-acquire

61 156 a quiescent homeostatic transcriptome as early as 7 days post-injury .

157

158 To evaluate exclusively the response of EOM MuSCs to a heterologous microenvironment while setting

159 aside modifications in transcriptome due to the transplantation process, we calculated a correction

160 coefficient based on post-graft TA samples and applied it to the transcriptome of post-graft EOM and

161 post-graft TA samples (see Methods). As expected, the corrected post-graft TA MuSCs clustered with

162 pre-graft TA MuSCs (Supplementary Figure 3E). Post-graft EOM MuSCs were then corrected similarly

163 and hereafter compared to pre-graft EOM and TA MuSCs (Figures 3B to 3F). We observed a global

164 shift in the transcriptome of post-graft EOM MuSCs towards a TA profile (Figure 3B). The majority of

165 TA-specific genes were upregulated in post-graft EOM MuSCs while EOM-specific genes were

166 downregulated (Figure 3B), indicating that a large proportion of the transcriptome was remodeled by the

167 cellular microenvironment. To further investigate these changes, we performed PCA analysis using

168 location-specific DEGs and observed that post-graft EOM MuSCs tended to cluster closer to pre-graft

169 TA MuSCs than to pre-graft EOM MuSCs (PC1, Figure 3C). The higher similarity of the post-graft EOM

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170 samples to pre-graft TA MuSCs was further corroborated by transcriptomic correlation and hierarchical

171 clustering analysis (Supplementary Figure 3F). We then classified location-specific genes in post-graft

172 EOM MuSCs according to their degree of change into three categories: responsive, intermediate, and

173 resistant (Figure 3D left). Genes were classified as resistant if they maintained their initial expression

174 level in EOM MuSCs post-graft, while genes were classified as responsive if they adopted a TA-like

175 expression level in EOM MuSCs post-graft. More specifically, responsive genes included EOM-specific

176 genes fully downregulated to pre-graft TA level or TA-specific genes fully upregulated to pre-graft TA

177 level; intermediate genes were those whose expression levels were between pre-graft EOM and TA

178 samples; and resistant genes included EOM-specific genes remained at pre-graft EOM level or TA-

179 specific genes remained at pre-graft EOM level. In agreement with the observed global shift of the

180 transcriptome (Figure 3B), the majority of the location-specific genes showed a responsive or

181 intermediate profile (~90% of the EOM-specific DEGs and ~80% of TA-specific DEGs) while only around

182 10-20% of DEGs were resistant after transplantation (Figure 3D right). Importantly, post-graft EOM

183 MuSCs upregulated the TA-specific markers Pax3, Tbx1 and Lbx1 while they down-regulated the EOM-

184 specific markers Alx4, Lmx1a and Mobp (Figure 3E), confirming their acquisition of a limb-like

185 transcriptional identity. Of note, the EOM-enriched Pitx2 gene was resistant to transplantation (Figure

186 3E). Strikingly, all TA-specific Hox genes were upregulated in post-graft EOM MuSCs, with intermediate

187 or responsive behaviours (Figure 3F). This observation is in agreement with previous reports showing

62,63 188 that Hox-negative tissues or cells adopt the Hox status of their new location upon grafting .

189

190 We then followed a similar approach to investigate the response of the epigenome to the different

191 cellular microenvironments in the absence of confounding factors. We first calculated a correction

192 coefficient based on the average DNA methylation differences between the methylomes of pre-graft and

193 post-graft TA MuSCs using windows covering the promoter and enhancer regions and applied it to the

194 methylome of the post-graft MuSCs. The correction coefficient was applied to promoters and enhancers

195 of post-graft TA and EOM MuSCs. After correction, we performed PCA analysis considering promoters

196 of location-specific genes (DEGs). Consistent with our previous results (Figure 2B), we observed no

197 substructure of the data, suggesting no clear changes in DNA methylation at the promoter level (Figure

198 4A). However, a clear pattern was observed at enhancer regions associated with EOM and TA DEGs.

199 PCA and clustering analysis of these regulatory regions showed that post-graft EOM MuSCs clustered

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200 closer to TA samples than to EOM samples (Figure 4B and Supplementary Figure 4A) indicating that

201 enhancer regions undergo global reprogramming after transplantation in response to the cellular

202 microenvironment. Overall, enhancers associated with EOM DEGs gained DNA methylation whereas

203 those associated with TA DEGs lost DNA methylation in post-graft EOM MuSCs (Figure 4C). These

204 epigenetic changes could contribute to some of the observed gene expression changes upon

205 heterotopic transplantation of EOM MuSCs. Some examples include the EOM DEGs Eya2 and Rtn1,

206 which gain DNA methylation at their associated enhancer and become repressed, and the TA DEGs Cit

207 and Enpep which lose DNA methylation at their associated enhancer and become more expressed in

208 the post-graft EOM MuSCs (Figure 4D). Interestingly, the TA-specific genes Lbx1 and Pax3 show

209 moderate changes of methylation at their associated enhancers while they become more expressed,

210 indicating that additional mechanisms regulate changes of expression observed in EOM MuSCs upon

211 heterologous transplantation. Of note, changes of enhancer methylation were also observed for some

212 genes showing little variation in expression upon grafting, as for Myod and Pitx2 (Supplementary Figure

213 4B). Interestingly, the HoxA gene cluster acquired a DNA methylation pattern resembling that of TA

214 MuSCs (Figure 4E), associated with an overall increased expression level (Figure 3F). The other Hox

215 clusters did not show much change in their DNA methylation patterns upon heterologous transplantation

216 (Supplementary Figure 4C). This was not unexpected as EOM and TA MuSCs were found to have

217 similar DNA methylation patterns across these regions. For the HoxC cluster, other mechanisms may

218 be responsible for the expression difference observed between EOM and TA MuSCs (Figure 1E) and

219 the gain of expression seen in EOM MuSCs after heterologous transplantation (Figure 3F). For the HoxB

220 and HoxD clusters, the relatively low methylation levels across these regions may be responsible for the

221 lack of expression in EOM MuSCs, TA MuSCs, and EOM MuSCs after heterologous transplantation

222 (Figures 1E and 3F).

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223 DISCUSSION

224 Here we examined the combined transcriptional and DNA methylation signatures of head (EOM) and

225 limb (TA)-derived MuSCs to assess the molecular determinants associated with their diversity and

226 inform on their functional differences such as susceptibility to disease and regenerative capacity. In this

227 context, we also investigated the extent to which a head-specific identity was determined cell-

228 autonomously through heterotopic transplantation into a limb environment, followed by transcriptome

229 and DNA methylation profiling of re-isolated cells.

230

231 Consistent with previous reports, we identified known EOM (e.g. Alx4, Pitx2) and TA (e.g. Pax3, Lbx1,

232 Hox genes)-specific genes. Surprisingly, we observed expression of HoxA and HoxC genes, while limb

233 development has been reported to rely mostly on HoxA and HoxD clusters, with minor contributions

64 234 from the HoxB and HoxC clusters . However, we detected expression of Hoxa11 but not Hoxa13 in TA

64 235 MuSCs, consistent with their differential roles in the proximal-to-distal patterning of the limb .

236 Interestingly, we identified Tbx1 as a TA-specific gene, although it is required for specification of

30 237 branchial-arch derived craniofacial muscles . This observation might also result from the different fiber-

238 type composition of EOM and TA muscles and the specific properties of myogenic progenitors derived

65 239 from different muscle fiber-types . Additionally, EOM and TA-specific genes were enriched for

240 developmental processes, which might reflect the persistence of molecular differences acquired during

241 embryogenesis through adulthood. When and to what extent the transcriptomes of head and limb

242 muscle progenitors initially diverge during embryogenesis remains to be addressed.

243

66 67 244 While several studies investigated the DNA methylation patterns of specific loci (Myod , Myogenin ,

68 69 70 43–45,71–77 245 Desmin , Six1 , α-smooth muscle actin ) or genome-wide during myogenic specification or

246 myoblast differentiation in vitro, we report, to the best of our knowledge, the first genome-wide DNA

247 methylation profiling of pure adult quiescent MuSCs. The adult quiescent MuSCs we analysed here

78 248 have much lower global methylation levels (~50%) than somatic cells (~70%) , notably muscle fibers

79 80 249 (~75%) , and other adult stem cells such as intestinal stem cells (78%) and hematopoietic stem cells

81 250 (84%) . Whether such a low methylation level is required for the stemness and quiescence properties

251 of adult MuSCs remains to be explored, as well as the dynamics of DNA methylation between MuSCs

252 and mature myofibers. Our analysis identified some distinct DNA methylation profiles between EOM and

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253 TA MuSCs, notably at enhancers associated with location-specific genes. These results suggest that

254 location-specific transcriptome signatures are determined by location-specific DNA methylation

255 patterns. We are not aware of any previous studies reporting a spatial address of enhancer methylation.

256 It is conceivable therefore that the enhancer epigenome not only registers developmental and tissue-

257 specificity but also the location (along the anterior-posterior axis in this case). Previous reports analysed

82,83 258 DNA methylation patterns of similar tissue samples across several anatomical locations , where

259 changes in cellular composition might be a confounding factor. We report here for the first time the co-

260 analysis of DNA methylome and transcriptome of pure populations of tissue-specific adult stem cells

261 between different anatomical locations.

262

263 Our analysis of EOM and TA MuSCs re-isolated after engrafting each population into injured TA muscle

264 followed by regeneration revealed a global reprogramming of EOM transcriptome towards a TA MuSC

265 transcriptome, indicating that the extracellular environment strongly determines the location-specific

84 266 signatures we identified. This observation is in agreement with a previous report , indicating that stem

267 cell-derived myogenic progenitors remodel partially their molecular signature towards an adult quiescent

268 MuSC transcriptome upon in vivo engraftment. Importantly, our data show that TA MuSCs re-isolated

269 after engrafting and regeneration display important transcriptome differences with their pre-grafting TA

270 MuSCs counterparts. This suggests that comparing engrafted cells re-isolated after regeneration to

84 271 adult quiescent MuSCs could underestimate the extent of transcriptome plasticity driven by the in vivo

272 environment.

273

274 Significantly, we identified different classes of EOM and TA-specific genes in EOM MuSCs depending

275 on their behaviour following in vivo engraftment: resistant, responsive, and intermediate. Genes with

276 intermediate responses could reflect i) the stable acquisition of new intermediary expression states due

277 to buffering from initial identity, ii) the existence of transcriptional changes with slow kinetics towards full

278 reprogramming or iii) the existence of different subpopulations of EOM MuSCs with variable

279 reprogramming efficiency. The first two hypotheses are favored due to the long-term transcriptional

280 changes associated with our experimental procedure and the existence of fully-responsive genes,

281 indicating a complete reprogramming of such genes in each cell.

282

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283 Transplantation of cranial-derived stem cells allowed us to assess positional information, given that EOM

284 MuSCs lack expression of Hox genes. Interestingly, we observed that all TA-specific Hox genes were

285 upregulated in EOM engrafted MuSCs, reflecting the acquisition of the Hox status of their new location,

63,85 286 as observed previously after transplantation of Hox-negative cells in a Hox-positive domain and

86 287 unlike myoblasts maintaining their axial identity in culture , indicating that factors derived from the in

288 vivo environment might determine the plasticity of the Hox status. Whether the initial Hox-free status of

289 EOM MuSCs is responsible for their superior regenerative capacity over Hox-positive limb-derived

290 MuSCs remains to be determined. The molecular mechanisms of the Hox expression and methylation

291 plasticity in adult stem cells remain to be identified, and their manipulation could be of interest in the

87 292 context of regenerative medicine .

293

294 We speculate that the limited set of EOM resistant genes could contribute to a niche-independent and

295 cell-intrinsic high engraftment potential of EOM MuSCs. Interestingly, these genes include Pitx2 and

296 genes involved in thyroid hormone signalling (Dio2, encoding the type-II iodothyronine deiodinase, a

297 thyroid hormone activator, and the Tsh hormone receptor Tshr). Of note, Pitx2 overexpression was

88 298 shown to increase the regenerative capacity of MuSCs and Pitx2;Pitx3 double mutant mice have

89 299 impaired muscle regeneration upon injury , while thyroid hormone signalling was reported to be critical

90,91 300 for MuSC survival and muscle regeneration . Therefore, EOM resistant genes could be of interest as

301 deterministic candidate regulators of EOM MuSC identity. Such determinants could be exploited for

39,92,93 302 improving cellular therapy of muscular dystrophies using the abundant limb MuSCs.

303

304 Finally, we identified plastic DNA methylation patterns in engrafted EOM MuSCs, notably at enhancers

305 associated with location-specific genes and at the HoxA gene cluster. Importantly, EOM enhancers were

58 306 annotated based on H3K27ac ChIP data from limb-derived MuSCs , which might not fully represent

307 actual enhancers in EOM MuSCs. These observations indicate that cell-extrinsic cues are relayed

308 through specific modifications of DNA methylation patterns to establish a MuSC transcriptome profile

309 matching the new location. Future studies will be required to identify key molecular determinants of this

310 interplay between the niche, the epigenome, and the transcriptome.

311

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312 In summary, we report the molecular profiling of two populations of adult MuSCs which were reported

313 to exhibit distinct regenerative capacities and disease susceptibility. We identify differences in the

314 methylation of enhancers and demonstrate plasticity upon exposure to a new microenvironment.

315 Therefore, the molecular characterization and functional analysis of MuSCs from multiple muscles and

316 physiopathological conditions will be highly informative for a more complete understanding of muscle

317 stem cell heterogeneity and plasticity.

318

319 FIGURES

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3 10 A Tg:Pax7-nGFP Figure 1 2 Extraocular muscles 10 (EOM) 1 MuSCs isolation 10

0 RNA/BS-seq 10

Tibialis PE fluorescence -1 anterior 10

(TA) -1 0 1 2 10 10 10 10 GFP fluorescence

Gene expression EOM vs TA MuSCs Gene expression EOM vs TA MuSCs MuSC Genes Regional Genes B C D Location *** *** * EOM EOM specific *** Location 10 TA EOM No significant difference 100 *** TA TA specific *** *** *** * 40 5

0 PC2 (16%) Hoxc10 (p-value) ransformed Counts 0 10 T g Eya2 Tbx1 Hoxa10 2 o

L 20 Lmx1a Myod Log -100 Lbx1 Hoxc9 -100 -50 0 50 100 Alx4 Hoxa7 Pax7 Pitx2 Myf5 Myod Alx4 Mobp Lbx1 Pax3 Tbx1 PC1 (18.5%) Pax7 Mobp Hoxa9 Pitx2 Enpep Cit 0 Rtn1 Myf5 Pax3

-10 -5 0 5 10 Log2 Fold Change

Gene expression in EOM MuSCs Gene expression in TA MuSCs E 7.5 HoxA HoxB HoxC HoxD 7.5 HoxA HoxB HoxC HoxD

5.0 5.0

2.5 2.5

0.0 0.0 Transformed Counts Transformed Counts Transformed 2 2 g g o o L L

-2.5 -2.5

Hoxa1Hoxa2Hoxa3Hoxa4Hoxa5Hoxa6Hoxa7Hoxa9 Hoxb1Hoxb2Hoxb3Hoxb4Hoxb5Hoxb6Hoxb7Hoxb8Hoxb9 Hoxc4Hoxc5Hoxc6Hoxc8Hoxc9 Hoxd1Hoxd3Hoxd4Hoxd8Hoxd9 Hoxa1Hoxa2Hoxa3Hoxa4Hoxa5Hoxa6Hoxa7Hoxa9 Hoxb1Hoxb2Hoxb3Hoxb4Hoxb5Hoxb6Hoxb7Hoxb8Hoxb9 Hoxc4Hoxc5Hoxc6Hoxc8Hoxc9 Hoxd1Hoxd3Hoxd4Hoxd8Hoxd9 Hoxa10Hoxa11Hoxa13 Hoxb13 Hoxc10Hoxc11Hoxc12Hoxc13 Hoxd10Hoxd11Hoxd12Hoxd13 Hoxa10Hoxa11Hoxa13 Hoxb13 Hoxc10Hoxc11Hoxc12Hoxc13 Hoxd10Hoxd11Hoxd12Hoxd13

320 Figure 1. Head and limb-derived MuSCs display specific transcriptome signatures.

321 (A) Experimental scheme. Head and limb MuSCs were isolated by FACS from EOM and TA muscles

322 respectively from Tg:Pax7-nGFP adult mice and processed through RNA- and bisulfite-sequencing.

323 N=10 mice, n=500 cells/mouse (Supplementary Table 1).

324 (B) PCA analysis of EOM and TA MuSCs transcriptomes. The first principal component separates

325 samples based on their anatomical location.

326 (C) Volcano plot showing the results of differential expression analysis of EOM and TA MuSCs. Genes

327 that demonstrated a fold change greater than 2 and a p-value less than 0.05 according to DESeq2 were

328 classified as differentially expressed and colour coded by their tissue specificity.

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329 (D) Selected markers and differentially expressed genes between EOM and TA MuSCs. Error bars

330 represent the standard deviation of the mean. p-values were determined by DESeq2. *** p < 0.001, **

331 p < 0.01, * p < 0.05.

332 (E) Gene expression analysis of throughout the HoxA, HoxB, HoxC and HoxD clusters in EOM (left) and

333 TA (middle) MuSCs. Genes were color-coded according to their antero-posterior expression domain in

57 334 mouse at embryonic day 12.5 (right, adapted from ).

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100 100 100 100 Figure 2 A Whole Genome IAP Promoters MuSCs Enhancers

75 75 75 75

50 50 50 50

25 25 25 25 Mean DNA Methylation level (%) Mean DNA Mean DNA Methylation level (%) Mean DNA Methylation level (%) Mean DNA

Mean DNA methylation level (%) Mean DNA 0 0 0 0 EOM TA EOM TA EOM TA EOM TA

B DNA methylation at all Promoters C DNA methylation at all Enhancers D ** * 0.4 0.03 0.2 Enhancers associated with: EOM-specific genes 0.2 TA-specific genes 0.0 0.02 Random genes PC2 (8.3%) PC2 (7.7%) 0.0

-0.2 Density

-0.2 0.01 -0.4 -0.25 0.00 0.25 0.50 -0.2 0.0 0.2 0.4 PC1 (24.6%) PC1 (15.3%) Location Location

EOM EOM 0.00 TA TA -40 0 40 Methylation difference (EOM - TA)

E Enhancers associated with EOM DEGs Enhancers associated with TA DEGs Sorbs2 Pitx2

75 75 Fam129b Me3 Rbm20 Enpep Glis3 Agap1 Thsd4 Significant Nr4a3 Vgll2 Significant 50 50 Tac4 Adamtsl1 Yes Plekha3 Yes St6galnac3 No No Adam19 Plk2 Atp2b1 in TA MuSCs (%) TA in

Adamtsl1 MuSCs (%) TA in Ttc7b 25 Foxp1 25 Gm13889 Sestd1 Cit DNA methylation level DNA Prex1 methylation level DNA Islr Gm20398 Lpar1 Sept8 Arrdc2 Rbm20 0 0 Fam198b

0 25 50 75 0 25 50 75 100 DNA methylation level DNA methylation level in EOM MuSCs (%) in EOM MuSCs (%)

335 Figure 2. Head and limb-derived MuSCs display specific DNA methylation patterns at enhancers.

336 (A) Mean DNA methylation levels of EOM and TA MuSCs across the whole genome, IAP repeat

337 elements, promoters and enhancers. Promoters were defined as -2000bp to 500bp of the TSS of

58 338 Ensembl genes. H3K27ac peaks were called using macs2 on H3K27ac ChIP-seq data obtained from .

339 Enhancers were defined as H3K27ac peaks that did not overlap promoters. Enhancers were linked to

15 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

340 genes based on proximity. Overall DNA methylation was similar between the two locations, though EOM

341 MuSCs had slightly higher levels of DNA methylation relative to TA MuSCs.

342 (B) PCA analysis of DNA methylation at promoters fails to separate EOM and TA samples based on

343 their anatomical location.

344 (C) PCA analysis of DNA methylation at enhancers separates EOM and TA samples based on their

345 anatomical location.

346 (D) Density plots showing methylation differences between EOM and TA MuSCs at enhancers

347 associated with location-specific genes (enhancers regions within 1 Mb to the closest transcription start

348 site of a DEG). Enhancers associated with EOM genes (n=198) were significantly hypomethylated in

349 EOM MuSCs relative to TA MuSCs when compared to a random subset of enhancers (n=350), while

350 enhancers associated with TA genes (n=346) were significantly hypermethylated in the same

351 comparison. ** p < 0.01, * p < 0.05 by Welch’s t test.

352 (E) Scatter plots comparing the mean DNA methylation levels of enhancers associated with location

353 DEGs in EOM and TA MuSCs. Enhancers were colour coded as significant if they were also found to

354 be differentially methylated by a rolling Z score approach (p < 0.05) when comparing the methylation

355 levels of all enhancers in EOM and TA MuSCs (Supplementary Figure 2B). The significant enhancers

356 have been labelled with their associated gene. Only a subset of enhancers associated with location

357 DEGs were differentially methylated. More enhancers associated with EOM DEGs were

358 hypermethylated in TA MuSCs than in EOM MuSCs. Likewise, more enhancers associated with TA

359 DEGs were hypermethylated in EOM MuSCs than in TA MuSCs.

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 3

Donor Tg:Pax7-nGFP Extraocular -/- -/- 3 Rag2 :ΥC 3 A muscles 10 10

2 2 10 10

1 1 10 10 escence Muscle escence r Injured TAs r

0 0 10 10 PE fluo PE fluo

-1 -1 10 10

-1 0 1 2 -1 0 1 2 Tibialis anterior 10 10 10 10 10 10 10 10 muscles FITC fluorescence FITC fluorescence

RNA/BS-seq RNA/BS-seq

Gene expression (Log Transformed Counts) Pre vs Post Graft using location DEGs B 2 C 20 Group 15 Pre graft EOM Post graft EOM (corrected)

Fold change Pre graft TA pre graft TA / pre graft EOM 0 10 More expressed in TA 4

5 0 -20 PC2 (13%) -4

Post graft EOM (corrected) More expressed in EOM 0

-40

0 5 10 15 -40 -20 0 20 Pre graft EOM PC1 (41.5%)

D Classification of responses of location-specific genes in post graft EOM MuSCs Response of location-specific genes in post graft EOM MuSCs EOM-specific genes TA-specific genes EOM-specific genes TA-specific genes

100100 100100 10% Resistant Responsive 20.4% Response 7575 7575 Resistant Intermediate 31.4% 47.5% 5050 Intermediate 5050 Intermediate 58.6% Responsive 32.2%

2525 2525 Responsive Resistant Normalised expression level (%) Normalised expression level (%)

00 00

Group Pre graft EOM Pre graft TA Post graft EOM (corrected)

E MuSC Genes Regional Genes Group p=0.66 *** Pre graft EOM 10 p=0.076 ns Pre graft TA Post graft EOM (corrected) * *** ** *** *** *** ns * 5 Transformed Counts Transformed 2 Log

0

Pax7 Pitx2 Myf5 Myod Alx4 Mobp Lbx1 Pax3 Tbx1

Post Graft EOM MuSCs (corrected) Post Graft TA MuSCs (corrected) F 7.5 7.5 HoxA HoxB HoxC HoxD HoxA HoxB HoxC HoxD

5.0 5.0

2.5 2.5

0.0 0.0 Transformed Counts Transformed Counts Transformed 2 2 g g o o 360L L -2.5 -2.5 17 Hoxa1Hoxa2Hoxa3Hoxa4Hoxa5Hoxa6Hoxa7Hoxa9 Hoxb1Hoxb2Hoxb3Hoxb4Hoxb5Hoxb6Hoxb7Hoxb8Hoxb9 Hoxc4Hoxc5Hoxc6Hoxc8Hoxc9 Hoxd1Hoxd3Hoxd4Hoxd8Hoxd9 Hoxa1Hoxa2Hoxa3Hoxa4Hoxa5Hoxa6Hoxa7Hoxa9 Hoxb1Hoxb2Hoxb3Hoxb4Hoxb5Hoxb6Hoxb7Hoxb8Hoxb9 Hoxc4Hoxc5Hoxc6Hoxc8Hoxc9 Hoxd1Hoxd3Hoxd4Hoxd8Hoxd9 Hoxa10Hoxa11Hoxa13 Hoxb13 Hoxc10Hoxc11Hoxc12Hoxc13 Hoxd10Hoxd11Hoxd12Hoxd13 Hoxa10Hoxa11Hoxa13 Hoxb13 Hoxc10Hoxc11Hoxc12Hoxc13 Hoxd10Hoxd11Hoxd12Hoxd13 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

361 Figure 3. Head-derived MuSCs adopt a limb-like transcriptome upon transplantation in limb

362 muscles.

363 (A) Experimental scheme. EOM and TA-derived MuSCs were engrafted into pre-injured recipient TA

364 muscles. After regeneration, engrafted MuSCs were re-isolated and processed through RNA- and BS-

365 sequencing. ‘Post-graft’ MuSCs were compared to their ‘Pre-graft’ counterparts. N=10 donor mice and

366 10 recipient mice. For each donor mouse, equal number of EOM and TA MuSCs (in the range of 10,000

367 cells) were transplanted to TA muscles of the same recipient mouse. For each donor muscle, 500 pre-

368 graft cells and 60 re-isolated post-graft cells (mean value) were analysed (Supplementary Table 1).

369 (B) Expression analysis of EOM post-graft and EOM pre-graft. Each dot represents a gene. Genes are

370 color-coded according to their fold change in pre-graft TA vs EOM MuSCs (from Figure 1, without

371 transplantation). Note that genes highly expressed in pre-graft TA MuSCs (from Figure 1, coloured in

372 pink) were mostly upregulated in EOM MuSCs following grafting, while genes highly expressed in pre-

373 graft EOM MuSCs (from Figure 1, coloured in blue) were mostly downregulated in EOM MuSCs

374 following grafting.

375 (C) PCA analysis of TA pre-graft, EOM pre-graft and EOM post-graft MuSCs using the expression

376 values of genes differentially expressed between EOM and TA pre-graft MuSCs. PC1 separates EOM

377 and TA MuSCs and shows that after engrafting EOM MuSCs into the TA muscle, they resemble

378 transcriptionally TA MuSCs more than EOM MuSCs.

379 (D) (left) Classification of EOM and TA-specific genes as resistant, responsive, or intermediate upon

380 heterotopic transplantation of EOM MuSCs. Determining the responsiveness of each gene to

381 transplantation was carried out in two steps. First, expression values for TA-specific genes (resp EOM-

382 specific) in pre-graft MuSCs were rescaled to 0-100, where 0 represented the mean expression in EOM

383 (resp TA) MuSCs and 100 represented the mean expression in TA (resp EOM) MuSCs. Next, the

384 expression of EOM and TA-specific genes were rescaled similarly in post-graft EOM MuSCs. Each dark

385 blue dot represents a gene with corresponding rescaled value in post-graft EOM MuSCs. EOM-specific

386 genes with rescaled values in post-graft EOM MuSCs less than 25 were classified as responsive,

387 between 25 and 75 as intermediate and above 75 as resistant. TA-specific genes with rescaled values

388 in post-graft EOM MuSCs less than 25 were classified as resistant, between 25 and 75 as intermediate

389 and above 75 as responsive. (right) Distribution of responses of EOM and TA-specific genes in post-

390 graft EOM MuSCs.

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391 (E) Expression of selected markers between TA pre-graft, EOM pre-graft and EOM post-graft MuSCs.

392 Many TA marker genes were upregulated to TA-like levels when EOM MuSCs were grafted into TA

393 muscle. In addition, some EOM marker genes such as Lmx1a and Mobp were downregulated in this

394 scenario. *** p < 0.001, ** p < 0.01, * p < 0.05 by Welch’s t test.

395 (F) Gene expression analysis throughout the HoxA, HoxB, HoxC and HoxD clusters in post-graft EOM

396 (left) and TA (middle) MuSCs. Genes were color-coded according to their antero-posterior expression

57 397 domain in mouse at embryonic day 12.5 (right, adapted from ). All TA-specific Hox genes were

398 upregulated in post-graft EOM MuSCs.

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Figure 4 A Methylation at location DEG Promoters B Methylation at location DEG enhancers C * 0.50 0.020 0.25

0.25 0.015

0.00

0.00 0.010 Density PC2 (8.4%) -0.25 PC2 (9.2%) -0.25 0.005

-0.50 -0.50 -0.8 -0.4 0.0 0.4 -0.6 -0.3 0.0 0.3 0.000 PC1 (20.8%) PC1 (17.1%) -50 0 50 Methylation change (Post graft EOM - Pre graft EOM) Group Group Pre graft EOM Pre graft EOM Enhancers associated with: Post graft EOM (corrected) Post graft EOM (corrected) Pre graft TA Pre graft TA EOM-specific genes Post graft TA (corrected) Post graft TA (corrected) TA-specific genes

Eya2 Methylation Eya2 Transcription Rtn1 Methylation Rtn1 Transcription Cit Methylation Cit Transcription 40 8 D 100 100

2 6 30 6 75 75

1

4 50 20 50 4 0

2 transformed counts transformed counts transformed counts

2 10 2 2 2 25 -1 25 Methylation level (%) Methylation level (%) Methylation level (%) Log Log Log

0 0 0 -2 0 0

Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.)

Enpep Methylation Enpep Transcription Lbx1 Methylation Lbx1 Transcription Pax3 Methylation Pax3 Transcription

100 100 100 5.0

4 4 75 75 75

2.5

2 2 50 50 50

0.0 transformed counts transformed counts transformed counts 2 2 25 0 25 2 0 25 Methylation level (%) Methylation level (%) Methylation level (%) Log Log Log

0 0 0 -2 -2.5 Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.)

4 5 6 10 E HoxA Gene: 1 2 3 7 9 11 13

100

75 Group Pre graft EOM 50 Pre graft TA Post graft EOM (corrected) 25 Methylation Level (%)

0 52150000 52175000 52200000 52225000 52250000 Chromosome 6 Coordinates

399

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400 Figure 4. DNA methylation at enhancers partially accounts for the transcriptome plasticity of

401 MuSCs upon heterotopic transplantation.

402 (A) PCA analysis of TA pre-graft, EOM pre-graft and EOM post-graft MuSCs DNA methylation at

403 promoters fails to separate samples based on anatomical location.

404 (B) PCA analysis of TA pre-graft, EOM pre-graft and EOM post-graft MuSCs DNA methylation at

405 enhancers separates samples based on anatomical location and demonstrates that EOM MuSCs after

406 grafting resemble TA MuSCs at an epigenetic level in the enhancer context.

407 (C) Density plots of the methylation difference between post-graft EOM MuSCs and pre-graft EOM

408 MuSCs at enhancers associated with EOM or TA upregulated genes. EOM enhancers became

409 hypermethylated in EOM MuSCs after grafting into the TA environment, while TA enhancers were

410 hypomethylated. * p < 0.05 by Student’s t test.

411 (D) Enhancer methylation and gene expression levels of selected EOM and TA DEGs that are

412 responsive to grafting.

413 (E) DNA methylation level across the HoxA gene cluster in pre-graft EOM, pre-graft TA and post-graft

414 EOM samples. The HoxA region was highly methylated in TA MuSCs but not in EOM MuSCs. Notably,

415 DNA methylation was gained across this region when EOM MuSCs were grafted into TA muscle.

21 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplementary Figure 1 Up regulated in EOM Up regulated in TA

Cell adhesion Multicellular organism development

Cell migration Anterior/posterior pattern specification

Kidney development Sterol biosynthetic process

Multicellular organism development Embryonic skeletal system development

Ureteric bud development Cholesterol biosynthetic process

Nervous system development Positive regulation of apoptotic process

Metanephric mesenchymal cell differentiation Angiogenesis

Negative regulation of axon extension Male gonad development

Cell differentiation Proximal/distal pattern formation

Adrenal gland development Muscle organ development

0 1 2 3 0 2 4 6 8 − Log10(p-value) − Log10(p-value)

416 Supplementary Figure 1. Gene ontology analysis of the genes upregulated in EOM or TA MuSCs. The

417 10 most significant categories are shown.

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Supplementary Figure 2

A

0.2

0.0 Location

EOM TA PC2 (9.6%) -0.2

-0.4

-0.50 -0.25 0.00 0.25 0.50 PC1 (21.8%)

B Methylation TA vs EOM MuSCs (all enhancers)

100

75

Significant

50 Yes No in TA MuSCs (%) TA in

DNA methylation level DNA 25

0

0 25 50 75 100 DNA methylation level in EOM MuSCs (%)

418

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419 Supplementary Figure 2. EOM and TA MuSCs display different DNA methylation patterns at

420 enhancers associated with anatomical location-specific genes.

421 (A) PCA analysis of DNA methylation of 544 enhancers associated with genes specifically expressed in

422 TA or EOM MuSCs. EOM MuSCs and TA MuSCs separate according to their location.

423 (B) Scatter plot comparing the mean DNA methylation levels of all enhancers in EOM and TA MuSCs.

424 Enhancers were colour coded as significant if they were found to be differentially methylated by a rolling

425 Z score approach (p < 0.05). See Figure 2E for genes with location-specific expression and enhancer

426 methylation.

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplementary Figure 3 A Gene expression in EOM / TA MuSCs, pre-graft / post-graft B Gene expression in pre-graft vs post-graft TA MuSCs 200 Group Group Pre graft EOM Pre graft TA Post graft EOM Post graft TA Pre graft TA 100 Post graft TA 100

0 PC2 (7.4%) PC2 (11.4%) 0

-100 -100 -200 -100 0 100 -200 -100 0 100 PC1 (30.9%) PC1 (28.8%) C Up regulated in Post graft TA Down regulated in Post graft TA Angiogenesis Branched-chain amino acid catabolic process Defense response to virus Positive regulation of mesenchymal cell proliferation Cellular response to interferon-beta Metabolic process Cell adhesion Skeletal muscle cell differentiation Immune system process Negative regulation of cell proliferation Cell migration Muscle organ development Negative regulation of viral genome replication Positive regulation of transcription, DNA-templated Innate immune response Skeletal muscle tissue development Response to virus Positive regulation of epithelial cell proliferation Positive regulation of cell migration Negative regulation of neuron apoptotic process 0 5 10 15 0 2 4 6 − Log10(p-value) − Log10(p-value)

D Whole Genome IAP Enhancers Promoters 100 100 100 100

75 75 75 75

50 50 50 50

25 25 25 25 Mean DNA methylation level (%) Mean DNA methylation level (%) Mean DNA methylation level (%) Mean DNA Mean DNA Methylation level (%) Mean DNA

0 0 0 0 Pre graft Post graft Pre graft Post graft Pre graft Post graft Pre graft Post graft TA TA TA TA TA TA TA TA

E Gene expression in TA MuSCs, pre-graft vs post-graft (corrected) F 150

100

50

PC2 (14.2%) 0

-50

-100 0 100 200 PC1 (15.3%) Color key Group Group Pre graft TA Pre graft EOM Post graft TA (corrected) 427 Post graft EOM (corrected) 0.5 0.7 0.9 Pre graft TA 25 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

428 Supplementary Figure 3. Transplantation of MuSCs induces long-term transcriptome and

429 epigenome modifications.

430 (A) Whole transcriptome PCA analysis of pre-graft and post-graft TA and EOM MuSCs. Samples

431 separate into before and after grafting.

432 (B) Whole transcriptome PCA analysis of pre-graft and post-graft TA MuSCs. Pre and post-graft samples

433 cluster away from each other suggesting there is a residual impact from the grafting procedure even

434 after the recovery period.

435 (C) GO categories of genes upregulated (left) and downregulated (right) in post-graft vs pre-graft TA

436 MuSCs. Upregulated categories suggest there was a remaining inflammatory response.

437 (D) Mean DNA methylation levels in pre-graft and post-graft TA MuSCs for whole genome, repeat

438 elements, promoters and enhancers. Overall there was a global increase in DNA methylation after

439 grafting.

440 (E) Whole transcriptome PCA analysis of pre-graft TA MuSCs and post-graft MuSCs after applying a

441 correction coefficient accounting for transcriptome modifications specifically induced by the

442 transplantation procedure (see Methods). Pre and post-graft samples no longer cluster separately.

443 (F) Hierarchical clustering analysis using Euclidean distance of Pearson correlation values between TA

444 pre-graft, EOM pre-graft and EOM post-graft MuSCs. The samples cluster separately based on

445 anatomical location. Notably, after engrafting EOM MuSCs into TA muscle they cluster with TA MuSCs

446 rather than EOM MuSCs.

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary Figure 4 1.5 A Group Pre graft EOM 1.0 Post graft EOM (corrected) Pre graft TA 0.5

0.0

Myod Methylation Myod Transcription Pitx2 Methylation Pitx2 Transcription B 100 100

9 7.5 75 75

6 5.0 50 50

transformed counts 3 transformed counts 2.5 25 2 25 2 Methylation level (%) Methylation level (%) Log Log

0 0 0 0.0

Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft Pre graft Pre graft Post graft EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.) EOM TA EOM (corr.)

8 C HoxB Gene: 13 9 7 6 5 4 3 2 1

100

75 Group Pre graft EOM 50 Pre graft TA Post graft EOM (corrected) 25 Methylation Level (%) 0 96200000 96250000 96300000 96350000 Chromosome 11 Coordinates

HoxC Gene: 13 12 11 10 9 8 6 5 4

100

75 Group Pre graft EOM 50 Pre graft TA Post graft EOM (corrected) 25 Methylation Level (%)

0

102950000 103000000 Chromosome 15 Coordinates

4 HoxD Gene: 13 12 11 10 9 8 3 1

100

75 Group Pre graft EOM 50 Pre graft TA Post graft EOM (corrected) 25 Methylation Level (%) 447 0 74675000 74700000 74725000 74750000 Chromosome 2 Coordinates 27 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

448 Supplementary Figure 4. DNA methylation changes in MuSCs upon heterotopic transplantation.

449 (A) Hierarchical clustering analysis using Euclidean distance of Pearson correlation values between TA

450 pre-graft, EOM pre-graft and EOM post-graft MuSCs overall clusters samples based on location.

451 Notably, post-graft EOM MuSCs cluster with TA MuSCs rather than with EOM MuSCs.

452 (B) Enhancer methylation and gene expression levels of Myod and Pitx2 prior to and following grafting.

453 (C) DNA methylation level across the HoxB (top), HoxC (middle) and HoxD (bottom) gene clusters in

454 pre-graft EOM, pre-graft TA and post-graft EOM samples. DNA methylation levels were similar between

455 EOM MuSCs and TA MuSCs. In addition, grafting EOM MuSCs into the TA muscle environment did not

456 substantially affect the DNA methylation across these regions.

28 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

RNA seq BS seq Sample Cell number Group Passed QC Reason for failing QC Total Read Count Forward Read Count Reverse Read Count Percent in Gene Percent in exons Percent in rRNA Percent Genes Measured Percent in MT Percent on sense strand Total Read Count Forward Read Count Reverse Read Count Passed BSseq QC Reason for failing BSseq QC Pre_Graft_1_EOM_Exp1 500 EOM No Below total read count threshold 2526 1258 1268 90,18987744 75,38339131 0,755699498 2,919620808 8,110751254 50,10508028 3984478 2396772 1587706 Yes Pre_Graft_1_TA_Exp1 500 TA No Below percent in exons threshold 2694738 1348117 1346621 73,03559015 61,4123425 0,432921764 65,05616187 4,05988112 50,10284173 3124624 1837702 1286922 Yes Graft_1_EOM_Exp1 156 EOM to TA No Below percent in exons threshold 8118000 4056772 4061228 74,31045775 59,86262126 0,403978425 65,88066598 4,578246078 50,09315883 5240168 3173582 2066586 Yes Graft_1_TA_Exp1 43 TA to TA No Below percent in exons threshold 8118128 4064844 4053284 67,22523366 51,09128276 0,262776243 64,12809687 3,718075046 50,20200634 2618435 1546105 1072330 Yes Pre_Graft_3_EOM_Exp1 500 EOM Yes 9714819 4855763 4859056 93,51584894 76,62899137 0,724236457 53,46929021 2,911528188 49,95224251 3262872 1959499 1303373 Yes Pre_Graft_3_TA_Exp1 500 TA Yes 9308622 4649308 4659314 93,07168382 74,87022883 0,908354716 55,6639847 3,926426543 50,11751135 3693672 2010695 1682977 Yes Graft_3_EOM_Exp1 100 EOM to TA No Below percent in exons threshold 9836285 4921322 4914963 67,80642539 55,76630088 0,244547533 60,69863778 5,391259804 49,95964551 1916836 1121556 795280 Yes Graft_3_TA_Exp1 24 TA to TA Yes 9865674 4931243 4934431 79,22706962 69,96656877 0,501624232 60,02549191 5,44851335 50,17973462 2944671 1766983 1177688 Yes Pre_Graft_4_EOM_Exp1 500 EOM Yes 7838450 3916739 3921711 92,94604627 75,6263681 0,745339286 56,0742452 3,044337031 49,97531637 5724331 3399684 2324647 Yes Pre_Graft_4_TA_Exp1 500 TA Yes 6582304 3290488 3291816 91,42184123 75,09392623 0,769314483 58,32072015 3,220825069 50,25973618 3471919 1924985 1546934 Yes Graft_4_EOM_Exp1 209 EOM to TA Yes 10526422 5256309 5270113 88,28918632 71,36606746 0,227194044 51,41400462 3,848697414 49,95126555 895 504 391 No Below total read count threshold Graft_4_TA_Exp1 28 TA to TA No Below percent in exons threshold 6768072 3378817 3389255 70,67912877 56,06331452 0,294868973 58,31275392 4,104152234 50,08825938 1607720 962806 644914 Yes Pre_Graft_5_EOM_Exp1 500 EOM Yes 9471491 4728932 4742559 93,58848502 75,55792704 0,994485877 54,45710189 3,643428772 50,15849241 3537638 2088006 1449632 Yes Pre_Graft_5_TA_Exp1 500 TA No Below total read count threshold 222822 111412 111410 91,19169941 78,77264236 0,526668098 36,25428184 2,53263049 49,88788362 12491074 7098514 5392560 Yes Graft_5_EOM_Exp1 105 EOM to TA No Below percent in exons threshold 9708205 4855043 4853162 76,02445475 62,1141577 0,286291 59,45192384 4,908080657 50,10830155 2329456 1369250 960206 Yes Graft_5_TA_Exp1 36 TA to TA No Below percent in exons threshold 1139937 569878 570059 70,97556352 61,13137404 0,278096141 48,43862025 4,859778773 49,70819874 2041521 1188335 853186 Yes Pre_Graft_1_EOM_Exp2 500 EOM Yes 8580937 4288170 4292767 93,92541256 79,59085786 1,167448329 53,31793197 7,021739897 49,99451211 4299795 2619022 1680773 Yes Pre_Graft_1_TA_Exp2 500 TA Yes 8888641 4462385 4426256 91,99653198 78,41363368 1,69199174 51,24273082 22,93827093 50,32893719 2429682 1368710 1060972 Yes Graft_1_EOM_Exp2 46 EOM to TA Yes 5517157 2759657 2757500 84,72944087 74,93079003 0,56734565 56,27738389 15,89844554 50,1478624 3287164 1944096 1343068 Yes Graft_1_TA_Exp2 26 TA to TA Yes 7373776 3706789 3666987 82,76502411 71,25249094 1,462189876 53,49318888 25,3478087 50,47282198 3744447 2230205 1514242 Yes Pre_Graft_2_EOM_Exp2 500 EOM Yes 14402975 7204134 7198841 92,90538725 76,91790527 1,42060353 50,50585517 8,16326724 49,81367102 2971898 1774302 1197596 Yes Pre_Graft_2_TA_Exp2 500 TA Yes 11372962 5683972 5688990 84,84251451 73,38443463 1,242364925 59,23683582 16,96431074 49,95241536 4383725 2485686 1898039 Yes Graft_2_EOM_Exp2 35 EOM to TA Yes 9472455 4742871 4729584 89,4076115 80,9335573 1,242123349 42,61132797 33,03771331 50,06935284 2135945 1240777 895168 Yes Graft_2_TA_Exp2 67 TA to TA Yes 9327101 4661691 4665410 89,88366732 73,80782874 0,658059026 48,48243448 16,42485708 50,08474135 2647527 1455104 1192423 Yes Pre_Graft_3_EOM_Exp2 500 EOM No Outlier in group 11093142 5539570 5553572 87,17112052 76,79700025 1,384625466 54,31370987 18,29608282 49,8062178 3118779 1872511 1246268 Yes Pre_Graft_3_TA_Exp2 500 TA Yes 9855767 4915867 4939900 90,21110607 77,75402091 0,948175304 46,02485462 14,42974675 49,87659585 1454312 785157 669155 Yes Graft_3_EOM_Exp2 16 EOM to TA Yes 6802637 3387031 3415606 91,70628905 80,12674953 9,438975386 47,16402454 59,62696 49,88199538 1492074 885602 606472 Yes Graft_3_TA_Exp2 16 TA to TA Yes 5378534 2669422 2709112 75,52384841 73,83395154 1,453079311 61,79399347 46,83940504 49,70965776 2967698 1807125 1160573 Yes Pre_Graft_4_EOM_Exp2 500 EOM Yes 13363659 6681346 6682313 93,2906331 74,95184777 0,872231419 54,8394806 4,331800788 50,27834165 9222445 5502069 3720376 Yes Pre_Graft_4_TA_Exp2 500 TA Yes 3326270 1662095 1664175 93,02931719 78,78842211 0,464837902 51,820282 4,225485665 49,76042663 8557770 4775275 3782495 Yes Graft_4_EOM_Exp2 9 EOM to TA Yes 7830185 3918610 3911575 80,02761509 72,92853341 1,302369123 55,03863618 37,06962635 50,1515974 2676672 1604720 1071952 Yes Graft_4_TA_Exp2 78 TA to TA Yes 8258350 4112506 4145844 89,28080937 74,99759842 1,454942548 48,6975225 32,27851736 49,64247798 3425477 1920639 1504838 Yes Pre_Graft_5_EOM_Exp2 500 EOM Yes 12301509 6143743 6157766 93,92563017 79,40682033 0,60683463 53,79988847 3,270604992 49,9812523 5723189 3372833 2350356 Yes Pre_Graft_5_TA_Exp2 500 TA Yes 9026147 4528852 4497295 92,82411688 75,80510025 0,849375467 55,55245758 6,994724008 50,24110001 3781453 2112089 1669364 Yes Graft_5_EOM_Exp2 60 EOM to TA Yes 5243925 2624030 2619895 83,32368228 77,60947927 2,348929483 58,04588545 45,33801121 49,98864409 4429479 2658831 1770648 Yes Graft_5_TA_Exp2 141 TA to TA Yes 7345185 3688828 3656357 79,82365201 69,01545455 1,237329435 58,20919302 19,33845234 50,3848372 2705658 1559481 1146177 Yes Pre_Graft_6_EOM_Exp2 500 EOM Yes 13514749 6760663 6754086 93,06693631 75,94415748 0,621321455 55,56839003 3,478315593 50,06965287 3875343 2322710 1552633 Yes Pre_Graft_6_TA_Exp2 500 TA Yes 11938050 5967137 5970913 90,10236216 75,70691336 1,618095056 55,97466741 17,37997732 50,04611728 2646683 1481954 1164729 Yes Graft_6_EOM_Exp2 33 EOM to TA Yes 8719716 4361861 4357855 77,47832187 68,66743098 1,2823181 60,9814387 24,83408691 50,08902471 4158705 2463848 1694857 Yes Graft_6_TA_Exp2 16 TA to TA Yes 9360048 4677495 4682553 88,92941297 79,37538471 1,48382525 46,73783159 28,899288 50,00912706 2137529 1284880 852649 Yes

457 Supplementary Table 1. RNA-seq and BS-seq quality control.

29 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

458 METHODS

459 Mice

460 Animals were handled according to national and European Community guidelines and an ethics

17 -/- 461 committee of the Institut Pasteur (CETEA) in France approved protocols. Tg:Pax7-nGFP and Rag2

-/- 59 462 ;gC mice were used in this study, on C57BL/6;DBA2 F1/JRj and C57BL/6J genetic backgrounds

463 respectively. 6 to 8-week-old male littermates were used.

464

465 Isolation of muscle stem cells

466 Mice were sacrificed by cervical dislocation. Tibialis anterior and extraocular muscles were

467 dissected and placed into cold DMEM (ThermoFisher, 31966). Muscles were then manually chopped

468 with scissors and put into a 15 ml Falcon tube containing 10 ml of DMEM, 0.08% collagenase D (Sigma,

469 11 088 882 001), 0.1% trypsin (ThermoFisher, 15090), 10 µg/ml DNase I (Sigma, 11284932) at 37°C

470 under gentle agitation for 25 min. Digests were allowed to stand for 5 min at room temperature and the

471 supernatants were added to 5 ml of foetal bovine serum (FBS; Gibco) on ice. The digestion was repeated

472 3 times until complete digestion of the muscle. The supernatants were filtered through a 70-µm cell

473 strainer (Miltenyi, 130-098-462). Cells were spun for 15 min at 515g at 4°C and the pellets were

474 resuspended in 1 ml cold freezing medium (10% DMSO (Sigma, D2438) in FBS) for long term storage

475 in liquid nitrogen or processed directly through FACS-isolation for transplantations.

476 Before isolation by FACS, samples were thawed in 50 ml of cold DMEM, spun for 15 min at

477 515g at 4°C. Pellets were resuspended in 300 µl of DMEM 2% FBS 1 µg/mL propidium iodide

478 (Calbiochem, 537060) and filtered through a 40-µm cell strainer (BD Falcon, 352235). Viable muscle

479 stem cells were isolated based on size, granulosity and GFP intensity using a MoFlo Astrios cell sorter

480 (Beckmann Coulter).

481 Cells were collected in 5 µl cold RLT Plus buffer (Qiagen, 1053393) containing 1U/µl RNAse

482 inhibitor (Ambion, AM2694), flash-frozen on dry ice and stored at -80°C, or in cold DMEM 2% FBS.

483

484 Muscle stem cell transplantations

94 485 Muscle injury was done as described previously . Briefly, mice were anesthetized with 0.5%

-/- -/- 486 Imalgene/2% Rompun. Both TAs of recipient immunocompromised Rag2 ;gC mice were injured with

487 50 µl of 10 µM cardiotoxin (Latoxan, L8102) in NaCl 0.9% 24 h before transplantation. Muscle stem cells

30 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

488 from freshly dissociated EOM and TA muscles of Tg:Pax7-nGFP mice were isolated by FACS, spun for

489 15 min at 515g at 4°C, resuspended in 10 µl of cold PBS and injected into recipient TAs. EOM and TA

490 muscle stem cells from a donor mouse were injected into separate TAs of the same recipient mouse,

491 and equivalent numbers of EOM and TA cells were injected. Transplanted muscle stem cells were re-

492 isolated by FACS based on GFP positivity after three to four weeks.

493

494 Bisulfite-seq and RNA-seq Library Preparation

495 RNA was separated from cell lysates and processed into libraries using the G&T method as

47 48 496 described in . DNA was also processed into libraries using the bulk protocol described in . Bisulfite-

497 seq libraries were sequenced on an Illumina HiSeq-2500 with 125bp paired end reads and RNA-seq

498 libraries were sequenced on an Illumina HiSeq-2500 with 75bp paired end reads.

499

500 Bisulfite-seq analysis

501 Adapter sequences and poor-quality calls were removed from the raw sequencing files using

502 Trim galore (version 0.4.2). Reads were then mapped to mouse genome GRCm38 and deduplicated

503 using Bismark (version 0.16.3).

504 Promoters were defined as -2000bp to 500bp of the TSS of Ensembl genes. Promoters were

505 split into CpG Islands (CGI) promoters and non-CGI promoters based on whether they overlapped a

58 506 CpG island. H3K27ac peaks were called using macs2 on H3K27ac ChIP-seq data obtained from .

507 Enhancers were defined as H3K27ac peaks that did not overlap promoters. Enhancers were linked to

508 genes based on proximity. At least two CpG sites needed to be covered for a promoter or enhancer to

509 be taken forward. In addition, promoters and enhancers needed to be covered in at least 3 samples of

510 each group to be considered in the analysis.

511

512 RNA-seq analysis

513 Adapter sequences and poor-quality calls were removed from the raw sequencing files using

514 Trim galore (version 0.4.4). Reads were then mapped to mouse genome GRCm38 using Hisat2 (version

515 2.1.0). Log2 transformed counts were generated using Seqmonk. Differential expression analysis was

516 carried out in R using DESeq and genes that demonstrated a fold change greater than 2 and a p-value

517 less than 0.05 were classified as differentially expressed.

31 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

518 Correcting for the effect of transplantation was performed by initially determining the expression

519 change for all genes between TA MuSCs before and after transplantation. This expression change was

520 then deducted from all post-graft samples to generate corrected values that excluded the effect of

521 transplantation alone.

522

523 ACKNOWLEDGMENTS

524 We would like to thank the Flow Cytometry Platform of the Center for Technological Resources and

525 Research (Institut Pasteur) and the Wellcome Trust Sanger Institute sequencing facility for assistance

526 with Illumina sequencing. This project was supported by grants from Institut Pasteur, Agence Nationale

527 de la Recherche (Laboratoire d'Excellence Revive, Investissement d'Avenir; ANR-10-LABX-73),

528 Association Française contre les Myopathies (21857), CNRS, the European Research Council

529 (Advanced Research Grant 332893), and the Biotechnology and Biological Sciences Research Council

530 (BBSRC, CBBS/E/B/000C0425). I.H.-H. was supported by a Marie Sklodowska-Curie Individual

531 Fellowship (751439).

532

533 AUTHOR CONTRIBUTIONS

534 B.E., T.S., S.T. and W.R. proposed the concept and designed the experiments. B.E. and G.C. performed

535 mouse dissections. B.E. and P.H.C. performed FACS. B.E. performed grafting experiments. T.S.

536 performed library preparation and sequencing. D.G., I.H.H. and T.S. developed the analysis

537 methodologies and analysed the experiments. B.E., D.G., I.H.H, S.T. and W.R. wrote the paper. All

538 authors read and agreed on the manuscript.

539

540 COMPETING INTERESTS

541 W.R. is a consultant and shareholder of Cambridge Epigenetix. T.S. is CEO and shareholder of

542 Chronomics. All other authors declare no competing interests.

543

544

545

546

547

32 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.20.107219; this version posted May 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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