U2OS were transfected with indicated siRNA oligos using protocol of screen fixed for immunofluorescence after 0 , 2 24 h after 5 Gy IR . Samples were stained with antibodies for γH2AX ( D ) or 53BP1 ( E ) analyzed through automated confocal microscopy specifically developed image software . From one experiment two to four separate with a total of at least 100 were analyzed for each knockdown . Shown are number of γH2AX ( D ) 53BP1 ( E ) foci per . In box plots , median with first third quartile is indicated , whiskers are drawn down to 10th percentile up to 90th .

B-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells were transfected with the indicated siRNA oligos using the protocol of the primary screen and fixed for immunofluorescence after 0, 2 and 24 h after 5 Gy IR. Samples were stained with antibodies for γH2AX (D) or 53BP1 (E) and analyzed through automated confocal microscopy and specifically developed image analysis software. From one experiment two to four separate images with a total of at least 100 cells were analyzed for each knockdown. Shown are the number of γH2AX (D) and 53BP1 (E) foci per cell. In the box plots, the median with the first and third quartile is indicated, and the whiskers are drawn down to the 10th percentile and up to the 90th.


U2OS were transfected with four different siRNA oligonucleotides targeting PHF6 , synchronized in G2 subsequent recovery after 5 Gy of IR addition of nocodazole was analyzed by flow cytometry with MPM2 PPM1D βTrCP were used as positive controls .

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U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 and subsequent recovery after 5 Gy of IR and the addition of nocodazole was analyzed by flow cytometry with MPM2 staining PPM1D and βTrCP were used as positive controls.


U2OS were transfected with four different siRNA oligonucleotides targeting PHF6 , synchronized in G2 or were lysed analyzed using western blotting with indicated

B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O

U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 or cells were lysed and analyzed using western blotting with the indicated antibodies


U2OS WT , PHF6 knockout PHF6 knock out that were reconstituted with GFP - PHF6 were synchronized in G2 recovery was determined after 2 Gy of IR using flow cytometry with pHH3

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U2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 and recovery was determined after 2 Gy of IR using flow cytometry with pHH3 staining


U2OS WT , PHF6 knockout PHF6 knock out that were reconstituted with GFP - PHF6 were synchronized in G2 were lysed analyzed using western blotting with indicated

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U2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 cells were lysed and analyzed using western blotting with the indicated antibodies


U2OS WT PHF6 knockout were irradiated using 3 Gy of IR were fixed at different time points for immunofluorescence . In ( F ) is presented 53BP1 IRIF . In ( G ) is shown number of γH2AX foci per .

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U2OS WT and PHF6 knockout cells were irradiated using 3 Gy of IR and cells were fixed at different time points for immunofluorescence. In (F) is presented 53BP1 IRIF intensity. In (G) is shown the number of γH2AX foci per cell.


U2OS WT PHF6 knockout were left untreated or irradiated with 3 Gy processed after 1 h for comet . comet tail moment was analyzed in at least 50 .

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U2OS WT and PHF6 knockout cells were left untreated or irradiated with 3 Gy and processed after 1 h for comet assay analysis. The comet tail moment was analyzed in at least 50 individual cells.


U2OS stably expressing GFP - PHF6 mCherry - NBS1 were - irradiated were analyzed by time - lapse imaging .

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U2OS cells stably expressing GFP-PHF6 and mCherry-NBS1 were laser-irradiated and cells were analyzed by time-lapse imaging.


U2OS stably expressing GFP - PHF6 were - irradiated were analyzed by time - lapse imaging . In ( B ) , PARP inhibitor Olaparib was added 30 min before - induced damage . At least 60 were analyzed in three experiments error bars represent SD ( lower panel ) . Scale bar is 5 μM .

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U2OS cells stably expressing GFP-PHF6 were laser-irradiated and cells were analyzed by time-lapse imaging. In (B), the PARP inhibitor Olaparib was added 30 min before laser-induced damage. At least 60 cells were analyzed in three individual experiments and error bars represent the SD (lower panel). Scale bar is 5 μM.


GC92 SV40 immortalized human fibroblasts containing I - SceI NHEJ reporter were transfected with indicated siRNA oligos I - SceI . 48 h later were fixed analyzed by flow cytometry . Represented is relative repair efficiency as compared to Luciferase control .

B-CELL_LINE B-ORGANISM O B-ORGANISM B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O

GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later the cells were fixed and analyzed by flow cytometry. Represented is the relative repair efficiency as compared to the Luciferase control.


Clonogenic survival assays of U2OS WT PHF6 knockout that were depleted for luciferase or XRCC4 treated with 2 , 3 or 4 Gy IR . Shown is relative survival as compared to undamaged control . Error bars represent of three experiments .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

Clonogenic survival assays of U2OS WT and PHF6 knockout cells that were depleted for luciferase or XRCC4 and treated with 2, 3 or 4 Gy IR. Shown is the relative survival as compared to the undamaged control. Error bars represent the SEM of three individual experiments.


GC92 SV40 immortalized human fibroblasts containing I - SceI NHEJ reporter were transfected with indicated siRNA oligos I - SceI . 48 h later genomic was extracted repair of I - SceI cut - sites analyzed through junction ( n = independently derived sequences , see materials methods for details ) .

B-CELL_LINE B-ORGANISM O B-ORGANISM B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later genomic DNA was extracted and repair of I-SceI cut-sites analyzed through junction analysis (n= independently derived sequences, see materials and methods for details).


A . RGFP966 ( 966 ) - dependent HDAC3 inhibition resulted in significant robust neurite outgrowth ( 24h in culture ) compared to HDAC class II , HDAC1 / 2 HDAC1 or 3 inhibitions via 69A , 963 233 respectively . Data information : Data expressed as mean fold change N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni ) .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

A. RGFP966 (966)-dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth (24h in culture) compared to HDAC class II, HDAC1/2 and HDAC1 or 3 inhibitions via 69A, 963 and 233 respectively. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test).


B , C . Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth ( average neurite outgrowth after 24h in culture , Tuj1 positive neurons ) on PDL - laminin or Myelin substrates . Scale bars , 100 μm . Data information : Data expressed as average neurite ± s . e . m . N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle Student ' s t - )

O O O O O O O O B-GENEPROD O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_TYPE O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

B, C. Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth (average neurite outgrowth after 24h in culture, Tuj1 positive neurons) on PDL-laminin or Myelin substrates. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)


D , E . Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle . Data information : Data expressed as mean fold change N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle Student ' s t - )

O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

D, E. Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)


F , G . AAV - mediated mutant HDAC3 ( Y298H , V5 ) infection of DRG neurons induces neurite outgrowth ( Tuj - 1 / V5 double positive neurons ) after 36h in culture , which is repressed by wildtype HDAC3 overexpression . Scale bars , 100 μm . Data information : Data expressed as average neurite ± s . e . m . N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus V5 vs AAV - GFP ANOVA followed by Bonferroni ) .

O O O O B-ORGANISM O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O B-GENEPROD O O O O O O

F, G. AAV-mediated mutant HDAC3 (Y298H, V5) infection of DRG neurons induces neurite outgrowth (Tuj-1/V5 double positive neurons) after 36h in culture, which is repressed by wildtype HDAC3 overexpression. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus V5 vs AAV-GFP ANOVA followed by Bonferroni test).


A . in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased , primarily in nucleus 24h after sciatic nerve axotomy ( SNA ) but not spinal dorsal column axotomy ( DCA ) . Scale bars , 100 μm .

O O O B-TISSUE O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O

A. IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased, primarily in the nucleus 24h after sciatic nerve axotomy (SNA) but not spinal dorsal column axotomy (DCA). Scale bars, 100 μm.


B - C . Bar graphs show mean fold change ( B ) or % of pHDAC3 + ( C ) ( pHDAC3 + threshold was set at minimum level of sham ) ± s . e . m . DAPI counter was used to label nuclei N = 9 - 10 biological replicates . ( * * * p < 0 . 005 ) indicate significant difference versus sham or laminectomy ( lam ) ( ANOVA followed by Bonferroni ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O

B-C. Bar graphs show mean fold change levels (B) or % of pHDAC3+ cells (C) (pHDAC3+ threshold was set at the minimum intensity level of sham cells) ± s.e.m. DAPI counter staining was used to label nuclei N= 9-10 biological replicates. (***p<0.005) indicate significant difference versus sham or laminectomy (lam) (ANOVA followed by Bonferroni test).


D . Western blot revealed that HDAC3 phosphorylation is decreased in DRG extracts but not in cytoplasm 24h after SNA . E . Bar graphs show measurement of vs cytoplasm immunoblot . Data is expressed as mean ± s . e . m . N = 3 biological replicates . ( * * * p < 0 . 005 ) indicate significant difference versus sham ( Student ´ s t - ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Western blot revealed that HDAC3 phosphorylation is decreased in DRG nuclear extracts but not in the cytoplasm 24h after SNA. E. Bar graphs show intensity measurement of nuclear vs cytoplasm immunoblot. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (***p<0.005) indicate significant difference versus sham (Student´s t-test).


F . HDAC3 activity ( HDAC3 IP activity ) is strongly reduced after SNA but not DCA . Data is expressed as mean ± s . e . m . N = 3 biological replicates . ( * p < 0 . 05 ) indicate significant difference versus sham ( ANOVA followed by Bonferroni ) .

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F. HDAC3 activity (HDAC3 IP and activity assay) is strongly reduced after SNA but not DCA. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (*p<0.05) indicate significant difference versus sham (ANOVA followed by Bonferroni test).


PP4 activities are induced after SNA but not DCA , are blocked by calcium chelator EGTA . Data is expressed as mean ± s . e . m . N = 3 - 4 biological replicates . ( * * * p < 0 . 001 ) indicate significant difference versus sham or lam ( Two - way ANOVA followed by Bonferroni ) .

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PP4 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).


PP2 activities are induced after SNA but not DCA , are blocked by calcium chelator EGTA . Data is expressed as mean ± s . e . m . N = 3 - 4 biological replicates . ( * * * p < 0 . 001 ) indicate significant difference versus sham or lam ( Two - way ANOVA followed by Bonferroni ) .

PP2 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).


C . SNA but not DCA significantly enhances calcium in DRG . Data is expressed as mean ± s . e . m . N = 4 biological replicates . ( * p < 0 . 05 ) indicate significant difference versus sham or lam ( ANOVA followed by Bonferroni ) .

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C. SNA but not DCA significantly enhances calcium levels in DRG. Data is expressed as mean ± s.e.m. N= 4 biological replicates. (*p<0.05) indicate significant difference versus sham or lam (ANOVA followed by Bonferroni test).


D - F . Immunoblotting of DRG homogenates shows that nerve injury - dependent HDAC3 dephosphorylation is reduced by calcium scavenger EGTA upon in vivo delivery on injured sciatic nerve ( Axo = Axotomy ) . pERK are used as control of axotomy - dependent signalling . ( E - F ) . Data is expressed as mean fold change of band ± s . e . m . N = 3 biological replicates . ( * * p < 0 . 01 , * * * p < 0 . 001 ) indicate a significant difference ( ANOVA followed by Bonferroni ) .

O O O O B-EXP_ASSAY O B-TISSUE O O O B-DISEASE I-DISEASE O O B-GENEPROD B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-DISEASE B-TISSUE I-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F. Immunoblotting of DRG homogenates shows that nerve injury-dependent nuclear HDAC3 dephosphorylation is reduced by the calcium scavenger EGTA upon in vivo delivery on the injured sciatic nerve (Axo=Axotomy). Cytoplasmic pERK levels are used as control of axotomy-dependent signalling. (E-F). Data is expressed as mean fold change of band intensity levels ± s.e.m. N= 3 biological replicates. (**p<0.01, ***p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).


Significant Pearson correlations between calcium ( Fluo4AM ) H3K9ac , r2 : 0 . 7802 ( G ) in cultured DRG neurons after KCl . Pearson correlations were disrupted by administration of Fostriecin ( calcium H3K9ac , r2 : 0 . 0007 Data is expressed as single . N = 50 per condition from 4 biological replicates .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O B-TISSUE B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Significant Pearson correlations between calcium levels (Fluo4AM) and H3K9ac, r2: 0.7802 (G) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin (calcium and H3K9ac, r2: 0.0007 Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.


Significant Pearson correlations between calcium ( Fluo4AM ) ; or pHDAC3 , r2 : 0 . 6366 ( H ) in cultured DRG neurons after KCl . Pearson correlations were disrupted by administration of Fostriecin calcium pHDAC3 , r2 : 0 . 3025 ) . Data is expressed as single . N = 50 per condition from 4 biological replicates .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

Significant Pearson correlations between calcium levels (Fluo4AM) ; or pHDAC3, r2: 0.6366 (H) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin calcium and pHDAC3, r2: 0.3025). Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.


J - L . Immunofluorescence ( J ) in DRG tissue sections 24h after injury in vivo shows that PP2a / PP4 inhibitor Fostriecin ( F ) inhibits injury - induced decrease in pHDAC3 ( K ) increase in H3K9ac ( L ) . Scale bar , 10 μm . ( K - L ) . Data is expressed as mean in arbitrary units ± s . e . m . N = 3 ( L ) or 4 ( K ) biological replicates ( * p < 0 . 05 ; * * p < 0 . 01 ) indicate significant difference versus sham ( ANOVA followed by Bonferroni ) .

O O O O B-EXP_ASSAY O O O O B-TISSUE O O O O B-DISEASE O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O B-DISEASE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-L. Immunofluorescence (J) in DRG tissue sections 24h after injury in vivo shows that the PP2a/PP4 inhibitor Fostriecin (F) inhibits the injury-induced decrease in pHDAC3 (K) and the increase in H3K9ac (L). Scale bar, 10 μm. (K-L). Data is expressed as mean fluorescence intensity in arbitrary units ± s.e.m. N= 3 (L) or 4 (K) biological replicates (*p<0.05; **p<0.01) indicate significant difference versus sham (ANOVA followed by Bonferroni test).


M - N . Pharmacological inhibition of PP2a / PP4 impairs conditioning - induced DRG neurite outgrowth ex vivo ( 12h in culture ) . M . DRG neurite outgrowth ( TUJ1 positive ) 24h after sham or sciatic nerve axotomy ( SNA ) prior to i . t . administration of vehicle ( V ) or Fostriecin ( F ) ( 240µM ) . Scale bar , 100µm . ( N ) Data is expressed as average neurite per neuron ± s . e . m . N = 3 biological replicates per condition , technical triplicate . * * * p < 0 . 005 indicate significant difference versus sham / vehicle ( One way ANOVA followed by Bonferroni ) .

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M-N. Pharmacological inhibition of PP2a/PP4 impairs conditioning-induced DRG neurite outgrowth ex vivo (12h in culture). M. DRG neurite outgrowth (TUJ1 positive cells) 24h after sham or sciatic nerve axotomy (SNA) prior to i.t. administration of vehicle (V) or Fostriecin (F) (240µM). Scale bar, 100µm. (N) Data is expressed as average neurite length per neuron ± s.e.m. N= 3 biological replicates per condition, technical triplicate. *** p<0.005 indicate significant difference versus sham/vehicle (One way ANOVA followed by Bonferroni).


A - E . PP4c gene silencing promotes HDAC3 phosphorylation in presence of KCl ( A - C , arrows ) , both in nucleus cytoplasm ( D ) . Scale bar , 20 μm . ( C , D ) Data is expressed as mean fold change vs scrambled ( scr ) siRNA of of pHDAC3 or PP4c in GFP positive ± s . e . m . N = 3 biological replicates . ( * * p < 0 . 01 ; * * * p < 0 . 005 ) indicate significant difference versus scrambled siRNA ( Student ' s t - ) . ( E ) PCR against PP4c demonstrates silencing of PP4c mRNA . F - G . PP2a gene silencing inhibits KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 ( F ) . ( G ) Data is expressed as mean fold change of immunoblot band ± s . e . m . N = 3 biological replicates ( * p < 0 . 05 ; * * * * p < 0 . 001 ) indicate significant difference versus control siRNA ( Student ' s t - ) .

O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-E. PP4c gene silencing promotes HDAC3 phosphorylation in the presence of KCl (A-C, arrows), both in the nucleus and the cytoplasm (D). Scale bar, 20 μm. (C,D) Data is expressed as mean fold change vs scrambled (scr) siRNA of fluorescent intensity of pHDAC3 or PP4c in GFP positive cells ± s.e.m. N=3 biological replicates. (**p<0.01; ***p<0.005) indicate significant difference versus scrambled siRNA (Student's t-test). (E) PCR against PP4c demonstrates silencing of PP4c mRNA. F-G. PP2a gene silencing inhibits the KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 (F). (G) Data is expressed as mean fold change of immunoblot band intensity ± s.e.m. N= 3 biological replicates (*p<0.05; ****p<0.001) indicate significant difference versus control siRNA (Student's t-test).


H - I . Genetic modification of activity - dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth . ( H ) DRG neurite outgrowth after transfection of control ( V5 control ) , WT HDAC3 - V5 , phospho - dead HDAC3 ( S424A ) - V5 or phospho - mimetic HDAC3 ( S424D ) - V5 after 24h in culture . Scale bar , 50µm . ( I ) Data is expressed as mean ± s . e . m . N = 8 - 9 biological replicates , approximately 35 V5 positive each . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 indicate significant difference ( One way ANOVA followed by Bonferroni ) .

O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H-I. Genetic modification of activity-dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth. (H) DRG neurite outgrowth after transfection of control (V5 control), WT HDAC3-V5, phospho-dead HDAC3 (S424A)-V5 or phospho-mimetic HDAC3 (S424D)-V5 and after 24h in culture. Scale bar, 50µm. (I) Data is expressed as mean ± s.e.m. N= 8-9 biological replicates, approximately 35 V5 positive cells each. * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference (One way ANOVA followed by Bonferroni).


A . graph shows number of differentially expressed ( DE ) genes 24h following SNA or DCA compared to their respective controls .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

A. The graph shows the number of differentially expressed (DE) genes 24h following SNA or DCA compared to their respective controls.


B . RNAseq read show differential occupancy of H3K9ac on specific gene regions , including an increased occupancy close to transcriptional start site ( TSS ) after SNA only , when compared to corresponding controls .

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B. RNAseq read counts show differential occupancy of H3K9ac on specific gene regions, including an increased occupancy close to the transcriptional start site (TSS) after SNA only, when compared to corresponding controls.


C . Bar graph shows number of genes that are upregulated or downregulated following SNA or DCA ( RNAseq ) with an increased or decreased H3K9ac occupancy ( ChIPseq ) , respectively . D . Odds of enrichment / depletion of genes with differential H3K9ac occupancy across each upregulated downregulated gene cluster in ( C ) ; numbers in red represent p value given by Fisher ' s exact ( ns = not significant ) .

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C. Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA (RNAseq) with an increased or decreased H3K9ac occupancy (ChIPseq), respectively. D. Odds ratio analysis of the enrichment/depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in (C); the numbers in red represent the p value given by Fisher's exact test (ns= not significant).


A , B . Co - immunofluorescence of anti - neuronal anti - regeneration associated in DRG following SCI treatment with 966 or vehicle . Shown is increased expression for regeneration - associated targets in NF200 positive DRG neurons ( arrows ) after 966 treatment ( 5 following SCI ) . Scale bar , 50 μm . ( B ) Data is expressed as fold change of in NF200 + , 966 versus vehicle ± s . e . m . N = 4 - 7 biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 indicate significant difference versus vehicle ( Student ' s t - ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O B-EXP_ASSAY O O B-TISSUE O B-DISEASE O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O B-TISSUE B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Co-immunofluorescence of anti-neuronal and anti-regeneration associated proteins in DRG following SCI and treatment with 966 or vehicle. Shown is increased protein expression for regeneration-associated targets in NF200 positive DRG neurons (arrows) after 966 treatment (5 weeks following SCI). Scale bar, 50 μm. (B) Data is expressed as fold change of fluorescence intensity in NF200+ cells, 966 versus vehicle ± s.e.m. N=4-7 biological replicates * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference versus vehicle (Student's t-test).


A - B . AAV - HDAC3 mutant ( Y298H ; dominant negative deacetylase inactive ) promoted DRG regenerative growth after spinal cord injury vs AAV - V5 control ( injected 5 prior to SCI in sciatic nerve bilaterally ) as shown by dextran - red traced axons across beyond lesion site ( GFAP , green ) ( dotted lines denote rostral caudal margins of scar around lesion ) . Scale bar , ( A - B ) 200 μm .

O O O O B-ORGANISM O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O B-DISEASE I-DISEASE I-DISEASE O B-ORGANISM O B-GENEPROD O O O O O O B-DISEASE O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-B. AAV-HDAC3 mutant (Y298H; dominant negative deacetylase inactive) promoted DRG regenerative growth after spinal cord injury vs AAV-V5 control (injected 5 weeks prior to SCI in the sciatic nerve bilaterally) as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, (A-B) 200 μm.


C - F . Immunofluorescence for H3K9ac . AAV - HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons , but not in surrounding nuclei of satellite . Scale bar , ( C - F ) 50 μm .

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C-F. Immunofluorescence for H3K9ac. AAV-HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons, but not in surrounding nuclei of satellite cells. Scale bar, (C-F) 50 μm.


G - J . New synaptic formations ( VGlut1 + ) from regrowing axons are shown beyond lesion site after injection of AAV - HDAC3mut . ( G ) Scale bar 200 μm , ( H - J ) High magnification insets depict co - localization of Dextran vGlut1 . Scale bar 50 μm .

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G-J. New synaptic formations (VGlut1+) from regrowing axons are shown beyond the lesion site after injection of AAV-HDAC3mut. (G) Scale bar 200 μm, (H-J) High magnification insets depict co-localization of Dextran and vGlut1 staining. Scale bar 50 μm.


K - L . of dextran positive axons shows that AAV - HDAC3mut promotes DRG regenerative growth across beyond spinal lesion site . Data is expressed as of dextran + axons at each vs dextran + axons at - 700μm from lesion margin ( K ) or from caudal margin of lesion to most rostral dextran + axon tip for each ( L ) . N = 10 per condition , ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 indicate a significant difference ( ANOVA followed by Bonferroni ) .

O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O B-ORGANISM O B-GENEPROD O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K-L. Quantification of dextran positive axons shows that AAV-HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site. Data is expressed as percentage of dextran+ axons at each distance vs dextran+ axons at -700μm from the lesion margin (K) or distance from the caudal margin of the lesion to the most rostral dextran+ axon tip for each animal (L). N= 10 animals per condition, ± s.e.m. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 indicate a significant difference (ANOVA followed by Bonferroni test).


A - B . Intrathecally administered RGFP966 ( 966 ) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran - red traced axons across beyond lesion site ( GFAP , green ) ( dotted lines denote rostral caudal margins of scar around lesion ) . Scale bar , 200 μm .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O B-SUBCELLULAR B-EXP_ASSAY O B-DISEASE I-DISEASE I-DISEASE O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

A-B. Intrathecally administered RGFP966 (966) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, 200 μm.


C - D . of axonal regeneration shows dextran axonal labelling across beyond spinal lesion site . Data is expressed as of dextran + axons vs dextran + axons at - 700 μm from lesion site ± s . e . m ( C ) or from caudal margin of lesion to last dextran + axon tip ( D ) . N = 6 - 10 per condition . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 ) indicate a significant difference ( ANOVA followed by Bonferroni ) .

O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site. Data is expressed as ratio of dextran+ axons vs dextran+ axons at -700 μm from the lesion site ± s.e.m (C) or distance from the caudal margin of the lesion to the last dextran+ axon tip (D). N= 6-10 animals per condition. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).


E . Immunofluorescence for H3K9ac Neurofilament 200 ( NF200 ) shows upregulated H3K9ac in vivo in DRG neurons after 966 . Scale bar , 50 µm .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-TISSUE B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O

E. Immunofluorescence for H3K9ac and Neurofilament 200 (NF200) shows upregulated H3K9ac in vivo in DRG neurons after 966. Scale bar, 50 µm.


F - G . Data is expressed as fold change of mean ( F ) or % of H3K9ac + ( G ) ( H3K9ac + threshold was set at mean level of vehicle treated , DAPI counter was used to label nuclei from NF200 + or NF200 - ) vs veh ± s . e . m . N = 11 - 12 biological replicates . ( * * * p < 0 . 005 ) indicate a significant difference vs respective Veh ( ANOVA followed by Bonferroni ) .

O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-G. Data is expressed as fold change of fluorescence intensity mean levels (F) or % of H3K9ac+ cells (G) (H3K9ac+ threshold was set at the mean intensity level of vehicle treated animals, DAPI counter staining was used to label nuclei from NF200+ or NF200- cells) vs veh± s.e.m. N= 11-12 biological replicates. (***p<0.005) indicate a significant difference vs respective Veh (ANOVA followed by Bonferroni test).


( B ) Western blot of Atxn1 in cerebellar granule neurons ( CGNs ) after knockdown of Zbtb7a / or Zbtb7b ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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(B) Western blot analysis of Atxn1 levels in cerebellar granule neurons (CGNs) after knockdown of Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.


( C ) Western blot of Atxn1 in cerebella of wild - type ( n = 3 ) , Zbtb7a + / - ( n = 4 ) , Zbtb7b + / - ( n = 3 ) , Zbtb7a + / - ; Zbtb7b + / - ( n = 4 ) mice . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Western blot analysis of Atxn1 levels in the cerebella of wild-type (n = 3), Zbtb7a+/- (n = 4), Zbtb7b+/- (n = 3), Zbtb7a+/-; Zbtb7b+/- (n = 4) mice. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.


( D ) Western blot of Atxn1 in CGNs after overexpressing Zbtb7a / or Zbtb7b ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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(D) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.


( E ) Genetic interaction of wild - type human ATXN1 ( 30Q ) with either ZBTB7A or ZBTB7B expressed in Drosophila eyes . Co - expression of ATXN1 ( 30Q ) with ZBTB7A or ZBTB7B severely disrupted external Drosophila eye structure increased ATXN1 . Scale bar : 100

O O O O B-EXP_ASSAY O O O O B-ORGANISM B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM B-TISSUE O O B-GENEPROD O O O O O

(E) Genetic interaction of wild-type human ATXN1(30Q) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes. Co-expression of ATXN1(30Q) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels. Scale bar: 100 μm


( C ) Western blot of ATXN1 after overexpression of ZBTB7B wild - type ( WT ) , zinc - finger domain mutant ( ZFmut ) or BTB domain mutants ( BTBmut1 BTBmut2 ) ( n = 3 ) . Data information : Mean ± SD , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild-type (WT), zinc-finger domain mutant (ZFmut) or BTB domain mutants (BTBmut1 and BTBmut2) (n = 3). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.


( D ) qRT - PCR results of Atxn1 mRNA in cerebella of wild - type ( n = 3 ) , Zbtb7b + / - ( n = 4 ) , Zbtb7b - / - mice ( n = 4 ) . Data information : Mean ± SD , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) qRT-PCR results of Atxn1 mRNA levels in the cerebella of wild-type (n = 3), Zbtb7b+/- (n = 4), and Zbtb7b-/- mice (n = 4). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.


( E ) MA plot ( M : log ; A : mean average ) of differentially expressed genes ( DEG ) from - seq samples of overexpressing either WT or ZFmut ZBTB7B . Four filters number of genes kept after applying each filter are described . Two genes that passed 4th filter are labeled .

O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(E) MA plot (M: log ratio; A: mean average) of the differentially expressed genes (DEG) analysis from RNA-seq samples of the cells overexpressing either WT or ZFmut ZBTB7B. Four filters and the number of genes kept after applying each filter are described. Two genes that passed the 4th filter are labeled.


( F ) Expression of 16 genes that passed 3rd filter . Color scale : row z - score .

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(F) Expression heatmap of the 16 genes that passed the 3rd filter. Color scale: row z-score.


( G ) qRT - PCR results of RSK3 mRNA across different CRISPR knockout lines ( n = 2 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(G) qRT-PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( H ) Epistatic relationship between ZBTB7B RSK3 in terms of ATXN1 regulation . ATXN1 were measured after overexpression of ZBTB7B in RSK3 knockout ( sgRSK3 ) control lines ( WT sgControl ) ( n = 3 ) . ZBTB7B + RSK3 : co - overexpression of both ZBTB7B RSK3 . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(H) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation. ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line (sgRSK3) and control cell lines (WT and sgControl) (n = 3). ZBTB7B+RSK3: co-overexpression of both ZBTB7B and RSK3. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( I ) qRT - PCR results of RSK3 mRNA after knockdown of ZBTB7B in Daoy ( n = 2 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(I) qRT-PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( J ) qRT - PCR results of Rsk3 mRNA in cerebella of Zbtb7b conditional knockout mice ( n = 6 ) . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - t - .

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(J) qRT-PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice (n = 6). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


( K ) Position frequency matrix of ZBTB7B binding a table of predicted ZBTB7B binding sites on human RSK3 promoter . Start End position numbers are described on TSS . seventy base pairs upstream RSK3 promotor includes top two potential binding sites was used for luciferase assays . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - t - .

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(K) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter. Start and End position numbers are described based on TSS. The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays. Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


( L ) Luciferase result of RSK3 promoter in HEK 293T HeLa transfected with either an empty or ZBTB7B expressing vector ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - t - .

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(L) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector (n = 3). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


( A ) Western blot of ATXN1 after knockdown of RSK3 in Daoy . Bottom left panel : qRT - PCR results of RSK3 mRNA . Bottom right panel : densitometry of western blot image in top panel ( n = 3 except for shATXN1 where n = 1 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(A) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells. Bottom left panel: qRT-PCR results of RSK3 mRNA. Bottom right panel: densitometry of the western blot image in the top panel (n = 3 except for shATXN1 where n = 1). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( B ) Western blot of ATXN1 after overexpression of RSK3 , ZBTB7B , or eGFP in Daoy ( n = 3 ) . Data information : RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3 . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(B) Western blot analysis of ATXN1 after overexpression of RSK3, ZBTB7B, or eGFP in Daoy cells (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( C ) Western blot of ATXN1 ( 82Q ) after overexpression of either wild - type or inactive RSK3 in Daoy expressing ATXN1 ( 82Q ) ( n = 3 ) . Data information : RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3 . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Western blot analysis of ATXN1(82Q) after overexpression of either wild-type or inactive RSK3 in Daoy cells expressing ATXN1(82Q) (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( D ) In vitro kinase of RSK3 with ATXN1 . MSK1 was used as a positive control . Asterisk : auto - phosphorylated RSK3 .

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(D) In vitro kinase assay of RSK3 with ATXN1. MSK1 was used as a positive control. Asterisk: auto-phosphorylated RSK3.


( E ) In vitro kinase of four different RSK members with ATXN1 .

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(E) In vitro kinase assay of four different RSK members with ATXN1.


( F ) In vitro kinase of RSK3 with N - terminal ( F1 ) , middle ( F2 ) , C - terminal fragment ( F3 ) of ATXN1 .

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(F) In vitro kinase assay of RSK3 with N-terminal (F1), middle (F2), and C-terminal fragment (F3) of ATXN1.


( G ) Mass spectrometry of phosphorylated residues in ATXN1 F3 after in vitro kinase with RSK3 .

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(G) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3.


( H ) Western blot of ATXN1 after a treatment with RSK3 inhibitor ( BI - D1870 ) on SCA1 iPSC - derived neurons ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(H) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( I ) Western blot of total phospho - S776 ATXN1 after a co - treatment with MSK1 ( SB - 747651A ) RSK3 inhibitor ( BI - D1870 ) on SCA1 iPSC - derived neurons ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(I) Western blot analysis of total and phospho-S776 ATXN1 after a co-treatment with MSK1 (SB-747651A) and RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( A ) of Drosophila eyes expressing human ATXN1 ( 82Q ) with either knockdown or overexpression of Drosophila RSK3 homolog Sk6II . Scale bar : 100 μm ( top ) 20 μm ( bottom ) .

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(A) Images of Drosophila eyes expressing human ATXN1(82Q) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II. Scale bar: 100 μm (top) and 20 μm (bottom).


( B ) Improvement of motor performance by knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1 ( 82Q ) in central nervous system . Mean ± , * P < 0 . 05 , Linear mixed - effect model ANOVA , n = 10 / genotype , four times of measurements .

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(B) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1(82Q) in the central nervous system. Mean ± SEM, *P < 0.05, Linear mixed-effect model ANOVA, n = 10/genotype, four times of measurements.


( C ) A representative image of a mouse brain 4 after AAV injection of shRsk3 into 12 old SCA1 mice ( Atxn1154Q / 2Q ) cerebellum . Scale bar : 2 mm .

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(C) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice (Atxn1154Q/2Q) cerebellum. Scale bar: 2 mm.


( D ) Western blot of wild - type ( 2Q ) mutant Atxn1 ( 154Q ) in cerebella of 16 old SCA1 mice dissected 4 after stereotaxic injection of AAV9 harboring shControl ( n = 3 ) , shRsk3 ( n = 3 ) , or shAtxn1 ( n = 2 ) . Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(D) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl (n = 3), shRsk3 (n = 3), or shAtxn1 (n = 2). Mean ± SD, *P<0.05, ***P<0.001 ****P<0.0001, one-way ANOVA.


( E ) Western blot of wild - type ( 2Q ) mutant Atxn1 ( 154Q ) in brainstem ( BS ) cerebella ( CBM ) of 8 old Atxn1154Q / 2Q ( n = 6 ) Atxn1154Q / 2Q ; Rsk3 + / - mice ( n = 7 ) . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , two - t - .

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(E) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the brainstem (BS) and cerebella (CBM) of 8 weeks old Atxn1154Q/2Q (n = 6) and Atxn1154Q/2Q; Rsk3+/- mice (n = 7). Mean ± SD, *P<0.05, **P<0.01, two-tailed t-test.


( F ) Rotarod motor performance result of Atxn1154Q / 2Q ; Rsk3 + / - mice at 11 - 12 old . Mean ± , * P < 0 . 05 , two - way ANOVA , n = 35 , 31 , 34 , 38 for WT , Rsk3 + / - , Atxn1154Q / 2Q , Atxn1154Q / 2Q ; Rsk3 + / - genotype , respectively .

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(F) Rotarod motor performance test result of Atxn1154Q/2Q; Rsk3+/- mice at 11-12 weeks old. Mean ± SEM, *P<0.05, two-way ANOVA, n = 35, 31, 34, 38 for WT, Rsk3+/-, Atxn1154Q/2Q, Atxn1154Q/2Q; Rsk3+/- genotype, respectively.


( A ) western blot of Atxn1 , Msk1 , Rsk3 in seven different brain regions of wild - type mice at 9 old ( n = 3 ) . Each were represented relative to in brainstem . Mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . Data information : BS : brainstem , CBM : cerebellum , MB : midbrain , ST : striatum , HC : hippocampus , CTX : cortex , OB : olfactory bulbs .

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(A) Fluorescent western blot analysis of Atxn1, Msk1, and Rsk3 in seven different brain regions of wild-type mice at 9 weeks old (n = 3). Each protein levels were represented relative to the protein levels in brainstem. Mean ± SD, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. Data information: BS: brainstem, CBM: cerebellum, MB: midbrain, ST: striatum, HC: hippocampus, CTX: cortex, OB: olfactory bulbs.


( B ) Representative BaseScope of Rsk3 in whole brain sagittal sections of wild - type ( top ) Rsk3 - / - mice ( bottom ) at 5 - 6 old . areas within black squares are magnified in right panels in each genotype . Scale bar = 2 mm in whole brain ; 50 μm in magnified . Data information : BS : brainstem , CBM :

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(B) Representative BaseScope assay images of Rsk3 in the whole brain sagittal sections of wild-type (top) and Rsk3-/- mice (bottom) at 5-6 weeks old. The areas within the black squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum


( C ) Representative IF of Msk1 in whole brain sagittal sections of wild - type ( top ) Msk1 - / - mice ( bottom ) at 6 months old . areas within squares are magnified in right panels in each genotype . Scale bar = 2 mm in whole brain ; 50 μm in magnified . Data information : BS : brainstem , CBM :

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(C) Representative IF images of Msk1 in the whole brain sagittal sections of wild-type (top) and Msk1-/- mice (bottom) at 6 months old. The areas within the white squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum


( D ) Western blot of total active form of Rsk3 Msk1 in cerebella brainstem of 12 old WT SCA1 mice ( n = 4 ) . Relative Rsk3 Msk1 were represented into log2 scale . of two kinases are significantly different between two brain regions . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - way ANOVA . Data information : BS : brainstem , CBM :

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(D) Western blot analysis of total and active form of Rsk3 and Msk1 in the cerebella and brainstem of 12 weeks old WT and SCA1 mice (n = 4). Relative Rsk3 and Msk1 levels were represented into log2 scale. The protein levels of the two kinases are significantly different between the two brain regions. Mean ± SD, *P<0.05, **P<0.01, ****P <0.0001, two-way ANOVA. Data information: BS: brainstem, CBM: cerebellum


( E ) Western blot of ATXN1 , RSK3 , pRSK3 , MSK1 , pMSK1 in postmortem cerebella , pons , medulla oblongata of human controls SCA1 ( n = 3 per genotype ) . Total was used for normalization , are represented relative to cerebellar in control group . C : Cerebellum , P : Pons , M : medulla oblongata . Values from P M are combined presented in BS . Mean ± , * P < 0 . 05 , * * P < 0 . 01 , two - way ANOVA , n = 3 ( CBM ) 6 ( BS ) . Data information : BS : brainstem , CBM :

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(E) Western blot analysis of ATXN1, RSK3, pRSK3, MSK1, and pMSK1 in the postmortem cerebella, pons, and medulla oblongata of human controls and SCA1 patients (n = 3 per genotype). Total protein was used for normalization, and protein levels are represented relative to cerebellar protein levels in control group. C: Cerebellum, P: Pons, M: medulla oblongata. Values from P and M are combined and presented in BS. Mean ± SEM, *P<0.05, **P<0.01, two-way ANOVA, n = 3 (CBM) and 6 (BS). Data information: BS: brainstem, CBM: cerebellum


( A ) Western blot of total pS776 wild - type mutant Atxn1 in brainstem of 8 old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA , n = 7 , 6 , 4 , 7 following order of genotypes in western blot , respectively .

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(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the brainstem of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, *P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.


( B ) Plethysmography data measuring breathing ability ( total ventilated volume per minute ) of 34 old mice with different genotypes . Mean ± SD , * P < 0 . 05 , two - t - , n = 12 , 13 , 15 , 13 , 15 , 9 , 15 , 13 following order of genotypes in graph , respectively .

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(B) Plethysmography data measuring breathing ability (total ventilated gas volume per minute) of 34 weeks old mice with different genotypes. Mean ± SD, *P<0.05, two-tailed t-test, n = 12, 13, 15, 13, 15, 9, 15, 13 following the order of genotypes in the graph, respectively.


( C ) Representative immunohistochemistry of Iba1 + reactive microglia in coronally sectioned hypoglossal nucleus of 36 old mice with different genotypes . Yellow arrows indicates Iba1 + microglia , larger pyramidal shaped are motoneurons . scale bar = 50 μm .

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(C) Representative immunohistochemistry images of Iba1+ reactive microglia in the coronally sectioned hypoglossal nucleus of 36 weeks old mice with different genotypes. Yellow arrows indicates Iba1+ microglia, and larger pyramidal shaped cells are motoneurons. scale bar = 50 μm.


( D ) of number of Iba1 + shown in ( C ) . Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from six sections per mouse .

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(D) Quantification of the number of Iba1+ cells shown in (C). Mean ± SD, *P<0.05, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.


( E ) Representative Hematoxylin Eosin stained motoneurons in coronally sectioned hypoglossal nucleus ( dashed ) of 36 old mice with different genotypes . motoneurons are purple with pyramidal shape within nucleus . C : central canal . Scale bar = 100 μm .

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(E) Representative Hematoxylin and Eosin stained motoneurons in the coronally sectioned hypoglossal nucleus (white dashed line) of 36 weeks old mice with different genotypes. The motoneurons are dark purple with pyramidal shape within the nucleus. C: central canal. Scale bar = 100 μm.


( F ) of number of motoneurons shown in ( E ) . Mean ± SD , * P < 0 . 05 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from six sections per mouse .

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(F) Quantification of the number of motoneurons shown in (E). Mean ± SD, *P<0.05, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.


( G ) Kaplan - Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases . data of Msk1 + / - , Rsk3 + / - , Msk1 + / - ; Rsk3 + / - mice overlap those of wild - type mice . * P < 0 . 05 , Gehan - Breslow - Wilcoxon , n = 15 , 15 , 12 , 13 , 22 , 11 , 20 , 19 following order of genotypes in legend on top of figure , respectively .

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(G) Kaplan-Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases. The data of Msk1+/-, Rsk3+/-, and Msk1+/-; Rsk3+/- mice overlap those of wild-type mice. *P<0.05, Gehan-Breslow-Wilcoxon test, n = 15, 15, 12, 13, 22, 11, 20, 19 following the order of genotypes in the legend on top of the figure, respectively.


( A ) Western blot of total pS776 wild - type mutant Atxn1 in cerebella of 8 old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA , n = 7 , 6 , 4 , 7 following order of genotypes in western blot , respectively .

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(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, **P<0.01, ***P<0.001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.


( B ) Rotarod motor performance result of 11 - 12 old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± , * P < 0 . 05 , two - way ANOVA , n = 18 , 12 , 13 , 11 , 19 , 13 , 15 , 13 following order of genotypes in legend on top of figure , respectively .

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(B) Rotarod motor performance test result of 11-12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SEM, *P<0.05, two-way ANOVA, n = 18, 12, 13, 11, 19, 13, 15, 13 following the order of genotypes in the legend on top of the figure, respectively.


( C ) Representative immunofluorescent ( IF ) of cerebellar molecular layer between cerebellum lobule V VI across different genotypes at 36 old , stained with anti - Calbindin 1 antibody . Scale bar = 100 μm .

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(C) Representative immunofluorescent (IF) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old, stained with anti-Calbindin 1 antibody. Scale bar = 100 μm.


( D ) of thickness of top bottom molecular layers shown in ( C ) . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from three sections per mouse .

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(D) Quantification of the thickness of top and bottom molecular layers shown in (C). Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from three sections per mouse.


( E ) Fold increase of Oct4 - GFP + colony number over control under forced expression of factors . n ≥ 5 , where each dot indicates an independent experiment . Box - plots show median , 1st 3rd quartile values . One - sample Wilcoxon p - values are as indicated , with p values < 0 . 05 shown in

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(E) Fold increase of Oct4-GFP+ colony number over control under forced expression of individual factors. n ≥ 5, where each dot indicates an independent experiment. Box-plots show median, 1st and 3rd quartile values. One-sample Wilcoxon test p-values are as indicated, with p values < 0.05 shown in red


( B ) of average gene expression normalised to β - actin over an EpiSC resetting time course in 2i + LIF . Each row is coloured according to unique minimum maximum for that gene . values shown are average expression of four independent

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(B) Heatmap of average gene expression normalised to β-actin over an EpiSC resetting time course in 2i+LIF. Each row is coloured according to the unique minimum and maximum for that gene. The values shown are average expression of four independent experiments


( C ) Gene expression for Stat3 , Klf2 , Esrrb Tfcp2l1 during EpiSC resetting relative to established mouse ESCs . β - actin serves as an internal control . Mean + / - , n = 4 independent experiments . * = p < 0 . 05 Student " s

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(C) Gene expression for Stat3, Klf2, Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs. β-actin serves as an internal control. Mean +/- SEM, n=4 independent experiments. * = p<0.05 Student"s t


( E ) Representative confocal ( top ) ( bottom ) of Oct4 - GFP reporter mean . indicated lines were treated with DOX for first 2 days imaged at Day 6 . Mean + / - SD of 9 technical replicates . One representative experiment of two is shown . Scale bars =

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(E) Representative confocal images (top) and quantification (bottom) of Oct4-GFP reporter mean intensity. The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6. Mean +/- SD of 9 technical replicates. One representative experiment of two is shown. Scale bars=300µm


( F ) Representative alkaline phosphatase ( AP ) ( left ) ( right ) of AP + colonies after clonal replating , as described in

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(F) Representative alkaline phosphatase (AP) staining images (left) and quantification (right) of AP+ colonies after clonal replating, as described in panel


( A ) Top : Predictions of number of regulation steps required for full activation of indicated gene under control or dual expression of Esrrb Tbx3 ( + E / T ) . Bottom : Gene expression of EpiSCs harbouring empty vector ( grey ) or Esrrb / Tbx3 ( orange ) , capture at D0 ( F / A ) , D2 D4 ( as described in Fig 3D ) . Dashed black : expression in ESCs maintained in 2i + LIF . Data normalised to empty vector cultures in F / A . Gapdh serves as an internal control . Mean + / - , n = 3 independent experiments . ( B ) As for panel A , comparison of control with dual expression of Esrrb Klf4 ( + E / K , green in bottom plot ) . Mean + / - , n = 3 independent experiments for bottom qRT - PCR

O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY

(A) Top: Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 (+E/T). Bottom: Gene expression of EpiSCs harbouring empty vector (grey) or Esrrb/Tbx3 (orange), capture at D0 (F/A), D2 and D4 (as described in Fig 3D). Dashed black line: expression levels in ESCs maintained in 2i+LIF. Data normalised to empty vector cultures in F/A. Gapdh serves as an internal control. Mean +/- SEM, n=3 independent experiments. (B) As for panel A, comparison of control with dual expression of Esrrb and Klf4 (+E/K, green in bottom plot). Mean +/- SEM, n=3 independent experiments for bottom qRT-PCR panels


( C ) Left : Flow cytometry profiles of resetting progression of EpiSCs stably transfected with Esrrb - T2A - Klf4 construct cultured in 2i + LIF with DOX for 2 4 days , with indicated fraction of sorted for colony formation . Since Venus reporter is under control of a DOX responsive element , emission spectra of Venus GFP overlap , Oct4 - GFP reporter could not be fully distinguished from Venus expression . Right : Number of AP + colonies formed from 250 sorted from indicated fractions . Data points represent two technical replicates of one out of two independent

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(C) Left: Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb-T2A-Klf4 construct and cultured in 2i+LIF with DOX for 2 and 4 days, with the indicated fraction of cells sorted for colony formation assay. Since the Venus reporter is under the control of a DOX responsive element, and the emission spectra of Venus and GFP fluorescence overlap, Oct4-GFP reporter could not be fully distinguished from Venus expression. Right: Number of AP+ colonies formed from 250 sorted cells from indicated fractions. Data points represent two technical replicates of one out of two independent experiments


( D ) of single - expression measured by qRT - PCR of major ESC EpiSC markers in un - induced EpiSCs ( black ) , established ESCs ( red ) , Day 2 High / Low ( blue ) , Day 4 High ( green )

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(D) Heatmap of single-cell expression measured by qRT-PCR of major ESC and EpiSC markers in un-induced EpiSCs (black), established ESCs (red), Day 2 High/Low (dark and light blue), and Day 4 High cells (green)


( E ) Scatterplots of relative expression of pluripotency transitional markers . Red bar corresponds to median gene expression , each dot represents a single

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(E) Scatterplots of the relative expression of pluripotency and transitional markers. Red bar corresponds to median gene expression, and each dot represents a single cell


Left : 0 . 782 cABN predictions of of gene activation between gene pairs ( , OFF ; blue , ON ) along resetting trajectory , compared to single gene expression measured by RT - qPCR . Each table summarises of single at indicated stage of resetting ( columns ) that have indicated expression state ( rows ) . Right : Scatterplots showing single coordinates on expression of gene

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Left: 0.782 cABN predictions of the sequence of gene activation between gene pairs (white, OFF; blue, ON) along the resetting trajectory, compared to single cell gene expression measured by RT-qPCR. Each table summarises the percentage of single cells at the indicated stage of resetting (columns) that have the indicated expression state (rows). Right: Scatterplots showing single cell coordinates based on the expression of the gene pair


( A ) 0 . 782 cABN predictions of essential or dispensable factors for EpiSC resetting , compared with experiment results shown in panel ( B ) . ( B ) siRNA knockdown effects measured by Oct4 - GFP + colony formation at Day 6 of resetting . n = 4 independent

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(A) 0.782 cABN predictions of essential or dispensable factors for EpiSC resetting, compared with the experiment results shown in panel (B). (B) siRNA knockdown effects measured by Oct4-GFP+ colony formation at Day 6 of resetting. n=4 independent experiments


( C ) Left , resetting capacity of Klf2 Klf4 KO EpiSCs measured by Oct4 - GFP + colony formation at Day 6 of resetting . Data points represent two independent experiments . Right , representative bright field of wild type Klf2 KO EpiSCs at Day 6 of resetting in 2i +

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(C) Left, resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4-GFP+ colony formation at Day 6 of resetting. Data points represent two independent experiments. Right, representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i+LIF


( D ) Expression of naïve pluripotency , transition somatic lineage markers in wild type Klf2 KO EpiSCs during a resetting time course in 2i + LIF . Expression is normalised to wild type EpiSCs in A / F , β - actin was used as internal control . values shown correspond to average expression of three technical replicates from one representative experiment out of

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(D) Expression of naïve pluripotency, transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i+LIF. Expression is normalised to wild type EpiSCs in A/F, and β-actin was used as internal control. The values shown correspond to the average expression of three technical replicates from one representative experiment out of two


( E ) Rescue of Klf2 KO EpiSC resetting by forced expression of network components measured by Oct4 - GFP + colony formation at Day 6 of resetting in 2i + LIF . Data points represent two independent

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(E) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4-GFP+ colony formation at Day 6 of resetting in 2i+LIF. Data points represent two independent experiments


( F ) Comparison between effect of single factor knockdowns on ESC maintenance ( n = 4 independent experiments ) EpiSC resetting using experimental

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(F) Comparison between the effect of single factor knockdowns on ESC maintenance (n=4 independent experiments) and EpiSC resetting using experimental results


( G ) EpiSC resetting in 2i + LIF measured by Oct4 - GFP + colony formation after Stat3 siRNA in wild type EpiSCs ( left ) , or Klf4 KO EpiSCs transfected with Tfcp2l1 Gbx2 siRNAs . n = 3 independent experiments in wild type ( box plots indicate min , median , max ) , n = 2 for Klf4 - / - ( box plots indicate min , mean , max )

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(G) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation after Stat3 siRNA in wild type EpiSCs (left), or Klf4 KO EpiSCs transfected with Tfcp2l1 and Gbx2 siRNAs. n=3 independent experiments in wild type cells (box plots indicate min, median, max), n=2 for Klf4-/- cells (box plots indicate min, mean, max)


( H ) EpiSC resetting in 2i + LIF measured by Oct4 - GFP + colony formation of Stat3 knockdown EpiSCs transiently transfected with Tfcp2l1 , Gbx2 Klf4 . n = 4 independent experiments : Student " s t - , p - value indicated on plot . Box plots indicate min , median , max . See also Appendix

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(H) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation of Stat3 knockdown EpiSCs transiently transfected with Tfcp2l1, Gbx2 and Klf4. n=4 independent experiments: Student"s t-test, p-value indicated on plot. Box plots indicate min, median, max. See also Appendix Fi


( B ) Comparison of predictions ( left ) experimental outcome ( right ) for potency of additional network factors in OSKM - driven MEF reprogramming in LIF containing medium n = 4 independent experiments , except Klf2 Nanog where n = 2 independent experiments . * = p < 0 . 05 Student " s t - . n . s . = not significant . Red dashed lines indicate empty vector + OSKM ( C ) Comparison of predictions ( left ) experimental outcome ( right ) for potency of additional network factors in OSKM - driven MEF reprogramming in 2i + LIF . n = 4 independent experiments , except Klf2 Nanog where n = 2 independent experiments . * = p < 0 . 05 Student " s t - . n . s . = not significant . Empty vector + OSKM reprogramming in LIF ( ' control no 2i ' ) was included as a control for effect of 2i addition . Red dashed lines indicate empty vector + OSKM control level . Box plots indicate min ,

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(B) Comparison of predictions (left) and experimental outcome (right) for the potency of additional network factors in OSKM-driven MEF reprogramming in LIF containing medium n=4 independent experiments, except Klf2 and Nanog where n=2 independent experiments. * = p<0.05 Student"s t-test. n.s. = not significant. Red dashed lines indicate empty vector + OSKM (C) Comparison of predictions (left) and experimental outcome (right) for the potency of additional network factors in OSKM-driven MEF reprogramming in 2i+LIF. n=4 independent experiments, except Klf2 and Nanog where n=2 independent experiments. * = p<0.05 Student"s t-test. n.s. = not significant. Empty vector + OSKM reprogramming in LIF ('control no 2i') was included as a control for the effect of 2i addition. Red dashed lines indicate empty vector + OSKM control level. Box plots indicate min, median


( D ) Recapitulation of gene activation kinetics during MEF reprogramming . Top , number of regulation steps required for permanent activation of indicated gene . Tfcp2l1 Sall4 are found to activate earlier than Nanog Esrrb . Bottom , gene expression measured from sorted populations of reprogramming intermediates from O " Malley et al . (

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(D) Recapitulation of the gene activation kinetics during MEF reprogramming. Top, the number of regulation steps required for permanent activation of the indicated gene. Tfcp2l1 and Sall4 are found to activate earlier than Nanog and Esrrb. Bottom, gene expression measured from sorted populations of reprogramming intermediates from O"Malley et al. (


A Heat map from - Seq data from FACS - isolated muscle single myofibers showing relative expression of Pax7 , Myf5 , MyoD , Myog Myf6 .

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A Heat map from RNA-Seq data from FACS-isolated muscle stem cells and single myofibers showing relative expression of Pax7, Myf5, MyoD, Myog and Myf6.


H Colormap of Myf5 , MyoD Myf6 peaks within 100kb of Transcription Start Sites ( TSS ) of myokines ( supplemental table EV3 ) ranging from zero ( black ) to six peaks ( red ) occupancy . onset of differentiation coincides with increased binding of MRFs to regulatory domains of myokines genes .

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H Colormap of Myf5, MyoD and Myf6 peaks within 100kb of the Transcription Start Sites (TSS) of myokines (supplemental table EV3) ranging from zero (black) to six peaks (red) occupancy. The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes.


A Gene expression of myokines ( table EV3 ) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media ( Ham ' s F10 supplemented with 20 % Bovine Serum , 1 % penicillin / streptomycin , 2 . 5 ng / ml basic Fibroblast Growth Factor ) to terminally differentiated myocytes ( 2 days in differentiation media , DMEM supplemented with 5 % horse serum ) to post mitotic multinucleated myotubes ( 5 days in differentiation media ) . Gene expression was assayed in biological triplicate by microarray ( Soleimani et al . , 2018 ; Soleimani et al . , 2012a ) . Expression of each gene was averaged across three replicates normalized to mean zero standard deviation of one . Expression values were then truncated at + / - 3 for color display . Blue indicates lower than average expression , while red indicates higher than average expression . B Distribution of Myf6 peaks within 100kb of TSS of myokine genes ( table EV3 ) , similar to shown in ( A ) .

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A Gene expression analysis of myokines (table EV3) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media (Ham's F10 supplemented with 20% Fetal Bovine Serum, 1% penicillin/streptomycin, 2.5 ng/ml basic Fibroblast Growth Factor) to terminally differentiated myocytes (2 days in differentiation media, DMEM supplemented with 5% horse serum) to post mitotic multinucleated myotubes (5 days in differentiation media). Gene expression was assayed in biological triplicate by microarray (Soleimani et al., 2018; Soleimani et al., 2012a). Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one. Expression values were then truncated at +/- 3 for color display. Blue indicates lower than average expression, while red indicates higher than average expression. B Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes (table EV3), similar to the analysis shown in (A).


C - E Snapshots of UCSC genome browser showing enrichment of Myf6 ChIP - Seq reads compared to control around EGF ( C ) , LIF OSM ( D ) , VEGFA ( E ) loci superimposed on reads for Histone H3 lysine 4 mono methyl ( H3K4me1 ) , Histone H3 lysine 4 tri methyl ( H3K4me3 ) total H3 in myoblasts ( pink ) myotubes ( green ) .

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C-E Snapshots of the UCSC genome browser showing enrichment of Myf6 ChIP-Seq reads compared to control around EGF (C), LIF and OSM (D), and VEGFA (E) loci superimposed on reads for Histone H3 lysine 4 mono methyl (H3K4me1), Histone H3 lysine 4 tri methyl (H3K4me3) and total H3 in myoblasts (pink) and myotubes (green).


F Snapshots of representative IGV tracks displaying - Seq data from WT whole muscle ( WM ) , single myofibers ( SF ) satellite ( SC ) showing expression of EGF , EGFR VEGFA . Ckm Myf6 are used as positive controls for whole muscle single . PDGFRA , a marker of fibro / adipogenic progenitor , is used as a control to show lack of niche in single myofiber samples .

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F Snapshots of representative IGV tracks displaying RNA-Seq data from WT whole muscle (WM), single myofibers (SF) and satellite cells (SC) showing the expression of EGF, EGFR and VEGFA. Ckm and Myf6 are used as positive controls for whole muscle and single fibers. PDGFRA, a marker of fibro/adipogenic progenitor cells, is used as a control to show the lack of niche cells in the single myofiber samples.


G - H - Sequencing Reads Per Million ( RPM ) for EGF ( G ) EGFR ( H ) in satellite single myofibers . - Sequencing libraries were prepared from 1000 satellite freshly - isolated by FACS or from a single myofiber as described in Material Methods . ( n = 3 biological replicates per group ) bars = mean + / - SD , two - t - ) .

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G-H RNA-Sequencing Reads Per Million (RPM) for EGF (G) and EGFR (H) in satellite cells and single myofibers. RNA-Sequencing libraries were prepared from 1000 satellite cells freshly-isolated by FACS or from a single myofiber as described in the Material and Methods. (n=3 biological replicates per group) bars=mean +/- SD, two-tailed t-test).


I Activation of EGFR in MuSCs on EDL myofibers treated with EGF compared to untreated control . Myofibers were cultured for 48 hours were treated with 40ng / ml of EGF for 10 minutes before fixing with antibodies for PAX7 phospho - EGFR . Scale bar = 30μm .

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I Activation of EGFR in MuSCs on EDL myofibers treated with EGF compared to untreated control. Myofibers were cultured for 48 hours and were treated with 40ng/ml of EGF for 10 minutes before fixing and staining with antibodies for PAX7 and phospho-EGFR. Scale bar=30μm.


J Activation of EGFR signaling in myoblasts treated with recombinant EGF . Myoblasts were treated with 2 ng / ml of recombinant in growth media ( Ham ' s F10 supplemented with 20 % FBS , 2 . 5 ng / ml bFGF ) for 15 minutes lysed in RIPA lysis buffer . Western Blots were performed with antibodies against EGFR phospho - EGFR ( pEGFR - Y1068 ) .

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J Activation of EGFR signaling in myoblasts treated with recombinant EGF. Myoblasts were treated with 2 ng/ml of recombinant protein in growth media (Ham's F10 supplemented with 20% FBS, 2.5 ng/ml bFGF) for 15 minutes and lysed in RIPA lysis buffer. Western Blots were performed with antibodies against EGFR and phospho-EGFR (pEGFR-Y1068).


K Depletion of Myf6 transcript by siRNAs in differentiated myotubes ( 5DM ) . ( n = 3 biological replicates ) , two - t - , bars = mean + / - SD L Depletion of Myf6 expression by siRNAs leads to a significant reduction in gene expression output of Epidermal Growth Factor ( EGF ) . ( n = 3 biological replicates ) , two - t - , bars = mean + / -

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K Depletion of Myf6 transcript by siRNAs in differentiated primary myotubes (5DM). (n=3 biological replicates), two-tailed t-test, bars = mean +/- SD L Depletion of Myf6 expression by siRNAs leads to a significant reduction in the gene expression output of Epidermal Growth Factor (EGF). (n=3 biological replicates), two-tailed t-test, bars = mean +/- SD


B Picture of 8 week - old Myf6 - KO WT mice .

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B Picture of 8 week-old Myf6-KO and WT mice.


C Relative expression of Myf6 transcript in hindlimb skeletal of Myf6 - KO WT mice measured by Quantitative Real - Time PCR ( RT - qPCR ) ( n = 4 mice ) . Boxes represent 1st 3rd quartile , central represents median , whiskers represent range , two - t - .

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C Relative expression of Myf6 transcript in the hindlimb skeletal muscles of Myf6-KO and WT mice measured by Quantitative Real-Time PCR (RT-qPCR) (n=4 mice). Boxes represent 1st and 3rd quartile, central line represents the median, whiskers represent the range, two-tailed t-test.


D Color map of genes that are up / down regulated in whole muscle transcriptome ( - Seq ) of injured uninjured hindlimb from Myf6 - KO WT counterparts . Differential expression is Log2 transformed truncated at + / - 3 . Color scale goes from blue ( - 3 = lower than mean ) , to yellow ( + 3 = higher than mean ) . E Heat map showing distribution of Myf6 peaks within 100kb of TSS of myokine genes ranging from zero ( black ) to six ChIP - Seq peaks ( red ) occupancy .

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D Color map of genes that are up/down regulated in the whole muscle transcriptome (RNA-Seq) of injured and uninjured hindlimb muscles from Myf6-KO and WT counterparts. Differential expression is Log2 transformed and truncated at +/-3. Color scale goes from blue (-3=lower than mean), to yellow (+3=higher than mean). E Heat map showing the distribution of Myf6 peaks within 100kb of the TSS of the myokine genes ranging from zero (black) to six ChIP-Seq peaks (red) occupancy.