CHAMP1 is an essential regulator for human myoblast fusion and muscle development

Domain: chemistry
Published: 2026-01-15
Editorial outcome: minor_revision

Abstract
Human skeletal muscle comprises myofibers formed by fusion of thousand of myoblasts. This process depends on tightly regulated, muscle-specific fusogens, but its genetic control remains poorly understood. Here, we identify CHAMP1 (Chromosome Alignment Maintaining Phosphoprotein 1) as essential for human myoblast fusion in vitro and in vivo. Genomic and protein-interaction assays reveal a noncanonical role for CHAMP1 as a MyoD cofactor that directly activates expression of the key muscle fusogen Myomaker. As established in prior clinical reports, CHAMP1 mutations in patients cause developmental delay, hypotonia, and muscle weakness. Consistently, patient-derived cells show fusion defects that can be fully rescued by restoring Myomaker expression. Structure and function analyses identify C2H2-type zinc-finger motifs on CHAMP1 protein that are both necessary and sufficient for MyoD interaction and Myomaker expression. These findings highlight a cell-autonomous role for CHAMP1 in muscle development and disease and point to therapeutic avenues for treating CHAMP1-related muscle development defects.

Introduction
A fundamental step in muscle development and regeneration is the fusion of muscle precursor cells or myoblasts to form multinucleated myofibers 1–7. Vertebrate myoblast fusion is controlled by the functional cooperation between membrane fusion proteins Myomaker (MYMK) and Myomixer (MYMX, also known as Minion and Myomerger) 8–12. Myomaker induces myoblast membrane fusion by forming a dimeric, GPCR-like structure with a conserved lipid-binding site that facilitates phospholipid insertion and enables transcellular interactions essential for fusogenic activity 13. Myomixer, through interaction with Myomaker, likely facilitates this process through its conserved hydrophobic motif that disrupts the stability of the plasma membrane 9,14. Importantly, mutations of MYMK or MYMX cause fusion myopathy in patients characterized by developmental delay, hypotonia, impaired muscle growth, and facial dysmorphism 15–17.

The expression of muscle fusogens Myomaker and Myomixer is uniquely restricted to skeletal muscle cells, transiently present only during the brief developmental window of myoblast fusion 8,9. Unlike most muscle proteins, their tightly controlled expression is essential because prolonged presence of these agents might disrupt sarcolemma stability and lead to muscle injury 18. Although the expression and activity of these fusogens are known to be regulated by myogenic factors including MyoD and MyoG, as well as several key signaling pathways 19–25 including Notch, ERK1/2, CaMKII, IRE1α, and TGFβ, the precise molecular mechanisms underlying the transcriptional activation of myoblast fusion program remain poorly understood.

We recently developed a CRISPR screening platform and identified a group of genes whose loss impairs human myoblast fusion ref. 26. CHAMP1 (Chromosome Alignment-Maintaining Phosphoprotein 1) emerged from this screen as a surprising hit, as its known role in chromosome alignment pertains to the mitotic phase of dividing cells, whereas myoblast fusion occurs in post-mitotic cells—a context in which CHAMP1 has not previously been studied. More importantly, mutations in CHAMP1 gene cause CHAMP1 syndrome 27–31 with patients consistently exhibit developmental delay, muscle weakness and hypotonia. Despite the significance, little is known about the precise molecular function of CHAMP1 in muscle development and disease. This study aims to close the gap through detailed functional and mechanistic analyses of CHAMP1 in human muscle cells.

We demonstrate that CRISPR-mediated deletion of CHAMP1 in human myoblasts abolishes myoblast fusion both in vitro and in vivo after transplantation into immunodeficient mice. Notably, fibroblasts derived from CHAMP1 patients also exhibit severe fusion defect when reprogrammed into muscle cells. Transcriptome analysis revealed a marked reduction of MYMK expression in CHAMP1 deficient cells. Importantly, the fusion defect, in both CRISPR mutants and patient cells, can be fully rescued by compensated expression of Myomaker. Mechanistically, chromatin binding and protein interaction assays uncovered a non-canonical function for CHAMP1 as a crucial cofactor of MyoD in directly activating MYMK expression. Structural and functional analyses identified key C2H2-type zinc finger motifs on CHAMP1 that are both necessary and sufficient for MyoD interaction, induction of MYMK and activation of human myoblast fusion. Together, these findings advance our understanding of the regulatory mechanisms governing human myoblast fusion and reveal an unexpected role for CHAMP1 in direct transcriptional regulation and human myogenesis, linking its activity to the muscle-autonomous component of a rare yet devastating congenital disorder caused by CHAMP1 mutations.

Results
The nuclear protein CHAMP1 is essential for human myoblast fusion in vitro and in vivo
As a first step to probe the molecular function of CHAMP1 protein in human muscle cells, we examined its expression and localization in myoblasts cultured in proliferation condition (growth medium, GM) or in myocytes at various time-points post myogenic induction (differentiation medium, DM). Immunostaining confirmed the robust inductions of myogenic differentiation makers including skeletal myosin proteins (detected by MF20 antibody) and myogenin (MyoG), and heavy multinucleations of cells after differentiation (Fig. 1a). In all these stages, CHAMP1 protein was consistently and exclusive detected inside the nucleus with exception of day three post differentiation when the CHAMP1 staining signal was observed additionally in the cytosol of multinucleated myotubes (Fig. 1a).

Using CRISPR-Cas9 and gRNAs targeting different coding regions (Fig. 1b), we generated a panel of clonally derived human CHAMP1 deficient (CHAMP1Δ) myoblasts. Genotyping and DNA sequencing confirmed the biallelic frame-shift mutations in all CHAMP1Δ clones (Supplementary Fig. 1). Immunostaining using an antibody raised against a C-terminus of CHAMP1 protein (amino acids 514–646, Fig. 1b) confirmed the complete depletion of CHAMP1 expression in CHAMP1Δ myoblasts (Fig. 1c). EdU incorporation assays showed similar proliferation rates between CHAMP1Δ and wildtype (WT) myoblasts, except for one CHAMP1Δ clone that exhibited a significant reduction in EdU incorporation (Supplementary Fig. 2a, b). Live/dead staining revealed comparable viability across all CHAMP1Δ and WT cells (Supplementary Fig. 2c, d). Karyotype analysis showed no gross chromosomal abnormalities for two CHAMP1Δ clones, while the third clone, which also exhibited reduced proliferation, showed minor regional chromosomal alterations (Supplementary Fig. 3). Importantly, since all CHAMP1Δ clones were derived from single cells following CRISPR-mediated gene inactivation and had undergone more than 20 divisions before karyotype analysis, these findings suggest that overall chromosome segregation during mitosis is largely preserved in CHAMP1Δ myoblasts.

Upon induction of myogenic differentiation, CHAMP1Δ myoblasts normally express myosin protein (Fig. 1c’) and exhibit a comparable differentiation index with that of WT cells (Fig. 1d). However, unlikely WT cells, which readily fuse into large and heavily multinucleated myotubes, the majority of CHAMP1Δ myoblasts fail to fuse after full-term induction of muscle differentiation (DM 6 days, Fig. 1c’, e). This defect persists after an extended period of culture in differentiation medium (DM 10 days, Fig. 1c”), suggesting an essential role of CHAMP1 for human myoblast fusion.

We then tested the fusion capacity of human CHAMP1Δ myoblasts in vivo by transplanting the cells into pre-injured muscle tissue of immunodeficient mice (Fig. 1f). To promote engraftment of human myoblasts, we adopted a previously established irradiation protocol 32 to treat the recipient mice that eliminates the regenerative function of endogenous satellite cells prior to muscle injury. After transplantation and tissue collection, human cells were identified by immunostaining for the expression of human-specific nuclear marker LAMIN A/C, while regenerating muscle cells were detected by immunostaining for embryonic myosin (MYH3). Notably, CHAMP1Δ myoblasts form markedly smaller myofibers compared to those formed by WT myoblasts (Fig. 1g, h). In both groups, no regenerating myonuclei of mouse origin (LAMIN A/C–/MYH3+/Hoechst+) were detected, confirming that regeneration by host satellite cells was effectively suppressed by their radiation. Together, these results suggest that despite normal myogenic differentiation, the fusion program of human myoblasts is severely impaired in vivo upon genetic disruption of CHAMP1 function.

CHAMP1 controls human myoblast fusion by activating Myomaker expression
To elucidate the molecular mechanism underlying the fusion phenotype of human CHAMP1Δ myoblasts, we conducted a time-course transcriptome analysis by comparing CHAMP1Δ myoblasts with a rescue control group in which full-length CHAMP1 protein was reintroduced into CHAMP1Δ myoblasts through retrovirus infection. This allowed a direct comparison between CHAMP1Δ cells and their rescued counterparts in an isogenic background, enabling unambiguous examination of CHAMP1’s molecular function. For both groups, we collected cells under growth medium conditions (DM 0 day), at the two day of differentiation (DM 2 days), and at a later stage of differentiation (DM 6 days).

Consistent with our EdU incorporation and live/dead cells staining assays, transcriptome analysis showed comparable expression of proliferation markers (Supplementary Fig. 4a) and apoptosis markers (Supplementary Fig. 4b) between CHAMP1Δ cells and their isogenic rescued counterparts. Muscle differentiation also appeared largely normal in CHAMP1Δ myoblasts, as reflected by similar expression of key myogenic transcription factors (e.g., MYF5, MYOD, MYOG, MEF2C), fusion protein MYMX, and structural genes (e.g., ACTA1, CACNB1, CAV3, CAVIN4, MYBPH, MYH2, MYH3, MYH7, MYH8, MYOT, SYNPO2, TNNI1, TNNT2, TTN) relative to rescue controls (Fig. 2a). Comparison with WT myoblasts revealed a delayed expression of these muscle structural genes in CHAMP1Δ cells; however, most of these changes were not affected by CHAMP1 re-expression in CHAMP1Δ cells, suggesting that these difference likely reflects clonal variations of CHAMP1Δ lines rather than a specific molecular function for CHAMP1.

Using a two-fold change threshold, no differentially expressed genes between CHAMP1Δ and the isogenic rescue control group were observed under the growth medium condition. In contrast, 52 genes were differentially expressed at day 2 of differentiation—all of which were downregulated—and 201 genes at day 6 of differentiation, with 176 of them downregulated in CHAMP1Δ cells compared to the rescue control (Supplementary Data 1). Among the genes commonly downregulated at both day 2 and day 6 of differentiation were CACNG1, CKM, MYMK, TNNC2, and TNNI2 (Fig. 2a).

Cross referencing of these differentially expressed genes with our previous myoblast fusion screen result revealed MYMK as the only gene standing out from both experiments. Notably, MYMK was the second most downregulated gene (by 7.1 folds) in CHAMP1Δ myoblasts at day 2 of differentiation, the peaking stage of myoblast fusion. Immunostaining confirmed the reduction of MYMK expression at protein level in CHAMP1Δ cells compared to WT cells (Fig. 2b). Notably, forced expression of Myomaker in CHAMP1Δ cells can rescue myoblast fusion, surpassing the fusion levels achieved by CHAMP1 rescue itself (Fig. 2c–e). By contrast, expressing another muscle fusogen Myomixer (MYMX), the function partner of Myomaker 9, or MyoD, an upstream regulator of Myomaker 24, did not improve fusion outcomes of CHAMP1Δ cells (Fig. 2d, e). These results highlight a crucial and specific role of Myomaker deficiency underlying the fusion defect of CHAMP1Δ cells.

CHAMP1 patient derived cells show reduced Myomaker expression and defective myoblast fusion which can be rescued by compensated Myomaker expression
De novo mutations of CHAMP1 gene cause CHAMP1 syndrome, a neurodevelopmental disorder showing muscle development delays, hypotonia, muscle weakness & facial dysmorphism 27,30,31,33. These symptoms were also observed in Carey-Fineman-Ziter syndrome caused by hypomorphic MYMK mutations 15. At present, the genetic connections between the two diseases remain unknown. A more critical question is whether the muscle defects in CHAMP1 disorders result from muscle-autonomous dysfunction.

We obtained skin primary fibroblasts from two CHAMP1 syndrome patients along with fibroblasts from their healthy family members as controls 34. Genotyping and DNA sequencing confirmed distinct heterozygous CHAMP1 mutations in the patient cells (Fig. 3a). In one patient, a nonsense variant that replaces a cytosine with thymine at position 1489 of the CHAMP1 coding sequence was detected (c.1489C>T), resulting in the substitution of arginin with a premature stop codon at amino acid position 497 (p.Arg497*); in the other patient, a frameshift variant caused by the deletion of two nucleotides (cytosine and thymine) at positions 542 and 543 was identified (c.542_543delCT), leading to a frameshift and a premature stop codon five residues downstream (p.Ser181Cysfs*5).

EdU incorporation and live/dead staining assays revealed similar proliferation rate (Supplementary Fig. 5a, b) and viability (Supplementary Fig. 5c, d) between patient cells and healthy control cells. Because fibroblasts cannot be directly used as a model to study muscle development, we first converted them into myoblast-like cells by retroviral expression of MyoD, the master regulator of myogenesis 35. Because retroviral transduction is limited to dividing cells and primary fibroblasts have relatively low mitotic activity, infection efficiency was modest (20%–25%, Supplementary Fig. 5e). Consequently, myogenic conversion was also limited (15%–30%, Supplementary Fig. 5f), as measured by the proportion of nuclei within MF20-positive cells relative to the total number of nuclei. To account for the myogenic conversion efficiency, we quantified cell fusion after normalizing to the muscle differentiation index.

In these assays, CHAMP1 patient-derived cells—unlike healthy controls—failed to fuse and did not form multinucleated myotubes (Fig. 3b). At the molecular level, the fusion defect of patient cells was accompanied by reduced MYMK expression for both patients (Fig. 3c) and can be rescued when the expression of Myomaker is ectopically compensated (Fig. 3b, d). These results suggest that CHAMP1 mutations may have cell-autonomously contributed to muscle development defects in CHAMP1 patients, and more importantly, compensated MYMK expression could be a therapeutic target to reverse such defects.

CHAMP1 controls Myomaker expression through interactions with MyoD
CHAMP1 protein is exclusively detected inside nucleus of differentiating myoblasts prior to myoblast fusion (Fig. 1a). Coincidently, all 52 genes including Myomaker, that were significantly changed in CHAMP1Δ myoblasts were in fact downregulated (Supplementary Data 1), suggesting that CHAMP1 might control human myogenesis by activating target gene expression.

Previously, we identified MyoD as a key factor for the transcriptional induction of MYMK expression and activation of human myoblast fusion 24. Consistent with this notion, RNA-seq analysis of human MyoDΔ myoblasts, generated previously by CRISPR-induced mutagenesis 24, revealed a complete loss of MYMK expression (Fig. 4a). As a confirmation of gene-specific CRISPR knockout effect, the MYMK expression can be robustly rescued upon re-expression of MyoD in MyoDΔ myoblasts (Fig. 4a). Likewise, expression of MYMK was greatly reduced in human CHAMP1Δ myoblasts, a change that was reversed upon re-expression of CHAMP1 in CHAMP1Δ myoblasts (Fig. 4a). Like Myomaker, majority of CHAMP1 target genes also showed consistent changes in MyoDΔ cells compared with the isogenic rescue group in which MyoD was restored in MyoDΔ cells (orange bars, Fig. 4b). Among these common targets for CHAMP1 and MyoD, many genes (e.g., MYMK) exhibited even greater reductions upon deletion of MyoD (Fig. 4b). These results suggest that CHAMP1 might regulate the expression of its target genes by working with MyoD.

At present, little is known about the role and mechanism by which CHAMP1 works as a transcriptional regulator to control tissue development. To examine the chromatin binding of CHAMP1 and its functional relationship with MyoD, we conducted CUT&Tag experiments for this duo using WT human myoblasts at peak of myoblast fusion (DM 2 days). Intriguingly, these analyses identified 33,324 binding peaks for CHAMP1 protein (Supplementary Data 2). Moreover, 72.6% of CHAMP1 binding peaks were also bound by MyoD protein (Fig. 4c, d). Likewise, 42.4% of the 57,069 MyoD peaks (Supplementary Data 3) showed binding by CHAMP1 (Fig. 4d, e). The discovered motifs for CHAMP1 peaks closely resemble those for MyoD, with the highest ranking motif for both proteins being the CAGCTG-type E-box (Fig. 4f), a canonical MyoD binding motif 36–38.

CHAMP1 and MyoD co-occupy a region in the intron 1 of MYMK that corresponds to an open chromatin region, as revealed by ATAC-seq (Fig. 4a’). This region is also enriched for the H3K27ac histone modification (Fig. 4a’), indicating it is an active enhancer. We also performed HiCAR (High-throughput Chromatin Accessibility and Regulatory interactions) to profile enhancer–promoter interactions in human myoblasts at day 2 post-induction. This analysis identified chromatin interactions at the MYMK locus (chr9:133,514,586–133,524,959), with the contact matrix revealing direct interactions between an intronic enhancer and the MYMK promoter within a single TAD (Supplementary Fig. 6), supporting their regulatory connection.

Importantly, the co-occupancy of CHAMP1 and MyoD on this enhancer appears to be dependent on each other because CUT&Tag signals for CHAMP1 binding were lost in MyoDΔ myoblasts; likewise, deletion of CHAMP1 also abolished MyoD binding to the same enhancer (Fig. 4a’). Such reciprocal effect is not simply due to a change in the expression or localization of one protein when the other factor is genetically deleted because the MyoD protein can be normally detected inside the nucleus of CHAMP1Δ myoblasts (Fig. 4g), and likewise, deletion of MyoD also did not affect the nuclear expression of CHAMP1 protein (Fig. 4g).

We then investigated whether CHAMP1 protein can directly interact with MyoD protein to control gene expression in human muscle cells. Indeed, super-resolution imaging revealed co-localization of this duo in differentiating WT myoblasts (Fig. 4g). Co-immunoprecipitation experiment and proximity ligation assay (PLA) confirmed the CHAMP1 and MyoD interaction in human myoblasts (Supplementary Fig. 7 and Fig. 4h). The protein-protein interaction signal was not detected when PLA was performed using either human CHAMP1Δ or MyoDΔ myoblasts, confirming the specificity of these interaction assays (Fig. 4h). Together, these results suggest that CHAMP1 protein functions as a key cofactor for MyoD in activating MYMK transcription and myoblast fusion.

C2H2-ZNF motifs, including those truncated from CHAMP1 patient mutations, are essential for CHAMP1 interaction with MyoD and activation of human myoblast fusion
We then performed the structure and function analysis of CHAMP1 protein for a deeper mechanistic understanding of its interaction with MyoD. CHAMP1 protein contains five C2H2-type zinc finger motifs 39 (C2H2-ZNF): two located at N-terminus (amino acid positions: 16–37; 64–85) and three at C-terminus (amino acid positions: 711–732; 740–760; 767–786) (Fig. 5a). The central region of the protein includes several other domains, with the FPE domain critical for chromosome alignment as initially characterized in HeLa cells 39. Patients with CHAMP1 syndromes frequently show truncations 29 (e.g., at amino acid positions 181 or 497, Fig. 3a) that often delete a region that includes the C-terminal C2H2-ZNF motifs yet the precise role of these motifs remains largely unknown.

We generated a series of CHAMP1 truncation mutants fused with Flag tag and confirmed their expression in CHAMP1Δ cells by Western blotting (Fig. 5b). Interestingly, deletion of the C2H2-ZNF motifs, but not any other domains, of CHAMP1 abolished its activity in the myoblast fusion test using CHAMP1Δ cells (Fig. 5c, d). Moreover, CHAMP1 mutant containing only the C2H2-ZNF motifs (5x ZNF, 45% of protein sequence) showed comparable activity with that of the full-length CHAMP1 protein in CHAMP1Δ cells (Fig. 5c, d). Thus, the five C2H2-ZNF motifs are both necessary and sufficient to carry out CHAMP1’s function in human muscle cells.

The C2H2-ZNF motif, which is abundant in transcription factors, coordinates a zinc ion via two cysteine (C2) and two histidine (H2) residues and can mediate various functions, including protein-protein interactions 40. To explore its role in CHAMP1, we then generated CHAMP1 mutants by changing the two histidine (H) residues in each C2H2-ZNF motif to alanine (A). These mutations are expected to cause major disruptions to the structure and function for each targeted C2H2-ZNF motif. Western blots confirmed the expression of these mutants after transduction into human CHAMP1Δ myoblasts (Fig. 5e). Interestingly, PLA tests showed that mutations of the 4th C2H2-ZNF motif (H756/760A) drastically reduced the CHAMP1’s interaction with MyoD (Fig. 5f). Importantly, this mutation abolished CHAMP1 activity in the myoblast fusion test using CHAMP1Δ cells (Fig. 5g). Together, these results confirm the crucial role of MyoD–CHAMP1 interaction for human myoblast fusion and reveal the key role of the 4th C2H2-ZNF motif on CHAMP1 for mediating this interaction.

Because CHAMP1 is a relatively large protein, it likely requires active nuclear import via nuclear localization signal (NLS) to access the nucleus and bind MyoD. Sequence analysis of CHAMP1 identified two NLS: NLS1 (residues 612–622, LFPSSKKLKKD) and NLS2 (residues 702–708, MKRGKGK) (Fig. 5a), which are retained in the 5× ZNF truncation mutant. Consistently, Flag immunostaining of CHAMP1 truncation mutants confirmed that the C-terminal 222 residues containing the two predicted NLS is required for the nuclear localization of CHAMP1 (Fig. 5h). Notably, deletion of NLS1 and NLS2, but not NLS1 alone, abolished CHAMP1’s nuclear localization, as shown by immunostaining (Fig. 5h) and nuclear fractionation followed by Western blotting analysis (Fig. 5i), this was accompanied by reduced CHAMP1 function in the myoblast fusion assay (Fig. 5j). Thus, CHAMP1 depends on NLS2 for its proper nuclear localization and function in human myoblast.

Methods
Human myoblast isolation, culture and karyotype analysis
Human myoblasts (AB1190) were isolated from paravertebral muscle of a healthy 16-year-old male donor 51. Briefly, the muscle biopsies were finely minced and explants were plated on oncoated Petri dishes in drops of fetal calf serum (Invitrogen, Carlsbad, CA). Primary cultures from muscle biopsies were co-transduced with two retroviral vectors expressing hTERT and CDK-4 cDNAs. Co-transduced cells were selected by neomycin and puromycin and then purified using magnetic beads coupled to antibodies directed against the myogenic marker CD56 (NCAM1). Individual clones with high myogenic and fusion capacity were selected. Human myoblasts are available upon request from Dr. Vincent Mouly at vincent.mouly@upmc.fr. Human MyoDΔ myoblasts were generated by CRISPR mutagenesis and authenticated previously 24. Immortalized human myoblasts are maintained in skeletal muscle cell basal medium (PromoCell, C-23260) supplemented with 15% fetal bovine serum (FBS; Sigma-Aldrich, F2442), 5% growth medium supplement mix (PromoCell, C-39365), GlutaMAX, and 1% gentamicin sulfate. For differentiation, myoblasts were cultured in differentiation medium comprising DMEM supplemented with 2% horse serum, 10 μM DAPT (Cayman, 13197), and 1% penicillin/streptomycin. Karyotype analysis was performed by KaryoStat™ Assay using Genechip Probe Array containing 1.1 million probes across somatic and sex chromosomes.

Culture of patient and healthy controls skin fibroblasts
Primary human skin fibroblasts—healthy control 1 (male, 32 years; GM27968), patient 1 (male, 23 months; GM27353), healthy control 2 (female, 30 years; GM27961), and patient 2 (female, 2 years; GM27963)—were obtained from the Coriell Institute and have been described previously 34. No primary isolation was performed in this study. Cells are cultured according to the supplier’s recommended protocol. Cells were maintained in Eagle’s Minimum Essential Medium (EMEM) with Earle’s salts, non-essential amino acids, and 2 mM L-glutamine, supplemented with 15% non–heat-inactivated fetal bovine serum. All experiments were performed using cells between passages 3 and 5. For the sequencing validation of the CHAMP1 genotypes, genomic DNA was extracted and used for PCR amplification with the two pairs of primers. F1 (GCCCTCCTCTTCCTGAACAC) and R1 (GGATGGTCCCCAAGGTTCTG) for the authentications of GM27961 and GM27961 cells. F1 & R1 primer pair amplifies 458 bp for WT; F2 (CCCACCACTATCCCCAGAGA) and R2 (GGAGTTCAGGGAAAAGGGCA) for the authentications of GM27353 and GM27961 cells. F2 & R2 primer pair amplifies 422 bp for WT.

EdU incorporation assay
Cells were seeded at around 30% confluency one day before EdU incorporation assay. For the EdU assay, cells were cultured in growth medium containing 40 μM EdU for 3.5 h. EdU staining was performed by click chemistry following a published protocol 52. Briefly, cells were fixed in PFA and were rinsed with PBS, permeabilized with 0.1% Triton X-100 for 10 min at room temperature, and then incubated for 30 min in the dark with a freshly prepared click reaction mixture containing 100 mM Tris, 1 mM CuSO₄, 1 μM Azide-Fluor 545, and 100 mM ascorbic acid. Nuclei were counterstained with Hoechst.

Cell viability assay
The viability of human myoblasts and primary fibroblasts was assessed by flow cytometry using the LIVE/DEAD™ Fixable Violet Stain Kit (ThermoFisher, L34963) according to the manufacturer’s protocol. Cells were detached with TrypLE (Gibco, 12605028), washed once with PBS, and resuspended at 1×10⁶ cells/ml in PBS containing 1 μl of dye per ml. Samples were incubated on ice for 20 min, washed once with PBS, and resuspended in 1% BSA/PBS prior to analysis on a CytoFLEX S Flow Cytometer (Beckman Coulter, Cat. No. B75408). Cells were gated by FSC-H and SSC-H to exclude debris and enrich for the main population. A dead cell control (freeze–thawed at –80°C) was used to define LIVE/DEAD dye distribution; live cells were gated by excluding the ~10% tail overlapping with the dead cell population. Flow cytometry data were analyzed with FlowJo v10.8.1 to quantify the percentage of live and dead cells.

Fibroblast mixing and fusion experiment
C3H/10T1/2 fibroblasts (ATCC, CCL-226, Clone 8) were transduced with GFP-retrovirus together with either empty-retrovirus, myomaker-retrovirus, or CHAMP1-retrovirus. After infection, cells were thoroughly washed, detached with TrypLE, mixed with human WT myoblasts at a 1:1 ratio, and cultured in a mixed medium composed of equal volumes of 10% FBS–DMEM and human myoblast medium. Mixed cultures were allowed to attach and then switched to myogenic differentiation medium for three days, after which cells were subjected to myosin immunostaining and imaging.

Human myoblast transplantation experiment
All animal procedures were conducted with approval from the Institutional Animal Care and Use Committee (IACUC) at the University of Georgia. 2–3 months age NSG mice of both male and female (Jackson Laboratory, 005557) were anesthetized with isoflurane vapor during the entire procedure to perform targeted X-ray irradiation of the hind limbs (Rad320 X-ray, 1100 Rad) 32. The mice’s main bodies were shielded with a lead covering, leaving only the hind limbs exposed to irradiation. One day after irradiation, a localized muscle injury was induced by intramuscular injection of 20 µL of 1.2% barium chloride into the periphery of the tibialis anterior (TA) muscle. On the third day post-injury, 0.5×10⁶ cultured human cells (WT or CHAMP1 knockout), suspended in 10 µl of sterile saline containing 0.5% BSA, were transplanted into the injured TA muscle of randomly allocated NSG mice using a Hamilton syringe. Prior to transplantation, human cells were confirmed negative for rodent pathogens (IMPACT Panel A). Mice remained anesthetized with isoflurane during the transplantation procedure. Thirty days after transplantation, TA muscles were harvested, sectioned, stained, and analyzed to evaluate muscle fiber contributions from the transplanted cells, following established protocols. Each experimental group consisted of four independent transplantations.

Gene knockout experiments in human myoblasts by lenti-CRISPR-Cas9
Three gRNAs were designed for CHAMP1: gRNA1 (GGAAGAGGTGTCTGTAGCTC), gRNA2 (TGCTGGACTACGGATCTCTG), and gRNA3 (GGGAAAAGGGCATGCTTCCG), and was individually cloned into plentiCRISPRv2-Blast gRNA vector (Addgene, 83480) in Golden Gate assembly. DNA sequencing validated the correct insertion of gRNA. For lentivirus production, Lenti-X 293T cells (Clontech, 632180) were cultured in DMEM (containing 1% penicillin/streptomycin, 10% FBS). Transfection was performed using FuGENE 6 (Promega, #E2692) with psPAX2 plasmid, pMD2.G plasmid. Two days later, lentivirus-containing supernatants were filtered and concentrated with Lenti-X Concentrator (Clontech, PT4421-2) following manufacturer’s protocol. psPAX2 vector was a gift from Didier Trono (Addgene plasmid # 12260). pMD2.G vector was a gift from Didier Trono (Addgene plasmid # 12259). Human myoblasts were infected with lentivirus in growth medium. Following blasticidin selection, single clones were isolated, expanded, and genotyped using PCR and subsequent Sanger sequencing for validation of mutations. CHAMP1 genotypes were determined by sequencing after PCR amplifications using the following primers. F1: GCCCTCCTCTTCCTGAACAC and R1: GGATGGTCCCCAAGGTTCTG; F1 & R1 primer pair amplifies a 458 bp band in WT; F2: ATCAGTCTCA CCCAGCTCCT and R2: GAGAACCACCACGGGAACTT; F2 & R2 primer pair amplifies a 311 bp band in WT; F3: GCCTAGAAAACCTGCCCTGT and R3: CCCATGGGCTGCTACATGAT; F3 & R3 primer pair amplifies a 712 bp band in WT.

Retroviral expression vector cloning and retrovirus preparation
The retroviral expression vector pMXs-Puro (Cell Biolabs, RTV-012) was used to construct gene expression vectors for this study. The CHAMP1 open reading frame and its mutants were codon-optimized and synthesized by Integrated DNA Technologies, with sequences verified through Sanger sequencing or whole plasmid sequencing. The expression vectors for Myomxier, Myomaker and MyoD were reported previously 24. For rescue experiments, sgRNA-insensitive DNA cassettes with silent mutations destroying the protospacer or PAM sequence were designed and utilized. All retrovirus vectors used in this study have been sequenced to verify the correct insertions of DNA sequences. Retrovirus production was performed by transfecting human embryonic kidney 293 cells with the retroviral plasmid using FuGENE 6 (Promega, E2692). Viral supernatant was harvested two days post-transfection, filtered, and used to infect target cells in the presence of polybrene (Sigma-Aldrich, TR1003-G). Infected cells were switched to fresh culture medium 24 h post-infection.

RNA extraction and bulk RNA sequencing
Total RNA was extracted from human myoblast cells using TRIzol reagent (ThermoFisher Scientific, 15-596-018) according to the manufacturer’s protocol. RNA quality and concentration were assessed using a TapeStation, and only samples with an RNA Integrity Number (RIN) above 9 were used for library preparation using the NEBNext Ultra II Directional RNA Library Prep Kit (E7760L) with poly(A) selection. Next-generation sequencing was performed on an Illumina platform by Admera, generating 150 bp paired-end reads with a depth of approximately 40 million reads per sample.

Bulk RNA sequencing data analysis
Raw sequencing data was processed with initial quality control (QC) using FastQC (version 0.11.9: http://www.bioinformatics.babraham.ac.uk/projects/fastqc) to assess read quality and MultiQC 53 (version 1.14) was used to aggregate and visualize quality control metrics across all samples. After QC, raw sequencing reads were processed using fastp 54 (version 0.23.2) for adaptor trimming and reads filtering with the following filtering parameters: quality score threshold: 20, maximum proportion of unknown bases (N): 10, maximum percentage of unqualified bases: 30. A reference human hg38 genome index was constructed using HISAT2 (version 2.2.1) with the provided genome sequence 55. Cleaned reads were aligned to the reference genome using HISAT2. Alignment outputs were converted to sorted BAM files using SAMtools 56 (version 1.6), and BAM files were indexed for downstream analysis. Aligned reads were converted to normalized genome browser tracks (BigWig files) using deepTools 57 (version 3.5.2) with 1 bp bin size for track visualization. Gene-level read counts were quantified using featureCounts 58 (Subread version 2.0.3) and differential expression analysis was performed using DESeq2 59 (version 1.40.2) in R (version 4.3.1) combined with Benjamini-Hochberg multiple testing correction. Gene Ontology (GO) enrichment analysis was conducted using clusterProfiler 60 (version 4.8.3) and the Homo sapiens annotation database (org.Hs.eg.db, version 3.17.0) in R. RNA-seq data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database under accession number: GSE307319.

Duo-link proximity ligation assay (PLA)
Proximity Ligation Assay (PLA) experiments were conducted using Duo-link kits (Sigma-Aldrich, DUO92013, DUO92001, DUO92005, DUO82049, DUO82040, DUO92202). Myoblasts with the indicated genotypes were cultured on chamber slides and washed with 1× PBS before fixation in 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min at RT and then washed with 1× PBS. Duolink II Blocking Solution (1×) was added to each well, and slides were incubated in a pre-heated humidity chamber at 37°C for 1 h. Primary antibodies used for PLA experiments are anti-MyoD (Novus Biologicals, NBP1-54153, NB100-56511, 1.0 mg/ml, dilution 1:500), anti-CHAMP1 (Thermofisher Scientific, HPA008900, 0.1 mg/ml, dilution 1:200) and anti-FLAG (Sigma-Aldrich, F3165, 3.8-4.2 mg/ml, dilution 1:500). Primary antibodies were diluted in Duolink II Antibody Diluent (1×), and 40 μl of the antibody solution was applied per well, followed by overnight incubation at 4°C. The next day, PLA probe incubation, oligonucleotide ligation, and amplification steps were performed following the manufacturer’s instructions. Finally, ~100 μl of Duolink II Mounting Medium with DAPI was added, and coverslips were applied. Images were captured using a Zeiss LSM980 confocal microscope with Airyscan 2.

Assay for transposase-accessible chromatin sequencing (ATAC-seq)
ATAC-seq experiments were conducted following a published protocol 61, with minor modifications. Specifically, Adaptor A and Adaptor B were prepared at a concentration of 50 µM by mixing 100 µM Adaptor A-ME oligo (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’) or Adaptor B-ME oligo (5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’) with 100 µM pMENTs oligo (5’- [Phos] CTGTCTCTTATACACATCT-3’) in a 1:1 ratio, followed by annealing in a thermocycler (95°C for 5 min, then ramping down at −0.1°C/s to 20°C). Annealed adaptors were combined with recombinant Tn5 protein (0.5 mg/mL) in a 1:6 ratio, incubated at room temperature for 30 min, and then placed on ice for 10 min. Tn5-Adaptor A and Tn5-Adaptor B were then mixed in a 1:1 ratio and stored at −20°C for long-term use. For ATAC-seq, cells were permeabilized with ice-cold Nuclei Isolation Buffer (5% BSA, 0.2% IGEPAL CA-630, 1x cOmplete protease inhibitor cocktail in PBS) for 5 min. A total of 50,000 cells were collected in a PCR tube by centrifugation at 500 xg, 4°C for 5 min, then resuspended in 20 µL transposition mixture (10 µL 2x TD buffer, 6.6 µL PBS, 2 µL H2O, 0.2 µL 10% Tween-20, 0.2 µL 1% digitonin, 1 µL Tn5-Adaptor A/B mix). The transposition reaction was carried out in a ThermoMixer at 37°C, 550 rpm for 30 min. Tagmented DNA was purified using a Qiagen MinElute kit following the manufacturer’s protocol and eluted in 20 µL Qiagen Buffer EB. Subsequently, 25 µL NEBNext Ultra II Q5 Master Mix, 2.5 µL 10 µM Nextera i5 indexing primer, and 2.5 µL 10 µM Nextera i7 indexing primer were added to the eluted DNA, followed by PCR amplification using the following conditions: 72°C for 5 min; 98°C for 30 s; 9 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min; final extension at 72°C for 2 min; hold at 4°C. The PCR-amplified product was purified using the Qiagen MinElute kit and eluted in 30 µL Qiagen Buffer EB. The quality of ATAC-seq libraries was assessed using the Agilent Tapestation 4200 system, quantified by qPCR, and sequenced on an Illumina NextSeq 2000 using the following parameters: Read 1 – 51 cycles, Index 1 – 8 cycles, Index 2 – 8 cycles, Read 2 – 51 cycles.

Cleavage under targets and tagmentation (CUT&Tag)
CUT&Tag experiments were performed as previously described 62, with minor modifications. Specifically, annealed adaptors (as described above for ATAC-seq) were loaded onto recombinant Protein A/G-Tn5 (0.7 mg/mL) using the same procedure. Myoblasts (differentiated for two days) and Drosophila S2 cells were subjected to light crosslinking with 0.2% formaldehyde in PBS on ice for 5 min, followed by quenching with 0.125 M glycine. Crosslinked cells were permeabilized with ice-cold Nuclei Isolation Buffer for 5 min. A total of 300,000 human myoblasts and 30,000 Drosophila S2 cells (spike-in control) were collected by centrifugation at 1000 xg, 4°C for 5 min and resuspended in 75 µL MED1 buffer (300 mM NaCl, 2% BSA, 20 mM HEPES, 2 mM EDTA, 1 mM DTT, 1x cOmplete protease inhibitor cocktail, 0.5 mM spermidine, 0.01% digitonin, 0.01% IGEPAL CA-630 in nuclease-free H2O). The following antibodies were used for overnight incubation at 4°C (1 µg per reaction): anti-Drosophila histone H2Av (Active Motif #61751), anti-CHAMP1 (Atlas Antibodies #HPA008900), anti-MyoD (Novus #NBP1-54153), or anti-H3K27ac (Abcam #ab4729). After primary antibody incubation, the nuclei were washed once with 75 µL MED1 buffer, resuspended in 75 µL MED1 buffer with 2 µg secondary antibody (Rabbit anti-Mouse IgG, Invitrogen #A27022; or Guinea Pig anti-Rabbit IgG, Novus bio #NBP1-72763, depending on the host species of the primary antibody), and incubated on a rotator at room temperature for 45 min. For H3K27ac CUT&Tag, secondary antibody incubation step was skipped. The nuclei were then washed once with 75 µL MED1 buffer, resuspended in 75 µL MED1 buffer with 2 uL loaded Protein A/G-Tn5, and further incubated on a rotator at room temperature for 45 min. After Protein A/G-Tn5 incubation, the nuclei were washed 3 times with 75 µL MED2 buffer (300 mM NaCl, 2% BSA, 20 mM HEPES, 1 mM DTT, 1x cOmplete protease inhibitor cocktail, 0.5 mM spermidine, 0.01% digitonin, 0.01% IGEPAL CA-630 in nuclease-free H2O) and resuspended in 75 µL MED2 buffer. The tagmentation reaction was initiated by adding 3 µL 250 mM MgCl2 and incubating at 37°C, 550 rpm for 1 h. Tagmented DNA was purified using a Qiagen MinElute kit and eluted in 20 µL Qiagen Buffer EB. Library preparation was performed as described for ATAC-seq, using PCR amplification with 15 cycles for transcription factor CUT&Tag and 10 cycles for H3K27ac CUT&Tag. Libraries were purified, quality-controlled, and sequenced using the same procedures and parameters as above described for ATAC-seq.

HiCAR library preparation
HiCAR was performed as previously described 63,64. Briefly, 100,000 crosslinked cells were lysed in 400 μl NPB buffer (PBS with 5% BSA, 1 mM DTT, 0.2% IGEPAL, Roche Complete Protease Inhibitor, 12.5 μl RNaseOUT) at 4°C for 15 min to isolate nuclei. Nuclei were resuspended in 350 μl 2X TB buffer (66 mM Tris-acetate pH 7.8, 132 mM potassium acetate, 20 mM magnesium acetate, 32% DMF), 335 μl water, and 15 μl assembled Tn5 transposome and rotated at 37°C for 1.5 h. Reactions were quenched with 350 μl 40 mM EDTA, nuclei washed with 0.075% BSA, and treated with 32.5 μl water, 5 μl 10X NEBuffer 3.1 (NEB, B7203S), and 12.5 μl 2% SDS at 62°C for 10 min; supernatants containing nuclear RNA were saved. Nuclei were resuspended in 100 μl H2O, 14 μl 10X NEBuffer 3.1, and 25 μl 10% Triton X-100, and incubated at 37°C for 15 min. For CviQI digestion and ligation, nuclei were washed with 1 ml 1.1X NEBuffer 3.1, incubated with 90 μl 1.1X NEBuffer 3.1 containing 100 U CviQI (NEB, R0639L) and 1 μl 20 μM Truseq R1 oligo at room temperature for 2 h, followed by ligation with 48 μl 10X T4 ligation buffer, 6 μl T4 DNA ligase (400 U/μl, NEB, M0202S), 2.4 μl 20 mg/ml BSA (NEB, B9000S), 40 μl 10% Triton X-100, and 283.6 μl H2O for 4 h at room temperature. After centrifugation, nuclei were resuspended in 200 μl 2X RCB (100 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.4% SDS) and incubated at 68°C for ≥1.5 h to reverse crosslink, and DNA was recovered by ethanol precipitation, washed with 80% ethanol, and dissolved in 21 μl 10 mM Tris-HCl (pH 8.0). DNA gap repair was performed with 4 μl 10 mM dNTPs, 5 μl 10X CutSmart buffer, 1.5 μl T4 DNA polymerase (NEB, M0203L), and 20.5 μl H2O at room temperature for 30 min, followed by heat inactivation at 75°C for 20 min. Samples were digested with 1 μl NlaIII (NEB, R0125L) at 37°C for 1 h, purified with 0.9X SPRI beads (Beckman, B23319), and dissolved in 80 μl 10 mM Tris-HCl. DNA was diluted to 1 ng/μl and circularized in T4 ligation buffer with T4 DNA ligase (400 U/μl, NEB, M0202S) for ≥2 h at room temperature, then purified with DNA Clean & Concentrator kit (Zymo, D4013) and eluted in 16 μl H2O. For PmeI digestion and PCR, 14 μl DNA was incubated with 1.7 μl 10X CutSmart buffer and 1.3 μl PmeI at 37°C for 1 h, followed by PCR with 16 μl 5X Q5 buffer, 1.6 μl 10 mM dNTPs, 1.6 μl Nextera-pcr-i7–10-L primer, 1.6 μl NEB primer i501 (both 10 μM), 0.8 μl Q5 polymerase (NEB, M0491L), and 58.4 μl H2O using the following program: 72°C for 5 min, 98°C for 30 s; then 11 cycles of 98°C for 10 s, 59°C for 30 s, 72°C for 45 s; followed by 72°C for 5 min and hold at 4°C. Amplified DNA (300–750 bp) was gel-extracted with DNA Recovery Kit (Zymo, D4002) for deep sequencing.

ATAC-seq, CUT&Tag and HiCAR data analysis
Quality control of the raw sequencing reads were performed using MultiQC (v1.14) 53. Reads were quality trimmed using trim-galore (v0.6.7) 65. Sequencing reads were mapped to GRCh38 (hg38) reference genome using Bowtie 2 (v2.4.4) 66. Duplicated sequencing reads were removed using picard (v2.26.11, https://broadinstitute.github.io/picard/). Peak calling was performed for deduplicated sequencing reads using MACS2 (v2.2.7.1) 67. For ATAC-seq and H3K27ac CUT&Tag, genome alignment was normalized by RPKM using deeptools (v3.5.3) 68, and visualized using IGV (v2.15.4) 69. For transcription factor CUT&Tag, genome alignment was normalized using deeptools bamCoverage 68, with a scaling factor calculated as [Control Drosophila reads]/[Sample Drosophila reads], and visualized using IGV. Motif analysis was performed using HOMER (v4.11.1) 70. HiCAR sequencing data were processed using the nf-core/hicar pipeline under default instructions 71. Raw reads were subjected to quality control, adapter trimming, alignment to hg38, and extraction of valid paired-end interactions. Downstream analysis including ATAC-seq peak calling, chromatin loops identification, and genomic interaction annotation was performed using default setting. Processed outputs were converted into multiple visualization formats, and final contact matrices and ATAC-seq tracks were visualized with pyGenomeTracks 72.

Immunostaining and microscopy
Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature, permeabilized with 0.5% Triton X-100 in PBS, and blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Primary antibodies (1:500 dilution) used for immunostaining experiments are anti-MyoD (Novus Biologicals, NBP1-54153, NB100-56511, 1.0 mg/ml), anti-MYOG (DSHB, F5D), anti-myosin (DSHB, MF20), MYH3 (DSHB, F1.652), anti-laminin (Novus Biologicals, NB300-144, 1.0 mg/ml), anti-Lamin A/C (Thermofisher Scientific, MA3-1000, 1.0 mg/ml), anti-CHAMP1 (Thermofisher Scientific, HPA008900, 0.1 mg/ml), anti-Myomaker (mouse monoclonal antibody) and anti-FLAG (Sigma-Aldrich, F3165, 3.8-4.2 mg/ml). Following blocking, cells were incubated with the primary antibody overnight at 4°C, then with an Alexa Fluor–conjugated secondary antibody (1:1,000 dilution, Thermofisher Scientific, A21127, A21242, A27034, A27036, A28177). Nuclei were counterstained with Hoechst dye. Phase-contrast images were captured using a BioTek Lionheart FX automated microscope, and fluorescence images were acquired using either the BioTek system or an Olympus FLUOVIEW FV1200 confocal laser scanning microscope. Superresolution imaging was conducted with a Zeiss LSM980 confocal microscope with Airyscan 2.

Differentiation index and fusion index measurements
The differentiation index was calculated as the proportion of nuclei within MF20-positive cells relative to the total number of nuclei. The fusion index was determined as the proportion of nuclei within myotubes (cells containing ≥3 nuclei) relative to the total number of myosin expressing nuclei. Both indices were derived from manual cell counts, with treatment information blinded to maintain objectivity.

Western blotting analysis
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, R0278) supplemented with cOmplete protease inhibitor (Sigma-Aldrich, 04693159001) and incubated on ice for 15 min. The lysates were centrifuged at 15,000 xg for 10 min at 4°C, and the supernatant was collected and mixed with 4× Laemmli sample buffer (Bio-Rad, 161-0747). A total of 30 µg of protein was resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Sigma-Aldrich, ISEQ00010). The membrane was blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies diluted 1 in 1000 in 5% milk: GAPDH (Santa Cruz Biotechnology, sc-32233), FLAG (Sigma-Aldrich, F3165), and CHAMP1 (Sigma-Aldrich, HPA008900), anti-Lamin A/C (Thermofisher Scientific, MA3-1000). After washing with Tris-buffered saline containing 0.1% Tween-20 (TBST), the membrane was incubated with secondary antibodies (1 in 2500 dilution)—goat anti-mouse IgG–HRP (Invitrogen, A28177) and goat anti-rabbit IgG–HRP (Invitrogen, A27036)—in blocking buffer for 1 h at room temperature. Proteins were detected using Western Blotting Luminol Reagent (Thermofisher Scientific, 34075). Band intensities from the Western blot images were quantified using ImageJ (version 1.44p). Uncropped, unprocessed scans of all blots are provided in Supplementary Fig. 9.

Statistics & reproducibility
All mice and human cells were randomly allocated to the experimental group. All measurements were taken from distinct samples. For RNA-seq analysis, the differentially expressed genes were identified with a cutoff of fold change greater than 2 and p value < 0.05. Sample sizes are provided in the figure legends. Data were processed using GraphPad Prism 9 software. Error bars in all bar graphs represent standard deviation. Experiments were not randomized, and investigators were not blinded to sample allocation or outcome assessment. No statistical method was used to predetermine sample size. No data were excluded from the analyses.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript titled CHAMP1 is an essential regulator for human myoblast fusion and muscle development described the\nrole of CHAMP1 in human myoblast fusion. The extra data, clearer explanations and the splitting of the paper in two have\ngreatly improved the paper.\nIn this paper, the authors describe the new role of one of these new proteins previously identified. They choose CHAMP1 to\ninvestigate its role in myoblast fusion in vitro and in vivo. Then, they investigated the molecular mechanism and found\nCHAMP1 controls myoblast fusion by activating Myomarker, one gene important for the myoblast fusion, expression. Finally,\nthey showed, by different approaches that CHAMP1 interacts with MyoD and is a co-activator of MyoD inducing Myomaker\nexpression.\nOverall, the paper is clear, easier to understand and well written. We can follow correctly the paper with all the new\nnecessary information. While CHAMP1 has been described as a key factor for chromosome alignment, heterochromatin\nassembly and homologous recombination, this paper highlight a new role in CHAMP1 in muscle and provides some insights\ninto its role in CHAMP1 syndrome.\nThe authors have addressed the previous comments however thare are additional points requiring attention:\n-The quality of pictures is not great (blurry), due perhaps to exportation problems.\n-Figure 1, for the transplantation pictures, please also provide a lower magnification to see the whole section. This will help\nto see the results. How can the authors explain the presence of fiber with no Lamin A/C staining since the endogenous SCs\nare not present after irradiation?\n-In Figure 2, the authors should also compare the champ1Δ to control myoblast and not only to the rescued myoblast? This\ncontrol is essential.\n-Figure 3: how can the authors explain the low myogenic conversion efficiency? Is this within the normal range?\n-Figure 4, To improve the understanding of CHAMP1-MyoD interaction the authors can set up a Co-IP experiment to gain\ninsight into the direct interaction of these two proteins and their potential interaction in a complex.", "reviewer_2": "The manuscript by Zhang and colleagues present the surprising role of CHAMP1 as an upstream activator of human\nmyoblast fusion. This gene was identified in a CRISPR screen developed and now reported by the authors in a separate\nstudy. Typically, CHAMP1 mutations in humans are associated with a neurological disorder characterized by intellectual\ndisability, unique facial features, and more. Building upon their CRISPR screen, which identified CHAMP1 as one of the top\nhits, the authors generated several CHAMP1-KO myoblast lines, which showed muscle fusion defects both in vitro and upon\ntransplantation into mice in vivo. They further report that these myoblasts exhibit reduced MYMK expression and that\nCHAMP1 overexpression can rescue the observed deficiency in myoblast fusion. Furthermore, overexpression of MyoD in\nfibroblasts derived from patients with CHAMP1 mutations failed to induce fusion, unless MYMK was overexpressed,\nsupporting the notion that CHAMP1 activates MYMK expression. Cut & Tag datasets revealed shared binding sites between\nMyoD and CHAMP1, and through a proximity ligation assay (PLA), the authors propose that CHAMP1 and MyoD directly\ninteract. Finally, through elegant experiments using CHAMP1-KO myoblasts and truncated CHAMP1 isoforms, the authors\nidentify the C2H2-ZNF motif within CHAMP1 as important for its role in myoblast fusion.\nThe decision to split the original submission into 2 manuscripts improved the clarity of the narrative for each study. The\nadditional data included during revision supports the role for CHAMP1 in myoblast fusion. However, the interpretation of\nthese results can still be explained by alternative ways, as mentioned in my former review, and additional experimental\nverification and controls are necessary to substantiate the authors’ conclusions regarding the role of CHAMP1 in\nmyogenesis before publication of the study.\nMajor points:\n1. The authors reiterate on several occasions throughout the new manuscript (lines 23-24, 58-60, 160-162, 286-293) that\npatients with CHAMP1 mutations exhibit features of a muscle fusion myopathy. To support this claim, they cite several\nstudies describing patients with CHAMP1 mutations. In these works, no indication that CHAMP1 patients exhibit hallmarks\nof skeletal muscle pathology can be found, a pre-requisite for the definition of a myopathy. While these patients do show\nhypotonia, motor delay and unique facial features, these are attributed to the underlying severe neurological defect rather\nthan a skeletal muscle pathology (PMID: 26340335). As such, the authors should clarify how they reconcile these\nobservations with their claim. If indeed there is no skeletal muscle pathology documented in CHAMP1 patients, the claims\nmade in the manuscript would be inaccurate and should be removed or significantly toned down. Alternatively, providing\nmuscle sections from CHAMP1-deficient patients, demonstrating a fusion defect (i.e. small, immature and mononucleated\nfibers) as typically observed in patients with a fusion myopathy (see for example PMID: 28681861) would significantly\nstrengthen the manuscript.\n2. An alternative explanation for the reduced fusion in CHAMP1-KO myoblasts is diminished myoblast proliferation, which\nreduces the frequency of cell-cell interactions in the culture dish, an essential occurrence for myoblast fusion. The authors\naddress this in lines 97-99, stating that doubling time of CHAMP1-KO myoblasts is equivalent to WT cells, however no data\nis provided in the manuscript to support this claim (i.e. “data now shown”). Please add the respective dataset that supported\nthis sentence. Ideally, this should be via Ki67 immunostaining or EDU proliferation analysis via FACS of mutated and\nparental immortalized myoblast lines. Furthermore, the reduced fusion of patient-derived fibroblasts shown in Figure 3B can\nalso be explained by impaired proliferation, requiring a proliferation assay for these fibroblasts vs. control cells. Finally,\nanother alternative explanation is that CHAMP1-KO myoblasts exhibit increased cell death. An assay assessing cell viability\nin CHAMP-KO cells vs. parental myoblast lines would be desirable to address this possibility. Given CHAMP1 canonical\nrole in cell division, these alternative possibilities need to be assessed and clarified.\n3. The authors reported in the rebuttal letter that CHAMP1 overexpression does not induce fusion in fibroblasts, unlike\nMyoD or its downstream target MYMK. This is a relevant finding and the results should be included in the manuscript.\n4. The authors report that CHAMP1 and MyoD show physical protein proximity via a PLA assay and as such “directly\ninteract” (line 231). The PLA assay is insufficient to support claims that these proteins are direct binding partners, as it only\nshows close proximity and consequently an indirect interaction. To establish a binding interaction, a pull-down of MyoD or\nCHAMP1 followed by Western blot or equivalent assay would be required to demonstrate whether they are indeed true\nbinding partners.\nMinor comments:\n1. Lines 130-131: please indicate in the main text how CHAMP1 was overexpressed in the cells. In addition, the\ntransduction efficiency should be specified as different transduction levels may affect the outcome of the fibroblast-to-\nmyogenic conversion for the different overexpressed genes.\n2. Figure 2A: please add a proliferation marker to the heatmap. If the expression is the same between WT and mutated\nmyoblasts, it would reduce concerns that a diminished proliferation rate rather than fusion defect is associated with the\nobserved phenotype.\n3. Lines 58-60: the sentence correlates fusion myopathy to patients with a CHAMP1 mutation, however no citations are\nprovided for this claim.\n4. Citation 33: the correct citation is PMID: 3690668.\n5.Line 210- “litter”-> “little”\n6. Can the authors clarify whether, based on their results, they deem CHAMP1 to be a novel myogenic transcription factor as\nit can bind DNA (i.e. CATCTG E-box, MYMK enhancer), and if so- by which DNA binding motif? Incorporating the HiCAR\ncontact matrix results to the new manuscript could be relevant to the proposed new role of CHAMP1 as a myogenic\ntranscriptional regulator."}, "author_response": "Point-by-Point Response To Reviewers’ Comments\nWe thank all reviewers for their careful and thoughtful evaluation of our manuscript. We\nare pleased that the previous major revision and manuscript split were viewed as\nimprovements that clarified the narrative of each study and strengthened our findings. We\nhave dedicated substantial resources to perform ALL of the suggested experiments and\nanalyses to address the new questions raised in the last round of review. We hope the\nreviewers will find this revision thorough and satisfactory for publication.\nSummary of the Major Revision and New Results:\nWe performed ALL your suggested experiments and analyses. Many of these new results\nhave been incorporated into the revised manuscript (Fig. 2a, Extended Data Figures 2,\n4, 5 6, 7) as well as in this Response to Reviewer. An overview of these new data is\nprovided in the table below. The Methods section has also been updated to include details\nfor the newly added experiments.\nFigure in Relates to\nFigure in Revised\noriginal What is the change comments\nmanuscript\nmanuscript by\nAdded the RNA-seq results for\nFig. 2a Fig. 2a Reviewer 1\nWT samples\nAdded the EdU proliferation\nassay (panel a, b) and the cell\nExtended Data Fig. 2 N/A Reviewer 2\ndeath assay (panel c) for\nCHAMP1∆ myoblasts\nAdded RNA-seq results for the\ncell proliferation markers (panel\nExtended Data Fig. 4 N/A Reviewer 2\na) and cell apoptosis markers\n(panel b)\nAdded the EdU proliferation\nassay (panel a, b) and the cell\nExtended Data Fig. 5a–c N/A Reviewer 2\ndeath assay (panel c) for patient\nfibroblasts\nThe retroviral transduction\nefficiency (panel d) and the\nReviewers\nExtended Data Fig. 5d, e N/A\nmyogenic conversion efficiency\n1&2\n(panel e) in human primary\nfibroblasts\nHiCAR analysis result to show\nthe functional linkage of the\nExtended Data Fig. 6 N/A Reviewer 2\nMyomaker enhancer and the\npromoter\nCo-IP results confirming the\nReviewers\nN/A CHAMP1-MyoD protein\n1&2\nExtended Data Fig. 7 interactions in human myoblasts\nFibroblast expression and cell\nmixing assay to test the effect of\nExtended Data Fig. 8 N/A Reviewer 2\nCHAMP1 on fibroblast fusion\nwith myoblasts\nRNA-seq, ATAC-seq and\nFor rigor & reproducibility, we deposited\nCUT&Tag results were uploaded Author\nall sequencing data to NCBI and is\nto NCBI GEO (accession Voluntary\nready to be released\nGSE307319)\nAlternative transplantation\nexperiments tested modified\nResponse to Reviewer injury conditions that allowed\nN/A Reviewer 1\nFig. 1 greater engraftment but resulted\nin adverse health effects in the\nanimals.\nREVIEWER COMMENTS\nTo facilitate your re-evaluation, author responses [AU1–17] were highlighted in blue,\nPubMed hyperlinks were added to all literature cited in this Response file. New text in the\nrevised manuscript was highlighted in green.\nReviewer #1 (Remarks to the Author):\nThe manuscript titled CHAMP1 is an essential regulator for human myoblast fusion and muscle\ndevelopment described the role of CHAMP1 in human myoblast fusion. The extra data, clearer\nexplanations and the splitting of the paper in two have greatly improved the paper. In this paper,\nthe authors describe the new role of one of these new proteins previously identified. They choose\nCHAMP1 to investigate its role in myoblast fusion in vitro and in vivo. Then, they investigated\nthe molecular mechanism and found CHAMP1 controls myoblast fusion by activating Myomarker,\none gene important for the myoblast fusion, expression. Finally, they showed, by different\napproaches that CHAMP1 interacts with MyoD and is a co-activator of MyoD inducing Myomaker\nexpression.\nOverall, the paper is clear, easier to understand and well written. We can follow correctly the\npaper with all the new necessary information. While CHAMP1 has been described as a key factor\nfor chromosome alignment, heterochromatin assembly and homologous recombination, this paper\nhighlight a new role in CHAMP1 in muscle and provides some insights into its role in CHAMP1\nsyndrome.\nThe authors have addressed the previous comments however there are additional points requiring\nattention:\n-The quality of pictures is not great (blurry), due perhaps to exportation problems.\n[AU1] We thank the reviewer for the reminder. The lower image quality was due to file size\nconstraints when merging the manuscript for submission. We will ensure that the final published\nversion includes original high-resolution images that meet the journal’s requirements.\n-Figure 1, for the transplantation pictures, please also provide a lower magnification to see the\nwhole section. This will help to see the results.\n[AU2] We revisited the tissue slides to capture lower-magnification images. However, the\nfluorescence signals on the stored slides were too dim, likely due to storage conditions and time.\nTo address this, we repeated the immunostaining on additional sections from the same mice. A\nrepresentative lower-magnification (larger area) image of the muscle section is now provided in\nResponse Fig. 1a.\nWe agree that engrafted areas of human cells are near the edge of the tibialis anterior (TA) muscle\nfor both WT and CHAMP1 KO cells (Response Fig. 1a). This was intentional, as we induced only\na partial TA muscle injury controlled by BaCl2 injection volume and site. Based on our prior\nexperience and also published work with the same human myoblast lines (see Fig. 3A in PMID:\n22040608), we expect the repair of muscle injury, either in tissue structure or muscle function,\nfrom human grafts to be limited. In another pilot experiments where larger areas of muscle injury\nwere induced with BaCl₂ (Response Fig. 1b) prior to human wild-type myoblast transplantation,\nall recipient NSG mice (N = 6) developed severe scaling and crusted dermatitis of the hind feet and\ntail about one week post-transplantation (Response Fig. 1c), despite more permissive human cell\nengraftment (Response Fig. 1d). After consultation with the veterinarian and the IACUC\n(Institutional Animal Care and Use Committee), we believe this adverse outcome likely resulted\nfrom the greater BaCl₂ exposure diffusing to distal muscles, such as those in the feet and toe.\nBecause transplanted human cells were restricted to the tibialis anterior, they could not repair\ndistal tissue damage. Due to these negative health effects, the IACUC did not approve this more\naggressive injury-transplantation strategy.\nWe want to emphasize that, in contrast, all NSG mice in our main experiments (used to generate\nFig. 1f–h) were apparently active, alert and healthy. This is important because it eliminates\npotential confounding effects on data interpretation that could arise from systemic illness (e.g.,\nsevere dermatitis), particularly in the immunodeficient NSG background. Furthermore, our\nstatistical analysis of human muscle size was based on all muscle regions containing human cells\nand incorporated multiple biological replicates (N = 4 per genotype of transplanted cells).\nIn summary, the level of engraftment we reported is consistent with prior studies using the same\nmyoblast line and protocol (PMID: 22040608). At the same time, we devoted substantial effort to\nexploring strategies for improving engraftment and observed higher efficiency in pilot studies.\nHowever, these approaches also produced unanticipated complications that compromised animal\nhealth and could also confound result interpretation. Together, these efforts reflect our\ncommitment to improving the model, while also underscoring the importance of adhering strictly\nto IACUC-approved protocols in all animal studies.\nHow can the authors explain the presence of fiber with no Lamin A/C staining since the\nendogenous SCs are not present after irradiation?\n[AU3] This is a great question. The myonuclei only occupies a relatively small portion of the cell.\nTherefore, given our tissue section is only 10 microns, the nuclei can be missed from the tissue\nsection.\n-In Figure 2, the authors should also compare the champ1Δ to control myoblast and not only to\nthe rescued myoblast? This control is essential.\n[AU4] We believe the reviewer referred to Fig. 2a, since the other panels already included WT\ncontrols (e.g., Fig. 2b; Fig. 2e quantifies results from Fig. 2d). The purpose of Fig. 2c was to confirm\nsuccessful re-expression of CHAMP1, and to show that CHAMP1 expression was not indirectly\ninduced by overexpression of other proteins such as MyoD.\nRegarding Fig. 2a, we have now added the WT parental line as another control in the RNA-seq\nanalysis. The results confirm a strong reduction of MymK expression—the central focus of this\nstudy—in the KO line compared with WT. Importantly, this reduction is rescued by CHAMP1 re-\nexpression, as shown in the revised Fig. 2a. For the expression of muscle structural genes, we have\nupdated the text accordingly: “Comparison with WT myoblasts revealed a delayed expression of\nthese muscle structural genes in CHAMP1∆ cells; however, most of these changes were not\naffected by CHAMP1 re-expression in CHAMP1∆ cells, suggesting that these differences likely\nreflect clonal variations of CHAMP1∆ lines rather than a specific molecular function for CHAMP1”\n(Lines 163–167). Please note that the gene list in Fig. 2a has also been updated (e.g., gene names\nwere reordered alphabetically, and some redundant muscle structural genes were removed). The\ncomplete DE gene list is provided in Supplementary Table 1.\nWe would also like to explain why we designed our RNA-seq experiments with a focus on the\nisogenic rescue control. While the WT parental line is an obvious and convenient reference, the\nisogenic rescue offers distinct advantages. In this approach, the only variable is the presence or\nabsence of the gene of interest, thus providing a genetically defined system in which to evaluate\nmolecular changes. Similar preference is seen in other studies where the disease phenotype was\nrigorously studied, e.g., an earlier study by Jaenisch group (PMID: 21757228). The use of isogenic\ncontrol minimizes confounding variables and ensures that observed differences are attributable\nspecifically to CHAMP1 molecules.\nBy contrast, the parental WT line carries a critical limitation: clonal KO derivation itself introduces\nopportunities for unintended genetic variation. A single KO clone undergoes ~20 cell doublings\nbefore it can be expanded for analysis, during which spontaneous mutations may occur. Indeed, a\nrecent study demonstrated that clonally derived cells can harbor DNA sequence variations even\nwhen compared with the parental line (PMID: 37071670). Such clonal background effects become\nespecially problematic in RNA-seq, where we are interrogating late-stage differentiation\nphenotypes that are subtle and highly sensitive to genetic background.\nThus, comparing KO cells only to the parental WT line risks identifying false positives (DE genes)\nthat reflect clonal variation rather than bona fide effects of CHAMP1 loss. The isogenic rescue\ncontrol mitigates this issue, as re-expression of CHAMP1 within the same KO background allows\nus to isolate the true molecular consequences of CHAMP1 deletion. This principle has been\nsupported by other studies as well (e.g., PMID: 36307508; PMID: 32029722; PMID: 31503414).\nTherefore, we agree with the reviewer that both WT parental and isogenic rescue controls have\nvalues though the isogenic rescue provides the most rigorous and reliable framework for\ninterpreting RNA-seq data in clonally derived KO models, and therefore represents the most\nrelevant control for defining authentic CHAMP1-dependent changes.\n-Figure 3: how can the authors explain the low myogenic conversion efficiency? Is this within the\nnormal range?\n[AU5] This is a great question. We have used retrovirus infection which is known to only infect\nactively dividing cells which is relatively low percentage for primary human fibroblasts as shown\nin the newly added data (Extended Data Fig. 5a, d). Consistent with the idea, the retrovirus,\nprepared by the same method, always yield high infection rate in a more actively dividing\nfibroblast cell lines (10T1/2, e.g., Extended Data Fig. 8b). We have added this important discussion\nof this experiment in the manuscript “Because retroviral transduction is limited to dividing cells\nand primary fibroblasts have relatively low mitotic activity, infection efficiency was modest (20%–\n25%, Extended Data Fig. 5d). Consequently, myogenic conversion was also limited (15%–30%,\nExtended Data Fig. 5e), as measured by the proportion of nuclei within MF20-positive cells relative\nto the total number of nuclei. To account for the myogenic conversion efficiency, we quantified\ncell fusion after normalizing to the muscle differentiation index” (Line 209–215).\nRelated to this discussion, we want to emphasize that the viral fusogen would NOT affect the assay\nof cell fusion. First, we previously confirmed by immunostaining that the viral fusogen VSV-G is\ncleared from infected cells within two days post-infection, prior to the cell fusion assay. Moreover,\ncontrol retroviruses (e.g., GFP-expressing or empty control) have never induced cell fusion in any\nexperimental context, either fibroblast or myoblast of various genotypes (MymK KO, MymX KO,\nMyoD KO, MyoG KO, CHAMP1 KO etc) we have previously tested.\n-Figure 4, To improve the understanding of CHAMP1-MyoD interaction the authors can set up a\nCo-IP experiment to gain insight into the direct interaction of these two proteins and their\npotential interaction in a complex.\n[AU6] This is a great suggestion. We have performed the Co-IP experiment which clearly showed\nthe interaction of the CHAMP1 with MyoD protein. This new result was added as revised\nExtended Data Fig. 7. This new result is consistent with results from two other independent\nexperiments (super-resolution imaging and the PLA assay) that also showed the protein\ninteractions between the duo.\nWe respectfully encourage cross-reviews and read the comments from the other\nreviewer along with our responses\nReviewer #2 (Remarks to the Author):\nThe manuscript by Zhang and colleagues present the surprising role of CHAMP1 as an upstream\nactivator of human myoblast fusion. This gene was identified in a CRISPR screen developed and\nnow reported by the authors in a separate study. Typically, CHAMP1 mutations in humans are\nassociated with a neurological disorder characterized by intellectual disability, unique facial\nfeatures, and more. Building upon their CRISPR screen, which identified CHAMP1 as one of the\ntop hits, the authors generated several CHAMP1-KO myoblast lines, which showed muscle fusion\ndefects both in vitro and upon transplantation into mice in vivo. They further report that these\nmyoblasts exhibit reduced MYMK expression and that CHAMP1 overexpression can rescue the\nobserved deficiency in myoblast fusion. Furthermore, overexpression of MyoD in fibroblasts\nderived from patients with CHAMP1 mutations failed to induce fusion, unless MYMK was\noverexpressed, supporting the notion that CHAMP1 activates MYMK expression. Cut & Tag\ndatasets revealed shared binding sites between MyoD and CHAMP1, and through a proximity\nligation assay (PLA), the authors propose that CHAMP1 and MyoD directly interact. Finally,\nthrough elegant experiments using CHAMP1-KO myoblasts and truncated CHAMP1 isoforms, the\nauthors identify the C2H2-ZNF motif within CHAMP1 as important for its role in myoblast fusion.\nThe decision to split the original submission into 2 manuscripts improved the clarity of the\nnarrative for each study. The additional data included during revision supports the role for\nCHAMP1 in myoblast fusion. However, the interpretation of these results can still be explained\nby alternative ways, as mentioned in my former review, and additional experimental verification\nand controls are necessary to substantiate the authors’ conclusions regarding the role of CHAMP1\nin myogenesis before publication of the study.\n[AU7] We thank the reviewer for the thorough summary of our work, the extensive effort on\nexamining our revised manuscript, and for the constructive and encouraging comments that\nimproved our work.\nMajor points:\n1. The authors reiterate on several occasions throughout the new manuscript (lines 23-24, 58-60,\n160-162, 286-293) that patients with CHAMP1 mutations exhibit features of a muscle fusion\nmyopathy. To support this claim, they cite several studies describing patients with CHAMP1\nmutations. In these works, no indication that CHAMP1 patients exhibit hallmarks of skeletal\nmuscle pathology can be found, a pre-requisite for the definition of a myopathy. While these\npatients do show hypotonia, motor delay and unique facial features, these are attributed to the\nunderlying severe neurological defect rather than a skeletal muscle pathology (PMID: 26340335).\nAs such, the authors should clarify how they reconcile these observations with their claim.\nIf indeed there is no skeletal muscle pathology documented in CHAMP1 patients, the claims\nmade in the manuscript would be inaccurate and should be removed or significantly toned down.\nAlternatively, providing muscle sections from CHAMP1-deficient patients, demonstrating a fusion\ndefect (i.e. small, immature and mononucleated fibers) as typically observed in patients with a\nfusion myopathy (see for example PMID: 28681861) would significantly strengthen the manuscript.\n[AU8] We agree with the reviewer that directly proving fusion myopathy would require carefully\nconducted histopathological studies, which are currently lacking (as also confirmed in consultation\nwith the CHAMP1 Foundation and patient families). While we are motivated to engage clinicians\nand CHAMP1 patients, such studies will take considerable time due to the requirements for IRB\napproval and patient recruitment.\nAs suggested by the reviewer, we have removed ALL references to fusion myopathy in CHAMP1\npatients (in this as well as in the Screen manuscript). In addition, we have expanded the discussion\nof our study’s limitations to include: “However, confirming fusion myopathy in patients requires\ncareful histopathological evaluation. Findings from individuals with Myomaker mutations suggest\nthat fusion defects are associated with a pronounced imbalance in fiber size, with hypertrophied\ntype II (fast-twitch) fibers and atrophic type I (slow-twitch) fibers, resembling congenital fiber\ntype disproportion (Lines 339–343).”\n2. An alternative explanation for the reduced fusion in CHAMP1-KO myoblasts is diminished\nmyoblast proliferation, which reduces the frequency of cell-cell interactions in the culture dish,\nan essential occurrence for myoblast fusion. The authors address this in lines 97-99, stating that\ndoubling time of CHAMP1-KO myoblasts is equivalent to WT cells, however no data is provided\nin the manuscript to support this claim (i.e. “data now shown”). Please add the respective dataset\nthat supported this sentence. Ideally, this should be via Ki67 immunostaining or EDU proliferation\nanalysis via FACS of mutated and parental immortalized myoblast lines. Furthermore, the reduced\nfusion of patient-derived fibroblasts shown in Figure 3B can also be explained by impaired\nproliferation, requiring a proliferation assay for these fibroblasts vs. control cells. Finally, another\nalternative explanation is that CHAMP1-KO myoblasts exhibit increased cell death. An assay\nassessing cell viability in CHAMP-KO cells vs. parental myoblast lines would be desirable to\naddress this possibility. Given CHAMP1 canonical role in cell division, these alternative\npossibilities need to be assessed and clarified.\n[AU9] We apologize for omitting these results. Following the reviewer’s suggestion, we have added\nnew data from EdU incorporation assays and FACS-based cell viability analyses for both human\nmyoblasts and primary fibroblasts. These results are described in Line 114–118 (myoblasts) and\nLine 205–207 (fibroblasts) of the revised manuscript.\nIn these assays, CHAMP1 KO cells and patient fibroblasts showed some variation in proliferation\nrate and viability. However, impaired cell fusion was a consistent phenotype across all these cells.\nMoreover, given the well-established, specific role of Myomaker in cell fusion (instead of cell\nproliferation and death PMID: 23868259), one would NOT predict that Myomaker expression\nwould rescue fusion defects if the proliferation or viability significantly contributed to the fusion\nphenotype.\nTo share more context about the cell viability analysis, we used the LIVE/DEAD™ Fixable Dead\nCell Stain Kit (ThermoFisher, L34963), which assesses membrane integrity by flow cytometry. Per\nthe user manual, the reactive fluorescent dye penetrates compromised membranes of necrotic cells\nand binds both intracellular and surface amines, producing strong fluorescence. In contrast, in\nviable cells, only surface amines are labeled, resulting in weaker staining as we have observed\n(Extended Data Fig. 2c and Extended Data Fig. 5c). Therefore, it is largely expected that the live\ncells also showed staining, compared with non-stain controls.\n3. The authors reported in the rebuttal letter that CHAMP1 overexpression does not induce\nfusion in fibroblasts, unlike MyoD or its downstream target MYMK. This is a relevant finding\nand the results should be included in the manuscript.\n[AU10] We agree with the reviewer that this is a relevant finding. The results are now added as\nExtended Data Fig. 8. Description of the result is seen in Line 389–391.\n4. The authors report that CHAMP1 and MyoD show physical protein proximity via a PLA assay\nand as such “directly interact” (line 231). The PLA assay is insufficient to support claims that\nthese proteins are direct binding partners, as it only shows close proximity and consequently an\nindirect interaction. To establish a binding interaction, a pull-down of MyoD or CHAMP1\nfollowed by Western blot or equivalent assay would be required to demonstrate whether they\nare indeed true binding partners.\n[AU11] This is a great suggestion. We have performed the Co-IP experiment which clearly showed\nthe interaction of the CHAMP1 with MyoD protein. This new result was added as revised\nExtended Data Fig. 7 and is consistent with results from two other independent experiments\n(super-resolution imaging and the PLA assay) that also showed the protein interactions between\nthe duo.\nMinor comments:\n1. Lines 130-131: please indicate in the main text how CHAMP1 was overexpressed in the cells. In\naddition, the transduction efficiency should be specified as different transduction levels may affect\nthe outcome of the fibroblast-to-myogenic conversion for the different overexpressed genes.\n[AU12] We apologize for omitting this important detail. Both the genetic rescue of CHAMP1 KO\nmyoblasts and the protein expression in human fibroblasts were achieved using retroviral infection.\nThis information has now been explicitly included in the revised manuscript (now Line 151 and\n208).\nIn response to the reviewer’s suggestion, we have also provided the fibroblast-to-myogenic\nconversion rate (measured as the differentiation index) for all expression groups (revised Extended\nData Fig. 5e). Additionally, please note that our cell fusion quantification was normalized to the\nconversion efficiency, as shown in Fig. 3d.\nRelated to this discussion, we want to emphasize that the viral fusogen would NOT affect the assay\nof cell fusion. First, we previously confirmed by immunostaining that the viral fusogen VSV-G is\ncleared from infected cells within two days post-infection, prior to the cell fusion assay. Control\nretroviruses (e.g., GFP-expressing or empty control) have never induced cell fusion in any\nexperimental context, either fibroblast or myoblast of various genotypes (MymK KO, MymX KO,\nMyoD KO, MyoG KO, CHAMP1 KO etc) that we previously analyzed.\n2. Figure 2A: please add a proliferation marker to the heatmap. If the expression is the same\nbetween WT and mutated myoblasts, it would reduce concerns that a diminished proliferation rate\nrather than fusion defect is associated with the observed phenotype.\n[AU13] Following the reviewer’s suggestion, we added heatmaps to show the expression of\nproliferation and apoptosis markers in CHAMP1 KO myoblasts. Because of space limitations in Fig.\n2a, these results are included as Extended Data Fig. 4 (panel a for proliferation markers and panel\nb for apoptosis markers). The description of these results was added in Line 156–159: “Consistent\nwith our EdU incorporation and live/dead cell staining assays, transcriptome analysis showed\ncomparable expression of proliferation markers (Extended Data Fig. 4a) and apoptosis markers\n(Extended Data Fig. 4b) between CHAMP1∆ cells and their isogenic rescued counterparts.”\n3. Lines 58-60: the sentence correlates fusion myopathy to patients with a CHAMP1 mutation,\nhowever no citations are provided for this claim.\n[AU14] We have removed this or similar statement throughout the manuscript. We agree with the\nreviewer that the confirmation of fusion myopathy needs muscle histopathology evidence.\n4. Citation 33: the correct citation is PMID: 3690668.\n[AU15] We apologize for this mistake. The correct citation (PMID: 3690668) was added (now\nreference #34) in the revised manuscript.\n5.Line 210- “litter”-> “little”\n[AU16] Thanks for the meticulous check. This typo was corrected (now Line 244).\n6. Can the authors clarify whether, based on their results, they deem CHAMP1 to be a novel\nmyogenic transcription factor as it can bind DNA (i.e. CATCTG E-box, MYMK enhancer), and if\nso- by which DNA binding motif? Incorporating the HiCAR contact matrix results to the new\nmanuscript could be relevant to the proposed new role of CHAMP1 as a myogenic transcriptional\nregulator.\n[AU17] This is a great question. Our data suggest that CHAMP1 functions primarily as a\ntranscriptional cofactor of MyoD, rather than binding directly to E-box sequences itself. This is\nsupported by the finding that in MyoD KO cells, CHAMP1 no longer binds DNA in CUT&Tag\nresults, suggesting CHAMP1 binds chromatin in a manner dependent on MyoD. Description of the\nresults is seen in Line 263–270.\nFollowing the reviewer’s suggestion, we have now incorporated the HiCAR data into Extended\nData Fig. 6, with description of the results provided in Line 257–262 of the revised manuscript.\nWe respectfully encourage cross-reviews and read the comments from the other\nreviewer along with our responses"}, {"round": 2, "reviewer_comments": {"reviewer_1": "THe authors have addressed the concerns raised by the reviewers.", "reviewer_2": "The authors have thoroughly addressed the points I have raised in the second review round, resulting in a significantly\nimproved manuscript on CHAMP1. I have no further comments."}, "author_response": ""}]

Decision letter