ATF3 Induction Prevents Precocious Activation Of Skeletal Muscle Stem Cell By Regulating H2B Expression - Nature Communications

ATF3 is rapidly and transiently induced during SC early activation

To explore the transcriptional regulatory events governing the SC quiescence-to-activation transition, we re-analyzed our previously published transcriptomic profiling datasets³⁶ acquired from SCs in the lineage progression. Briefly, muscles from Pax7-nGFP mice³⁷ were fixed in situ by PFA before subjecting to the standard 3 h long cell dissociation and isolation process, and FACS sorting to obtain QSCs¹³, or without prior fixation to obtain freshly isolated early activating SCs (FISC) (Fig. 1a and Supplementary Fig. 1a); FISCs were cultured in vitro for 24, 48 or 72 h to obtain fully activated (ASC-24h), proliferating (ASC-48h) or differentiating (DSC-72h) cells. Consistent with the findings from other recent reports^38,39,40, AP-1 family genes including Fos, Atf, Jun and Maf sub-families were dramatically induced in FISCs compared to QSCs (Fig. 1b). Notably, Atf3 was sharply induced (42.4 fold) in FISCs but was then rapidly decreased at ACS-24h (Fig. 1c, d); this was also confirmed by RT-qPCR (Fig. 1e), indicating that it is a rapid and transient responsive gene to early activation induced by the isolation. Furthermore, immunofluorescence (IF) staining demonstrated the concomitant induction of ATF3 protein in FISCs and a decrease in ASCs (Fig. 1f). Even on the freshly isolated single myofiber that is believed to preserve the quiescent niche to some extent and minimize SC activation^41,42, the associated SCs displayed high expression level of ATF3 protein immediately after 2 h isolation which diminished at 24 h in culture (Fig. 1g), reinforcing the notion that ATF3 is rapidly induced in early activated SCs. To further confirm the ATF3 induction in vivo, BaCl2 was injected into the Tibialis Anterior (TA) muscles of C57BL/6 mice to induce acute muscle damage. In this injury model^36,43,44, tissue degeneration with abundant immune cell infiltrates is normally observed at 1 day post injury (dpi); SCs are rapidly activated to expand as proliferating myoblasts which then fuse to form small new myofibers expressing embryonic myosin heavy chain (eMyHC) and characterized by centrally localized nuclei (CLN); these eMyHC+ fibers are readily seen at 5 dpi. At 7dpi, the muscle is mainly composed of larger regenerated myofibers with down-regulated eMyHC expression; muscle damage and inflammatory cells are largely cleared at 14 dpi while the regenerated myofibers continue to grow in size and mature to achieve full regeneration around 30 dpi. As expected, ATF3 protein was not detected on uninjured TA muscles by IF but readily seen at 1dpi; some staining did not merge with Pax7+ cells, which is in line with its reported ubiquitous induction in multiple cells after injury⁴⁵. At 2dpi ATF3 expression in Pax7+ cells was highly increased, concomitant with the full activation of SCs at this stage (Fig. 1h). Several other AP-1 family members, Atf4, Fos, FosB, and JunB, shared a similar dynamic expression profile (Fig. 1b–d), which was also confirmed by RT-qPCR (Supplementary Fig. 1b) and immunofluorescent staining (Supplementary Fig. 1c) results. Taken together, our findings show that ATF3 and several other AP-1 family members are rapidly and transiently induced during early SC activation, suggesting their potential roles in the regulation of SC early activation.

Short-term Atf3 deletion accelerates acute injury-induced muscle regeneration

To facilitate the dissection of ATF3 function in SCs, we crossed the Atf3^flox allele⁴⁶, in which two LoxP sites were inserted onto Atf3 exon2, with Pax7^{CreERT2/CreERT2}-R26R^YFP/YFP mouse⁴³ to generate control (Ctrl) (Atf3^+/+– Pax7^{CreERT2/CreERT2}-R26R^YFP/YFP) and inducible knockout mice of Atf3 (Atf3 iKO) (Atf3 ^flox^/flox– Pax7^{CreERT2/CreERT2}-R26R^YFP/YFP) (Supplementary Fig. 2a). After consecutive 5 days of intraperitoneal (IP) injection of Tamoxifen (TMX) (Fig. 2a), successful deletion of ATF3 in FISCs was confirmed by WB (Supplementary Fig. 2b) or IF (Supplementary Fig. 2c); its depletion was further confirmed by IF staining of SCs on single myofibers (Supplementary Fig. 2d). Morphologically, the 2-month-old iKO showed no apparent difference from the Ctrl littermate mice; no changes in body size and weight were detected either (Supplementary Fig. 2e).

Considering the rapid induction of ATF3 during early SC activation, we reasoned that its loss might impact SC-mediated muscle regeneration. To test this notion, BaCl₂ was injected into the TA muscles of Ctrl or iKO mice to induce acute muscle damage 5 days after TMX deletion (Fig. 2a). By examining the above injured TA muscles at 3, 5, 7 and 30 dpi (Fig. 2a), the iKO muscles displayed a highly accelerated rate of regeneration. By H&E staining (Fig. 2b), at 5 dpi, regenerating fibers with CLNs were already present in the iKO but not the Ctrl muscles. At 7 dpi the damaged muscle was nearly repaired with homogenous fibers and no sign of immune cells in the iKO and the fiber size was also significantly larger compared to the Ctrl (the quantification on the right). These experimental observations were further supported by IF staining of eMyHC (Fig. 2c); eMyHC+ fibers were readily observed in the iKO even at 3 dpi (91.64 in iKO vs. 29.86 in Ctrl) but sharply decreased by 7 dpi. Consistently, the number of Pax7+ cells at 3 dpi was significantly higher in iKO vs. Ctrl but lower at 5 dpi and 7 dpi (Fig. 2h and Supplementary Fig. 2f). In addition, the TA muscles collected from the iKO mice at 30 dpi had increased muscle weight compared to the Ctrl (43.7 vs. 48.4 mg) (Supplementary Fig. 2g) and enlarged muscle fiber size (1137.4 vs. 862.8 μm²) (Supplementary Fig. 2h), reflecting the enhanced repair.

Surprisingly, the enhanced regenerative ability of Atf3 iKO SCs persisted after one more round of BaCl₂-induced acute injury given one month later (Fig. 2a). As shown in Fig. 2d, e, the accelerated regeneration remained equally strong based on the increased fiber size at 7 dpi and the number of eMyHC+ fibers at 3 dpi after the 2nd injury. At 30 dpi the iKO muscle also showed increased muscle weight compared to the Ctrl (61.7 vs. 55.8 mg) (Supplementary Fig. 2i) and slightly enlarged muscle fiber size (1724.8 vs. 1633.8 μm²) (Supplementary Fig. 2j). The 3rd round of injury was then given (Fig. 2a) and the expedited repair was still evident (Fig. 2f, g), as the number of eMyHC+ fibers at 3 dpi was still higher in the iKO vs. Ctrl muscles (89.9 vs. 45.7) (Fig. 2g) and a higher number of larger fibers was observed at 7 dpi (Fig. 2f). However, despite slightly increased TA muscle weight in the iKO compared to the Ctrl 30 day post-3rd injury (Supplementary Fig. 2k), there was no significant difference in the average muscle fiber size (Supplementary Fig. 2l), suggesting that the accelerated regeneration in the iKO muscle eventually disappeared after the 3rd injury.

The engraftment assay was then conducted to further validate the enhanced regenerative ability of the iKO SCs. As illustrated in Fig. 2i, 21 days after YFP+ SCs from the Ctrl or iKO mice were injected into the pre-injured TA muscles of receptor nude mice, the TA muscles were collected to evaluate donor cells’ regenerative ability based on YFP expression. Indeed, we observed a higher number of YFP+ myofibers in the receptor mice transplanted with the iKO vs. Ctrl SCs (Fig. 2j). Altogether, our results demonstrate that Atf3 deletion enhances acute injury-induced muscle regeneration.

Atf3 deletion provokes premature SC activation and pseudo-regeneration in homeostatic muscle

To dissect the underlying cause for the improved regenerative capacity upon ATF3 loss, we speculated that the iKO SCs underwent rapid activation upon BaCl₂ injury. Indeed, when plotting the dynamics of MyoD+ SCs along the course of the three rounds of injury/regeneration. a much higher number of MyoD+ cells were detected at 3 dpi (Fig. 2k and Supplementary Fig. 2m), but the cell number quickly declined at 5 dpi. Such phenomenon existed during the first two rounds of injuries but disappeared in the third round (Fig. 2k and Supplementary Fig. 2m). Compared to the Ctrl, the faster activation of iKO cells upon acute injury implies the quicker transition from quiescence to activation. Indeed, FISCs from the Atf3-iKO mice displayed a strikingly increased propensity for cell cycle entry based on EdU incorporation 24 h after culture (~23% increase) (Fig. 3a). In addition, Pax7+MyoD+ cells in the iKO exhibited 10% increase compared to the Ctrl 24 h after culture (Fig. 3b). Furthermore, the FISCs from the iKO muscles were also slightly larger in size than the Ctrl ones (86.1 vs. 72.9) (Fig. 3c). Consistently, isolated single myofibers from the iKO muscles had significantly higher EdU incorporation (14% increase) than the Ctrl ones 36 h after culture (Fig. 3d), reinforcing the faster cell cycle entry of the iKO SCs. The iKO SCs on freshly isolated single myofibers also produced 1.90-fold and 2.08-fold more Pax7+MyoD+ cells than those Ctrl SCs without culturing or with 3 h (Fig. 3e, f). Altogether, these results indicate that the loss of ATF3 results in the release from quiescence and thus rapid SC activation without acute injury.

The enhanced regenerative ability of iKO SCs prompted us to further test if the ATF3 loss may lead to spontaneous activation and pseudo-regeneration in muscles without injury. Compared to the Ctrl, there was no increase of Pax7+ SCs in the iKO TA muscles at 5 days post-TMX injection but a significant increase of Pax7+ cells in the iKO TA muscles at 21 (13.4 vs. 9.58) and 28 days (11.7 vs. 8.9) (Figs. 3g and S3a), indicating the occurrence of spontaneous activation and expansion of iKO SCs in homeostatic muscles. Interestingly, by 56 days post-TMX injection, the number of Pax7+ cells in the iKO muscles finally declined compared to the Ctrl (Figs. 3g and S3a); this was accompanied by readily seen CLN+ fibers (Fig. 3h), indicating the fusion of the activated SC-derived myoblasts to existing fibers, which is known as pseudo-regeneration⁴⁷. To confirm these findings, we performed five doses of EdU injections at 5 days post-TMX injection to show that there were more EdU+ cells on the muscle sections of the iKO than the Ctrl at 21 days post-EdU injection (Fig. 3i). Similarly, with a single dose of EdU administrated, EdU+ cells were only detected on the isolated iKO myofibers or TA muscles 12 h later but not on the Ctrl ones (Supplementary Fig. 3b). Altogether, these results suggest that Atf3 deletion provokes the precocious activation of SCs in homeostatic muscles thus ATF3 may function to actively suppress SC activation. This was further strengthened by overexpressing ATF3 in FISCs by a lentivirus (Supplementary Fig. 3c); it significantly inhibited SC activation (Fig. 4a, 20.9% reduction of Pax7+MyoD+ cells and Fig. 4b, 21.3% reduction of EdU+ cells). Moreover, overexpressing ATF3 in vivo by intramuscular injection of the lentivirus particles at 1 dpi (Fig. 4c) delayed the regeneration in Ctrl mice and also blunted the accelerated regeneration in iKO mice (Figs. 4d–h and S3d, e).

**Fig. 4: ATF3 re-expression in iKO reverts the accelerated SC activation and muscle regeneration.**

In addition, we observed there was 24% increase of MyoD+ MyoG+ differentiating cells in the isolated iKO muscle fibers compared to the Ctrl after 72 h culture (Supplementary Fig. 3f), suggesting that Atf3 deletion may accelerate SC progeny differentiation. This was confirmed by staining for MyoD and MyoG on SCs cultured for 72 h; 9% increase in the double positive cells was detected in the iKO vs. Ctrl (Supplementary Fig. 3g). Interestingly, there was also a slight decrease of Pax7+MyoD− cells in the iKO muscle fibers compared to the Ctrl (10.13% vs. 8.48%) (Supplementary Fig. 3h), suggesting that Atf3 loss may also lead to impaired self-renewal. Consistently, we also noticed a progressive decline in SC number after each round of injury (Fig. 2h and Supplementary Fig. 2f).

Long-term Atf3 deficiency depletes SC pool and impairs muscle regeneration

Next, to test if long-term Atf3 deficiency causes SC exhaustion and impairs muscle regeneration, we induced muscle injury by BaCl₂ administration 30 days or 120 days after deleting Atf3 from SCs and examined the injured TA muscles at 3, 5 and 7 dpi (Fig. 5a). Thirty days after the deletion, the muscle regeneration (based on the muscle fiber size and the eMyHC+ fiber number) showed no significant changes compared to the Ctrl mice (Fig. 5b, c). The number of Pax7+ SC was also comparable at 3 and 7 dpi and only slightly lower in the iKO at 5 dpi (Fig. 5d and Supplementary Fig. 4a). In contrast, 120 days after the deletion, the muscle regeneration was obviously compromised in the iKO compared to the Ctrl mice. A significant increase of smaller fibers was observed at 7 dpi (Fig. 5e); the number of eMyHC+ fibers was much lower at 5 dpi but higher at 7 dpi (Fig. 5f); the number of Pax7+ SCs was also significantly reduced at both 3 and 7 dpi (Fig. 5g and Supplementary Fig. 4b). These results demonstrate that the long-term Atf3 deficiency is detrimental to regeneration by shrinking the SC pool. Interestingly, when the same number of YFP+ cells isolated from the Ctrl or iKO mice 4 months after TMX injection were used in the engraftment assay (Fig. 5h), a higher number of YFP+ myofibers were observed in the recipient nude mice transplanted with the iKO cells (59.28 vs. 31.72, Fig. 5i), suggesting that enhanced regenerative ability of the iKO cells persists after long term ATF3 loss and the impaired muscle regeneration indeed arises from the reduced cell pool.

To further establish the importance of Atf3 in SC establishment and maintenance, we successfully deleted Atf3 in Pax7+ myogenic progenitors by crossing the Atf3^flox allele with a non-inducible Pax7^Cre-R26R^YFP transgenic mouse strain⁴⁸, in which the Cre recombinase is expressed in Pax7+ progenitor cells in as early as E9.5 (Supplementary Fig. 5a–c). Interestingly, the cKO muscles exhibited 84% increase of Pax7+ SCs compared to the Ctrl around 1 month after birth when SC pool was established⁴⁹, but 79% decrease in two-month-old adult mice (Supplementary Fig. 5e), indicating that ATF3 is required for SC maintenance at adulthood but dispensable for SC establishment. A decreased TA muscle weight was detected in cKO at 1-month-old (29.42 mg vs. 34.88 mg, Supplementary Fig. 5f) but no significant difference in the body weight (12.66 mg vs. 12.42 mg, Supplementary Fig. 5d) and fiber size (861.96 μm² vs. 848.71 μm², Supplementary Fig. 5g), it is thus hard to conclude whether muscle hypertrophy occurs in the young cKO mice. Expectedly, when the adult SCs were isolated and cultured, cKO cells displayed a much higher propensity for activation assessed by both Pax7+Myod+ staining (Supplementary Fig. 5h, 23.8% increase in cKO vs. Ctrl) and EdU assay (Supplementary Fig. 5i, 99.9% increase). Correspondingly, the regenerative capacity after acute injury was evidently compromised in cKO muscles (Supplementary Fig. 5j–l). Therefore, the loss of ATF3 induces precocious SC activation and leads to the eventual reduction of the SC pool.

Atf3 deletion enhances SC activation during voluntary and endurance exercises

Short term and non-strenuous voluntary exercise (VE) fails to cause SC activation but endurance exercise (EE) activates SCs^{50,51,52,53,54}. To further prove the function of ATF3 in preventing SC activation, we sought to determine if the ATF3 depletion influences the VE-induced SC activation. Immediately after five doses of TMX injection, Ctrl and iKO mice were subject to an established voluntary wheel running regime; the mice were provided with access to free rotating running wheels; a stable VE routine was reached within 7 days of training and continued for another 21 days long VE (Fig. 6a). The daily running distance was recorded and no significant difference was detected between Ctrl (14.38 km) vs. iKO (13.78 km) mice (Supplementary Fig. 6a, b).

**Fig. 6: *Atf3* deletion induces SC activation during voluntary and endurance exercise.**

Expectedly, the expressions of AP-1 family members were induced by the VE in Ctrl SCs (Supplementary Fig. 6c). Also consistent with prior reports⁵⁴, the VE did not induce SC activation in the Ctrl mice as no MyoD+ cells were detected on the muscle sections before and after the VE (Fig. 6b and Supplementary Fig. 6d); consistently, Pax7+MyoD+ staining of FISCs (Fig. 6c) or EdU staining of ASCs cultured for 24 h (Fig. 6d) did not reveal increased activation rate before and after the VE. In contrast, the iKO cells were readily activated by the VE (Fig. 6b–d and Supplementary Fig. 6d). Consistently, in the iKO muscles, we also detected a significantly increased number of CLN fibers (136% increase) (Fig. 6e) and evident hypertrophy (fiber size increased by 43.3%) after the VE (Fig. 6f). In the Ctrl muscles, the number of CLN was increased after the VE but not enough to cause hypertrophic growth (Fig. 6e, f). The above findings thus reinforce the notion that ATF3 loss induces rapid activation of SCs by the VE. As a result, there was reduced SC pool (38.2 % decrease of Pax7+ FISCs) at the end of the VE regime (Fig. 6g and Supplementary Fig. 6e).

Since EE is known to cause SC activation and muscle hypertrophy^50,51,52,53, we next examined if the Atf3 deficiency affects the EE-induced SC activation. To this end, Ctrl and iKO mice were subject to a treadmill running regime^55,56 in which the treadmill was set at a 5° incline and a speed of 20 cm/s for 60 min. After five doses of TMX injection, the mice were trained for a 5-day adaption period, then followed by a 10-day endurance training (+EE) or, as the control condition, without any training (NE) (Fig. 6h). As expected, the expressions of AP-1 family members were rapidly induced by the EE in SCs (Supplementary Fig. 6c). Furthermore, the EE caused evident SC activation in the Ctrl muscles, but the activation was much stronger in the iKO muscles (Fig. 6i–k and Supplementary Fig. 6f). Consistently, CLN fibers were induced in both Ctrl and iKO muscles after the EE; and a higher increase of CLN fibers was seen in the iKO compared to the Ctrl muscles (Fig. 6l). In addition, both Ctrl and iKO muscles showed hypertrophic growth after the EE but no difference was detected (Fig. 6m). At the end of the EE regime, the number of Pax7+ FISCs was significantly increased in the Ctrl but decreased in the iKO (Fig. 6n and Supplementary Fig. 6g), indicating the rapid exhaustion of the iKO SC pool. Altogether, these findings indicate that the loss of ATF3 provokes SC activation by VE and also enhances SC activation by EE.

ATF3 regulates H2B gene expression and nucleosome patterning

Altogether the above results suggest an essential role of ATF3 induction in preventing precocious SC activation, to illuminate the underlying molecular mechanism, we profiled the ATF3 transcriptional output by performing RNA-Seq in FISCs collected from the Ctrl and iKO muscles (Fig. 7a). A total of 1866 transcripts (red dots) (79.9%) were up-regulated in the iKO compared to the Ctrl, while 469 (blue dots) (20.1%) were down-regulated (Fig. 7b, Supplementary Fig. 7a and Supplementary Data 1), suggesting that the ATF3 loss induced global transcriptional activation. The GO analysis revealed that the up-regulated genes were enriched for “mitochondrion” “extracellular matrix”, etc. (Fig. 7c and Supplementary Data 1). Of note, elevated mitochondrial activity and extracellular matrix expression have previously been described in the G_Alert cells¹⁰, suggesting that ATF3 functions to repress the expression of these activating genes, thus preventing premature SC activation. The down-regulated genes were interestingly enriched for “nucleosome” and “nucleosome assembly”, etc. (Fig. 7d and Supplementary Data 1). Notably, the genes encoding Histone proteins were highly represented among the down-regulated genes (Fig. 7e), which was also confirmed by RT-qPCR (19–46% decrease) (Supplementary Fig. 7b). Histone encoding genes are typically organized into multigene clusters and H2B protein is encoded by 2 gene clusters with 15 on Chr13 forming a Hist1h2b cluster and 2 on Chr3 forming a Hist2h2b cluster (Supplementary Fig. 7c). Remarkably, 13 out of the 17 H2b genes were down-regulated in the iKO SCs (Fig. 7e). Additionally, we also performed RNA-Seq on FISCs isolated from the Ctrl and Atf3-cKO mice and similar results were obtained (Supplementary Fig. 7d–g and Supplementary Data 3). These results demonstrate that the ATF3 loss decreases H2b gene expression.

Next, we performed ChIP-Seq on the C2C12 myoblasts with exogenous ATF3 over-expression as a surrogate system to define direct binding targets of ATF3 (Fig. 7a and Supplementary Fig. 7h). A total of 2871 binding peaks were identified in 869 genes with 60%, 28% and 9% located in intergenic regions, introns and promoters, respectively (Fig. 7f and Supplementary Data 2). These peaks were enriched for the known ATF3 binding motif, TGACTCA (Fig. 7g), attesting to the good data quality. GO analysis of these ATF3 bound genes indicated remarkable enrichment for the terms related to nucleosome (Fig. 7h and Supplementary Data 2). By integrating the RNA-Seq and ChIP-Seq results, 72 down-regulated genes contained ATF3 binding sites and again they were enriched for nucleosome related GO terms and many were histone genes (Fig. 7i and Supplementary Data 2). Notably, 8 of the 13 down-regulated H2b genes had ATF3 binding sites in their promoters (Fig. 7i, j, Supplementary Fig. 7i and Supplementary Data 2). Meanwhile, 112 up-regulated genes possessed ATF3 binding and were enriched for mitochondrion related GO terms (Supplementary Fig. 7j and Supplementary Data 2). The Western blot (Fig. 7k) and IF staining (Fig. 7l) results confirmed a substantial reduction or near loss of H2B protein in the iKO FISCs while other histone proteins such as H3, H4 and H2A remained unaltered. These results demonstrate that H2b genes are bona fide transcriptional targets of ATF3.

Global diminution of histones leads to decreased amount of nucleosomes, augmented nucleosome spacing, and alters nucleosome occupancy thus affecting transcription^57,58,59, to determine if the H2B loss in the iKO SCs alters nucleosome positioning and occupancy, CUT&RUN assay^60,61 was performed to map the genomic localization of H2B in Ctrl and iKO cells (Fig. 7a). A total of 272590 bins were defined genome-wide from the three replicates (Supplementary Data 4). Unexpectedly, the average H2B CUT&RUN signals were largely unaltered in the iKO vs. Ctrl with only 3429 bins (1.4%) showing changes (Fig. 7m, n). Nevertheless, 3220 out of the altered bins indeed showed decreased H2B enrichment in various genomic regions, including promoter (12%), gene body (39%) and intergenic regions (49%) (Fig. 7m). Since the nucleosome is partly dissembled through the removal of one H2A-H2B dimer to facilitate Pol II transcription⁶¹ (Fig. 7o), we hypothesized that the local loss of H2B could facilitate nucleosome destabilization to promote the transcription of associated genes. Indeed, by intersecting with RNA-Seq data, 137 up-regulated genes showed decreased H2B signals on their promoters or gene bodies (Fig. 7p, q and Supplementary Data 4). These H2B target genes were enriched for GO terms related to muscle differentiation and also included Id3, IGF-1 and Jun, etc. (Fig. 7r, s), which was consistent with the activation state of the iKO SCs^11,62,63. Taken together, these findings demonstrate that the decreased H2B level could mediate the precocious SC activation following the ATF3 loss.

H2B mediates ATF3 function in SC activation and muscle regeneration

To solidify the functional link between ATF3 and H2B in SC activation and muscle regeneration, we found that overexpressing H2B by transfecting a pcDNA-H2B plasmid in FISCs indeed repressed the accelerated activation of Atf3 iKO cells but no impact on Ctrl cells (Fig. 8a, b). And in vivo, we also overexpressed H2B (Supplementary Fig. 7k) by intramuscular injection of lentiviral particles at 1dpi (Fig. 8a). As shown in Fig. 8c–g, this also restored the acute injury induced regeneration in the Atf3-iKO mice: a significantly lower number of eMyHC+ fibers were observed 5 dpi (Fig. 8d, e), which was accompanied by reduced numbers of Pax7+ cells (Fig. 8f) and MyoD+ cells (Fig. 8g). Altogether these findings validate that H2B loss mediates the precocious SC activation and enhanced muscle regeneration observed upon ATF3 loss.

H2B loss increases DNA damage and senescence in Atf3 iKO SCs

To further elucidate the consequence of H2B loss in SCs, we examined genome instability and cellular senescence considering histone proteins are important for chromatin integrity and genome stability^57,59. FISCs from iKO or Ctrl muscles were treated with various dosages (0, 1000 or 2000J) of UV light and comet assay was performed. Indeed, a substantial increase in the comet tail length was observed under all three dosages in the iKO as compared to the Ctrl (Fig. 9a), suggesting that H2B decrease causes genomic instability. This was further confirmed by a much higher percentage of γH2AX+ cells in the cultured iKO cells compared to the Ctrl (Fig. 9b, c). To examine if the genomic instability leads to cellular senescence, cells were cultured in growth medium for 9 days and β-Gal staining was performed; a 19% increase of β-Gal+ cells was detected in the iKO compared to the Ctrl (Fig. 9d). Consistently, a significant up-regulation of p16, p21, and p53 mRNAs (Fig. 9e) and a higher level of p21 and p53 proteins were also detected in the iKO (Fig. 9f). Altogether, the above findings demonstrate that H2B loss leads to increased genome instability and cellular senescence in the Atf3 iKO cells. Expectedly, H2B over-expression in the iKO cells reduced the DNA damage accumulation and inhibited cellular senescence as assessed by decreased p21 protein (Fig. 9g) as well as p16, p21 and p53 mRNAs (Fig. 9h). These results indicate that the decreased H2B expression causes SCs to undergo replicative senescence.

Screening of other functional AP-1 family members in SCs and regeneration

After the above holistic characterization of the function and mechanism of ATF3 in SCs, we sought to expand the study by asking if other AP-1 family TFs such as ATF4, FOS, FOSB, and JUNB, also play functional roles in SCs considering their similar rapid and transient induction dynamics during SC early activation (Figs. 1b–d and S1c). To test the idea, we conducted a functional screening using our recently developed Pax7^Cas9/AAV-sgRNA mediated in vivo genome editing platform⁶⁴, which is based on a Cre-dependent Cas9 knock-in mice and AAV-mediated sgRNAs delivery. Briefly, one pair of sgRNAs targeting each TF were selected and packaged into AAV9 virus particles; 4 × 10¹¹ viral genomes (vg)/mouse of AAV9-sgRNA was intramuscularly (IM) injected into the TA muscles of Pax7^Cas9 mice at postnatal (P) age of 10 days (P10). For the control (Ctrl) group, the same dose of AAV9 virus containing pAAV9-sgRNA vector backbone without any sgRNA insertion was injected. The mice were then sacrificed for SC isolation and analysis after eight weeks (Fig. S8a). Successful DNA editing efficiency was confirmed for all four TFs (Supplementary Fig. 8b–e); consistently, knock down (KD) of each protein to various degrees (52.4%–83.6%) was detected by WB (Supplementary Fig. 7b–e) or IF (Supplementary Fig. 8f) despite unaltered mRNA levels (Supplementary Fig. 8g).

To examine the effect of the above AP-1 TF KD on SC activation, FISCs from each mutant were cultured for in vitro EdU assay (Supplementary Fig. 8a). Similar to Atf3 KO, the Fos-KD cells showed accelerated SC activation while the JunB-KD displayed blunted activation; the Atf4 or FosB-KD, on the other hand, did not have any impact (Supplementary Fig. 8h). When measuring Pax7+ cells on the ~2 month-old uninjured muscles, interestingly, reduced numbers were observed upon the Atf4, Fos or JunB-KD (Supplementary Fig. 8i), suggesting a loss of SC pool. Since the Atf4 or JunB-KD did not lead to precocious SC activation, the SC number loss may be caused by other reasons considering the deletion occurred very early in the postnatal stage⁶⁴. The FosB-KD did not have any impact on the Pax7+ SC pool.

To further investigate the impact on muscle regenerative ability, BaCl₂ was injected 7 weeks after the AAV injection (Supplementary Fig. 8a) and the regeneration was assessed by H&E (Supplementary Fig. 8j), eMyHC (Supplementary Fig. 8k), Pax7 (Supplementary Fig. 8l) and MyoD (Supplementary Fig. 8m) staining at 5 dpi. An impaired regeneration was revealed in the Atf4 and Fos-KD presumably because of the exhaustion of SCs after its long-term deletion; The regeneration in the JunB-KD was also compromised probably due to a reduced SC pool; consistently, no impact on the regeneration was observed in the FosB-KD. Altogether, our results that these AP-1 family members may play diverse functions in SC activation and muscle regeneration.

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Source: https://www.nature.com/articles/s41467-023-40465-w

Generative Data Intelligence

ATF3 induction prevents precocious activation of skeletal muscle stem cell by regulating H2B expression – Nature Communications

ATF3 is rapidly and transiently induced during SC early activation

Short-term Atf3 deletion accelerates acute injury-induced muscle regeneration

Atf3 deletion provokes premature SC activation and pseudo-regeneration in homeostatic muscle

Long-term Atf3 deficiency depletes SC pool and impairs muscle regeneration

Atf3 deletion enhances SC activation during voluntary and endurance exercises

ATF3 regulates H2B gene expression and nucleosome patterning

H2B mediates ATF3 function in SC activation and muscle regeneration

H2B loss increases DNA damage and senescence in Atf3 iKO SCs

Screening of other functional AP-1 family members in SCs and regeneration

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