The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.
- ribosome biogenesis
- skeletal muscle hypertrophy
- ribosomal deoxyribonucleic acid transcription
skeletal muscle hypertrophy requires an increase in the number of ribosomes to cope with the anabolic demands of the growth process. Enhanced ribosome production seems necessary as blockage of de novo rRNA synthesis severely restricts muscle hypertrophy in vivo (10) and in vitro (20). Despite the importance of ribosome production during hypertrophy, very little is known about the mechanisms involved in this process and how this is modulated by mechanical loading.
Synthesis of functional ribosomes requires the coordinated expression of four different rRNA subunits and ∼80 ribosomal proteins. All rRNA subunits are transcriptionally regulated, and of all of these, the 5.8S, 18S, and 28S rRNA subunits are processed from a single 45S pre-rRNA, which originates from transcription of ribosomal DNA (rDNA) genes by RNA polymerase I (Pol I) (13). Pol I transcription of rDNA genes is rate limiting for growth (19), and its association with the rDNA promoter depends, to a large extent, on the formation of a preinitiation complex (PIC) (25). This complex is composed of multiple factors that operate in synergy to modulate transcriptional efficiency through enhanceosome formation, chromatin remodeling, and promoter regulation. Transcript levels of the PIC factors have recently been shown to decrease coordinately during granulocyte differentiation at the time when ribosome production also decreases, suggesting that the expression of Pol I accessory factors is adjusted according to the growth demands of the cell (24). Interestingly, the coordinated regulation of Pol I factors, collectively known as “Pol I regulon,” is driven by c-Myc (24). Because c-Myc directly controls rDNA transcription (3, 11, 23, 28), it is a likely candidate for the synchronized expression of the Pol I machinery and rDNA genes to facilitate enhanced ribosome production during muscle adaptation. c-Myc is an early response gene that is essential for embryonic development and postnatal growth (27), and, although skeletal muscle c-Myc expression gradually decreases to undetectable levels during postnatal development, its expression is reinduced by hypertrophic stimuli. For example, our laboratory has previously shown that c-Myc mRNA is rapidly targeted (∼6 h) to the translational pool following an acute bout of resistance exercise in rats (8), indicating that the reexpression of c-Myc is an early event in skeletal muscle hypertrophy. In addition to regulating the expression of PIC factors, c-Myc also regulates upstream binding factor (UBF) transcription (23), which is needed for efficient Pol I transcription (5), and its availability is necessary for in vitro myotube hypertrophy (20). Because both UBF and the Pol I regulon are important for Pol I-mediated rDNA transcription, we hypothesize that c-Myc may orchestrate the expression of the Pol I machinery to modulate ribosome production during the early adaptive responses to skeletal muscle overload. In addition to transcriptional control, rDNA genes are also subjected to epigenetic regulation (17). Recent studies have identified the Williams syndrome transcription factor (WSTF) as a key component of the B-WICH chromatin-remodeling complex (6). WSTF belongs to a group of ATP-dependent nucleosome remodeling factors that alter chromatin structure to regulate promoter accessibility, maintain transcriptionally competent chromatin, and thereby facilitate gene expression (9, 22).
Thus the goal of the present study was to investigate the regulation of rDNA gene transcription in response to mechanical loading-induced skeletal muscle hypertrophy. We found that, as early as 3 days following the imposition of mechanical loading, augmented rDNA transcription was associated with the induction of c-Myc and its downstream target UBF. In addition, the Pol I regulon was also induced, consistent with the expression of c-Myc. Similarly, WSTF expression was modulated by loading, and, collectively, these factors were functionally associated with the rDNA promoter. In addition to transcriptional regulation, an increase in histone H3 acetylation in lysine 9 (H3K9-Ac) was also detected at the rDNA promoter, demonstrating that both transcriptional and epigenetic mechanisms take place to regulate rDNA gene activity and ribosome production at the onset of muscle hypertrophy.
Animals and surgical procedures.
Ten-week-old C57Bl/6J mice (Charles River, Wilmington, MA) were kept on a 12:12-h light-dark cycle, had unlimited access to water, and were fed a standard rodent chow diet ad libitum. Animals were operated under surgical depth anesthesia, induced, and maintained with isofluorane (Baxter, Norfolk, UK). Functional overload was imposed by bilaterally removing both soleii and gastrocnemii muscles. Control animals underwent a sham operation, where muscles were separated by blunt dissection, paying special attention to avoid any tissue damage. All procedures were approved by the local ethics committee and were carried out following Federation of Laboratory Animal Science Associations guidelines for animal experimentation.
Following the indicated times, muscles were collected from live animals under surgical depth anesthesia. Following dissections, animals were euthanized with an intraperitoneal injection of pentobarbital sodium (Allfatal Vet, Omnidea, Stockholm, Sweden). Dissected muscles were blotted to remove excess blood, cleaned from any nonmuscle tissue, and weighed on a precision scale (Sartorius Acculab ATL-84, Göttingen, Germany). After weighing, muscles were either snap frozen in liquid nitrogen or mounted in OCT and frozen in isopentane cooled on liquid N2. All tissues were stored at −80°C until further use.
Homogenization, protein quantification, and Western blot.
Muscles were homogenized in lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 2.5 mM EGTA, 0.5% Igepal, 0.5% Triton X-100, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, and 0.1 mM NaVO3 supplemented with one tablet of complete mini and Phospho STOP; Roche Diagnostics, Indianapolis, IN) using a 5-mm generator coupled to a Polytron tissue disruptor (Kinematica, Kriens, Switzerland). All chemicals were purchased from Sigma, unless otherwise stated (Sigma-Aldrich, St. Louis, MO). Crude homogenates were transferred to 1.5 ml Eppendorf tubes and rotated on a spinning wheel for 20–30 min at 4°C. Thereafter, tubes were vortexed and spun down, and the supernatant transferred to a new tube. Protein concentration was measured using the DC Protein assay (Biorad, Hercules, CA). Protein homogenates were diluted with lysis buffer, if needed, and mixed 1:1 with 2× Laemmli buffer (Biorad) containing 5% β-mercaptoethanol. Samples were boiled at 95°C for 10 min and immediately cooled on ice and stored at −20°C until further use. Samples were separated by SDS-page on 7.5–12.5% polyacrylamide gels, depending on the size of the protein studied, and transferred to polyvinylidene difluoride membranes activated in 100% methanol. Western blotting was performed using standard techniques, membranes were blocked in a protein containing buffer, and washes were performed with 0.1% Tris-buffered saline-Tween. Primary and secondary antibodies were diluted in either 5% BSA or 5% milk diluted in 0.1% Tris-buffered saline-Tween. Primary antibodies were used as follow: c-Myc (1:500) and UBF (1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA), and WSTF (1:1,000) (Abcam, Cambridge, UK). Pol I (1:1,000) was provided by Dr. T. Moss (Laval University, Quebec, Canada). All membranes were stained following ECL visualization using Commassie blue to ensure equal loading.
RNA extraction, cDNA synthesis, and quantitative RT-PCR.
RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). rRNA quantity was determined spectrophotometrically using a Nanodrop ND-1000 (Saveen Werner AB, Malmö, Sweden) at 260 nm and by 260-to-280-nm ratio and agarose gel electrophoresis, respectively. cDNA was synthesized using Superscript VILO cDNA synthesis kit (Invitrogen) from 2 μg RNA, according to the manufacturer's recommendations. Quantitative RT-PCR was performed using GoTaq qPCR Master Mix (Promega, Fitchburg, WI) on a CFX384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Each sample was run in triplicate for each gene analyzed. A melting curve analysis was performed for each primer pair to ensure that a single product was efficiently amplified with each primer set. Relative expression levels of transcripts from genes of interest were normalized by using the comparative Ct (ΔΔCt) method using glyceraldehyde-3-phosphate dehydrogenase as housekeeping gene. This normalization target was used as it was not modulated by the overload period. Data were analyzed using the Bio-Rad CFX manager software (version 2.0). Primer sequences will be made available upon request.
Overloaded and sham-operated muscles were collected bilaterally as described above and processed in parallel. Muscles were minced in cross-linking buffer (1 M NaCl, 10 mM EDTA, 5 mM EGTA, 500 mM HEPES, pH 8) containing 1% formaldehyde. Muscle slurries were incubated for 30 min, and the reaction was stopped by the addition of 1.5 ml of 1 M glycine solution (final concentration of 0.1 M). Chromatin was then sheared by sonication to a DNA size of 500–1,500 bp, and fragments were verified by agarose gel electrophoresis. DNA was quantified spectrophotometrically and adjusted to a concentration of 10 ng/μl. Chromatin (180 μl) was incubated with the corresponding antibodies for 18 h with rotation and protein-chromatin fragments precipitated in IP buffer (1% Triton-100 and 0.1% deoxycholate). The antibody-chromatin fragments were precipitated in IP buffer by adding 15 μl protein A/G agarose beads. The IP was subsequently washed five times with RIPA buffer containing 150 mM NaCl. The cross-linked protein-DNA complexes were reversed overnight by incubating the samples at 65°C. DNA was extracted with phenol-chloroform-isoamylalcohol, washed with 75% EtOH, and used for SYBR green-based quantitative PCR (KAPA SYBR FAST, KAPA Biosystems, Woburn, MA) targeted against the mouse rDNA promoter. The primers (2.5 μM) used for detecting DNA sequences were as follow: rDNA promoter forward (5′-GGT ATA TCT TTC GCT CCG AG-3′) and reverse (5′-AGC GAC AGG TCG CCA GAG GA-3′). An aliquot of 40 μl of chromatin solution was used to extract DNA for the input RT reaction. Samples were run under the following conditions, hold 95°C for 3 min, followed by cycles of 95°C for 3 s, 60°C for 20 s, and 72°C for 3 s, and analyzed using the Rotor-Gene 6000 series software 1.7. A reaction consisting of no antibody and IgG was used for background correction. Because the Ct values were similar between these two groups, the results were averaged and subtracted from the specific antibody IP signals and subsequently divided by the corrected input DNA signal. Each control and overloaded sample was run in triplicates and used to calculate the 2ΔCt. Changes in H3 acetylation were determined by carrying out chromatin immunoprecipitation (Ch-IP) reactions using a H3K9-Ac antibody (Abcam cat no. 10812). Chromatin bound to H3K9-Ac values were normalized to the quantitative PCR signal obtained by chromatin bound to H3 (Abcam cat no. 1791), as previously described (29). This approach allowed us to account for any differences in H3 occupancy in sham vs. overloaded muscle so that any differences in H3K9-Ac were due to posttranslational modifications in H3K9 and not due to differences in H3 content.
Sections (10 μm) from overloaded and sham-operated plantaris muscles were sliced on a cryostat and fixed in 4% formaldehyde for 20 min after being allowed to air dry at room temperature. Sections were permeabilized in PBS containing 0.1% Triton 100X for 10 min and blocked with 2% rabbit serum (DAKO, Carpinteria, CA) and mouse-on-mouse blocking agent (Vector Laboratories, Burlingame, CA) in PBS-Triton 100X. After blocking for 45 min, sections were incubated overnight with primary antibodies against UBF (1:100), WSTF (1:200), and dystrophin (1:50) (Novacastra, Newcastle upon Tyne, UK), followed by anti-rabbit, CF633-conjugated or anti-mouse, CF488A-conjugated (Biotium, Hayward, CA) secondary antibodies for 45 min (1:200). Sections were mounted in Vectashieled Mounting Media containing 4,6-diamidino-2-phenylindole (Vector Laboratories) and examined using a Leica DM RXA2 fluorescent microscope. Images were merged using Image J software (National Institutes of Health, Bethesda, MD).
Values are reported as means ± SD, unless stated otherwise. Differences between groups for muscle-to-body weight ratio, rRNA-to-muscle weight ratio, and quantitative RT-PCR data were determined using a one-way ANOVA with the Tukey post hoc test. Western blot quantification and Ch-IP data were compared using a two-tailed student t-test. The level of significance was set at P < 0.05 for all statistical comparisons.
Mechanical loading increased rDNA gene activity and rRNA content at the onset of skeletal muscle hypertrophy.
As expected, functional overload of the plantaris muscle resulted in a progressive increase in wet mass during the course of the experiment (Fig. 1A). Since ∼80–90% of cellular RNA is rRNA (31), we determined changes in rRNA by measuring total RNA levels. The concentration of rRNA was significantly elevated at all time points relative to controls (Fig. 1B), indicating a steady increase in ribosome production during skeletal muscle hypertrophy. To determine the early responses to overload, rDNA transcription rates were estimated by determining 45S pre-rRNA levels. Quantification of both external transcribed and internal transcribed spacers showed a significant elevation at 3 days postloading, as indicated by a three- to fourfold increase in both external and internal transcribed spacers (P < 0.001) (Fig. 1, C and D).
Mechanical loading induced a Pol I regulon and accessory factors involved in Pol I-mediated rDNA transcription.
Consistent with the increase in rDNA gene activity, all mRNAs coding for the Pol I regulon factors (POL1RB, TIF-1A, PAF53, TTF1, and TAF1C) were coordinately induced, and their expression significantly elevated 3- to 6.5-fold (P < 0.05) 3 days following mechanical loading (Fig. 2). In most cases, except for PAF53, all transcripts began to decrease to baseline levels by 7 days following mechanical loading. In view of the coordinated Pol I regulon expression, we sought to determine whether c-Myc, which has previously been demonstrated to be a key regulator of the Pol I regulon, was induced in a similar temporal fashion. c-Myc mRNA levels increased significantly by 6.5-fold (P < 0.05) at 3 days, remained elevated by 7 days, and returned to baseline by 14 days (Fig. 3A). In line with its transcriptional regulatory functions, c-Myc protein levels were also markedly increased by 3 days (Fig. 3B). As expected, c-Myc protein was below detection levels in sham-operated control, indicating that the surgical intervention did not affect c-Myc protein expression. In addition to the Pol I regulon, another downstream target of c-Myc involved in rDNA transcription, UBF, showed a similar temporal expression pattern, consistent with the transcriptional activity of c-Myc. UBF mRNA was significantly increased threefold at day 3 postloading (P < 0.05), remaining elevated for at least another 4 days (Fig. 3C).
Increased chromatin remodeling following mechanical loading.
In view of the increase in rDNA gene activity and coordinated expression of Pol I factors, we investigated whether mechanical loading also modulated the expression and association of factors regulating rDNA transcription and chromatin remodeling at the rDNA promoter. UBF and WSTF protein content increased three- and fourfold (P < 0.05) with overload, respectively, but Pol I protein levels were unchanged compared with control (Fig. 4). Because of the complex structural organization of skeletal muscle, we sought to determine whether the expression of UBF and WSTF was localized to myonuclei. Both UBF and WSTF were expressed in myonuclei, as determined by the localization within the myofiber boundary (Figs. 5 and 6). To define the functional significance of the increase in transcriptional and chromatin remodeling factors, we carried out Ch-IP to assess their binding to the rDNA promoter. We found that all three factors, in addition to c-Myc, were significantly enriched at the rDNA promoter. c-Myc, Pol I, UBF, and WSTF enrichment was 4.5-, 9.5-, 3.5- (P < 0.01), and 2.5-fold (P < 0.05), respectively, 3 days following mechanical loading (Fig. 7A). Furthermore, to establish whether these factors contributed to changes in chromatin structure, we determined changes in histone modifications, reflective of open chromatin and active gene transcription. Ch-IP analysis revealed a 27.5-fold increase (P < 0.05) in H3K9-Ac relative to H3 in overloaded muscles compared with sham-operated controls (Fig. 7B).
The goal of the present study was to investigate the regulation of rDNA gene transcription in response to mechanical loading-induced skeletal muscle hypertrophy. Enhanced ribosome production during hypertrophy has been known for several decades (12, 15, 26), but the mechanisms involved in this process remain largely unknown. The importance of this response is highlighted by the fact that muscle protein synthesis is a major determinant of muscle mass and is mainly regulated by ribosome content (18). Given the relatively slow turnover of ribosomes, any rapid increase in ribosome content is due to increased ribosome synthesis. This is a critical adaptive response as blocking de novo rRNA synthesis severely impairs skeletal muscle hypertrophy in vivo (10) and myotube hypertrophy in vitro (20). In the present study, the initial increase in rRNA observed following mechanical loading was maintained during the overload period. Because the relative rRNA concentration remained steady during the 2-wk overload, we interpret that the initial transcriptional response induced by mechanical loading was sufficient to maintain the steady increase in ribosome content required to sustain the anabolic demands of the hypertrophying muscle. In this model, the onset of muscle hypertrophy is defined as the time point(s) preceding the significant increase in dry mass, which has been previously reported to be between 10 and 14 days following the imposition of the functional overload (1, 16, 21). Thus the early increase in rRNA concentration seems to be a key event in the development of work-induced skeletal muscle hypertrophy. Similarly, it is important to recognize that an increase in translational efficiency at earlier time points may play an important role in the initiation of rDNA transcription and hence rRNA accumulation (15).
Enhanced ribosome production is mainly determined by Pol I-mediated transcription of rDNA genes (13, 19). In the present study, we assessed Pol I-dependent rDNA transcription by determining the expression of 45S pre-rRNA. rDNA transcription was elevated as early as 3 days following mechanical loading, indicating that Pol I-mediated rDNA transcription is an early event at the onset of skeletal muscle hypertrophy. Because Pol I transcription is regulated by a number of accessory factors that control its assembly and activity (13, 24, 25), we determined whether the Pol I regulon was induced by mechanical loading. We found that the coordinated expression of the Pol I regulon genes increased in a temporal fashion, consistent with the increased expression of 45S pre-rRNA. This suggests that the Pol I machinery responds to mechanical loading by increasing several of its components to support the increased demand for ribosomes typical of the hypertrophic response and is in agreement with previous studies demonstrating that Pol I activity is enhanced in hypertrophying muscle nuclei (26). Because the coordinated expression of the Pol I regulon is under the control of c-Myc (24), we determined whether c-Myc expression was elevated at this early time point. Consistent with the expression of the Pol I regulon and rDNA gene activity, both c-Myc mRNA and protein were elevated at 3 days following mechanical overload, indicating that c-Myc may regulate enhanced rDNA transcription by inducing the Pol I regulon. To further support the early transcriptional activity of c-Myc, we determined the expression of another direct c-Myc target (23). UBF mRNA was also induced in a similar temporal fashion, which suggests that c-Myc may orchestrate the expression of the Pol I machinery and accessory factors to regulate enhanced ribosome production during skeletal muscle hypertrophy. Our laboratory has previously shown that c-Myc mRNA translation is an early event (6 h) following resistance exercise in rats (8), a response that is conserved across species (2, 30). Thus, although the early induction of c-Myc during muscle hypertrophy has previously been reported, the expression of the Pol I regulon and UBF, together with the increased association of c-Myc at the rDNA promoter, provide new evidence demonstrating the involvement of c-Myc in the regulation of the Pol I machinery and rRNA synthesis at the onset of skeletal muscle hypertrophy.
In addition to the expression of c-Myc and UBF, which directly modulate transcription of rDNA genes (3, 11, 14), we determined the expression of Pol I and WSTF, of which the latter is a chromatin remodeling factor that modulates rDNA genes by epigenetic mechanisms (22, 29). As expected, UBF and WSTF protein expression was induced in a similar temporal fashion with the increase in rDNA transcription, and both factors were localized in myonuclei, consistent with previous data demonstrating c-Myc localization during mechanical loading-induced hypertrophy (4). Contrary to our expectations, Pol I protein expression did not increase, which indicates that not all components of the transcriptional machinery are similarly regulated. A possible explanation for this is that, because Pol I is a core component of the “basal” transcriptional machinery (13, 19, 25), its protein expression is not transcriptionally regulated as the factors involved in “enhanced” Pol I-mediated transcription. Given the increased rDNA gene activity and Pol I regulon expression, we determined whether these regulatory factors were functionally involved in active rDNA transcription. For this, we have determined their enrichment at the rDNA promoter by Ch-IP in 3-day overloaded and control muscles. Our results demonstrate that c-Myc, UBF, and Pol I were associated with the rDNA promoter, indicating that the expression of factors involved in Pol I-dependent rDNA transcription is modulated by mechanical loading and is functionally associated with rDNA gene activity during enhanced ribosome production. Because rDNA transcription can also be regulated by epigenetic mechanisms, we determined whether the expression of WSTF, a core factor in the chromatin remodeling complex B-WICH (29), was functionally associated with the rDNA promoter. WSTF binding to the rDNA promoter was also enriched 3 days post-overload compared with control muscles. This finding is consistent with previous reports demonstrating increased WSTF binding during enhanced rDNA transcription in serum-stimulated cells, which correlates with an open chromatin conformation at key regulatory regions upstream of the rDNA promoter (−1 kb to +300) (14, 19). This region includes the upstream core element, where UBF association to rDNA exerts regulatory functions (7) and is in agreement with the observed increase in UBF binding. Another important finding supporting WSTF enrichment and the epigenetic modulation of rDNA gene activity by mechanical loading is the increase in H3K9-Ac. Histone modifications play an important role in gene regulation (9), specifically H3K9-Ac is generally associated with transcription initiation and unfolded chromatin, thereby positively influencing gene expression. We have found that 3 days of overload exerted a dramatic increase in H3K9-Ac relative to H3, indicating a marked epigenetic modulation of the rDNA promoter. This is consistent with previous data demonstrating that WSTF modulates chromatin structure at the rDNA promoter to induce histone modifications, in particular of H3K9-Ac (6, 29). Because downregulation of WSTF by small interfering RNA results in a decrease in H3K9-Ac and transcriptional silencing of rDNA genes, we conclude that increased WSTF loading, together with H3K9-Ac, reflect an open chromatin state and active rDNA gene transcription to favor enhanced ribosome production.
In summary, our results provide novel data indicating that mechanical loading modulates the Pol I transcriptional machinery. Furthermore, the expression of the Pol I machinery is accompanied by an increase in factors that contribute to the transcriptional regulation and epigenetic state of the rDNA genes. This indicates that both transcriptional and epigenetic mechanisms may induce an active chromatin conformation competent for efficient rDNA transcription to support enhanced ribosome production at the onset of skeletal muscle hypertrophy.
The work presented herein was supported by grants from the Swedish Research Council (VRK2008-67X-20797-01-04), Centrum för Idrottsforskning (P2011-01-0133), Kung Gustaf V:s 80 Årsfond (FAI2009-0065), and Reumatikerforbundet (R-21211) to G. A. Nader.
No conflicts of interest, financial or otherwise, are declared by the author(s).
F.v.W. and G.A.N. conception and design of research; F.v.W., V.C., A.-K.Ö.F., and G.A.N. performed experiments; F.v.W., A.-K.Ö.F., and G.A.N. analyzed data; F.v.W., A.-K.Ö.F., and G.A.N. interpreted results of experiments; F.v.W., A.-K.Ö.F., and G.A.N. prepared figures; F.v.W., A.-K.Ö.F., and G.A.N. drafted manuscript; F.v.W., A.-K.Ö.F., and G.A.N. edited and revised manuscript; F.v.W., A.-K.Ö.F., and G.A.N. approved final version of manuscript.
We thank Dr. Tom Moss for the kind gift of Pol I antibody.
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