Generation of a conditional bitransgenic mouse expressing PC4 in skeletal muscle. A, transgene CMV-βactin-rtTA contains the CMV enhancer/chicken β-actin promoter, followed by the doxycycline rtTA and the rabbit β-globin polyadenylation site. The TRE-PC4 transgene contains TRE fused downstream to the minimal CMV promoter, the PC4 ORF, and the SV40 late polyadenylation site. The rtTA protein binds the TRE and activates transcription of the PC4 transgene in the presence of doxycycline. B, analysis of the PC4 protein expressed in skeletal muscle by the three lines, B, G, and H, of PC4 bitransgenic mice, activated or not with doxycycline supplied in the drinking water since P30. C, tissue expression analysis of the PC4 protein by Western blot. D, PC4 mRNA by real time PCR in line G of bitransgenic mice treated or not with doxycycline (Doxy). PC4 mRNA fold expression was calculated relative to the level of expression in untreated mice. Tg PC4 mice used for experiments were 2 months old and isogenic, being the progeny of at least six generations of interbreeding. Skm, skeletal muscle; H, heart; B, brain; L, liver; K, kidney; S, spleen; TA, tibialis anterior; EDL, extensor digitorum longum; Dia, diaphragm.

shRNA-mediated inhibition of PC4 expression impairs myoblast differentiation. A, analysis of PC4 and myosin heavy chain (MHC) protein expression in proliferating (GM) or differentiating (DM) C2C12 myoblasts, infected with retroviruses generated by the pSUPER-retro vector expressing the PC4-specific shRNA sequences PC4/2 and PC4/7 (RS/2 and RS/7, respectively) or with the insertless retrovirus (RS). After infection, cells were selected for 96 h with puromycin and then cultured in proliferating (GM) or differentiating conditions (DM), as indicated. B, Northern analysis of C2C12 myoblasts infected with the retrovirus expressing PC4 shRNA (RS/2) or with the insertless control retrovirus (RS). Total RNA was analyzed with a ³²P-labeled PC4 or GAPDH probe, as a measure of the amount and integrity of the mRNA. C, C2C12 myoblasts, infected with the retrovirus expressing PC4 shRNA (RS/2) or with the insertless control retrovirus (RS), were cultured in DM for 96 h; cells were then fixed and stained for immunofluorescence detection with anti-MHC antibody through secondary goat anti-rabbit FITC-conjugated antibody. Nuclei were detected by Hoechst 33258 dye (corresponding photomicrographs on the right). Bar, 140 μm. RS/2-infected cultures show a clear impairment of differentiation. Differentiation index (percentage of cells labeled by MHC) (D) and fusion index (percent ratio of the number of nuclei detected in MHC-labeled cells to the total number of nuclei) (E) were both significantly decreased. The numbers of myoblasts analyzed were 2067 and 1920 for RS- or RS/2-infected cultures, respectively. *, p < 0.01; **, p < 0.001, Student's t test.

Deprivation of PC4 results in inhibition of muscle gene mRNA expression and enhanced expression of cell cycle genes. Real time PCR analysis of the indicated muscle and cell cycle mRNAs from C2C12 myoblasts, infected with the retrovirus expressing the shRNA to PC4 (RS/2) or with the control retrovirus (RS). Cells were cultured in GM or DM as indicated. Average ± S.E. values are from three independent experiments and are shown as fold change relative to control sample (RS-infected cells in GM), which was set to unit. TATA-binding protein mRNA was used as endogenous control for normalization. *, p < 0.05; **, p < 0.01 versus the corresponding time point of control-infected cells (RS), Student's t test.

Reduced transcriptional activity of MyoD and MEF2C in the absence of PC4. A–C, C3H10T1/2 cell cultures were infected with the shRNA retrovirus expressing shRNA to PC4 (RS/2) or the control retrovirus (RS); following selection with puromycin for 96 h, cells were seeded in 35-mm dishes (7 × 10⁵) and cotransfected the next day with the pCDNA3-FLAG-MyoD construct (0.1 or 0.2 μg) or the empty vector and either the 4RE-LUC reporter (0.1 μg) (A), MCK-LUC reporter (0.3 μg) (B), or 3×MEF2-LUC reporter (0.2 μg) (C). After 24 h, the cultures were shifted to DM and harvested for analysis 48 h later. Luciferase (LUC) activity from cell extracts was expressed as fold induction relative to the activity of the RS-infected control sample not transfected with MyoD (that was set to unit), and it resulted in significantly reduced cultures deprived of PC4, with respect to control RS cultures. Bars represent the average fold induction ± S.E. determined in four independent experiments, each performed in duplicate. *, p < 0.05 or **, p < 0.01 (Student's t test). Parallel C3H10T1/2 cell cultures treated as above were analyzed for endogenous and exogenous MyoD protein expression by Western blot (D) and for MyoD mRNA expression by real time PCR (E), which is shown as average ± S.E. fold expression relative to the RS-infected control sample not transfected with MyoD, set to unit. TATA-binding protein mRNA was used as endogenous control for normalization. *, p < 0.05 (Student's t test). F, C2C12 cells infected with the RS/2 retrovirus targeting PC4 or the control RS retrovirus were plated in duplicate in 35-mm culture dishes (5 × 10⁴) and cotransfected the following day with the 3×MEF2-LUC reporter (0.1 μg), the pCDNA1-MEF2C (0.025 μg), and the pSCT-PC4 (0.5 μg) expression vectors, as indicated. Cells were maintained in GM and harvested 48 h after transfection. Luciferase activities are expressed as fold induction relative to the activity of the RS-infected control sample not transfected with MEF2C or PC4. Bars represent the average fold induction ± S.E. from three independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01 (Student's t test). F′, parallel cultures were analyzed for MEF2 protein expression by Western blot. G, C3H10T1/2 cells infected with the RS/2 or the control RS shRNA retroviruses were transfected with the 3×MEF2-LUC reporter (0.1 μg) and increasing concentrations of pCDNA1-MEF2C expression vector, as indicated. Cells were maintained in GM and harvested 48 h after transfection. Luciferase activities are expressed as fold induction relative to the activity of the RS-infected control sample. Bars represent the average fold induction ± S.E. from four independent experiments performed in duplicate. *, p < 0.05 (Student's t test). G′, parallel cultures were analyzed for MEF2 protein expression by Western blot.

PC4 represses the NF-κB transactivation function by promoting HDAC-dependent deacetylation of p65. A and B, PC4 synergizes with HDAC4 or HDAC3 in inhibiting NF-κB activity. C2C12 cells, plated in duplicate 35-mm culture dishes (7 × 10⁴ cells), were cotransfected the next day with the NF-κB-LUC reporter (0.1 μg), the pSCT-PC4 (0.1–0.3 μg), and the pcDNA3-myc-HDAC4 (10–30 ng) (A) or pcDNA3-myc-HDAC3 expression constructs (10–30 ng) (B). The pSCT and/or pcDNA3 empty vectors were included where necessary to normalize for DNA content. Cells were maintained in GM and harvested 48 h after transfection. Luciferase activity from cell extracts is calculated as fold change relative to the activity of the GM control sample transfected with the empty vectors. Bars represent the average fold activity ± S.E. determined from four independent experiments performed in duplicate. *, p < 0.05 versus the corresponding condition, as indicated (Student's t test). A′ and B′, C2C12 cells transfected as in A and B were analyzed for PC4, myc-HDAC4, or myc-HDAC3 protein expression by Western blot. C, increased acetylation and nuclear localization of p65 in C2C12 cells deprived of PC4. Western blot analysis of nuclear extracts from C2C12 cells infected with the RS/2 retrovirus expressing shRNA to PC4 or the control retrovirus (RS), cultured in proliferating (GM) or differentiating conditions (DM 24 or 48 h). The same filter was probed with antibodies against PC4, p65, p65 acetylated at lysine 310 or MyoD, as well as c-Jun and GAPDH, markers of nuclear and cytoplasmic localization, respectively. D, decreased acetylation and nuclear localization of p65 in primary myoblasts isolated from adult muscle of the Tg PC4 mouse. Nuclei were prepared from primary myoblasts derived from the Tg PC4 mouse, cultured in proliferating (GM) or differentiating conditions (DM 12 h). PC4 transgene was induced by administration of doxycycline to Tg PC4 mice since conception and by addition of doxycycline to the medium (20 ng/ml) of primary myoblasts. The same filter was probed with antibodies against PC4, p65, p65 acetylated at lysine 310, MyoD, c-Jun or GAPDH. E, PC4 forms complexes with HDAC3 and p65. C2C12 cells (clone S4 constitutively overexpressing PC4) were transfected with pcDNA-Myc-HDAC3 or the empty vector and cultured in GM or DM for 48 h as indicated. Cell lysates were immunoprecipitated with either anti-p65 antibody or control rabbit IgG, covalently bound to agarose beads. Immuno-complexes (IP: a-p65 and control IP) and input cell lysates were analyzed by Western blotting (WB) with anti-Myc, anti-PC4 or anti-p65 antibodies. Input lysates: 1/15 of lysates used for immunoprecipitation.

PC4 up-regulation in skeletal muscle increases myogenic gene expression and satellite cells number and potentiates regeneration. A, induction of MyoD, myogenin, MEF2C, MHC, Pax7, c-met, nestin, and Myf5 mRNA levels in TA muscle from 2-month-old Tg PC4 mice with transgene activated since P30 (TG), relative to control mice (CT). n = 3 for each group. B, decrease of the number of myofibers per area in cross-sections of TA from P60 Tg PC4 mice with transgene activated since P30 (left); increase of the number of myofibers per area in TA of P45 Tg PC4 mice with transgene activated since conception (right). C, increase of the number of satellite cells, measured as Pax7⁺ cells/myofiber in cross-sections from TA muscle of P45 Tg PC4 mice with transgene activated since conception. The experiments shown in B and C were performed with n = 3 animals for both CT and TG groups. D, increase of the number of regenerating myofibers in TA muscle of P60 Tg PC4 mice with transgene activated at P30, identified by the presence of central nuclei. Injury was induced by injecting cardiotoxin in TA 5, 7, and 20 days before killing mice at P60 for analysis. E, cross-sectional area of regenerating myofibers analyzed in D showed a decrease in Tg PC4 mice that became significant in the 20 days post-lesion group. n = 3 for CT and n = 4 for TG groups 5 and 7 days post-lesion; n = 3 for both CT and TG groups 20 days post-lesion. *, p < 0.05, or **, p < 0.01 CT versus the Tg PC4 mice group, Student's t test. F and G, representative confocal microscopy images of the regenerating myofibers at 5 and 7 days after injury, respectively. Immunofluorescence staining for laminin was used to identify the basal lamina surrounding each myofiber; nuclei were visualized by Hoechst 33258. Bar indicates 100 μm. A–G, CT mice were bitransgenic mice that did not inherit the TRE-PC4 transgene, exposed to doxycycline (Doxy) as the activated bitransgenic mice (TG).

Inhibition of muscle-specific proteins and induction of cell cycle proteins following deprivation of PC4 in myoblasts. A, expression analysis of the indicated muscle and cell cycle proteins in proliferating or differentiating C2C12 myoblasts deprived of PC4. Representative Western blots are shown (out of a total of three experiments). Myoblast cultures infected with the retrovirus expressing the PC4-targeting RS/2 shRNA or with the insertless retrovirus were selected with puromycin for 96 h and then cultured in GM or in DM for the indicated times. B, densitometry analysis of Western blots is shown in A; values are presented as fold change of protein expression in PC4-deprived cells relative to RS control cells, after normalization to the corresponding values of α-tubulin expression (the control base line is set to 0).

MyoD-dependent delay of cell cycle exit in C2C12 myoblasts deprived of PC4. A, flow cytometry analysis of proliferating or differentiating C2C12 myoblasts infected with the retrovirus expressing shRNA to PC4 (RS/2) or with the control retrovirus (RS). The infected cells were selected with puromycin for 96 h and then cultured in GM or DM for the indicated times. The DNA content was analyzed after staining with propidium iodide, by flow cytometry. Data from five independent experiments are shown as means ± S.E. of the changes in the percentage of PC4-silenced cells in G₀/G₁, S, or G₂/M cycle phase, relative to the percentage of cells infected with the control virus. RS/2-infected cells show a significant decrease of the G₁, and an increase of the S population, 12 and 48 h after shift to DM. *, p < 0.05 versus the corresponding control group at the same time point, Student's t test. B and C, flow cytometry analysis of C3H10T1/2 fibroblasts deprived of PC4, induced or not to differentiate into myotubes by ectopic MyoD. C3H10T1/2 cell cultures were infected with PC4-targeting shRNA retrovirus (RS/2) or with control retrovirus (RS), selected with puromycin for 96 h, and then infected with a retrovirus expressing MyoD (pBABE-MyoD) (B), or infected with the control retrovirus (pBABE) (C). Infected cells were cultured in GM or DM as indicated. Data from three independent experiments are shown as means ± S.E. of the changes in the percentage of PC4-silenced cells in the different cell cycle phases, with respect to control cells. The absence of PC4 causes cell cycle changes similar to those observed in C2C12 myoblasts only in C3H10T1/2 cells expressing MyoD. *, p < 0.05 versus the corresponding control group (RS-infected) of the same time point, Student's t test.

PC4 represses the NF-κB transactivation function. A, C2C12 cells were plated in duplicate 35-mm culture dishes (7 × 10⁴ cells) and cotransfected the next day with the NF-κB-LUC reporter (0. 1 μg) and either the pSCT-PC4 expression construct or the empty pSCT vector (0.5 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later. TNF (10 ng/ml) was added 6 h before harvesting, where indicated. Luciferase activity from cell extracts is expressed as fold induction relative to the activity of the GM control sample (transfected with the empty vector and not treated with TNF). Bars represent the average fold induction ± S.E. determined from five independent experiments, each performed in duplicate. *, p < 0.05 (Student's t test). B, C2C12 cells transfected as in A were analyzed for PC4 protein expression by Western blot. C, C2C12 cells infected with the RS/2 retrovirus expressing shRNA to PC4 or the control retrovirus (RS) were plated in duplicate 35-mm culture dishes (7 × 10⁴ cells) and transfected the next day with the NF-κB-LUC reporter (0.1 μg). Cells were either maintained in GM or switched to DM 24 h after transfection and harvested 48 h later; where indicated, TNF (10 ng/ml) was added 6 h before harvesting. Luciferase activity is expressed as fold induction relative to the activity of the GM RS-infected control sample not treated with TNF. Bars represent the average fold induction ± S.E. determined from five independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01 (Student's t test).

MyoD is recruited to the PC4 gene promoter. A, ChIP analysis of MyoD binding to the mouse PC4 promoter in proliferating (GM) or differentiating (DM) C2C12 myoblasts. The scheme above the graph illustrates the first exon of the mouse PC4/Tis7 gene and the promoter region analyzed, located 780 nt before the transcription start. The amounts of PC4 promoter region retrieved from immunoprecipitates obtained with anti-MyoD antibody (black columns) or with normal rabbit serum (gray columns) are expressed as percentage of the amounts of promoter region from input cell lysates. The same amount of chromatin was immunoprecipitated in ChIPs with a-MyoD or normal rabbit serum. B, ChIP analysis of MyoD binding to the NeuroD1 promoter in C2C12 myoblasts, performed as negative control. C, PC4 mRNA levels, detected by real time PCR in duplicate cultures of myoblasts analyzed by ChIP, expressed as fold values relative to the mRNA amount present in GM cultures. A–C, the average ± S.E. values are from three experiments.