Myofibrillar gene expression is increased in IκBα-SR-expressing C2C12 myoblasts. (A) EMSA for NF-κB binding activity in TNF-α-treated vector and IκBα-SR-expressing C2C12 myoblasts. (B and C) Confirmation of microarray results (Table 1) was performed with semiquantitative RT-PCR (B) and real-time PCR (C) analyses of myofibrillar genes expressed in vector and IκBα-SR-expressing C2C12 myoblasts. Real-time PCR data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. (D) C2C12 vector and IκBα-SR cells were cultured in 12-well plates and transiently transfected in triplicate with a TnI-Luc reporter plasmid along with LacZ-expressing plasmids. Cell extracts were prepared 48 h posttransfection, and relative luciferase units were determined by normalizing to β-galactosidase (β-Gal) activity. (E and F) Proposed models for how NF-κB functions to repress myofibrillar gene expression in proliferating skeletal myoblasts, either through direct DNA binding (E) or indirectly through the regulation of an unknown downstream transcriptional repressor (F).

YY1 expression is regulated by NF-κB. C2C12 (A) or 10T1/2 (B) cells were plated in 10-cm plates and treated with 10 ng/ml of TNF-α. Total RNA was prepared at the indicated times, and the amount of YY1 mRNA was quantified by real-time PCR and normalized to GAPDH. For comparison, real-time PCR was repeated while probing for a known NF-κB target gene, IκBα. Induction changes were calculated by setting YY1 or IκBα levels in untreated cells to a value of 1. 10T1/2 cells were also treated with 10 ng/ml of IL-1β (C) or 50 ng/ml LPS (D). The amounts of YY1 mRNA were quantified by real-time PCR. (E) Nuclear extracts were prepared from C2C12 at the indicated times in differentiating myoblasts (DM), and EMSAs were performed with radiolabeled probes corresponding to the MHC-I probe. The amounts of YY1 mRNA at the indicated times were quantified by real-time PCR.

YY1 is regulated by p65 in vivo. (A) Tibialis anterior muscles were isolated from 4-week-old TNF^−/−; p65^+/+ (p65^+/+) and TNF^−/−; p65^−/− (p65^−/−) mice, and YY1 was measured by RT-PCR (upper panel) and Western blot analysis (lower panel). (B) YY1 measurement from quadriceps or gastrocnemius muscles from p65^+/+ and p65^−/− mice. (C) YY1 RNA expression from nonmuscle tissues (spleen, thymus, heart, and lung) isolated from 4-week-old p65^+/+ and p65^−/− mice. (D) Tibialis anterior muscles from control mice were injected with CTX and, at indicated days, Western blot analysis was performed with probes for YY1 and α-tubulin. (E) Analysis of YY1 from tibialis anterior muscles from p65^+/+ and p65^−/− after 6 days of CTX injections by Western blotting. (F) Uninjected (control) or 6-day CTX-injected tibialis anterior muscles from p65^+/+ and p65^−/− mice were sectioned and immunostained for YY1 (red) and nuclei (Hoechst; blue) and photographed at ×20 magnification. Arrows indicate the muscle fibers with YY1 expression localized in the nuclei. Scale bar = 50 μM. (G) Percentage of central nucleation (%CLN) was determined for 6-day CTX-treated tibialis anterior muscles from control and p65-null littermate pairs (n = 2) by counting the number of central nuclei in randomly chosen fields from a minimum of 1,500 fibers; P < 0.05, Student's t test.

YY1 binds and represses the Tnni2 enhancer. (A) Schematic illustration of the murine Tnni2 first intron region containing a MEF2 binding site, two E-boxes, CAGG, CCAT regulatory elements, and three putative CCAT YY1 binding sites (diamonds; sites A, B, and C). (B) Nuclear extracts were isolated from C2C12 myoblasts (MB) or myotubes (MT), and EMSA was performed with probes containing either the known YY1 binding site in the MyHCIIb promoter or the putative wild-type YY1 site A (Tnni2) or mutant site A [Tnni2(Mut)] in the Tnni2 enhancer. Supershift EMSAs with no antibody (−) or with YY1 antibodies (+) were used to confirm YY1 binding complexes. The arrow denotes a supershift complex. (C) ChIP assays with either an YY1 antibody or control IgG were performed on chromatin isolated from C2C12 myoblasts. The precipitated DNA fragments were amplified with specific olionucleotides spanning regions A, B, or C of the Tnni2 enhancer. Total input is indicated. (D) C2C12 cells were plated in 12-well plates and transfected with 0.2 μg of MyHC-Luc, TnI-Luc, or TnI-Mut-Luc reporter plasmid along with 0.05 μg of MyoD and indicated amounts of a YY1 expression plasmid. Luciferase activities were determined 48 h posttransfection and normalized to β-Gal protein. (E) C2C12 myoblasts were cotransfected with siRNA YY1 and a Tnni2 reporter plasmid, and reporter activity was determined by luciferase assays after 2 days in differentiation conditions. (F) ChIP assays were performed with C2C12 myoblasts (MB) or myotubes (MT) with antibodies against YY1, Ezh2, HDAC1, trimethyl-histone H3-k27, PCAF, or acetyl-histone H3-K9. Primers specific to Tnni2 intron region A were used for PCR amplification. Total inputs are indicated.

A model for how NF-κB negatively regulates troponin and other myofibrillar genes. The model depicts basal NF-κB activity regulating YY1 expression in undifferentiated myoblasts. YY1 expression, in turn, binds to the promoter or enhancer elements of various myofibrillar genes (troponin; Tnn is used as an example), thereby recruiting the HDAC1 corepressor and the Polycomb silencer complex to inhibit myogenic differentiation. At the onset of differentiation, YY1 is displaced from the chromatin and replaced by serum response factor (SRF), MyoD, and PCAF, which permits the initiation of transcription and subsequent myogenesis.

NF-κB does not directly bind to the Tnni2 promoter. (A) Schematic illustration of the tnni2 promoter. Five putative NF-κB binding sites, A to E, are indicated as black boxes upstream of the transcriptional start site, and sequences are shown. (B) Nuclear extracts were prepared from C2C12 myoblasts either untreated (−) or treated (+) with TNF-α for 15 min, and EMSAs were performed with radiolabeled probes corresponding to putative NF-κB sites A to E or to the MHC-I probe containing a bona fide NF-κB binding site. (C) Supershift EMSAs were performed with TNF-treated C2C12 nuclear extracts preincubated with IgG (lanes 1, 5, and 9) or antisera specific for p65 (lanes 2, 6, and 10) or p50 subunits (lanes 3, 7, and 11). For competition EMSAs, extracts were preincubated with a 100-fold molar excess of unlabeled oligonucleotides containing MHC-I NF-κB binding sites (lanes 4, 8, and 12). Arrows denote p65- and p50-containing complexes and supershift complexes.

YY1 regulation is p65 dependent. (A) Total RNA and protein were prepared from C2C12 myoblasts stably expressing vector or p65, and YY1 RNA was measured by real-time PCR. The inset shows the expression of Flag-tagged p65 normalized to tubulin. (B) Total RNA and protein were prepared from vector control (V) and IκBα-SR (SR)-expressing C2C12 myoblasts, and YY1 RNA and protein were measured by real-time PCR and Western blot analysis. (C) 293T cells were plated in 10-cm plates and transfected with either vector control, p65 shRNA, or a negative control shRNA construct (Cont. shRNA). Total RNA was prepared from the same transfected plate, and RT-PCR was performed to measure the expression of YY1 normalized to GAPDH mRNA. Total protein was also extracted, and Western blots were performed to detect the expression of p65. The blot was reprobed for α-tubulin as a loading control. (D and E) p65^+/+ and p65^−/− or p50^+/+ and p50^−/− MEF cells were treated with TNF-α for 1 or 2 h and quantified for YY1 mRNA by real-time PCR.

NF-κB binds and transcriptionally regulates the YY1 promoter. (A) Schematic illustration of the murine YY1 promoter containing two putative NF-κB consensus binding sequences (diamonds) and a Sp1 binding site (oval) upstream of the transcription start site. (B) C2C12 myoblasts were either untreated (−) or treated (+) with TNF-α for 15 min, and EMSA was performed with radiolabeled probes containing putative YY1-A, YY1-B, or YY1-A-mutant NF-κB binding sites. Complexes formed were identified by supershift EMSA with antisera specific to p65 (lanes 3, 8, and 13), p50 (lanes 4, 9, and 14) or IgG (lanes 5, 10, and 15). Arrows denote supershift complexes. (C) ChIP analysis with p65 or control IgG was performed with chromatin derived from either vector control (V) or IκBα-SR (SR)-expressing C2C12 myoblasts. The precipitated DNA fragments were amplified with specific oligonucleotides containing the putative NF-κB binding site on the YY1 promoter (upper panel). As a control, ChIP assays were repeated with the IκBα promoter (bottom panel). Total inputs are indicated. (D) 10T1/2 cells were plated in triplicate in 12-well plates; the next day, transient transfections were performed with DNA consisting of 0.2 μg of YY1-Luc reporter plasmids along with 0.2 μg of a LacZ expression plasmid and 0.01 μg of a p65 or p50 expression plasmid. At 48 h posttransfection, cell extracts were prepared and relative luciferase units were determined by normalizing to β-Gal protein. (E) C2C12 myoblast cell lines were generated to stably express luciferase reporters under the control of the YY1 promoter, either wild type (WT) or mutated (Mut) in the NF-κB binding site. Luciferase reporter assays were then performed in cells untreated or treated with TNF-α (10 ng/ml) for 6 h.

YY1-mediated repression of myofibrillar gene expression by NF-κB. (A) ChIP assays were performed with chromatin from C2C12 vector (V) or IκBα-SR (SR) myoblasts with antibodies against YY1 or IgG. Primers specific to Tnni2, MyHCIIb, α-skeletal actin, cTnnt, or Tnnc promoters/enhancers were used for PCR amplification. Total input DNA is indicated. (B) Total RNA was prepared from C2C12 IκBα-SR myoblasts transiently transfected with vector or YY1, and Tnni2 and MyHCIIb RNA were measured by real-time PCR. (C) Total RNA was prepared from C2C12 vector (V) or IκBα-SR (SR) myoblasts, and MyoD levels were measured by semiquantitative RT-PCR. (D) Western blot of MyoD C2C12 IκBα-SR myoblasts transfected with control or MyoD siRNA. (E) Cells were transfected as for panel D, and Tnni2 and MyHCIIb expression was measured by real-time PCR.