Journal: eLife

Article Title: MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle

doi: 10.7554/eLife.43017

Figure Lengend Snippet: ( A ) UCSC genome browser visualization (mm10 genome) of MYOD1 binding tags within the Bmal1 locus. ( B ) Representative BMAL1 western blots from C2C12 myoblasts transiently transfected with 150 ng pGEM empty vector (-) or MYOD1 expression vector (+). Densitometric values are expressed as average fold-change of BMAL1 over the Tubulin loading control which was unchanged with MYOD1 transfection ± SEM (n = 3). Results were analyzed with one-way ANOVA, * indicates a p-value less than 0.05 (p = 0.025). Bmal1P -Luc luminescence in C2C12 myotubes (2 C , n = 4 biological replicates) and skeletal muscle primary myotubes ( D , n = 3 biological replicates) with transient transfection of pGEM control, MYOD1, or MYOD1mut expression vectors. Luciferase activity for each co-transfection is plotted as average fold-change in relation to the pGEM empty vector control ± SEM. Results were analyzed using one-way ANOVA, ** indicates a p-value less than 0.001. # indicates a p-value less than 0.01 comparing MYOD1 vs MYOD1mut. ( E ) Bmal1P -Luc luminescence in electroporated skeletal muscle. Bmal1P -Luc activity was normalized to Renilla luciferase as an electroporation control with the right-leg receiving MYOD1 expression vector (red circles) and the left leg receiving the pGEM empty vector control (black circles). The p-value statistic was calculated by performing a Mann-Whitney non-parametric t-test. Note, one outlier was removed from each group based upon the Robust regression and outlier removal (ROUT) test, with a false discovery rate of <0.01. Bmal1P -Luc Dual-Luciferase activities in skeletal muscle primary myotubes ( F-H ) Representative Bmal1P -Luc driven bioluminescence recordings in synchronized C2C12 myotubes co-transfected with pGEM control (F, n = 7), MYOD1 (G, n = 6), or MYOD1mut (H, n = 4) expression vectors. Luminescence recordings are expressed as average counts/sec (base-line subtracted) ( I ) Average Bmal1P -Luc amplitudes ± SEM calculated by JTK_CYCLE from 1.5 to 4.5 days post-synchronization. Results were analyzed with one-way ANOVA, ** indicates p-value less than 0.001, # indicates p-value less than 0.002 (n = 4 biological replicates per group).

Article Snippet: Proteins were transferred with a semidry apparatus onto PVDF membrane and then blotted with anti-BMAL1 Ab (Sigma SAB4300614, rabbit source, 1:1000) or anti-tubulin (Sigma T6557, mouse source, 1:1000) antibodies.

Techniques: Binding Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Cotransfection, Electroporation, MANN-WHITNEY

Journal: eLife

Article Title: MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle

doi: 10.7554/eLife.43017

Figure Lengend Snippet: Interactions were visualized in C2C12 cells by bimolecular fluorescence complementation assay (BiFC). ( A ) VN-HDAC5 and VC-MATR3; ( B ) VC-BMAL1 and VN-MATR3; ( C ) VN-HDAC5 and VC-vector; ( D ) VC-MEF2C and VN-vector; ( E ) VN-HDAC5 and VC-MEF2C; ( F ) VN-BMAL1 and VC-vecctor; ( G ) VC-BMAL2 and VN-vector; ( H ) VC-MYOD1 and VN-vector; ( I ) VN-CLOCK and VC-vector; ( J ) VN-BMAL1 and VC-vector.

Article Snippet: Proteins were transferred with a semidry apparatus onto PVDF membrane and then blotted with anti-BMAL1 Ab (Sigma SAB4300614, rabbit source, 1:1000) or anti-tubulin (Sigma T6557, mouse source, 1:1000) antibodies.

Techniques: Bimolecular Fluorescence Complementation Assay, Plasmid Preparation

Journal: eLife

Article Title: MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle

doi: 10.7554/eLife.43017

Figure Lengend Snippet: Representative images from the BiFC assay performed in C2C12 myoblasts co-transfected with ( A ) VN-CLOCK and VC-BMAL1, ( B ) VN-BMAL1 and VC-BMAL2, ( C ) VN-CLOCK and VC-MYOD1, and ( D ) VN-BMAL1 and VC-MYOD1. mCherry expression plasmids (red fluorescence signal) were co-transfected in each experiment to visualize the myoblasts and ensure successful transfection. Yellow fluorescence signals indicate positive co-localization via the formation of the Venus Luciferase.

Article Snippet: Proteins were transferred with a semidry apparatus onto PVDF membrane and then blotted with anti-BMAL1 Ab (Sigma SAB4300614, rabbit source, 1:1000) or anti-tubulin (Sigma T6557, mouse source, 1:1000) antibodies.

Techniques: Bimolecular Fluorescence Complementation Assay, Transfection, Expressing, Fluorescence, Luciferase

Journal: eLife

Article Title: MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle

doi: 10.7554/eLife.43017

Figure Lengend Snippet: ( A ) TcapP -Luc Dual-Luciferase activity responses from C2C12 myotubes co-transfected with BMAL1 +CLOCK, BMAL1mut + CLOCK, and BMAL1 +CLOCKmut vectors (n = 4 biological replicates/group). Luciferase activity for each co-transfection is plotted as average fold-change in relation to the pGEM empty vector control ± SEM. Results were analyzed using one-way ANOVA, * indicates p = 0.029. ( B ) TcapP -Luc Dual-Luciferase activity responses from C2C12 myotubes co-transfected with MYOD1 alone, MYOD1 +BMAL1+CLOCK, or variations of BMAL1mut, CLOCKmut, and MYOD1mut vectors. Luciferase activity for each co-transfection is plotted as average fold-change in relation to the pGEM empty vector control ± SEM (n = 4). Results from MYOD1 and MYOD1 +BMAL1:CLOCK co-transfections were analyzed using one-way ANOVA, ** indicates a p-value less than 0.001 in relation to the pGEM control. Results from the mutant co-transfection experiments (red) were analyzed using one-way ANOVA. # indicates a p-value less than 0.01 relative to the MYOD1 +BMAL1:CLOCK result. ( C–E ) Representative TcapP -Luc driven bioluminescence recordings in synchronized C2C12 myotubes co-transfected with the pGEM control ( C ), MYOD1 ( D ), or MYOD1mut ( E ) expression vectors. Luminescence recordings are expressed as average counts/sec (base-line subtracted). ( F ) Average TcapPmut -Luc amplitudes calculated by JTK_CYCLE from 1.5 to 4.5 days post-synchronization. Data are displayed as average amplitude ± SEM (n = 3 biological replicates/group). Results were analyzed with one-way ANOVA, * indicates p-value less than 0.05. ( G ) Tcap temporal mRNA expression profiles from muscles of MYOD1-CE (dotted red) or C57BL/6J (solid black) mice. Dark shading indicates relative dark/active phase as these mice were reared in DD at the time of collection. At each time-point RT-PCR expression values are displayed as average fold-change relative to the Rpl26 house-keeping gene ± SEM (n = 3). Results were analyzed with one-way ANOVA comparing WT vs. MYOD1-CE at each time-point, * indicates a p-value less than 0.05. ( H ) JTK_CYCLE statistics for the RT-PCR results corresponding to Tcap ’s temporal expression values. ‘BH.Q’ column reports false discover rates and ‘ADJ.P’ reports adjusted p-values.

Article Snippet: Proteins were transferred with a semidry apparatus onto PVDF membrane and then blotted with anti-BMAL1 Ab (Sigma SAB4300614, rabbit source, 1:1000) or anti-tubulin (Sigma T6557, mouse source, 1:1000) antibodies.

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle

doi: 10.7554/eLife.43017

Figure Lengend Snippet: Relative mRNA expression levels for Bmal1 , Tcap , and MYOD1 in C2C12 myotubes transfected with either Bmal1 siRNA (black bars) or a control non-targeting siRNA-A vector (white bar). Results are displayed as average fold-change relative to the control siRNA-A ± SEM (n = 4 biological replicates). Results were analyzed with one-way ANOVA, ** indicates a p-value less than 0.001.

Article Snippet: Proteins were transferred with a semidry apparatus onto PVDF membrane and then blotted with anti-BMAL1 Ab (Sigma SAB4300614, rabbit source, 1:1000) or anti-tubulin (Sigma T6557, mouse source, 1:1000) antibodies.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: eLife

Article Title: MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle

doi: 10.7554/eLife.43017

Figure Lengend Snippet: ( A ) Graphical representation of the TcapP -Luc promoter:reporter construct. TSS indicates transcript start site. E1 E-box is located at −103 and E2 tandem E box is located −272 (E2 3’) and −278 (E2 5’) from the TSS. ( B ) Dual-Luciferase activity responses from the wildtype TcapP -Luc and the Tcap E-box mutants in C2C12 myotubes co-transfected with BMAL1:CLOCK. Luciferase activity for each co-transfection is plotted as average fold-change in relation to the pGEM empty vector control ± SEM (E2-3’ and E2-5’ n = 4, E1 n = 3). Results were analyzed using one-way ANOVA, ** indicates p-value less than 0.01 in relation to the WT TcapP -Luc response, * indicates a p-value less than 0.05 in relation to the pGEM control vector. ( C ) Dual-Luciferase activity responses from the Tcap E-box mutants with co-transfection of MYOD1 alone or MYOD1 +BMAL1:CLOCK. Luciferase activity for each co-transfection is plotted as average fold-change in relation to the pGEM empty vector control ± SEM (n = 3). Results were analyzed using one-way ANOVA. In comparison to the pGEM control all co-transfections were significantly elevated (p-value < 0.05), ** indicates a p-value of less than 0.01 in relation to the pGEM control, and * indicates a p-value of less than 0.05 comparing TcapP -Luc with over-expression of MYOD1 vs MYOD1+BMAL1:CLOCK. In comparison to the pGEM control all co-transfections were significantly elevated (p-value<0.05). . No statistical differences were observed for each of the Tcap mutant reporters comparing MYOD1 alone to MYOD1 +BMAL1:CLOCK. ( D-F ) Representative bioluminescence recordings from the TcapP-E1 -Luc ( D ), TcapP-E2-3’ -Luc ( E ), and TcapP-E2-5’mut -Luc ( F ) in synchronized C2C12 myotubes. (G) Representative TcapP-Luc (black, n = 3 biological replicates) and TcapPmut-E2-5’-Luc (red, n = 3 biological replicates) driven bioluminescence recordings in synchronized skeletal muscle primary myotubes. Luminescence recordings are expressed as average counts/sec (base-line subtracted).

Article Snippet: Proteins were transferred with a semidry apparatus onto PVDF membrane and then blotted with anti-BMAL1 Ab (Sigma SAB4300614, rabbit source, 1:1000) or anti-tubulin (Sigma T6557, mouse source, 1:1000) antibodies.

Techniques: Construct, Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Over Expression, Mutagenesis

Journal: eLife

Article Title: MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle

doi: 10.7554/eLife.43017

Figure Lengend Snippet: ( A-B ) Chromatin Immunoprecipitation-PCR with anti-MYOD1 and -BMAL1 antibody pulldowns (and IgG controls) to detect binding of MYOD1 and BMAL1 within Tcap ’s E1 E-box element ( A, B ) or the tandem E-box ( C, D , primers contain 3’ and 5’ Eboxes). Pull-downs were performed with extracts from adult mouse quadricep muscles collected at ZT 2 (2 hours after lights on). Data are displayed as an average % of input ± SEM (n = 3 samples/group). Results were analyzed with a one-way ANOVA and * denotes a p-value ranging from 0.02 to 0.037. ( E ) Graphical model of depicting the role of MYOD1 in modulating core clock gene expression and working as a co-factor with BMAL1:CLOCK to amplify downstream circadian genes in skeletal muscle. MYOD1 activity is modulated by extracellular signals and it amplifies Bmal1 expression via direct transcriptional activation. BMAL1:CLOCK in turn form a positive feedback loop to regulated the circadian expression of MyoD1 by targeting the core-enhancer element. MYOD1 and BMAL1:CLOCK work in a synergistic fashion to amplify the expression of circadian genes.

Article Snippet: Proteins were transferred with a semidry apparatus onto PVDF membrane and then blotted with anti-BMAL1 Ab (Sigma SAB4300614, rabbit source, 1:1000) or anti-tubulin (Sigma T6557, mouse source, 1:1000) antibodies.

Techniques: Chromatin Immunoprecipitation, Binding Assay, Expressing, Activity Assay, Activation Assay