Figure 1.
YY1 binding sites are enriched in both up- and down-regulated miRNAs.
(A). miRNAs in DM cells are rank-ordered by the degree of up-regulation (red) and down-regulation (green) relative to GM cells. (B). The two plots (left and right) show moving average of the frequency of probes for genes that have YY1 or MyoD -binding sites in a 300-probe sliding window. The dashed line indicates the expected average (background level or the ratio of the number of probes for YY1 or MyoD targets over the total number of interrogated gene probes).
Table 1.
Up-regulated miRNAs with predicted YY1 binding sites on promoter.
Figure 2.
YY1 negatively regulates miR-1/133 expression both in vitro and in vivo.
(A) C2C12 myoblasts were grown in growth medium (GM) or differentiation medium (DM) for 2, 3 or 5 days. Total RNAs or proteins were extracted and used for real-time RT-PCR assay (left) or Western blot analysis (right panel), respectively. (B) Primary myoblasts were isolated from limb muscles of 1 week old C57/BL6 mice and maintained in GM or induced to differentiate in DM. Cell morphology was visualized under light microscopy (left). Real-time PCR was performed to measure the expression levels of miR-1 and miR-133 normalized to U6 (middle). Semi-quantitative RT-PCR analysis was performed to measure the expression levels of Troponin, MyHC, α-Actin and YY1. Water (H2O) was used as negative control and GAPDH was used as a normalization. (C) Total proteins were isolated from lower limb muscles at post-natal day (P) 3 and 8 or tibialis anterior (TA) muscles from 2, 4, or 5 week old C57/BL6 background mice and Western blotting was used to probe for YY1 protein expression with GAPDH as a loading control (left). Total RNAs were isolated and qRT-PCR was subsequently performed to measure the expression of miR-1 and miR-133, normalized to U6 (middle and right). Expression folds are shown with respect to 3 day old mice where miR-1 and miR-133 levels were set to a value of 1. (D) TA muscles were isolated from 3 w, 4 w, 5 w, 8 w and 10 w old C57BL/6 wild type mice or mdx mice. RNAs were extracted and used for qRT-PCR assay of miR-1 (left), miR-133 (middle) or YY1 (right). Expression folds are shown with respect to wild type where miR-1, miR-133 or YY1 levels were set to a value of 1. (E) C2C12 myoblasts or (F) primary myoblasts were transfected with either negative control (siNC) or siRNA oligos against YY1 (siYY1). Cells were then cultured for 48 hours, at which time miR-1 and miR-133 expressions were measured by qRT-PCR and normalized to U6. Expression folds are shown with respect to siNC where miR-1 and miR-133 levels were set to a value of 1. (G) Expression of the primary transcripts of miR-1-2/miR-133a-1 was detected by qRT-PCR in C2C12 transfected with siYY1 or siNC oligos, and normalized to GAPDH. All quantitative data are represented as mean ± S.D. The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0.001.
Figure 3.
YY1 repression of miR-1/133 is mediated through multiple enhancers.
(A) Two conserved enhancers (E1 and E2) were identified in the promoter region and intragenic region of miR-1-2/miR-133a-1 cluster, respectively. Three putative YY1 binding sites, A, B and C, were identified. (B) One conserved enhancer (E3) was identified in between miR-1-1 and miR-133a-2 with a putative YY1 binding site, D, identified. (C) One conserved enhancer (E4) was identified upstream of miR-206 and miR-133b cluster with a putative YY1 binding site, E, identified. Binding sites for MyoD, MEF2 and SRF were also shown. (D) C2C12 cells were transfected with 250 ng of E1, E2, E3 or E4 reporter plasmid along with Renilla and control vector (YY1 0 ng) or 50 ng, 200 ng, 500 ng YY1 expressing plasmid. Cells were then cultured for 48 h at which time luciferase activities were determined and normalized to Renilla protein. The data represent the average of three independent experiments ± S.D. (E) C2C12 cells were transfected with 0.25 µg of E1, E2, E3 or E4 reporter plasmid along with Renilla luciferase vector and siYY1 or siNC oligos. Luciferase activity was determined as in (D). (F) Chromatins were harvested from C2C12 myoblasts growing in growth medium (GM) or myotubes maintained in differentiation medium (DM) and subjected to ChIP-PCR analysis. Primers were designed to amplify regions encompassing putative YY1 binding sites A, B, C, D, or E. MyHC and Tnni2 are known YY1 targets and used as positive controls. A genomic region that contains no YY1 binding sites was included as a negative control (NC). (G) Site A (Mut A) or both A and B (Mut A+B) were mutated in E1 luciferase reporter plasmid and luciferase reporter assay was performed to measure the response of mutants to YY1 over-expression as in (D). Relative luciferase unit (RLU) is shown with respect to Vector transfection where luciferase activities were set to a value of 1. (H) ChIP-PCR for Ezh2 or H3K27me3 was performed as in (F). The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0.001.
Figure 4.
YY1 represses miR-1 functionally during C2C12 myogenesis.
(A) C2C12 cells were transfected with the indicated combination of NC or miR-1 oligos and Vector or YY1 expression plasmids. Cells were then differentiated (DM) for 2 days, at which time cells were immunostained for MyHC. Cell morphology was visualized by phase-contrast microscopy. (B) MyHC positive cells were quantified by counting positively stained cells from 10 randomly chosen fields and are represented as mean ± S.D. (C) Total proteins were isolated from the above transfected cells and Western blotting was performed to probe for α-Actin. α-Tubulin was used as a loading control. (D) Total RNAs were extracted from the above transfected cells and used for qRT-PCR analysis of myogenic markers, MyHC, Troponin, α-Actin, and Myogenin normalized with GAPDH. YY1 and miR-1 levels were also measured to show the transfection efficiency. Quantitative values are represented as mean ± S.D. The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0,001.
Figure 5.
miR-1 targets Pax7 during C2C12 differentiation.
(A) Predicted target sites, A and B, of miR-1 in the 3′UTR of mouse Pax7. (B) A luciferase reporter plasmid was generated by cloning a ∼800 bp region of Pax7 3′UTR encompassing both site A and B downstream of the luciferase (Luc) reporter gene. The reporter construct was then transfected into C2C12 cells with negative control (NC) or miR-1 oligos along with Renilla luciferase plasmid. Luciferase activities were determined at 48 h post-transfection and normalized to Renilla readings. The data represent the average of three independent experiments ± S.D. (C) A mutation was introduced in either site A (Mut A) or site B (Mut B) or both (Mut A+B). Their responses to miR-1 over-expression were tested as above. (D) C2C12 myoblasts were transfected with either NC or miR-1 oligos. Both miR-1 and Pax7 mRNAs levels were then measured 48 hr post-transfection. (E) HDAC4 or Pax7 proteins were probed in extracts from cells 48 hr after transfection. Blots were stripped and reprobed for α-Tubulin as the loading control. (F) Proteins extracted from C2C12 differentiated (DM) for 0 d, 1 d, 3 d and 5 d were used for Western blotting assay of Pax7. α-Tubulin was used as a loading control. (G) Left: C2C12 myoblasts were transfected with Vector or YY1 expression plasmid. Pax7 mRNA expression was then measured in extracts from cells 48 hr after transfection using GAPDH as normalization. Right: C2C12 myoblasts were transfected with siNC or siYY1 oligos. Pax7 mRNA expression was then measured in extracts from cells 48 hr after transfection using GAPDH as normalization. Expression folds are shown with respect to Vector or siNC control where Pax7 levels were set to a value of 1. Values are represented as mean ± S.D. The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0,001.
Figure 6.
YY1 negatively regulates miR-1 during CTX-induced muscle regeneration.
(A) Tibialis anterior (TA) muscles from six-week old C57/BL6 background mice were injected with 10 µM cardiotoxin (CTX). RNAs and proteins were then extracted from injected muscles at the indicated days post-injection, and qRT-PCR was performed to measure the expression of miR-1 and miR-133, normalized to U6. Expression folds are shown with respect to day 0 where miR-1 and miR-133 levels were set to a value of 1. Quantitative values are represented as means ± S.D. (B) YY1 expression was measured by Western blotting. α-Tubulin was used as a loading control. Numbers below indicates the quantification by densitometry. (C) TA muscles from 6 week C57/BL6 background mice were injected CTX at day 0, followed by injection with siNC (left leg) and siYY1 oligos (right leg) 6 hours later. And re-injection of siRNA oligos was performed every other day for two more times. The injected muscles were harvested at the indicated days. N = 6 for each group. (D) Expressions of miR-1 and miR-133 were detected by qRT-PCR in CTX/siRNA injected muscles at day 2, 4 and 6, normalized to U6. Expression folds are shown with respect to siNC where miR-1 and miR-133 levels were set to a value of 1. (E) Western blotting was performed to analyze the expression of YY1, Pax7, MyoD and Myogenin. α-Tubulin was used as a loading control. Data is representative of 6 mice. (F) Expression of Pax7 and MyoD RNA levels were also detected by qRT-PCR normalized with GAPDH. Expression folds are shown with respect to siNC where Pax7 and MyoD levels were set to a value of 1. Quantitative values are represented as mean ± S.D. The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0,001.
Figure 7.
miR-1 inhibits YY1 expression through targeting its 3′UTR.
(A) Predicted target site of miR-1 in the 3′UTR of mouse YY1. (B) A wild type (WT) luciferase reporter plasmid was generated by fusing a ∼500 bp fragment of the YY1 3′UTR encompassing the miR-1 binding site downstream of the luciferase (Luc) reporter gene. The mutant plasmid was generated by mutating the miR-1 binding site from ACAUUCU to GGGCCUU. WT or Mutant reporter construct was transfected into C2C12 cells with indicated miRNA oligos and Renilla luciferase reporter plasmid. Luciferase activities were determined at 48 h post-transfection and normalized to Renilla readings. Relative Luciferase Unit (RLU) is shown with respect to wild type and NC transfection where luciferase activities were set to a value of 1. The data represent the average of three independent experiments ± S.D. (C) Upper: C2C12 myoblasts were transfected with either NC or miR-1 oligos. Total RNAs were used to detect YY1 expression level with GAPDH as normalization. Expression folds are shown with respect to negative control where YY1 levels were set to a value of 1. Quantitative values are represented as mean ± S.D. Lower: YY1 protein was then probed in extracts from cells 48 hr after transfection. Blots were stripped and reprobed for α-Tubulin as the loading control. The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0.001.
Figure 8.
A model of NF-κB-YY1-miR-1-Pax7 circuit in skeletal myogenesis.
The model depicts the role of the NF-κB-YY1-miR-1 regulatory circuit in myogenic differentiation. This circuit involves the constitutive activity of NF-κB in myoblasts that regulates YY1 expression which subsequently suppresses miR-1 expression epigenetically and maintains cells in an undifferentiated state. As differentiation is initiated, the down-regulation of the NF-κB-YY1 pathway leads to concomitant up-regulation of miR-1 that in turns further decreases YY1 as well as Pax7 levels to ensure proper differentiation into myotubes. (B) Network visualization depicting YY1-centered circuitry drawn by Cytoscape. Findings from the current studies demonstrate a repression of miR-1, miR-133 and miR-206 by YY1. Together with previous findings, these interactions constitute an YY1-miRNA regulatory network. YY1, Yellow octagon; miRNAs, red diamonds; Experimentally validated miRNA targets, grey circles; red blunted arrows, transcriptional repression; black blunted arrows, post-transcriptional repression.