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. 2006 Feb 8;25(3):502-11.
doi: 10.1038/sj.emboj.7600958. Epub 2006 Jan 26.

Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters

Yi Cao  1 , Roshan M Kumar, Bennett H Penn, Charlotte A Berkes, Charles Kooperberg, Laurie A Boyer, Richard A Young, Stephen J Tapscott
Affiliations

Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters

Yi Cao et al. EMBO J. .

Abstract

We used a combination of genome-wide and promoter-specific DNA binding and expression analyses to assess the functional roles of Myod and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that Myod and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, Myod can bind and recruit histone acetyltransferases. At early targets, Myod is sufficient for near full expression, whereas, at late expressed genes, Myod initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without Myod; however, transcriptional activation requires the combined activity of Myod and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by Myod.

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Figures

Figure 1
Figure 1
Myod and Myog bind to a similar set of promoters. (A) Representative scatter plots showing enrichment of Myod- and Myog-bound promoters by genome-wide location analysis. Confidence thresholds of P=0.001 and P=0.01 are shown. (B) Venn diagrams indicating the degree of overlap between Myod and Myog targets in MDER cells. The overlap increases when confidence thresholds are lowered to include more targets. (C) Venn diagrams indicating the degree of overlap between Myod and Myog targets in C2C12 myotubes differentiated for 36 h. (D) E-box motifs identified from lists of Myod- and Myog-bound promoters.
Figure 2
Figure 2
Myod and Myog are associated with genes that RNA Pol II is recruited to during differentiation. Genome-wide location analysis was performed on the initiating form of RNA Pol II in MDER cells maintained in growth medium (GM) or differentiated for 36 h (DM). The x-axis represents the difference in the negative log of the P-value for RNA Pol II binding in DM versus GM, so that points on the right side of the axis show an increase in Pol II occupancy during the course of differentiation. The y-axis indicates the negative log of the P-value for either Myod or Myog binding, so that points higher on the axis are more likely to be bound by Myod or Myog. The dashed line indicates the 0.001 P-value significance threshold for Myod or Myog binding. There is a statistically significant (P-value of 5.3 × 10−8 for Myod, 9.9 × 10−5 for Myog) enrichment of points in the upper right-hand quadrant of the graphs (bound by Myod/Myog and increase in Pol II occupancy upon shift to DM).
Figure 3
Figure 3
Exogenous Myog is expressed at levels comparable to endogenous Myog at 24 h of differentiation. MDER (Cells 1) or MDER-Myog (Cells 2) cells were induced for the indicated time in DM in the presence or absence of β-estradiol (B-e). MDER was activated by the addition of β-estradiol. Exo-Myod and Exo-Myog indicates whether the cells were expressing exogenous Myod or Myog. (A) Four independent RNA samples were isolated at the indicated time and the total amount of Myog transcripts in each sample was determined by real-time PCR using a probe against the Myog coding region. The relative expression levels were normalized to Timm17b in the same samples. (B) Cell lysates collected at the indicated time were subjected to SDS–PAGE and immunoblotted with anti-Myog, anti-Myod, or anti-α-tubulin antibodies. The multiple bands in Myod and Myog blots are consistent with known phosphorylation of Myod and Myog.
Figure 4
Figure 4
Myod and Myog cooperate to activate a set of genes normally expressed late in differentiation. (A) Myog activates a subset of Myod-activated genes. Venn diagrams show the overlap of Myod- and Myog-activated genes at different time points. At 12 h, Myod alone activates a substantially larger number of genes than Myog alone, whereas at the 24 and 48 h time points, the combination of Myod and endogenous Myog activates a larger number of genes than Myog alone. (B) Precocious expression of Myog together with Myod shifts the expression of normally late-activated genes to an earlier time point. Venn diagrams show that the 12 h Myod+Myog-activated genes encompass the set of genes activated by Myod alone at 12 h (left panel) and are a subset of genes activated by Myod at 12, 24, or 48 h (right panel) (note that 24 and 48 h Myod targets are activated in the presence of endogenous Myog).
Figure 5
Figure 5
Myod and Myog have distinct and sequential roles in regulating late gene expression. Cells expressing MDER (Cells 1) or MDER+Myog (Cells 2) were induced for the indicated time as described in Figure 3. Exo-Myod and Exo-Myog indicate whether the exogenous Myod or Myog is expressed in the cells. For each gene studied, the left panel shows the mRNA levels quantified by quantitative real-time PCR (A, C–E) or Northern blot (B), the middle panel shows the ChIP results performed with acetyl-H3 and acetyl-H4 antisera, and the right panel shows the ChIP results with Myod and Myog antisera. The relative expression levels of real-time PCR were normalized to the amount of Timm17b mRNA in the same samples, and the data shown are the means±s.e. of reactions from four independent samples. The endogenous Myog mRNA levels were detected by Northern blot with a probe against the 3′-UTR region, and the same blot reprobed with 18S rRNA was used as a loading control. For the ChIP assays, pancreatic amylase 2 (Amy2) was used as an internal control in multiplex PCR for the indicated promoters. The fold enrichments were calculated relative to the amount of input chromatin and the basal histone acetylation or Myod or Myog binding at the 0 h time was subtracted to determine fold change relative to uninduced cells. The error bars indicate standard errors of the mean for the particular experiments performed in triplicate.
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