Volume 9, Issue 3, Pages 587-600 (March 2002) Promoter-Specific Regulation of MyoD Binding and Signal Transduction Cooperate to Pattern Gene Expression  Donald A. Bergstrom, Bennett H. Penn, Andrew Strand, Robert L.S. Perry, Michael A. Rudnicki, Stephen J. Tapscott  Molecular Cell  Volume 9, Issue 3, Pages 587-600 (March 2002) DOI: 10.1016/S1097-2765(02)00481-1 Copyright © 2002 Cell Press Terms and Conditions

Figure 1 S1 Nuclease and Northern Analysis of MyoD-Responsive Genes (A) S1 nuclease analysis of gene expression in MyoD-ER cells (MDER) and control cells (Con) induced for 24 hr in differentiation medium with β-estradiol. The MyoD-ER, which is expressed at levels comparable to wild-type MyoD in the C2C12 cell line (data not shown), induces expression of MHC, Myog, and Des and upregulates expression of p21 (compare lanes 1 and 2). Addition of basic-fibroblast growth factor (+bFGF) preferentially inhibits the induction of MHC (lanes 3 and 4), whereas SB 203580 (SB) preferentially inhibits MHC and Des (lanes 5 and 6). (B) MDER cells were induced to differentiate for the indicated time, and Northern analysis showed different expression times for MyoD targets. (C and D) Northern analysis of RNA from control (Con) or MDER cells following 12 hr induction with cycloheximide (+CHX). MHC, Myosin heavy chain (Myh3); Myog, Myogenin; Des, Desmin; p21, Cdkn1a; Cdh-15, M-Cadherin; Ckmm, muscle creatine kinase (MCK); Mylf, Myosin light chain 1/3; Rb1, retinoblastoma protein; Prdc, protein related to DAC and Cerebrus; Fstl, Follistatin-like gene. Molecular Cell 2002 9, 587-600DOI: (10.1016/S1097-2765(02)00481-1) Copyright © 2002 Cell Press Terms and Conditions

Figure 2 Genes Regulated by MyoD Form Distinct Kinetic Clusters (A) Graph of the averaged log value of the normalized expression ratio for genes in each cluster based on a K-means clustering algorithm. (B) Representation of the subset of named genes in each cluster based on the functional assignment. The number of genes with a functional assignment relative to the total number of genes in that cluster is as follows: 1, 19/37; 2, 15/46; 3, 16/37; 4, 19/46; 5, 19/35; 6, 6/21; 7, 10/30; 8, 31/115; 9, 11/38. Adh/Mat, adhesion and matrix; CC/DNA, cell cycle and DNA replication/repair; Gr. Fact., growth factors/ligands; Met, metabolism; NRF, nuclear regulatory factors; PAS, proteolysis/apoptosis/stress/chaperone; Rec/Sig, receptors/signaling; Str/Cyt, structural/cytoskeletal. Molecular Cell 2002 9, 587-600DOI: (10.1016/S1097-2765(02)00481-1) Copyright © 2002 Cell Press Terms and Conditions

Figure 3 Histone Hyperacetylation Is Correlated with the Time of Gene Activation (A) Target genes were analyzed by ChIP with antisera specific for diacetylated histone H3, and sequences were detected by quantitative duplex PCR analysis coamplifying sequences of MyoD-regulated genes and the amylase gene, an internal control sequence. A titration of DNA was performed to demonstrate a linear detection range. (B) Fold-enrichment of the normalized muscle-specific regulatory sequence in the IP relative to the lysate. (C and D) ChIP analysis of the IgH enhancer E boxes. Nonspecific antisera control is shown in Figure 4. All ChIP assays were performed multiple times, and representative results are shown. Molecular Cell 2002 9, 587-600DOI: (10.1016/S1097-2765(02)00481-1) Copyright © 2002 Cell Press Terms and Conditions

Figure 4 MyoD Binding Is Correlated with the Time of Gene Activation (A) ChIPs were performed using antisera specific for MyoD or nonspecific antisera (NS Ab). Note that an underexposure of the Mylf signal is shown for the ChIP and titration panels due to the degree of enrichment in the IP. (B) Fold-enrichment of the normalized muscle-specific regulatory sequence in the IP relative to the lysate. (C and D) ChIP analysis of the IgH enhancer E boxes. All ChIP assays were performed multiple times, and representative results are shown. Molecular Cell 2002 9, 587-600DOI: (10.1016/S1097-2765(02)00481-1) Copyright © 2002 Cell Press Terms and Conditions

Figure 5 Inhibition of p38-MAPK Specifically Alters the Expression of Subsets of MyoD Target Genes For each MyoD target gene, the effect of the p38 inhibitor SB 203580 on gene expression was calculated by subtracting the average expression value for the gene in 6 μM SB 203580 (n = 3) from the average expression in carrier (n = 3). All normalized expression ratios are in log2 space. (A) The average effect of SB 203580 on genes assigned to each cluster in the time-course analysis. The averaged expression of clusters 5 and 6 is reduced significantly in the presence of the p38 inhibitor at both 24 and 48 hr. (B) SB 203580 effects relative to the assigned function of MyoD target genes. Genes in the cytoskeleton/structural category are more affected by inhibition of p38 than genes in other categories (ANOVA p < .0001). Molecular Cell 2002 9, 587-600DOI: (10.1016/S1097-2765(02)00481-1) Copyright © 2002 Cell Press Terms and Conditions

Figure 6 p38 Regulates Targets Independently of MyoD Binding (A) Northern analysis demonstrating that MyoD induces expression of Tncs with delayed kinetics. (B) Inhibition of p38 kinase activity by SB 203580 prevents MyoD-mediated expression of Tncs. (Note that the Tncs probing in this panel was overexposed relative to [A] to show the faint signal in the SB sample.) For reference, Cdh-15 is shown, a gene induced by MyoD but not inhibited by SB 203580. (C) ChIP analysis demonstrating MyoD binding (middle panel) and histone H3 acetylation (lower panel) at the Tncs enhancer at the indicated times following induction with fold-enrichment of the normalized muscle-specific regulatory sequence in the IP relative to the lysate. (D) Inhibition of p38 kinase does not alter MyoD binding to the Tncs regulatory region as assessed by ChIP analysis of SB 203580-treated samples. All ChIP assays were performed multiple times, and representative results are shown. Molecular Cell 2002 9, 587-600DOI: (10.1016/S1097-2765(02)00481-1) Copyright © 2002 Cell Press Terms and Conditions