Volume 14, Issue 4, Pages 465-477 (May 2004) Pbx Marks Genes for Activation by MyoD Indicating a Role for a Homeodomain Protein in Establishing Myogenic Potential  Charlotte A Berkes, Donald A Bergstrom, Bennett H Penn, Karen J Seaver, Paul S Knoepfler, Stephen J Tapscott  Molecular Cell  Volume 14, Issue 4, Pages 465-477 (May 2004) DOI: 10.1016/S1097-2765(04)00260-6

Figure 1 The H/C and Helix III Domains of MyoD Are Essential for Activating Expression of a Restricted Subset of Gene Targets (A) Schematic diagram of the functional domains of MyoD; point mutations used in subsequent experiments are indicated. (B) Venn diagram representing genes regulated by the H/C, helix III, or N-terminal acidic domains of MyoD. (C) Northern confirmation of H/C- and helix III-dependent MyoD targets. MyoD−/−Myf5−/− embryonic fibroblasts expressing MyoD-ER or MyoD-ERW/C/S were induced for 0, 12, 24, or 48 hr, and RNA was hybridized with the indicated probes, including GAPDH as a loading control. (D) ChIP of MyoD-ER or MyoD-ERW/C/S at specific MyoD target loci. Cells were induced to differentiate for 0 hr (−) or 12 hr (+). Target genes were analyzed by ChIP with antisera specific for MyoD, and sequences were detected by quantitative duplex PCR coamplifying the MyoD-regulated genes and a gene that is not regulated by MyoD (amylase), an internal control sequence. The amount of the endogenous amylase gene in the IP sample represents the background level of nonspecific DNA precipitation. To demonstrate a linear detection range, a titration of input DNA was performed. Molecular Cell 2004 14, 465-477DOI: (10.1016/S1097-2765(04)00260-6)

Figure 2 The H/C and Helix III Domains of MyoD Are Required for Activating Myogenin Expression but Not for Binding to Promoter E Boxes (A) Transcriptional activity of luciferase reporters driven by either four multimerized E boxes and the thymidine kinase promoter (4RTK), or upstream regulatory sequence from the myogenin or muscle creatine kinase (MCK) genes. Reporters were cotransfected into NIH 3T3 cells with expression vectors for either wild-type MyoD, a H/C mutant (MyoDW96A/C98A), a helix III mutant (MyoDS253P), or empty vector. Reporter activity was measured following 24 hr in DM and normalized to CMV-βgal expression to control for transfection efficiency. Activity is expressed as a percentage of the activity of wild-type MyoD on each reporter construct. (B) EMSA analysis comparing binding of wild-type and MyoDW/C/S to the myogenin E1 E box. (C) EMSA analysis comparing binding of wild-type MyoD, MyoDW96A/C98A, MyoDS253P, and MyoDW/C/S to a probe containing the right MCK E box. Molecular Cell 2004 14, 465-477DOI: (10.1016/S1097-2765(04)00260-6)

Figure 3 The H/C and Helix III Domains of MyoD Functionally Interact with a cis Element in the Proximal Myogenin Promoter NIH3T3 cells were cotransfected with the indicated luciferase reporters and wild-type or mutant MyoD expression vectors. Luciferase activity is expressed as fold increase in comparison to activity observed in the absence of MyoD and normalized to an internal CMV-βgal control. (A) The right MCK E box, plus 7 bp upstream and downstream flanking sequence, was inserted in place of the myogenin E1 E box within the myogenin-luciferase reporter. (B) Transcriptional activity of myogenin-luciferase constructs harboring mutations within E1, E2, or both sites. (C) Transcriptional activity of serially truncated myogenin-luciferase reporters. Molecular Cell 2004 14, 465-477DOI: (10.1016/S1097-2765(04)00260-6)

Figure 4 Pbx-Meis Heterodimers Bind to an Evolutionarily Conserved Site within the Myogenin Promoter (A) Alignment of sequences of the H/C- and helix III-responsive element in the myogenin promoter from multiple species. The portion of the mouse sequence used as a probe in subsequent EMSA experiments is indicated. (B) EMSA using nuclear extracts from C2C12 myoblasts or differentiated myotubes and the Mgn −123/−97 probe. An excess of unlabeled probe effectively competes the shifted species, whereas a mutant competitor does not. (C) Supershift analysis demonstrating that Pbx and Meis proteins bind to the myogenin −123/−97 probe. Incubation of myotube nuclear extract with probe and Pbx or Meis antisera results in loss of bands A and B, whereas incubation with preimmune sera does not. Additional analysis shows that this antiserum does not disrupt or supershift specific factor-DNA complexes that do not contain Meis (data not shown). (D) Supershift analysis demonstrating that MyoD is present in a complex binding to the −123/−97 probe. Incubation of myotube nuclear extract with MyoD antisera results in depletion of band C, whereas incubation with preimmune sera does not. Molecular Cell 2004 14, 465-477DOI: (10.1016/S1097-2765(04)00260-6)

Figure 5 MyoD and E12 Form a Complex with Pbx and Meis on the Myogenin Promoter (A) Pbx1b, Meis1, E12, and wild-type or mutant MyoD proteins were generated by in vitro translation using a reticulocyte lysate and subjected to EMSA with the myogenin −123/−97 probe. Wild-type MyoD or the indicated MyoD mutants were incubated with probe and E12 alone or with E12, Pbx1b, and Meis1. (B) The indicated probes were mixed with in vitro-translated MyoD, E12, Pbx1b, and Meis1 proteins and subjected to EMSA. Mutation of the Pbx site eliminated formation of the Pbx-Meis complex (note that mutation of the Pbx site creates a new nonspecific shifted species that migrates slightly faster than the Pbx-Meix complex). The Pbx mutation slightly decreases binding of MyoD-E12. Mutation of the noncanonical E boxes prevents binding of MyoD-E12 dimers, and the high molecular weight species formed in the presence of all four proteins does not form on either of the mutant probes. (C) Transcriptional activities of luciferase constructs driven by five multimerized copies of the sequences shown in (B). (D) Transcriptional activities of wild-type MyoD or MyoDW/C/S on luciferase constructs driven by the myogenin promoter with deletions of the Pbx and noncanonical E boxes, as indicated. Molecular Cell 2004 14, 465-477DOI: (10.1016/S1097-2765(04)00260-6)

Figure 6 Pbx and Meis Proteins Associate with the Myogenin Promoter In Vivo C2C12 myoblasts were differentiated for 0, 6, or 12 hr, and extracts were immunoprecipitated with Pbx or Meis antisera, or nonspecific rabbit IgG. Muscle-specific primers amplifying myogenin, M-cadherin, Six1, or MCK sequences were used in duplex PCR reactions with primers amplifying IgH, an internal control sequence used to detect background genomic DNA in the immunoprecipitate. PCR reactions were performed in triplicate; a representative set is shown, and the fold enrichment of the normalized target sequence in the IP relative to the input is shown below each panel. A titration of input DNA was performed to demonstrate a linear detection range. Molecular Cell 2004 14, 465-477DOI: (10.1016/S1097-2765(04)00260-6)