Multiple roles for the MyoD basic region in transmission of transcriptional activation signals and interaction with MEF2

Abstract

Establishment of skeletal muscle lineages is controlled by the MyoD family of basic helix-loop-helix (bHLH) transcription factors. The ability of these factors to initiate myogenesis is dependent on two conserved amino acid residues, alanine and threonine, in the basic domains of these factors. It has been postulated that these two residues may be responsible for the initiation of myogenesis via interaction with an essential myogenic cofactor. The myogenic bHLH proteins cooperatively activate transcription and myogenesis through protein-protein interactions with members of the myocyte enhancer factor 2 (MEF2) family of MADS domain transcription factors. MyoD-E12 heterodimers interact with MEF2 proteins to synergistically activate myogenesis, while homodimers of E12, which lack the conserved alanine and threonine residues in the basic domain, do not interact with MEF2. We have examined whether the myogenic alanine and threonine in the MyoD basic region are required for interaction with MEF2. Here, we show that substitution of the MyoD basic domain with that of E12 does not prevent interaction with MEF2. Instead, the inability of alanine-threonine mutants of MyoD to initiate myogenesis is due to a failure to transmit transcriptional activation signals provided either from the MyoD or the MEF2 activation domain. This defect in transcriptional transmission can be overcome by substitution of the MyoD or the MEF2 activation domain with the VP16 activation domain. These results demonstrate that myogenic bHLH-MEF2 interaction can be uncoupled from transcriptional activation and support the idea that the myogenic residues in myogenic bHLH proteins are essential for transmission of a transcriptional activation signal.

FIG. 1

Double-DNA binding assay for MEF2C and myogenin. Biotin-labeled E-box probe and ³²P-labeled MEF2 site probe (A) or biotin-labeled MEF2 site probe and ³²P-labeled E-box probe (B) were mixed with either wild-type (wt) or mutant MEF2C and myogenin-E12 proteins transcribed and translated in vitro. A dash indicates the use of unprogrammed reticulocyte in place of MEF2, E12, or myogenin-containing lysate. Complexes were immunoprecipitated with avidin-conjugated agarose and were washed three times. Radioactive counts from the ³²P-labeled probes were measured in a scintillation counter. ³²P-labeled probe can be precipitated by the avidin-conjugated agarose only if protein-protein interaction occurs. The data are expressed as the counts per minute precipitated minus the background counts per minute precipitated in the presence of unprogrammed lysate alone. The background in panel A was 1,174 cpm, and the background in panel B was 385 cpm. The results shown are from representative experiments. For the experiments shown in both panels, similar results were obtained in three separate immunoprecipitations using three separate preparations of in vitro-translated proteins and labeled probes. The MEF2C mutant used (KR23,24ID) interacts with myogenin-E12 heterodimers (4) but is incapable of binding DNA (30). The schematic representations at the right show how the ³²P-labeled probes are coimmunoprecipitated by the avidin-conjugated agarose in these experiments.

FIG. 2

Amino acid sequences of the basic regions examined in this study. The basic domains of MyoD, MyoD-E12basic, MyoD-E12basic(AT), and E12 are indicated at the top. The myogenic residues alanine-114 and threonine-115 of the MyoD basic region and the corresponding asparagine residues of the E12 basic domain are boxed. The junction sequence of the first helix follows the basic domain.

FIG. 3

Interaction between myogenic bHLH proteins and MEF2C detected by an in vivo trihybrid assay. (A) Schematic representation of the trihybrid assay used in these experiments. In panel B, 10T1/2 cells were transfected with the GAL4-dependent CAT reporter plasmid pG5E1bCAT and the indicated expression plasmids. Plasmids included are indicated by name or with a plus sign and are described in Materials and Methods. A minus sign indicates that the parent expression vector without a cDNA insert was used. The results in panel B show the fold activation in CAT activity compared to that for reporter plus GAL(DBD)-E12 bHLH alone. Extracts were serially diluted such that each sample yielded activity in the linear range of the assay. Total extract was held constant in the serial dilutions by using lysate from untransfected 10T1/2 cells. Results of a representative experiment are shown; similar results were achieved in three independent transfections and analyses. MyoD-wt, wild-type MyoD.

FIG. 4

In vivo trihybrid analysis of transcriptional synergy mediated by the MEF2C amino terminus. (A) Schematic representation of the trihybrid assay used in these experiments. In panel B, 10T1/2 cells were cotransfected with the GAL4-dependent CAT reporter plasmid pG5E1bCAT, GAL(DBD)-MEF2C, which encodes amino acids 1 to 143 of MEF2C, and E12 expression plasmid. Also included were the indicated bHLH expression plasmids. The presence of GAL(DBD)-MEF2C and E12 is indicated with a plus sign. The absence of a cDNA-encoding plasmid and the presence of the parent expression vector are denoted by a minus sign. The mean fold activation in CAT activity compared to that for reporter alone from four independent transfections and analyses is shown.

FIG. 5

Transcriptional activation of an E-box-dependent reporter by MyoD and MEF2. 10T1/2 cells were transiently transfected with the E-box-dependent reporter 4RtkCAT along with the indicated expression vectors. Plasmids are described in Materials and Methods. (A) Ability of MyoD or the MyoD mutants to activate the reporter as either full-length or VP16 fusion proteins. The data show that the activation defect in MyoD-E12basic is overcome by the fusion of the VP16 activation domain. Values are expressed as the fold induction of CAT activity over the activity of the reporter alone. The results shown represent the mean fold activation obtained in four independent transfections and analyses. (B) Ability of either MEF2C or MEF2-VP16 to activate transcription in collaboration with MyoD or the MyoD mutants bound to the E boxes in the reporter. Wild-type MEF2C was unable to activate transcription through the MyoD-E12basic mutant bound to DNA (lane 4), whereas MEF2-VP16 activated transcription in collaboration with MyoD-E12basic to the same extent as with wild-type MyoD (lane 8). The results shown are from a representative experiment. Similar results were obtained in two independent transfections and analyses. The diagrams at the right illustrate how we envision that transcriptional activation occurs.

FIG. 6

Hypothetical model of transcriptional activation mediated by MEF2 bound to MyoD. (A) MEF2 relays its activation signal to the myogenic bHLH factor, which then transmits both its own activation signal and that of MEF2 to the transcription initiation complex. This transcriptional transmission is dependent on the myogenic residues, alanine and threonine, in the myogenic factor’s basic domain. (B) MEF2 sends its activation signal to the basic domain of E12 substituted into the myogenic bHLH factor (E12basic), but the E12 basic domain substitution is unable to transmit that activation signal or its own activation signal to the initiation complex due to a conformational defect. (C) VP16 directly activates the transcription initiation complex, thus bypassing the transcriptional block caused by E12 basic domain. The E12basic mutant is unable to transmit its own activation signal because it lacks the myogenic residues.