MOLECULAR AND CELLULAR BIOLOGY, Sept. 1994, p. 6232-6243 Vol. 14, No. 9 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology Dimerization through the Helix-Loop-Helix Motif Enhances Phosphorylation of the Transcription Activation Domains of Myogenin JUMIN ZHOU AND ERIC N. OLSON* Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Received 6 December 1993/Returned for modification 4 February 1994/Accepted 6 June 1994 The muscle-specific basic helix-loop-helix (bHLH) protein myogenin activates muscle transcription by binding to target sequences in muscle-specific promoters and enhancers as a heterodimer with ubiquitous bHLH proteins, such as the E2A gene products E12 and E47. We show that dimerization with E2A products potentiates phosphorylation of myogenin at sites within its amino- and carboxyl-terminal transcription activation domains. Phosphorylation of myogenin at these sites was mediated by the bHLH region of E2A products and was dependent on dimerization but not on DNA binding. Mutations of the dimerization- dependent phosphorylation sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. The ability ofE2A products to potentiate myogenin phosphorylation suggests that dimerization induces a conformational change in myogenin that unmasks otherwise cryptic phosphorylation sites or that E2A proteins recruit a kinase for which myogenin is a substrate. That phosphorylation of these dimerization-dependent sites diminished myogenin's transcrip- tional activity suggests that these sites are targets for a kinase that interferes with muscle-specific gene expression. A variety of transcription factors bind DNA and activate nin, myf5, and MRF4 (reviewed in references 15, 36, 37, and transcription as heterodimers. Interactions among heterolo- 49). These myogenic bHLH proteins bind DNA weakly by gous transcription factors provide opportunities for positive themselves but acquire high affinity for the E box consensus and negative control of gene transcription and dramatically sequence upon heterodimerization with several ubiquitous expand the regulatory potential of different classes of tran- bHLH proteins, referred to as E proteins. Among the E scription factors. In many cases, each component of a het- proteins that have been shown to serve as partners for myo- erodimer binds DNA weakly on its own but shows enhanced genic bHLH proteins are E12 and E47, encoded by the E2A DNA-binding activity when associated with the appropriate gene, and the HEB gene product (9, 21, 25, 34). Activation of partner. While it is conceivable that heterodimerization could muscle-specific transcription ensues when heterodimers of also alter the properties of the transcription factors them- myogenic bHLH proteins and E proteins bind to E boxes in the selves, there is as yet little evidence for this type of cooperat- control regions of muscle-specific genes. ivity among dimerization partners. Intracellular growth factor signals interfere with the ability The helix-loop-helix (HLH) family of transcription factors of myogenic bHLH proteins to activate muscle-specific tran- includes a wide range of regulatory proteins that control gene scription through several different mechanisms (reviewed in transcription in species ranging from Drosophila melanogaster reference 37). Recently, we showed that protein kinase C to humans (reviewed in reference 23). The HLH motif medi- (PKC) can block myogenesis through phosphorylation of a site ates dimerization and brings together the basic regions of HLH in the DNA-binding domain of myogenin that is specific to and proteins to form a composite DNA-binding domain that conserved in all myogenic bHLH proteins (28). Phosphoryla- recognizes the consensus sequence CANNTG (where N is any tion of site attenuates the of nucleotide), known as an E box (2, 33). Most HLH proteins this transcriptional activity preferentially form heterodimers with specific HLH protein myogenin by inhibiting its ability to bind DNA. Protein kinase partners (10, 34). The ability of individual basic HLH (bHLH) A (PKA) also phosphorylates the myogenin DNA-binding proteins to recognize distinct half-sites within the dyad sym- domain, but phosphorylation by PKA does not appear to metry of the E box consensus sequence therefore allows influence myogenin's ability to activate muscle transcription different combinations of HLH proteins to discriminate be- (26). In addition to sites for phosphorylation by PKC and PKA, tween different E boxes (2). HLH proteins lacking basic myogenin contains several other phosphorylation sites, the regions can inhibit the activities of bHLH proteins by forming locations of which remain to be determined (26, 28). MyoD, heterodimers that are incapable of binding DNA (1). myf5, and MRF4 are also highly phosphorylated, but the sites Skeletal muscle gene expression is controlled by a family of for phosphorylation in these proteins have likewise not yet muscle-specific bHLH proteins which includes MyoD, myoge- been mapped (17, 25, 32, 46, 52). Here we show that dimerization with E2A gene products results in an increase in the phosphorylation of myogenin at * Corresponding author. Mailing address: Department of Biochem- sites within its amino- and carboxyl-terminal transcription istry and Molecular Biology, Box 117, The University of Texas M. D. activation domains. Replacement of these sites with neutral Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. amino acids that prevent phosphorylation enhances the ability Phone: (713) 792-3648. Fax: (713) 791-9478. of myogenin to activate E box-dependent reporter genes and 6232 VOL. 14, 1994 DIMERIZATION-DEPENDENT PHOSPHORYLATION OF MYOGENIN 6233 potentiates the activity of myogenin's transcription activation and loaded onto a 10% polyacrylamide-SDS gel. Following domains when they are fused to the DNA-binding domain of electrophoresis, the gel was dried and processed for autora- the yeast protein GAL4. The ability of E2A products to diography. enhance phosphorylation of myogenin suggests that dimeriza- Phosphopeptide mapping and phosphoamino acid analysis. tion induces a conformational change in myogenin that ex- For phosphoamino acid analysis, 32P-labeled myogenin was poses otherwise cryptic phosphorylation sites or that E2A excised from SDS gel slices, which were then hydrated in water products recruit a kinase for which myogenin is a substrate. and rinsed in 50 mM NH4HCO3. The labeled protein was That phosphorylation of the dimerization-dependent sites di- ground into pieces and boiled for 3 min in 1 ml of 50 mM minishes myogenin's activity also suggests that the dimeriza- NH4HCO3 with 5 ,ul of and 10 p.l of tion-dependent kinase plays a negative role in regulating the ,B-mercaptoethanol 10% myogenic program. SDS. After a 5-h incubation at 37°C, the eluted proteins were precipitated in cold 15% trichloroacetic acid in the presence of 20 p.g of heat-treated RNase A. After overnight incubation on MATERIALS AND METHODS ice, the trichloroacetic acid precipitates were collected by Cell culture and transfections. Transfections were per- centrifugation for 10 min at 4°C and washed with 500 p.l of formed by calcium phosphate precipitation as described before ice-cold absolute ethanol. The pellet was then dried in air and (28). Briefly, COS-1 or 1OT1/2 cells were plated at 50% subjected to peptide mapping as described elsewhere (28) or confluency, and 1 day later, each plate of cells was refed with resuspended in 6 N HCl for phosphoamino acid analysis. Dulbecco's minimal essential medium (DMEM) containing Protein was then hydrolyzed for 2.5 h, and the partial acid 10% fetal bovine serum (2 ml for 60-mm dishes and 4 ml for hydrolysates were mixed with 3 p.g of phosphoserine, phospho- 100-mm dishes) and transfected within 4 h. Calcium phos- threonine, and phosphotyrosine (Sigma) and resolved by two- phate-DNA precipitates were prepared by adding 250 ,ul of 2x dimensional electrophoresis, with the first dimension at pH 1.9 HBS (0.818% NaCl, 0.594% HEPES [N-2-hydroxyethylpipera- and the second dimension at pH 3.5 (acetic acid-pyridine- zine-N'-2-ethanesulfonic acid], 0.02% Na2HPO4 [pH 7.12]) to water, 50:5:945). The thin-layer chromatography plate was first 250 p.l of 250 mM calcium chloride plus DNA (5 pLg per 60-mm visualized by autoradiography, and the phosphoamino acid plate). At 4 to 16 h after transfection, the medium was replaced standards were visualized after the plate was sprayed with with fresh DMEM containing 10% fetal bovine serum. For 0.25% ninhydrin in acetone and incubated for 10 min at chloramphenicol acetyltransferase (CAT) assays, transfected 650C. cells were refed with DMEM containing 2% horse serum 24 h Myogenin and E47 mutants. Site-directed mutagenesis was following transfection and allowed to differentiate for 2 days performed by PCR. All mutations were confirmed by DNA before being harvested. CAT activity was determined on aliquots of cell extract normalized to,-galactosidase activity as sequencing. described before (28). All assays were linear with respect to The following myogenin deletion mutants were made: protein concentration. DM1-33 was made by insertion of a SmaI fragment from the The reporter plasmid MCK-CAT contains the basal pro- myogenin cDNA (14), extending from codon 33 to the 3' end moter and muscle-specific enhancer from the mouse muscle of the transcript, into the SmaI site of the pECE-Flag vector creatine kinase (MCK) gene (45). Plasmid 4R-tkCAT (gener- (IBI), creating a Flag epitope-tagged protein. An NcoI-XbaI ously provided by A. Lassar) contains four tandem copies of fragment that encompassed the myogenin coding region was the high-affinity right-hand E box from the MCK enhancer isolated from this vector, the ends were filled in with de- linked to the thymidine kinase (tk) gene basal promoter (48). oxynucleotide triphosphates and Klenow polymerase, and the The GAL4 reporter plasmid pG5E1bCAT contains five copies fragment was subcloned into a filled EcoRI site of the vector of the GAL4 binding site upstream of the Elb promoter (29).