Inhibition of MuSK expression by CREB interacting with a CRE-like element and MyoD

Abstract

The type I receptor-like protein tyrosine kinase MuSK is essential for the neuromuscular junction formation. MuSK expression is tightly regulated during development, but the underlying mechanisms were unclear. Here we identified a novel mechanism by which MuSK expression may be regulated. A cyclic AMP response element (CRE)-like element in the 5'-flanking region of the MuSK gene binds to CREB1 (CRE-binding protein 1). Mutation of this element increases the MuSK promoter activity, suggesting a role for CREB1 in attenuation of MuSK expression. Interestingly, CREB mutants unable to bind to DNA also inhibit MuSK promoter activity, suggesting a CRE-independent inhibitory mechanism. In agreement, CREB1 could inhibit a mutant MuSK transgene reporter whose CRE site was mutated. We provide evidence that CREB interacts directly with MyoD, a myogenic factor essential for MuSK expression in muscle cells. Suppression of CREB expression by small interfering RNA increases MuSK promoter activity. These results demonstrate an important role for CREB1 in the regulation of MuSK expression.

FIG. 1.

Identification of a CRE-like element in the MuSK promoter. (A) Schematic diagram of M715-Luc. The CRE-like element, located between −648 and −641, has the consensus sequence of the CRE element (24). (B) Interaction of the CRE-like element with CREB1 in ChIP assays. Chromatin preparations of C2C12 myotubes were incubated without (No Ab) or with antibodies against CREB1 or p300. Immunopurified DNA was used as the template for PCR amplification using primers against the indicated products. (C) Binding of the CRE-like element to purified CREB1. A 50-bp DNA fragment flanking the CRE-like element of the MuSK promoter was labeled with [γ-³²P]ATP by PNK. The labeled probe was incubated with various amounts of purified His-CREB1 without or with excess wild-type unlabeled probes. The reaction mixture was resolved on a nondenaturing polyacrylamide gel. (D) Supershift of the CRE probe-CREB1 complex in the presence of anti-CREB1 antibodies.

FIG. 2.

Mutation of the CRE-like element enhances promoter activity. The CRE-like element in M715(mt)-Luc was mutated to 5′G GCG TGA C. C2C12 myoblasts were transfected with pRL-CMV and M715-Luc or M715(mt)-Luc. Firefly and Renilla luciferase activities were assayed in C2C12 myotubes. Shown is the relative luciferase activity (firefly/Renilla, mean ± the standard error of the mean) from a representative experiment done in duplicate and repeated three times with similar results.

FIG. 3.

Inhibitory effects of KCREB and CREB on MyoD transactivation of the MuSK promoter. (A) Down-regulation by KCREB and CREB of MyoD transactivation. M715-Luc or M715(mt)-Luc was cotransfected with pRL-CMV and pCMV-CREB or pCMV-KCREB. Luciferase activities were assayed as in Fig. 2. (B) Identification of theE box 1 element as the site by which CREB or KCREB inhibits MuSK promoter activity. M215-Luc contains the 215-bp 5′-flanking region and the luciferase gene. CRE-M215-Luc contains the CRE element (5′GGG AGA GAT TGC CTG ACG TCA GAG AGC TAG GGT AC), the 215-bp 5′-flanking region, and the luciferase gene. In CRE(mt)-M215-Luc, the CRE element was mutated to 5′GGG AGA GAT TGC CGA CCA TAG GAG AGC TAG GGT AC (mutated portion underlined). Luciferase activities were assayed as in panel A. Shown is relative luciferase activity (firefly/Renilla, mean ± the standard error of the mean) from a representative experiment done in duplicate and repeated three times with similar results. (C) No CREB1-binding activity was detected in the −215 5′-flanking region. DNA fragments encompassing the −215 5′-flanking region (M215A-D; see Materials and Methods for sequence information) or the CRE cis element were labeled as in Fig. 1 and incubated with purified CREB1. The reaction was resolved on nondenaturing polyacrylamide gel.

FIG. 4.

MyoD coimmunoprecipitated with multiple domains of CREB1. (A) Schematic diagram of CREB1 constructs and their binding activities. Putative binding domains are underlined; thin lines indicate indirect binding, whereas the thick line indicates direct binding (see Fig. 6). Co-IP binding, coimmunoprecipitation data from panels B and C; pull-down binding, data from Fig. 6; NT, not determined. (B and C) Coimmunoprecipitation between MyoD and CREB1 and deletion mutants. COS-7 cells were transfected with Myc-tagged MyoD and HA-tagged CREB1 or its deletion mutants. Twenty-four hours after transfection, cells were lysed and subjected to immunoprecipitation with anti-Myc antibody. CREB proteins in the immunocomplex were revealed by immunoblotting with anti-HA antibody. The bottom two panels show inputs of HA-CREB1 constructs and Myc-MyoD, respectively. IP, immunoprecipitation; IB, immunoblotting.

FIG. 5.

MyoD interacts with CREB1 via the basic domain. (A and B) Coimmunoprecipitation between CREB1 and MyoD and its deletion mutants. COS-7 cells were transfected with plasmids expressing the indicated proteins, i.e., HA-tagged CREB_100-341, which contains the C-terminal region, and Myc-tagged MyoD or its deletion mutants. Immunoprecipitation was performed as in Fig. 4. CREB proteins in the immunocomplex were revealed by immunoblotting with anti-HA antibody. The bottom two panels show inputs of HA-CREB1 and Myc-MyoD constructs, respectively. (C) Schematic diagram of MyoD constructs and their CREB1 binding activities. IP, immunoprecipitation; IB, immunoblotting.

FIG. 6.

Direction interaction between MyoD and CREB1. (A) CREB1 and the indicated deletion proteins were generated by in vitro translation in the presence of [³⁵S]methionine using the TNT SP6 coupled reticulocyte lysate system (Promega). ³⁵S-labeled proteins were revealed by autoradiography. (B) Direct interaction of MyoD with the bZIP domain in CREB1. ³⁵S-labeled CREB1 constructs were incubated with recombinant GST-MyoD protein (full length) immobilized on agarose beads. After washing, bead-bound proteins were resolved by SDS-polyacrylamide gel electrophoresis and autoradiography. (C) GST-beads did not interact with ³⁵S-labeled CREB1 constructs. (D) Interaction between endogenous MyoD and CREB1 in muscle cells. Lysates of C2C12 cells were incubated with anti-CREB1 antibody or an irrelevant antibody (control), and resulting complexes were probed with anti-MyoD antibody. IP, immunoprecipitation; IB, immunoblotting.

FIG. 7.

Regulation of MuSK promoter activity by endogenous CREB1. (A) CREB1-siRNA inhibited CREB1 expression. COS-7 cells were transfected with pKH3-CREB1 together with CREB1-siRNA or control siRNA (Con-siRNA) oligonucleotides. Expression of transfected CREB1 was revealed by immunoblotting with anti-HA antibody. (B) Effects of suppression of CREB1 expression on muscle differentiation. Transfected C2C12 myotubes were collected at different times after being switched to the differentiation medium and lysed. Lysates were probed for expression of MHC. (C) Increased MuSK promoter activity in C2C12 myotubes cotransfected with CREB1-siRNA. M715-Luc was cotransfected with pRL-CMV and pCMV-MyoD with CREB1-siRNA or Con-siRNA. Luciferase activities were assayed as in Fig. 2. Shown is relative luciferase activity (firefly/Renilla, mean ± the standard error of the mean) from a representative experiment done in duplicate and repeated three times with similar results. (D) Expression of MyoD99-170 reversed CREB1 inhibition of MyoD transactivation activity. M715-Luc was cotransfected with pRL-CMV and pCMV-MyoD with MyoD99-170. Luciferase activities were assayed as in Fig. 2. Shown is relative luciferase activity (firefly/Renilla, mean ± the standard error of the mean) from a representative experiment done in duplicate and repeated three times with similar results. (E). Expression of CREB1 inhibited dimerization of MyoD. Flag- or Myc-tagged MyoD was coexpressed without or with HA-CREB1 in COS-7 cells. Lysates were incubated with anti-Flag antibody and resulting lysates probed with anti-Myc antibody. IP, immunoprecipitation; IB, immunoblotting.

FIG. 8.

Effects of forskolin and bromo-cAMP on expression of the M715-Luc transgene and MuSK mRNA. (A) C2C12 myotubes transfected with M715-Luc and pRL-CMV were treated with forskolin, bromo-cAMP, or vehicle (−) for 24 h. Luciferase activities were assayed as in Fig. 2. Shown is relative luciferase activity (firefly/Renilla, mean ± the standard error of the mean) from a representative experiment done in duplicate and repeated three times with similar results. (B) C2C12 myotubes were treated with forskolin for 24 h and lysed for RNA purification. RNase protection assay was performed as described in Materials and Methods. Shown are representative blots (top) and histograms summarizing three independent experiments (mean ± the standard error of the mean).