Abstract
Myogenin and its upstream regulator MyoD are known to be required for myogenic cell differentiation. Although both of them can be expressed in rhabdomyosarcoma-derived RD cells, the cells are unable to undergo full-scale terminal myogenic differentiation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) has been found to be functional in the induction of RD cell differentiation, whereas its mechanism is not fully understood. By using quantitative real-time-based chromatin immunoprecipitation and real-time reverse transcription-PCR-based promoter activity assays, we examined the activation mechanism of the myogenin gene during TPA-induced differentiation of the RD cells. We have shown that a histone acetyltransferase PCAF and ATPase subunit BRG1 of the SWI/SNF chromatin remodeling complex are sequentially recruited to the promoter of the myogenin gene. Both PCAF and BRG1 are also involved in the activation of the myogenin gene. In addition, we have found that the p38 mitogen-activated protein kinase is required for BRG1 recruitment in TPA-mediated myogenin induction. We propose that there are two distinct activation steps for the induction of myogenin in TPA-induced early differentiation of RD cells: 1) an early step that requires PCAF activity to acetylate core histones and MyoD to initiate myogenin gene expression, and 2) a later step that requires p38-dependent activity of the SWI/SNF remodeling complex to provide an open conformation for the induction of myogenin. Our studies reveal an essential role for epigenetic regulation in TPA-induced differentiation of RD cells and provide potential drug targets for future treatment of the rhabdomyosarcoma.
Highlights
Rhabdomyosarcoma is a malignant tumor of childhood that is thought to derive from muscle precursor cells
We have shown that a histone acetyltransferase PCAF and ATPase subunit BRG1 of the SWI/SNF chromatin remodeling complex are sequentially recruited to the promoter of the myogenin gene
SWI/SNF Complex Is Recruited to the Myogenin Promoter at a Later Stage in TPA-treated RD Cells—To examine whether the TPA-induced expression of myogenin requires the participation of other chromatin remodeling molecules, we focused on the mammalian SWI/SNF complex as it has been implicated in myogenin gene expression (9, 10)
Summary
Cell Culture—RD cells were purchased from ATCC and maintained in Dulbecco’s modified Eagles’s medium (Invitrogen) supplemented with 10% fetal calf serum, 0.37% NaHCO3, and sodium penicillin and streptomycin sulfate (100 units/ml each) in a 5% CO2 humidified atmosphere at 37 °C. The transfection control plasmid pCMV--gal was generated by amplifying the -galactosidase gene from pRSV--gal plasmid (constructed in our laboratory) with primer pairs of the 5Ј primer: 5Ј-CCCAAGCTTTTCGTCTGGGACTGGGTG-3Ј and the 3Ј primer: 5Ј-GCTCTAGAGGTCGGGATAGTTTTCTTGC-3Ј, followed by insertion into the XbaI/HindIII-digested pRc/CMV vector (Invitrogen). Expression plasmids of p38 and BRG1 genes were individually co-transfected with pREP4m-myog-CAT and pCMV-gal into RD cells. TPA (100 nM) was added to the medium after transient transfection for 16 h and incubated for another 24 h or the time interval indicated followed by cell harvesting. Quantitative Real-time RT-PCR Analysis—Total RNA was extracted from cells and followed by reverse transcription with a first-strand RT-PCR kit (Promega) per the manufacturer’s instructions. To quantitate products generated in the chromatin immunoprecipitation assays or mRNA expression, we used the following primers: primers for myogenin gene, 5Ј-ATGGAGCTGTATGAGACATCCCC-3Ј (forward, ϩ1/ϩ23) and 5Ј-GGACACCGACTTCCTCTTACAC-3Ј(reverse, ϩ237/ϩ216).
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