Abnormal expression of the transcription factor BERF-1/ZBP-89/Zfp148 in C2.7 myoblasts alters the myogenic differentiation program in a MyoD-dependent manner

The aim of our work is to investigate the functional role of the zinc finger transcription factor BERF-1/ZBP- 89/Zfp148 in myogenic differentiation. For this purpose we have generated C2.7 myoblasts stable lines overexpressing BERF-1 as a transgenic protein fused to the tamoxifen-sensitive estrogen receptor (ER) binding site. As expected, ERBERF- 1 cell clones showed a significant accumulation of the transgenic protein into the nucleus and more interesting cells were not able to fuse and to form myotubes when induced to differentiate. Western Blot analysis of ER-BERF-1 cell clones revealed the lack of expression of the MyoD protein, the regulatory factor necessary to trigger the cascade of events that leads to muscle cells differentiation, as well as of early and late differentiation markers like myogenin and myosin heavy chain (MHC). Semi-quantitative RT-PCR analysis of total RNA extracted from ER-BERF-1 cell clones maintained either in proliferation or in differentiation medium showed no expression of MyoD mRNA suggesting that the block of myogenic differentiation is due to a BERF-1-dependent negative regulation of the MyoD gene. RNAinterference in C2.7 cells confirmed our hypothesis: downregulation of BERF-1 leads to an increased expression of MyoD protein and as a consequence to a strikingly enhanced myotubes formation compared to wild-type cells. Our data demonstrate that the correct expression of BERF-1 is necessary for the normal execution of the myogenic differentiation program and candidate BERF-1 as an upstream transcriptional regulator of the MyoD gene