alpha-Sareoglycan striated muscle-specific protein is a member of the sarcoglycan-sarcospan complex. Positive and negative transcriptional regulation of sarcoglycan genes are important in sarcoglycan's intracellular localization and sarcolemmal stability. In the present work we assessed the function of NFI transcription factors in the regulation of alpha-sarcoglycan promoter through the C2C12 cell line differentiation. NFI factors act alternatively as activators and negative modulators of alpha-sarcoglycan promoter activity. In myoblasts NFI-A1.1 and NFI-B2 are activators, whereas NFI-C2 and NFI-X2 are negative regulators. In myotubes, all NFI members are activators, being NFI-C2 the less potent. We identified the alpha-sarcoglycan promoter NFI-C2 response element by testing progressive deletion constructs and point mutations in C2C12 cells over-expressing NFI-C2. Gel-shift and chromatin immunoprecipitation experiments demonstrated that NFI factors are indeed interacting in vitro and in vivo with the binding sequence. These results suggest a NFI role in C2C12 cell differentiation. (C) 2004 Elsevier Inc. All rights reserved.