Regulated proteolysis has been postulated to be critical for proper control of cell functions. Muscle development, in particular, involves a great deal of structural adaptation and remodeling mediated by proteases. The transcription factor YY1 represses muscle-restricted expression of the sarcomeric ␣-actin genes. Consistent with this repressor function of YY1, the nuclear regulator is down-regulated at the protein level during skeletal as well as cardiac muscle cell differentiation. However, the YY1 message remains relatively unaltered throughout the myoblast-myotube transition, implicating a post-translational regulatory mechanism. We show that YY1 can be a substrate for cleavage by the calciumactivated neutral protease calpain II (m-calpain) and the 26 S proteasome. The calcium ionophore A23187 destabilized YY1 in cultured myoblasts, and the decrease in YY1 protein levels could be prevented by calpain inhibitor II and calpeptin. Treatment with the proteasome inhibitors MG132 and lactacystin resulted in the stabilization of YY1 protein, which is consistent with the finding that YY1 is readily polyubiquitinated in reticulocyte lysates. We further show that proteolytic targeting by calpain II and the proteasome involves different structural elements of YY1. This study thus illustrates two proteolytic pathways through which the transcriptional regulator can be differentially targeted under different cell growth conditions. Many cellular processes are known to be controlled by shortlived proteins, including products of the proto-oncogenes, cell cycle regulators, and developmentally regulated transcription factors (1-3). The fast turnover of these regulatory proteins reflects a metabolic requirement for rapidly changing their concentrations and is presumably mediated by a complex interplay among various proteases and protease inhibitors. Selective degradation of transcriptional activators and repressors, in particular, may provide efficient regulatory mechanisms contributing to the rapid shut-off and turn-on of gene activity, respectively. The recent study of the pleiotropic transcription factor NF-B has revealed that the ubiquitinproteasome pathway can function not only in the complete degradation of proteins but also in the regulated processing of precursors into active transcription factors (4, 5). Calpains represent the other major class of nonlysosomal proteases functioning in a calcium-dependent fashion (6, 7). Interestingly, both the proteasome and calpains have been found to play a regulatory role in the function and/or stability of c-Fos and the tumor suppressor protein p53 (8 -11). Thus, rapid degradation or processing of specific transcription factors can underlie a wide range of dynamic cellular and developmental processes.Muscle development involves a great deal of structural adaptation and remodeling mediated by induced protein synthesis and degradation (12)(13)(14). Although protein turnover must be highly selective if it is to be developmentally useful, little is known concerning the regulatory mech...