Yeast two-hybrid cloning of a novel zinc finger protein that interacts with the multifunctional transcription factor YY1

Muscle-restricted transcription of sarcomeric actin genes is negatively controlled by the zinc finger protein YY1, which is down-regulated at the protein level during myogenic differentiation. To identify cellular proteins that might mediate the function/stability of YY1 in muscle cells, we screened an adult human muscle cDNA library using the yeast two-hybrid cloning system. We report the isolation and characterization of a novel protein termed YAF2 (YY1-associated factor 2) that interacts with YY1. The YAF2 cDNA encodes a 180 amino acid basic protein (pl 10.5) containing a single N-terminal C-2-X(10)-C-2 zinc finger. Lysine clusters are present that may function as a nuclear localization signal, Domain mapping analysis shows that the first and second zinc fingers of YY1 are targeted for YAF2 protein interaction, In contrast to the down-regulation of YY1, YAF2 message levels increase during in vitro differentiation of both rat skeletal and cardiac muscle cells. YAF2 appears to have a promyogenic regulatory role, since overexpression of YAF2 in C2 myoblasts stimulates myogenic promoter activity normally restricted by YY1. Co-transfection of YY1 reverses the stimulatory effect of YAF2. YAF2 also greatly potentiates proteolytic cleavage of YY1 by the calcium-activated protease m-calpain, The isolation of YAF2 may help in understanding the mechanisms through which inhibitors of myogenic transcription may be antagonized or eliminated by proteolysis during muscle development.