YEAST 2-HYBRID CLONING OF A NOVEL ZINC-FINGER PROTEIN THAT INTERACTS WITH THE MULTIFUNCTIONAL TRANSCRIPTION FACTOR YY1

Muscle-restricted transcription of sarcomeric actin genes is negativel y controlled by the zinc finger protein YY1, which is down-regulated a t the protein level during myogenic differentiation. To identify cellu lar proteins that might mediate the function/stability of YY1 in muscl e cells, we screened an adult human muscle cDNA library using the yeas t two-hybrid cloning system. We report the isolation and characterizat ion of a novel protein termed YAF2 (YY1-associated factor 2) that inte racts 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. Lys ine clusters are present that may function as a nuclear localization s ignal, Domain mapping analysis shows that the first and second zinc fi ngers of YY1 are targeted for YAF2 protein interaction, In contrast to the down-regulation of YY1, YAF2 message levels increase during in vi tro differentiation of both rat skeletal and cardiac muscle cells. YAF 2 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 ef fect of YAF2. YAF2 also greatly potentiates proteolytic cleavage of YY 1 by the calcium-activated protease m-calpain, The isolation of YAF2 m ay help in understanding the mechanisms through which inhibitors of my ogenic transcription may be antagonized or eliminated by proteolysis d uring muscle development.