Sequence-specific DNA binding transcription factors play a central role in gene regulation processes by activating or repressing he rate of transcriptional initiation of specific larger genes. The domains of transcription factors which mediate activation and their mechanisms have been extensively characterized. These mechanisms include either direct interaction with the basal transcription machinery or via a number of co- activator/adaptor/mediator proteins. However, little is known of the nature of eukaryotic repression domains and their mechanisms. We have previously identified and characterized a highly conserved repression domain which is encoded by approximately one third of all zinc finger genes in the human genome. The Kruppel-Associated Box (KRAB) domain is a about 75 amino acid module composed of two amphipathic helices which functions as a potent transcriptional repression domain. in order to characterize the mechanisms of kRAB-mediated repression we have purified a protein (KRAB-domain-Associated Protein-1;KRAP-1) which directly interacts with the KRAB domain, but fails to interact with mutants which lack repression function. We have cloned the cDNA encoding kRAP-1. The KRAP- 1protein contains a RING finger, B1, B2 boxes, a coiled-coil. WD40 and PHD motifs as well as a bromo-like domain. We hypothesize that the KRAP01 molecule is an """"""""adaptor"""""""" which mediates KRAB domain repression. We propose to test this hypothesis and decipher the mechanisms of KRAB-KRAP-1-mediated transcriptional repression by performing the following specific aims. Specifically, we will: 1. Reconstitute the KRAB-KRAP-1 interaction in vitro and in vivo and map the domains/specific amino acids in each protein critical for the interaction. 2. Define and characterize the requirement for KRAP-1 in KRAB-mediated transcriptional repression function in vivo. 3. Identify and biochemically characterize endogenous cellular KRAP-1 and KRAB-KRAP-1 complexes. 4. Identify the domains of kRAP-1 required for repression and define the mechanism (s) of KRAB-KRAP-1 mediated transcriptional repression using purified cell-free transcription systems. The experiments described in these specific aims represent a comprehensive analysis of a unique repressor-co-repressor interaction and should provide new insights into the mechanisms of gene regulation utilized by the about 200 KRAB domain encoding genes ina the human genome.
