Molecular determinants for targeting heterochromatin protein 1-mediated gene silencing: Direct chromoshadow domain-KAP-1 corepressor interaction is essential

The KRAB domain is a highly conserved transcription repression module commo nly found in eukaryotic zinc finger proteins. KRAB-mediated repression requ ires binding to the KAP-1 corepressor, which in turn recruits members of th e heterochromatin protein 1 (HP1) family. The HPI proteins are nonhistone c hromosomal proteins, although it is unclear how they are targeted to unique chromosomal domains or promoters. In this report, we have reconstituted an d characterized the HP1-KAP-1 interaction using purified proteins and have compared KAP-1 to three other known HP1 binding proteins: SP100, lamin B re ceptor (LBR), and the p150 subunit from chromatin assembly factor (CAF-1 p1 50), We show that the chromoshadow domain (CSD) of HP1 is a potent repressi on domain that binds directly to all four previously described proteins. Fo r KAP-1, we have mapped the CSD interaction region to a 15-amino-acid segme nt, termed the HP1BD, which is also present in CAF-1 p150 but not SP100 or LBR, The region of KAP-1 harboring the HP1BD binds as a monomer to a dimer of the CSD, as revealed by gel filtration, analytical ultracentrifugation, and optical biosensor analyses. The use of a spectrum of amino acid substit utions in the human HP1 alpha CSD revealed a strong correlation between CSD -mediated repression and binding to KAP-1, CAF-1 p150, and SP100 but not LB R, Differences among the HP1 binding partners could also be discerned by fu sion to a heterologous DNA binding domain and by the potential to act as do minant negative molecules. Together, these results strongly suggest that KA P-1 is a physiologically relevant target for HP1 function.