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 commonly found in eukaryotic zinc finger proteins. KRAB-mediated repression requires binding to the KAP-1 corepressor, which in turn recruits members of the heterochromatin protein 1 (HP1) family. The HP1 proteins are nonhistone chromosomal proteins, although it is unclear how they are targeted to unique chromosomal domains or promoters. In this report, we have reconstituted and characterized the HP1-KAP-1 interaction using purified proteins and have compared KAP-1 to three other known HP1 binding proteins: SP100, lamin B receptor (LBR), and the p150 subunit from chromatin assembly factor (CAF-1 p150). We show that the chromoshadow domain (CSD) of HP1 is a potent repression domain that binds directly to all four previously described proteins. For KAP-1, we have mapped the CSD interaction region to a 15-amino-acid segment, 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 substitutions in the human HP1alpha CSD revealed a strong correlation between CSD-mediated repression and binding to KAP-1, CAF-1 p150, and SP100 but not LBR. Differences among the HP1 binding partners could also be discerned by fusion to a heterologous DNA binding domain and by the potential to act as dominant negative molecules. Together, these results strongly suggest that KAP-1 is a physiologically relevant target for HP1 function.
The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
M S
MS
Lechner
G E
GE
Begg
D W
DW
Speicher
F J
FJ
Rauscher
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000378:metabolism; D056504:Chromatin Assembly Factor-1; D002850:Chromatography, Gel; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014158:Transcription, Genetic; D000076183:Tripartite Motif-Containing Protein 28; D014461:Ultracentrifugation
D034961:Antigens, Nuclear; D001324:Autoantigens; C000715594:CBX5 protein, human; C533593:CHAF1B protein, human; C489780:CNOT8 protein, human; C533592:Chaf1a protein, mouse; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D000090266:Chromobox Protein Homolog 5; C066642:SP100 protein, human; C101116:TRIM28 protein, human; C103190:Trim28 protein, mouse; D000076183:Tripartite Motif-Containing Protein 28; D005982:Glutathione Transferase