PIAS1 regulates CP2c localization and active promoter complex formation in erythroid cell-specific alpha-globin expression.
Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In vitro, recombinant CP2c, CP2b and PIAS1 bound DNA as a stable CBP (CP2c/CP2b/PIAS1) complex. Following PIAS1 knockdown in MEL cells, however, the association of endogenous CP2c and CP2b with the α-globin promoter simultaneously decreased. By mapping the CP2b- and CP2c-binding domains on PIAS1, and the PIAS1-binding domains on CP2b and CP2c, we found that two regions of PIAS1 that interact with CP2c/CP2b are required for its co-activator function. We propose that CP2c, CP2b, and PIAS1 form a hexametric complex with two units each of CP2c, CP2b, and PIAS1, in which PIAS1 serves as a clamp between two CP2 proteins, while CP2c binds directly to the target DNA and CP2b mediates strong transactivation.
Kang
Ho Chul
HC
Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University, Seoul, 133-791, Korea.
Chae
Ji Hyung
JH
Jeon
Jinseon
J
Kim
Won
W
Ha
Dae Hyun
DH
Shin
June Ho
JH
Kim
Chan Gil
CG
Kim
Chul Geun
CG
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D041905:Erythroid Cells / Q000378:metabolism*; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic; D050828:Protein Inhibitors of Activated STAT / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D034622:RNA Interference; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation; D055542:alpha-Globins / Q000235:genetics*
D004268:DNA-Binding Proteins; D050828:Protein Inhibitors of Activated STAT; C501905:Tfcp2 protein, mouse; D014157:Transcription Factors; D055542:alpha-Globins