PIAS1 regulates CP2c localization and active promoter complex formation in erythroid cell-specific α-globin expression

Kang, Ho Chul
Chae, Ji Hyung
Jeon, Jinseon
Kim, Won
Ha, Dae Hyun
Shin, June Ho
Kim, Chan Gil
Kim, Chul Geun

¹Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University, Seoul, 133–791 and ²Department of Biotechnology, Konkuk University, Chungju 380–701, Korea

^*To whom correspondence should be addressed. Tel: +82 2 2220 0957; Fax: +82 2 2296 5996; Email: [email protected]

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

Received 28 January 2010; revised 1 April 2010; accepted 6 April 2010

Nucleic Acids Research 38(16):p 5456-5471, September 2010. | DOI: 10.1093/nar/gkq286

ABSTRACT

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.