A Novel Role for Protein Inhibitor of Activated STAT (PIAS) Proteins in Modulating the Activity of Zimp7, a Novel PIAS-like Protein, in Androgen Receptor-mediated Transcription

Yue Peng,Jane Lee,Chunfang Zhu,Zijie Sun

doi:10.1074/jbc.m109.079327

Abstract

The PIAS proteins (protein inhibitor of activated STAT) were originally identified as inhibitors of the JAK-STAT pathway. Subsequently, their roles on transcriptional regulation have been identified in modulation of the androgen receptor (AR) and other nuclear hormone receptor-mediated actions. Zimp7, also named Zmiz2, is a novel PIAS-like protein and functions as a transcriptional co-activator. In this study, we demonstrate an interaction between Zimp7 and PIAS proteins with higher preference for PIAS3. A modified mammalian one-hybrid assay showed that the NH(2)-terminal proline-rich domain of Zimp7 and the region spanning amino acids 321-486 of PIAS3 were the primary interaction segments. The interaction between Zimp7 and PIAS3 proteins was further confirmed by in vitro protein pull-down and co-immunoprecipitation assays with both exogenous and endogenous proteins. Expression of exogenous PIAS3 further enhances Zimp7-mediated augmentation of AR transcription. Knockdown of the endogenous PIAS3 protein using a specific PIAS3 small hairpin RNA reduced the augmentation of Zimp7 on AR-mediated transcription. Co-localization of Zimp7 and PIAS3 proteins was observed in the nuclei of cells by immunostaining. Exogenous PIAS3 expression enhances the stability of the Zimp7 protein. Using chromatin immunoprecipitation assays, we showed that PIAS3 is involved in the AR- and Zimp7-formed protein complex(es) in the AR downstream target promoter to facilitate androgen-induced transcription. Finally, we further demonstrated that loss of Zimp7 significantly impaired PIAS3-mediated enhancement on AR activity in mouse Zimp7 null (zimp7(-/-)) embryonic fibroblasts. Taken together, these results demonstrate a novel interaction between PIAS and PIAS-like proteins and elucidate a novel regulatory mechanism for PIAS proteins in AR-mediated transcription.

Highlights

CA70297. □S The on-line version of this article contains supplemental Fig. S1. 1 To whom correspondence should be addressed
We observed that the full-length Zimp proteins, when fused to the DNA-binding domain of GAL4, display a very limited activity compared with truncated mutants containing the COOH-terminal proline-rich domain only [11, 12]
Using a series of deletion mutants, we further demonstrated that the NH2-terminal domains of Zimp proteins inhibit the transcriptional activity of the COOH-terminal regions, suggesting a potential mechanism that may be involved in switching the Zimp proteins from an inactive form to an active form

Summary

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid System—Yeast two-hybrid experiments were performed as described previously [19]. The FLAG-PIAS3 plasmid was transfected with pcDNA3-HA-Zimp into CV-1 cells using Lipofectamine reagents (Invitrogen). Individual transfection experiments were done in triplicate, and the results reported as the mean Ϯ S.D. D, the full-length PIAS1, PIAS3, PIASx␣, or PIASx␤ were fused to pM vector, and co-transfected into CV-1 cells with full-length hZimp or Zimp proteins. The PIAS3 clone was cotransformed average size of 800 bp by sonication and diluted 10-fold in ChIP with various constructs containing either GAL4 DBD alone or dilution buffer Only moderate activity was observed in the samples cotransfected with PIAS3 and Zimp

To assess the interactions between

Mapping Interacting Regions for

Findings

DISCUSSION