Isolation and characterization of ZFM1, a human transcriptional repressor that interacts with G/Q-rich transcriptional activators

Abstract

Stage-specific activator protein (SSAP) is an enhancer-binding protein responsible for the transcriptional activation of the sea urchin late H1 gene expression at the mid-blastula stage of embryogenesis. It contains in its C-terminal half a novel glycine/ glutamine-rich domain (GQ domain) which functions as an extremely potent transcriptional activation domain (4-5 fold better than VP16) in a variety of mammalian cell lines. To understand the mechanism by which the GQ domain activates transcription, yeast two hybrid system was employed to identify several human cDNAs encoding proteins that interact with it from both HL60 and HeLa cell derived cDNA libraries, including ZFM1, EWS, Trip1 and novel proteins.;ZFM1 encodes a protein with a KH domain and a zinc knuckle motif and a long proline-rich region in its C-terminus. The interaction between ZFM1 and the GQ domain was observed in both two hybrid assays and GST pull-down experiments. Functional assays established ZFM1 as a transcriptional repressor. Overexpression of ZFM1 in HepG2 cells represses Gal4-GQ mediated transcription in an interaction-dependent manner. Furthermore, Gal4-ZFM1 functions as a direct repressor when tethered to the TK promoter and a repression domain of ZFM1 was mapped to its N-terminal 137 amino acids. The repression activity of ZFM1 is unique in that only activated but not basal transcription levels are affected.;The N-terminal domain (NTD) of EWS, a human protein involved in Ewing's sarcoma, resembles the GQ domain of SSAP in both amino acid composition and the potential to activate transcription. It was observed that ZFM1 interacts with the NTD of EWS and overexpression of ZFM1 in HepG2 cells represses Gal4-EWS-NTD mediated transcription. Furthermore, TLS and hTAF{dollar}\sb{lcub}\rm II{rcub}{dollar}68, which have extensive homology to EWS, also interact with ZFM1. Recently, it was found that native EWS, TLS and hTAF{dollar}\sb{lcub}\rm II{rcub}{dollar}68 may function in transcription as components of distinct TFIID complexes. The association of ZFM1 with these proteins implies that ZFM1 may negatively modulate transcription coordinated by these specific TAFs.