Itch interacts with the pre-initiation complex for gene transcription

Abstract

Itch, an E3 ubiquitin ligase, is highly expressed in the testis. Studieshave shown that cytoplasmic Itch interacts with a tight junctionprotein, occludin and is involved in the regulation of occludin deg-radation. However, the interacting partners of Itch in the nucleusremain enigmatic. We expressed and purified the GST-Itch fusionprotein. GST-Itch fusion protein was incubated with nuclearextract prepared from Sertoli cells followed by affinity chromatog-raphy and eluted proteins were subjected to mass spectrometry.Their identities are RNA pol II, nuclear actin and myosin I beta(NMIb) and they are the components of the pre-initiation complex(PIC) for gene transcription. To confirm an in vitro interaction ofItch with PIC components in mammalian cells, co-immunoprecipi-tation and immunostaining were performed in both TM4 cells(Sertoli cell cell line) and HeLa cells and confirmed that Itchindeed physically associates with these PIC components. Byexpression and purification of truncated Itch coupled with co-immunoprecipitation, we confirmed that the WW domain in Itchis responsible for the interaction with RNA pol II and NMIb. Toexamine whether Itch is involved in the RNA pol II-mediated genetranscription, in vitro gene transcription and in vivo BrUTP incor-poration assays were performed. It was found that in vitro tran-scription is significantly reduced in the presence of anti-Itchantibody, but not serum. Knockdown of Itch by siRNA in cul-tured cells reduced the BrUTP incorporation in the nascent tran-scripts. Taken together, Itch is a PIC component and is crucial forRNA pol II-dependent transcription.Acknowledgements: This work was supported by Hong KongResearch Grant Council (HKU7609/06M) and CRCG SeedFunding for Basic Research.