Research article

Characterization of sequences in human TWIST required for nuclear localization

Shalini Singh and Anthony O Gramolini

Department of Physiology, Charles H. Best Institute, University of Toronto, 112 College Street, Toronto, Ontario, M5G 1L6, Canada

author email corresponding author email

BMC Cell Biology 2009, 10:47doi:10.1186/1471-2121-10-47

Published:	17 June 2009

Abstract

Background

Twist is a transcription factor that plays an important role in proliferation and tumorigenesis. Twist is a nuclear protein that regulates a variety of cellular functions controlled by protein-protein interactions and gene transcription events. The focus of this study was to characterize putative nuclear localization signals (NLSs) ³⁷RKRR⁴⁰ and ⁷³KRGKK⁷⁷ in the human TWIST (H-TWIST) protein.

Results

Using site-specific mutagenesis and immunofluorescences, we observed that altered TWIST^NLS1 K38R, TWIST^NLS2 K73R and K77R constructs inhibit nuclear accumulation of H-TWIST in mammalian cells, while TWIST^NLS2 K76R expression was un-affected and retained to the nucleus. Subsequently, co-transfection of TWIST mutants K38R, K73R and K77R with E12 formed heterodimers and restored nuclear localization despite the NLSs mutations. Using a yeast-two-hybrid assay, we identified a novel TWIST-interacting candidate TCF-4, a basic helix-loop-helix transcription factor. The interaction of TWIST with TCF-4 confirmed using NLS rescue assays, where nuclear expression of mutant TWIST^NLS1 with co-transfixed TCF-4 was observed. The interaction of TWIST with TCF-4 was also seen using standard immunoprecipitation assays.

Conclusion

Our study demonstrates the presence of two putative NLS motifs in H-TWIST and suggests that these NLS sequences are functional. Furthermore, we identified and confirmed the interaction of TWIST with a novel protein candidate TCF-4.