Characterization of sequences in human TWIST required for nuclear localization.
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 TWISTNLS1 K38R, TWISTNLS2 K73R and K77R constructs inhibit nuclear accumulation of H-TWIST in mammalian cells, while TWISTNLS2 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 TWISTNLS1 with co-transfixed TCF-4 was observed. The interaction of TWIST with TCF-4 was also seen using standard immunoprecipitation assays.


CONCLUSIONS
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.
Singh
Shalini
S
Department of Physiology, Charles H, Best Institute, University of Toronto, 112 College Street, Toronto, Ontario, M5G 1L6, Canada. shalini.singh@utoronto.ca
Gramolini
Anthony O
AO
D000595:Amino Acid Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics; D051820:TCF Transcription Factors / Q000235:genetics / Q000378:metabolism; D000073940:Transcription Factor 4; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D051879:Twist-Related Protein 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C116115:MYCBP protein, human; D019913:Nuclear Localization Signals; D051820:TCF Transcription Factors; C532096:TCF4 protein, human; C544912:TCF7L1 protein, human; D000073940:Transcription Factor 4; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D051879:Twist-Related Protein 1; D001616:beta-Galactosidase