Developmentally regulated cytoplasmic retention of the transcription factor XMI-ER1 requires sequence in the acidic activation domain.
Xmi-er1 is a fibroblast growth factor regulated immediate-early gene that is activated during mesoderm induction in Xenopus embryonic explants. This gene encodes a nuclear protein with potent transcriptional regulator activity and overexpression of XMI-ER1 in Xenopus embryos inhibits mesoderm induction and leads to truncations along the anteroposterior axis. We showed previously that XMI-ER1 is retained in the cytoplasm during cleavage stages and only begins to appear in the nucleus at mid-blastula. Such developmentally regulated nuclear translocation may represent an important mechanism for regulating XMI-ER1 activity in the early embryo. Here, we investigate different mechanisms that might control nuclear translocation of XMI-ER1. Using alpha-amanitin to inhibit transcription, we show that nuclear localization is not dependent on zygotic transcription. Nor is it the result of a developmentally regulated import pathway, as the XMI-ER1 nuclear localization signal (NLS) fused to beta-galactosidase (betagal) was able to direct nuclear translocation prior to mid-blastula. Fusion of an additional, heterologous NLS to the N-terminus of XMI-ER1 was not sufficient to overcome cytoplasmic retention, indicating that retention does not involve NLS masking, but rather binding to a cytoplasmic anchor. The anchoring molecule is not an RNA, as microinjection of RNase A did not affect the timing of nuclear translocation. Western blot analysis using antibodies that recognize phosphorylated residues revealed that, while XMI-ER1 is not itself phosphorylated, it is associated with two differentially phosphorylated proteins, suggesting that the anchoring mechanism may involve interaction with a cytoplasmic protein(s). A series of XMI-ER1 deletion mutants was utilized to map the putative retention domain. Our analysis revealed that amino acids 144-175, containing the fourth acidic stretch of the acidic activation domain, are required for retention. These results suggest that XMI-ER1 is retained in the cytoplasm of the early embryo by interaction of the region containing amino acids 144-175 with a cytoplasmic anchor.
Terry Fox Cancer Research Laboratories, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Nfld, A1B 3V6, Canada.
Janine N
JN
Post
H Artee
HA
Luchman
F Corinne
FC
Mercer
Gary D
GD
Paterno
Laura L
LL
Gillespie
D000818:Animals; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002970:Cleavage Stage, Ovum / Q000166:cytology / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D008648:Mesoderm / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D015053:Zygote / Q000166:cytology / Q000378:metabolism*
D014157:Transcription Factors; D029867:Xenopus Proteins; C498111:transcription factor XMI-ER1, Xenopus laevis