Functional domains of the LIM homeodomain protein Xlim-1 involved in negative regulation, transactivation, and axis formation in Xenopus embryos.
The Xenopus LIM homeodomain protein Xlim-1 is specifically expressed in the Spemann organizer   region and assumed to play a role in the establishment of the body axis as a transcriptional activator. To   further elucidate the mechanism underlying the regulation of its transcriptional activity, we focused on the   region C-terminal to the homeodomain of Xlim-1 (CT239-403) and divided it into five regions, CCR1-5   (C-terminal conserved regions), based on similarity between Xlim-1 and its paralog, Xlim-5. The role of   Xlim-1 CT239-403 in the Spemann organizer was analyzed by assaying the axis-forming ability of a series   of CCR-mutated constructs in Xenopus embryos. We show that high doses of Xlim-1 constructs deleted   of CCR1 or CCR2 initiate secondary axis formation in the absence of its coactivator Ldb1   (LIM-domain-binding protein 1), suggesting that CCR1 and CCR2 are involved in negative regulation of   Xlim-1. In contrast, while Xlim-1 is capable of initiating secondary axis formation at low doses in the   presence of Ldb1, deletion of CCR2 (aa 275-295) or substitution of five conserved tyrosines in CCR2 with   alanines (CCR2-5YA) abolished the activity. In addition, UAS-GAL4 one-hybrid reporter assays in   Xenopus showed that CCR2, but not CCR2-5YA, with its flanking regions (aa 261-315) functions as a   transactivation domain when fused to the GAL4 DNA-binding domain. Finally, we show that none of the   known transcriptional coactivators tested (CBP, SRC-1, and TIF2) interacts with the Xlim-1 transactivation   domain (aa 261-315). Thus, Xlim-1 not only contains a unique tyrosine-rich activation domain but also   contains a negative regulatory domain in CT239-403, suggesting a complex regulatory mechanism   underlying the transcriptional activity of Xlim-1 in the organizer.
Hiratani
I
I
Department of Biological Sciences, Graduate School of Science,   University of Tokyo, Hongo, Tokyo, 113-0033, Japan.  
Mochizuki
T
T
Tochimoto
N
N
Taira
M
M
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D019521:Body Patterning; D017124:Conserved Sequence; D004625:Embryo, Nonmammalian / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000235:genetics; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D060850:LIM-Homeodomain Proteins; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009024:Morphogenesis; D020897:Organizers, Embryonic / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014443:Tyrosine; D014981:Xenopus / Q000196:embryology*; D029867:Xenopus Proteins
D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C543964:Lhx1 protein, Xenopus; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D029867:Xenopus Proteins; D014443:Tyrosine; D008156:Luciferases; D005982:Glutathione Transferase