Selective degradation of excess Ldb1 by Rnf12/RLIM confers proper Ldb1 expression levels and Xlim-1/Ldb1 stoichiometry in Xenopus organizer functions.
The Xenopus LIM homeodomain (LIM-HD) protein, Xlim-1, is expressed in the Spemann organizer and cooperates with its positive regulator, Ldb1, to activate organizer gene expression. While this activation is presumably mediated through Xlim-1/Ldb1 tetramer formation, the mechanisms regulating proper Xlim-1/Ldb1 stoichiometry remains largely unknown. We isolated the Xenopus ortholog (XRnf12) of the RING finger protein Rnf12/RLIM and explored its functional interactions with Xlim-1 and Ldb1. Although XRnf12 functions as a E3 ubiquitin ligase for Ldb1 and causes proteasome-dependent degradation of Ldb1, we found that co-expression of a high level of Xlim-1 suppresses Ldb1 degradation by XRnf12. This suppression requires both the LIM domains of Xlim-1 and the LIM interaction domain of Ldb1, suggesting that Ldb1, when bound to Xlim-1, escapes degradation by XRnf12. We further show that a high level of Ldb1 suppresses the organizer activity of Xlim-1/Ldb1, suggesting that excess Ldb1 molecules disturb Xlim-1/Ldb1 stoichiometry. Consistent with this, Ldb1 overexpression in the dorsal marginal zone suppresses expression of several organizer genes including postulated Xlim-1 targets, and importantly, this suppression is rescued by co-expression of XRnf12. These data suggest that XRnf12 confers proper Ldb1 protein levels and Xlim-1/Ldb1 stoichiometry for their functions in the organizer. Together with the similarity in the expression pattern of Ldb1 and XRnf12 throughout early embryogenesis, we propose Rnf12/RLIM as a specific regulator of Ldb1 to ensure its proper interactions with LIM-HD proteins and possibly other Ldb1-interacting proteins in the organizer as well as in other tissues.
Hiratani
Ichiro
I
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan.
Yamamoto
Naoko
N
Mochizuki
Toshiaki
T
Ohmori
Shin-ya
SY
Taira
Masanori
M
D000595:Amino Acid Sequence; D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005775:Gastrula / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D060850:LIM-Homeodomain Proteins; D008648:Mesoderm / Q000378:metabolism; D008969:Molecular Sequence Data; D020897:Organizers, Embryonic / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors; D014981:Xenopus; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C543964:Lhx1 protein, Xenopus; C492369:RLIM protein, Xenopus; D012097:Repressor Proteins; D014157:Transcription Factors; C486314:XLdb1 protein, Xenopus; D029867:Xenopus Proteins