Xlim-1 and LIM domain binding protein 1 cooperate with various transcription factors in the regulation of the goosecoid promoter.
The homeobox genes Xlim-1 and goosecoid (gsc) are coexpressed in the Spemann organizer and later in the prechordal plate that acts as head organizer. Based on our previous finding that gsc is a possible target gene for Xlim-1, we studied the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages, by using gsc-luciferase reporter constructs injected into animal explants. A 492-bp upstream region of the gsc promoter responds to Xlim-1/3m, an activated form of Xlim-1, and to a combination of wild-type Xlim-1 and Ldb1, a LIM domain binding protein, supporting the view that gsc is a direct target of Xlim-1. Footprint and electrophoretic mobility shift assays with GST-homeodomain fusion proteins and embryo extracts overexpressing FLAG-tagged full-length proteins showed that the Xlim-1 homeodomain or Xlim-1/Ldb1 complex recognize several TAATXY core elements in the 492-bp upstream region, where XY is TA, TG, CA, or GG. Some of these elements are also bound by the ventral factor PV.1, whereas a TAATCT element did not bind Xlim-1 or PV.1 but did bind the anterior factors Otx2 and Gsc. These proteins modulate the activity of the gsc reporter in animal caps: Otx2 activates the reporter synergistically with Xlim-1 plus Ldb1, whereas Gsc and PV.1 strongly repress reporter activity. We show further, using animal cap assays, that the endogenous gsc gene was synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene was activated by Xlim-1/3m, and this activation was suppressed by the posterior factor Xbra. Based on these data, we propose a model for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation.
Laboratory of Molecular Embryology, Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
T
T
Mochizuki
A A
AA
Karavanov
P E
PE
Curtiss
K T
KT
Ault
N
N
Sugimoto
T
T
Watabe
K
K
Shiokawa
M
M
Jamrich
K W
KW
Cho
I B
IB
Dawid
M
M
Taira
D000818:Animals; D001483:Base Sequence; D004247:DNA; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D051956:Goosecoid Protein; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D060850:LIM-Homeodomain Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D012097:Repressor Proteins; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D014981:Xenopus / Q000196:embryology; D029867:Xenopus Proteins
D017931:DNA Primers; D004268:DNA-Binding Proteins; D051956:Goosecoid Protein; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C543964:Lhx1 protein, Xenopus; D012097:Repressor Proteins; D014157:Transcription Factors; C486314:XLdb1 protein, Xenopus; D029867:Xenopus Proteins; D004247:DNA