Xlim-1 and LIM Domain Binding Protein 1 Cooperate with Various Transcription Factors in the Regulation of the goosecoid Promoter

• Published in: Developmental biology Vol. 224; no. 2; pp. 470 - 485
• Main Authors: Mochizuki, Toshiaki, Karavanov, Alexander A, Curtiss, Patricia E, Ault, Katherine T, Sugimoto, Naoshi, Watabe, Tetsuro, Shiokawa, Koichiro, Jamrich, Milan, Cho, Ken W.Y, Dawid, Igor B, Taira, Masanori
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.08.2000
• Subjects: Animals; Base Sequence; DNA; DNA Primers; DNA-Binding Proteins - metabolism; Gene Expression Regulation, Developmental; goosecoid gene; goosecoid promoter; Goosecoid Protein; Gsc; head organizer; homeobox gene; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Ldb1; Ldb1 gene; LIM domain; LIM-Homeodomain Proteins; luciferase; Molecular Sequence Data; Otx2; otx2 gene; Protein Binding; PV.1; Repressor Proteins; Sequence Homology, Nucleic Acid; Transcription Factors - metabolism; Xbra; Xenopus; Xenopus - embryology; Xenopus Proteins; Xlim-1; Xlim-1 gene; Xvent-1; Xenopus; Xbra; Xlim-1; Xvent-1; LIM domain; Ldb1; Gsc; head organizer; Otx2; homeobox gene; PV.1; goosecoid promoter
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1006/dbio.2000.9778

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.