Autoregulation of Xvent-2B; direct interaction and functional cooperation of Xvent-2 and Smad1.
Members of the Xvent-2 homeodomain transcription factor family are immediate response genes of BMP-4 signaling. The bone morphogenetic protein response element (BRE) of Xvent-2B was previously identified and characterized with respect to Smad1 and Smad4 binding sites. In this study, we further report on the transcriptional regulation of Xvent-2B. We provide evidence that Xvent-2B (Xvent-2) maintains its own expression through autoregulation. This activity was demonstrated for the endogenous gene by reverse transcriptase-PCR analysis and was found to be insensitive to cycloheximide. Localized by DNase I footprinting were several Xvent-2 binding sites within the proximal upstream region including the BRE. In the early Xenopus embryo, the BRE was shown to be sufficient to drive expression of a green fluorescent protein reporter in a similar pattern compared with the endogenous gene. Furthermore, Xvent-2B was able to activate the BRE in luciferase reporter assays, and in co-injection experiments Xvent-2B and Smad1 were found to synergistically activate the BRE. Moreover, glutathione S-transferase pull-down experiments demonstrated that Xvent-2B directly and specifically interacts with Smad1. This association was mediated by the MH1 domain of Smad1 and required the C-terminal domain of Xvent-2. The failure of an Xvent-2 mutant lacking the C terminus to stimulate the BRE underlines the significance of the C-terminal domain in the described autoregulatory loop.
Henningfeld
Kristine A
KA
Abteilung Biochemie, Universität Ulm, Albert-Einstein Allee 11, 89081 Ulm, Germany and the Abteilung Entwicklungsbiochemie, Universität Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.
Friedle
Henner
H
Rastegar
Sepand
S
Knöchel
Walter
W
D000818:Animals; D001483:Base Sequence; D018983:DNA Footprinting; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051785:Smad Proteins; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors; D014981:Xenopus; D029867:Xenopus Proteins
D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; C497402:MXD1 protein, Xenopus; D011993:Recombinant Fusion Proteins; D051785:Smad Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D029867:Xenopus Proteins; C099992:ventx2.1 protein, Xenopus; D049452:Green Fluorescent Proteins