Regulation of Smad7 promoter by direct association with Smad3 and Smad4.
Smad7 is a regulatory Smad protein that is able to antagonize signal transduction by transforming growth factor-beta (TGF-beta) and activin receptors. To characterize the regulation of Smad7 at the transcriptional level, we isolated the promoter region of the mouse Smad7 gene. When the Smad7 promoter luciferase reporter gene (-408 and +112 bp) was expressed in human hepatoma (HepG2) cells, its transcriptional activity was increased following TGF-beta or activin treatment. In addition, this region of the Smad7 promoter was stimulated by ectopic expression of Smad3 as well as constitutively active TGF-beta and activin receptors, indicating that Smad7 transcription was modulated by the signaling downstream those two receptors. A gel mobility shift assay indicated that a DNA fragment spanning -408 to -126 base pairs (bp) was able to directly bind purified Smad4. Furthermore, a consensus Smad3-Smad4 binding element (SBE) was discovered in this region of the promoter with a palindromic sequence of GTCTAGAC. A 33-bp Smad7 promoter fragment containing this SBE was able to bind Smad3 and Smad4. In human embryonic kidney 293 cells, the expression of constitutively active TGF-beta type I receptor was able to induce the formation of a Smad3- and Smad4-containing nuclear protein complex that bound the SBE. In HepG2 cells, TGF-beta1 treatment could induce the formation of an endogenous SBE-binding complex. Taken together, these data provided the first evidence that Smad7 transcription is regulated by TGF-beta and activin signaling through direct binding of Smad3 and Smad4 to the Smad7 promoter.
Nagarajan
R P
RP
Department of Medical Genetics, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Zhang
J
J
Li
W
W
Chen
Y
Y
D028341:Activins; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007265:Inhibins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D018125:Receptors, Transforming Growth Factor beta; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; C497806:SMAD7 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D051906:Smad7 Protein; C497807:Smad7 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D028341:Activins; D007265:Inhibins; D004247:DNA