Phylogenetic footprinting and genome scanning identify vertebrate BMP response elements and new target genes

Abstract

The complex gene regulatory networks governed by growth factor signaling are still poorly understood. In order to accelerate the rate of progress in uncovering these networks, we explored the usefulness of interspecies sequence comparison (phylogenetic footprinting) to identify conserved growth factor response elements. The promoter regions of two direct target genes of Bone Morphogenetic Protein (BMP) signaling in Xenopus, Xvent2 and XId3, were compared with the corresponding human and/or mouse counterparts to identify conserved sequences. A comparison between the Xenopus and human Vent2 promoter sequences revealed a highly conserved 21 bp sequence that overlaps the previously reported Xvent2 BMP response element (BRE). Reporter gene assays using Xenopus animal pole ectodermal explants (animal caps) revealed that this conserved 21 bp BRE is both necessary and sufficient for BMP responsiveness. We combine the same phylogenetic footprinting approach with luciferase assays to identify a highly conserved 49 bp BMP responsive region in the Xenopus Id3 promoter. GFP reporters containing multimers of either the Xvent2 or XId3 BREs appear to recapitulate endogenous BMP signaling activity in transgenic Xenopus embryos. Comparison of the Xvent2 and the XId3 BRE revealed core sequence features that are both necessary and sufficient for BMP responsiveness: a Smad binding element (SBE) and a GC-rich element resembling an OAZ binding site. Based on these findings, we have implemented genome scanning to identify over 100 additional putative target genes containing 2 or more BRE-like sequences which are conserved between human and mouse. RT-PCR and in situ analyses revealed that this in silico approach can effectively be used to identify potential BMP target genes.

The Proto-oncogene Transcription Factor Ets1 Regulates Neural Crest Development through Histone Deacetylase 1 to Mediate Output of Bone Morphogenetic Protein Signaling

“…However, the molecular mechanisms as to how the intermediate BMP activity is defined are not well understood. It has been reported that among the target genes of BMP signaling pathway, id3, a member of the Id gene family, is expressed in NC where it plays an essential role in mediating NC cell proliferation and cell survival (19,26).…”

Finding partners: How BMPs select their targets

“…Another link between vertebrate Schnurris and BMP signaling comes from examination of the cross-species behavior of a BRE from the Xenopus vent2 gene (Candia et al, 1997;Rastegar et al, 1999;Hata et al, 2000;Karaulanov et al, 2004;von Bubnoff et al, 2005). The BRE mediates BMP induction during frog gastrulation and is also found in the promoters of two other BMP target genes, id3 and bambi (Karaulanov et al, 2004;von Bubnoff et al, 2005).…”

“…The BRE mediates BMP induction during frog gastrulation and is also found in the promoters of two other BMP target genes, id3 and bambi (Karaulanov et al, 2004;von Bubnoff et al, 2005). Transgenic Drosophila lines bearing a vertebrate BRE-driven lacZ reporter gene have provided insight into how vertebrate target gene expression is regulated by BMPs.…”

Nanogel‐based scaffold delivery of prostaglandin E2 receptor–specific agonist in combination with a low dose of growth factor heals critical‐size bone defects in mice

“…Osteoblastic MC3T3-E1 cells were treated with BMP-2 (50 ng/ml), with or without EP4A (10 Ϫ7 M), or with vehicle (DMSO), and then were subjected to serum alkaline phosphatase assay (23) and luciferase assay, as described elsewhere (27).…”