von Bubnoff A et al. (2005), Phylogenetic footprinting and genome scanning i...

XB-ART-1899

Dev Biol 2005 May 15;2812:210-26. doi: 10.1016/j.ydbio.2005.02.014.

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

von Bubnoff A , Peiffer DA , Blitz IL , Hayata T , Ogata S , Zeng Q , Trunnell M , Cho KW .

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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.

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Species referenced: Xenopus laevis
Genes referenced: aadat ammecr1 babam2 bmp2 bmp4 csnk2a1 dach1 dlx5 flrt3 fst hoxa13 id3 kansl2 limk2 lrrn1 mef2a mpped2 olfm4 prkca prkd1 ptpn3 sall1 snrpe spdl1 stk3 tbx2 trps1 ventx2.2 wnt11 zeb2 znf423

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	Fig. 6. The Xvent2 and XId3 BREs direct expression of a GFP reporter gene to regions of BMP activity in transgenic frogs. Expression of XBMP4 compared to XId3 expression in gastrula, neurula, and tailbud stage embryos is shown in panels (A) and (DF), respectively. Unless otherwise indicated, for gastrula stages, dorsal is to the left and the animal pole up. For neurula stages, anterior is to the left and dorsal is up. For tailbud stages, lateral views are shown. (A, D) At the gastrula stage (10.5), both genes are expressed in the animal cap and excluded from part of the area above the dorsal blastopore lip (arrowheads). (B, E) At the neurula stage (14 5), both XId3 and XBMP4 are expressed in the future cement gland (arrowhead) and the ventral region. (C, F) At tailbud stages (2728), both genes are expressed in the proctodeum (p), the ventral branchial arch/heart region (vbr), the eyes (e), and the otic vesicle (ov). (G) Expression of the 8xXvent2-BRE/GFP-reporter gene analyzed in transgenic embryos. (G) Gastrula stage embryo (stage 10.511); arrowhead, dorsal blastopore lip. GFP mRNA is expressed on the ventral side only, including the ventral marginal zone and the ventral ectoderm. (H) Stage 32 embryo; stained with antisense GFP mRNA. (I) Stage 32 embryo, showing GFP protein fluorescence of the eye (e), ventral branchial arch/heart region (vbr), otic vesicle (ov), somites (s) and proctodeum (p). (J ) Expression of the 7xId3 BRE/GFP reporter gene in transgenic embryos; (J, M, Q) fluorescence of GFP protein; (K, L, N, O, P) expression of GFP mRNA. (J) Stage 11 embryo, vegetal view, dorsal is up. GFP protein is expressed ventrally and laterally but not dorsally. Note that there is also fluorescence in the yolk plug. (K) Stage 10.5 embryo; arrowhead, dorsal blastopore lip. GFP mRNA expression is excluded from the dorsal side. (L) Stage 10.5 embryo, uncleared, vegetal view, dorsal is up; white arrowheads show the limits of the ventral expression; red line, dorsal blastopore lip. Expression is observed only on the ventral side and in the yolk plug. (M) Neurula stage embryo (stage 14), dorsal view, anterior is to the left. GFP protein fluorescence is excluded from the neural plate. (N) Same embryo as in panel (M), dorsal view, anterior is to the left. GFP mRNA expression is excluded from the neural plate. GFP mRNA appears to be expressed in a slightly smaller domain than that of the GFP protein shown in panel (M) likely to due the fact that some of the mRNA disappears by this stage. (O) Same embryo as in panels (M, N), lateral view. GFP mRNA expression is observed only ventrally and is excluded from the neural plate. (P) Stage 32 embryo, stained with antisense GFP mRNA. (Q) Same embryo as in panel (P), but showing GFP protein expression.
	Fig. 7. Expression patterns of Xenopus orthologues to human genes with multiple BREs. Of the 26 representative orthologues from the 80 genes with available annotation in Xenopus, the expression patterns of 19 genes were analyzed by whole mount in situ hybridization. Expression patterns are organized based on the behavior of each gene in the RT-PCR experiments shown in Table 1 (strong, weak, no change, no data, not examined). Not shown are Id3 (Fig. 3) and BMP2 (Nishimatsu et al., 1992). Shown are the patterns at the neurula and tailbud stages for each gene; refer to Table 1 for a description of each pattern at the gastrula stage. Unless otherwise indicated, for neurula stages, anterior is to the left and dorsal is up. For tailbud stages, lateral views are shown.
	aadat (aminoadipate aminotransferase) gene expression in Xenopus laevis embryos, NF stage 34, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	ammecr1 (Alport syndrome, mental retardation, midface hypoplasia and elliptocytosis chromosomal region gene 1 ) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, dorsal view, anterior left.
	ammecr1 (Alport syndrome, mental retardation, midface hypoplasia and elliptocytosis chromosomal region gene 1 ) gene expression in Xenopus laevis embryos, NF stage 29, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	csnk2a1(casein kinase 2, alpha 1 polypeptide) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, dorsal view, anterior left.
	csnk2a1(casein kinase 2, alpha 1 polypeptide) gene expression in Xenopus laevis embryos, NF stage 29, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	dach1 (dachshund homolog 1) gene expression in Xenopus laevis embryos, NF stage 20, as assayed by in situ hybridization, dorsal view, anterior left.
	dach1 (dachshund homolog 1) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	kansl2 (KAT8 regulatory NSL complex subunit 2) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	kansl2 (KAT8 regulatory NSL complex subunit 2) gene expression in Xenopus laevis embryos, NF stage 29, as assayed by in situ hybridization, lateral view: dorsal up, anterior left.
	LRRN1 (leucine rich repeat neuronal 1) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, dorsal view, anterior left.
	LRRN1 (leucine rich repeat neuronal 1) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	olfm4 (olfactomedin 4) gene expression in Xenopus laevis embryos, NF stage 20, as assayed by in situ hybridization. Dorsal view: anterior left. Image extracted from XB-IMG-79890
	olfm4 (olfactomedin 4) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization lateral view, dorsal up, anterior left.
	prkca (protein kinase C, alpha) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, dorsal view, anterior left.
	ptpn3 (protein tyrosine phosphatase non-receptor type 3) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, dorsal viewd, anterior left.
	ptpn3 (protein tyrosine phosphatase non-receptor type 3) gene expression in Xenopus laevis embryos, NF stage 29, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	sall1 (spalt-like transcription factor 1) gene expression in Xenopus laevis embryos, NF stage 18, assayed by in situ hybridization, dorsal view, anterior left.
	sall1 (sal-like 1) gene expression in Xenopus laevis embryos, NF stage 34, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	snrpe (small nuclear ribonucleoprotein polypeptide E) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	snrpe (small nuclear ribonucleoprotein polypeptide E) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization., lateral view, dorsal up, anterior left.
	spdl1 (spindle apparatus coiled-coil protein 1) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, lateral view, dorsal up, anterior left. \
	spdl1 (spindle apparatus coiled-coil protein 1) gene expression in Xenopus laevis embryos, NF stage 29, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	stk3 (serine/threonine kinase 3) gene expression in Xenopus laevis embryos, NF stage 14, as assayed by in situ hybridization, dorsal view,anterior left.
	stk3 (serine/threonine kinase 3) gene expression in Xenopus laevis embryos, NF stage 29, as assayed by in situ hybridization, lateral view,dorsal up, anterior left.
	Supplemental Fig. 1. Alignments of candidate BREs among BMP targets. Shown is a screenshot capture of alignments for each BRE-like sequence and the flanking nucleotide sequences from a subset of the genes shown in Table 1. Each BRE-like element found is highlighted in dark green and other regions of homology are denoted by light green. Shown are approximately 30 base pairs of flanking sequence both upstream and downstream of each BRE-like element. Regions without homology are not shaded. For each gene, the nucleotide sequence for every BRE element found and adjacent sequence is shown. A Logo representation of the consensus sequence (Cons.) for the BRE elements and flanking sequence is shown below the alignments (Crooks et al., 2004). Note that, for SNRPE, only one BRE site is shown as this gene has a second BRE site on the complement strand with identical sequence to the other SNPRE site over the range shown.