Lee HS et al. (2011), Direct response elements of BMP within the PV.1...

XB-ART-43779

PLoS One 2011 Jan 01;68:e22621. doi: 10.1371/journal.pone.0022621.

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Direct response elements of BMP within the PV.1A promoter are essential for its transcriptional regulation during early Xenopus development.

Lee SY , Lee H , Hwang YS , Cha SW , Park S , Lee JY , Park JB , Kim S , Park MJ , Kim J .

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Xvent homeobox genes encode transcription factors that repress organizer genes and are essential for dorsoventral specification during early embryogenesis in Xenopus. In contrast to the Xvent-2 gene subfamily, Xvent-1 subfamily members, including PV.1A, have been proposed as indirect targets of Bone Morphogenetic Protein-4 (BMP-4) signaling. Because PV.1A is a critical downstream mediator of, and tightly regulated by, BMP-4 signaling, we hypothesized that its promoter contains a direct BMP-4 response element to effect this transcriptional regulation. We demonstrate that direct regulation by BMP-4 is necessary for transcription of PV.1A: its proximal promoter contains cis-acting binding elements for Smads and Oaz crucial to induction in response to BMP-4 signaling. In addition to these direct cis-acting BMP-4 responsive elements, an indirect Xvent-2 response element and several repressive elements exist in the PV.1A promoter to regulate its transcription. In summary, PV.1A undergoes combinatorial regulation during early Xenopus development as both the direct target of BMP-4 signaling and as the direct and indirect target of positive and negative regulatory factors.

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Species referenced: Xenopus laevis
Genes referenced: babam2 bmp4 gsc smad1 uqcc6 ventx1 ventx2.2 znf423

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	Figure 1. PV.1A is a direct target of BMP-4 signaling.(A, B) Animal caps injected with the indicated RNAs (0.5 ng/embryo) were dissected from stage 8.5 embryos and incubated until stage 11. Total RNA was isolated for RT-PCR and assayed to evaluate PV.1A expression. (C, D) Xenopus embryo animal poles were injected at the 2-cell stage with the specified promoter constructs (20 pg/embryo) in the presence or absence of BMP-4 (0.5 ng/embryo). At stage 7.5, embryos were treated with 25 Âµg/mL CHX in 0.5X MBS until control embryos reached stage 8.5. Animal caps were then dissected from the injected embryos and incubated to stage 11 in 0.5X MBS for RT-PCR analysis (C) or measurement of luciferase activity (D). Luciferase activity was measured as described in Materials and Methods. Gene expression was normalized to EF-1Î± transcription. Experiments were repeated three times using independent sample sets. Data are shown as mean Â± SD.
	Figure 2. Identification of a positive-regulatory element in the 5â²-flanking region of PV.1A.A putative element positively regulating PV.1A transcription was identified between â300 and â262 bp from the transcriptional start site. 2-cell stage embryos were injected at their animal poles with 20 pg of serially deleted PV.1A promoter constructs driving luciferase reporter transcription. Animal caps were dissected at stage 8.5 and incubated until stage 11 in 0.5X MBS for measurement of luciferase activity as described in Materials and Methods. Experiments were repeated three times using independent sample sets. Data are shown as mean Â± SD.
	Figure 3. Identification of a BMP-4-response element in the 5â²-flanking region of PV.1A.(A) Serially deleted promoter constructs were injected (20 pg/embryo) with or without BMP-4 (0.5 ng/embryo) into 2-cell stage embryos. Animal caps were dissected from injected embryos at stage 8.5 and incubated until stage 11 in 0.5X MBS for measurement of luciferase activity. A putative BRE was detected between â180 and â162 bp from the transcription start site based on reporter gene expression. The underlined sequence (CAGA) is a consensus binding-site for Smad proteins. Luciferase activity was measured as described in Materials and Methods. (B) 3BRE is a luciferase fusion construct with a triple BRE repeat. The 3BRE construct (20 pg/embryo) was co-injected with DN-BR or BMP-4 (each 0.5 ng/embryo) into 2-cell stage embryos. Animal caps were dissected at stage 8.5 and incubated until stage 11 in 0.5X MBS for measurement of luciferase activity as described in Materials and Methods. (C) The -180 WT and -180 MT constructs (20 pg/embryo) were injected with or without BMP-4 or Smad1 (each 0.5 ng/embryo) into 2-cell stage embryos. -180 MT indicates a construct with mutated BRE. Luciferase activity was measured as described in Materials and Methods. The sequences underlined indicate alterations in the original sequences. Experiments were repeated three times using independent sample sets. Data are shown as mean Â± SD.
	Figure 4. Oaz and BMP-4 cooperate to regulate PV.1A transcription.-180, -180 MT, and ORE(M)-180 constructs (20 pg/embryo) were injected with or without BMP-4 (0.5 ng/embryo) and Oaz (0.1 ng/embryo) into the animal poles of 2-cell stage embryos. Animal caps were dissected from injected embryos at stage 8.5 and incubated until stage 11 in 0.5X MBS for measurement of luciferase activity as described in Materials and Methods. The sequences underlined depict alterations to the original sequences. Experiments were repeated three times using independent sample sets. Data are shown as mean Â± SD.
	Figure 5. Positive regulation by Xvent-2 of the 5â²-flanking region of PV.1A.(A) Serial-deletion mutant constructs (â162, â136, and â103; 20 pg/embryo) of the PV.1A promoter were co-injected with Xvent-2 (0.5 ng/embryo) into the animal poles of 2-cell stage embryos. Animal caps were dissected from injected embryos at stage 8.5 and incubated until stage 11 in 0.5X MBS for measurement of luciferase activity (as described in Materials and Methods). A putative Xvent-2 binding site was detected between â136 and â103 bp from the major transcription initiation site. The underlined sequence, TAAT, is a consensus binding-site for homeobox proteins. (B) The â136 and XRE(M)-136 constructs were injected (20 pg/embryo) with or without Xvent-2 (0.5 ng/embryo) at the animal poles of 2-cell stage embryos. Luciferase activity was measured as described in Materials and Methods. The sequences underlined indicate alterations to original sequences. Experiments were repeated three times using independent sample sets. Data are shown as mean Â± SD.

References [+] :

Ault, A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos. 1996, Pubmed, Xenbase