Rehman ZU et al. (2022), Bmp4 Synexpression Gene, Sizzled, Transcription...

XB-ART-59454

Int J Mol Sci 2022 Nov 01;2321:. doi: 10.3390/ijms232113335.

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Bmp4 Synexpression Gene, Sizzled, Transcription Is Collectively Modulated by Smad1 and Ventx1.1/Ventx2.1 in Early Xenopus Embryos.

Rehman ZU , Tayyaba F , Lee U , Kim J .

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Sizzled (Szl) is a secreted frizzled protein, having a sequence homology with the extracellular cysteine-rich domain (CRD) of the Wnt receptor, 'Frizzled'. Contrary to the other secreted frizzled like proteins (Sfrps), szl belongs to the bone morphogenetic protein 4 (Bmp4) synexpression group and is tightly coexpressed with Bmp4. What is not known is how the szl transcription achieves its Bmp4 synexpression pattern. To address the molecular details of szl transcription control, we cloned a promoter of size 1566 base pairs for szl (bps) from the Xenopus laevis genomic DNA. Luciferase and eGFP reporter gene results of this szl promoter (-1566 bp) in its activation and repression patterns by Bmp4/Smad1 and a dominant negative Bmp4 receptor (DNBR) were similar to those of the endogenous szl expression. Reporter gene assays and site-directed mutagenesis of the szl promoter mapped an active Bmp4/Smad1 response element (BRE) and a cis-acting element, which competitively share a direct binding site for Ventx1.1 and Ventx2.1 (a Ventx response element, VRE). Smad1 and ventx2.1 alone increased szl promoter activity; in addition, the binding of each protein component was enhanced with their coexpression. Interestingly, Ventx1.1 repressed this reporter gene activity; however, Ventx1.1 and Ventx2.1 together positively regulated the szl promoter activity. From our analysis, Ventx2.1 binding was enhanced by Ventx1.1, but Ventx1.1 inhibitory binding was inhibited by co-injection of Ventx2.1 for the VRE site. The inhibitory Ventx1.1 co-injection decreased Smad1 binding on the szl promoter. In a triple combination of overexpressed Smad1/Ventx1.1/Ventx2.1, the reduced binding of Smad1 from Ventx1.1 was recovered to that of the Smad1/Ventx2 combination. Collectively, this study provides evidence of Bmp4/Smad1 signaling for a primary immediate early response and its two oppositely behaving target transcription factors, Ventx1.1 and Ventx2.1, for a secondary response, as they together upregulate the szl promoter's activity to achieve szl expression in a Bmp4 synexpression manner.

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Species referenced: Xenopus laevis
Genes referenced: babam2 bambi bmp4 bmp7 foxd4l1 gata2 myc smad1 szl ventx1 ventx2 ventx2.2

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	Figure 1. Bmp4 signaling modulates the isolated promoter activity of szl(-1566bp) in a stage-dependent manner: (a) Xenopus leavis embryos were injected with bmp4, dominant negative Bmp4 receptor (dnbr) and smad1 mRNAs (1 ng/embryo) at the one-cell stage and animal caps (10 AC/sample) were dissected at stage 8 and grown up to stage 11. Ventral marker genes, including szl mRNA expression, were assayed through quantitative RT-PCR analysis. (UI: uninjected.) The qPCR values were normalized to ODC. (b) The Szl(-1566) promoter was cloned into pGL3-luc+ vector. (c) Analysis of szl expression at various developmental stages in the presence of Bmp4, the reporter gene activity of the szl(-1566) promoter construct injected with bmp4 mRNA was assayed. Five sets of three, three embryos were used for each sample in each developmental stage. (a,c) Unpaired two-tailed Studentâs t-test or ANOVA were applied for statistical analysis. p â¤ 0.05 for , p â¤ 0.01 for , p â¤ 0.001 for , p â¤ 0.0001 for **, ns (non-significant) were the assignments for significance. (d) Szl(-1566) promoter was cloned into the pGL3-eGFP+ vector (luc was replaced with eGFP). (e) Embryos (30 embryos/sample) were injected with the reporter construct of szl(-1566)-eGFP+ with or without dnbr mRNA and their eGFP fluorescence was observed at stages 10.5, 12 and 24. (D: dorsal, V: ventral.). The arrows indicate the regions of eGFP fluorescence appeared with szl(-1566)-eGFP+ without dnbr mRNA and decreased with dnbr mRNA.
	Figure 2. Bmp4 signaling upregulates szl reporter gene activity via Smad1 regulatory response elements within the szl promoter: (a) Schematic diagram of serially deleted constructs of szl promoter. (b) Reporter gene activities of the serially deleted constructs of szl promoter injected with or without bmp4 and (c) smad1 mRNAs. (d) Conserved region of szl promoter consisting of a Bmp4 response element (BRE) (outlined within the dashed red box). (e) Luciferase reporter gene activity of szl(-370) wild type and mutated BRE constructs injected with or without smad1 mRNA were examined. (b,c,e) p â¤ 0.01 for , p â¤ 0.001 for , p â¤ 0.0001 for ***, ns (non-significant) were the assignments for significance. (f) Embryos were injected with 3flag-smad1 mRNA and quantitative ChIP-PCR was performed with anti-Flag immunoprecipitated genomic DNA using specific primers for the szl promoter fragment having the Smad1 binding site. Fold enrichment method used to normalize ChIP-qPCR. Smad1 binding scheme is given in the upper part of figure. (SRE: Smad1 response elements).
	Figure 3. Ventx1.1 and Ventx2.1 mediated szl transcriptional modulation acts as a secondary Bmp4 signaling response: (a) Green fluorescence of embryos injected with szl(-1566)-eGFP+ in combination of ventx1.1 and/or ventx2.1 mRNAs at the one-cell stage was observed at stage 11. (25 embryos were injected per sample) (b) Embryos were injected with, without or with some combination of ventx1.1/ventx2.1 mRNAs. Animal cap explants (10 AC/sample) were dissected at stage eight and relative expression of ventral specific marker genes, including szl at gastrula stage, through quantitative RT-PCR was performed. Ct values were normalized to ODC, a housekeeping gene. (c) Serially deleted constructs of the szl promoter were injected with or without ventx1.1 and (d) ventx2.1 mRNAs. Reporter gene activities were analyzed. (e) Szl (-1566)-expressing embryos were injected with, without or with some combination of ventx1.1/ventx2.1 mRNAs and analyzed for luciferase activity. (bâe) p â¤ 0.05 for , p â¤ 0.01 for , p â¤ 0.001 for , p â¤ 0.0001 for **, ns (non-significant) were the assignments for significance. (f) Fragment of szl promoter having the putative Ventx response elements (VRE, highlighted within the dashed red box).
	Figure 4. One VRE (ventx cis-acting response element) site for Ventx1.1/2.1 binding in szl promoter is required for szl transcription: (a) Szl(-370) wild type or szl(-370)mVRE were injected with the indicated mRNAs and their combination. Luciferase activities of the samples were analyzed. (b) Embryos (300 embryos/sample) were injected alone or with the combination of 3flag-ventx1.1 and myc-ventx2.1 mRNAs (1 ng/embryo) and cultured to the gastrula stage. ChIP-PCR analysis using specific primers for szl promoter region having the VRE site was performed. (c) Graph represents the relative band intensities of ChIP-PCR bands. (a,c) p â¤ 0.05 for , p â¤ 0.001 for , p â¤ 0.0001 for **, ns (non-significant) were the assignments for significance. (d) Immunoprecipitation assay were performed for 3F.Ventx1.1 and Myc.Ventx2.1 protein-protein interaction during szl promoter binding. (30 embryos/sample) (e) A model of Ventx1.1/2.1 alone and combination mediated regulation of szl transcription. The numbers indicate the number of lane shown on (e), indicating that each schematic model diagram is related with and based on the result(s) of lane(s) shown on (b).
	Figure 5. Smad1 enhances Ventx2.1-mediated szl activation and rescues Ventx1.1 mediated szl suppression. (a) Embryos were injected with smad1, ventx1.1 and ventx2.1 mRNAs individually and in different combinations. Animal cap explants (10 AC/sample) were dissected at stage eight and quantitative RT-PCR of ventral specific marker genes including szl at gastrula stage were analyzed. V1.1 = Ventx1.1, V2.1 = Ventx2.1 (b) Szl(-1566) promoter construct were injected with smad1, ventx1.1 and ventx2.1 mRNA alone and possible combinations and reporter gene activities were analyzed. (c) Xenopus leavis embryos (300 embryos/sample) were injected with ha.smad1, 3flag.ventx1.1, myc.ventx2.1 mRNAs alone and their possible combinations (0.5 ng/embryo each) and cultured to the gastrula stage. ChIP-PCR analysis using specific primers for the szl promoter region having the VRE site was performed. The asterisks of different colors indicate the difference of antibodies used for ChIP; red () for Myc.Ventx2.1, green () for Flag.Ventx1.1 and blue () for HA.Smad1. (d) Quantitative PCR analysis was performed with fold enrichment method and the experiment was repeated three times. (a,b,d) p â¤ 0.05 for , p â¤ 0.01 for , p â¤ 0.001 for , p â¤ 0.0001 for ***, ns (non-significant) were the assignments for significance. (e) A model of Smad1 and Ventx1.1/2.1 alone and in combinations mediated regulation of szl transcription. The numbers indicate the number of lane shown on (c), indicating that each schematic model diagram is related with and based on the result(s) of lane(s) shown on (c).
	Figure 6. Proposed model of the szl transcriptional regulation by Bmp4 signaling via Smad1/Ventx1.1/Ventx2.1 axis. This schematic overview of szl transcriptional regulation during Bmp4 synexpression fashion is based on our findings above. Szl promoter has two types of Bmp4 response elements required for synexpression; they are the Smad1 binding response element (SRE) and the Ventx binding response element (VRE). Smad1 binds to SRE (reported here as TCTG) while Ventx1.1 and Ventx2.1 bind to one VRE (described herein as TAAATT) to regulate szl expression. Ventx1.1 reduces szl expression, while Ventx2.1 induces it, and its combination further enhances it, compared to Ventx2.1 presence alone.
	Figure S1. Spatio-temporal expression pattern of szl and Bmp4 modulated promoter activities of synexpression genes. (a) Developmental expression of szl and ventx family transcription factors. (b) Dorso-ventral distribution of representative marker genes. (c and d) Bmp7.1.L (-3145).luc+ and bambi.S (-3073).luc+ promoters respectively were injected with and without dnbr mRNA at 1 cell stage of Xenopus leavis embryos to measure relative reporter gene activities.(c and d) Unpaired two-tailed Studentâs t-test or ANOVA were applied for statistical analysis. p â¤ 0.0001 for **, ns(non-significant) were the assignments for significance. a b c d e RLU bmp7.1.L(-3145) bmp7.1.L(-3145)+DNBR 0.0 0.5 1.0 1.5 bmp7.1.L (-3145) DNBR+ +- + bambi.S(-3073) bambi.S(-3073)+DN-BR 0.0 0.5 1.0 1.5 RLU (Fold) bambi.S (-3073) DNBR + + - + ** (e) The bambi(-3073).eGFP promoter were injected with and without dnbr mRNAs to visualize fluorescence at developmental stages 11, 15 and 24 and the number of embryos are indicated in the down left corner.
	Figure S2. Bmp4 signaling directly targets szl gene expression during gastrulation.(a) RT-PCR of ventral specific genes including szl expression in the animal cap explants treated with bmp4 mRNA, cycloheximide and combination to check the direct and indirect targets of Bmp4 signaling. (b) Relative reporter gene assay was performed from Xenopus embryos treated with szl serially deleted constructs alone and in combination with dnbr mRNA. p â¤ 0.0001 for ****, ns(non-significant) were the assignments for significance.
	Figure S3. One VRE site (work as a secondary BRE site) is required for Ventx1.1/Ventx2.1 mediated szl transcriptional regulation. (a) Ventx1.1 and Ventx2.1 negatively and positively regulate szl transcription respectively while together ventx1.1/2.1 over expression enhances the szl transcription in whole embryo during early gastrulation. (b) Bmp4/Smad1 response elements (BRE) and Ventxs response elements (VRE) are highlighted in szl(-370) construct.

References [+] :

Bijakowski, Sizzled is unique among secreted frizzled-related proteins for its ability to specifically inhibit bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases. 2012, Pubmed, Xenbase

	Figure 1. Bmp4 signaling modulates the isolated promoter activity of szl(-1566bp) in a stage-dependent manner: (a) Xenopus leavis embryos were injected with bmp4, dominant negative Bmp4 receptor (dnbr) and smad1 mRNAs (1 ng/embryo) at the one-cell stage and animal caps (10 AC/sample) were dissected at stage 8 and grown up to stage 11. Ventral marker genes, including szl mRNA expression, were assayed through quantitative RT-PCR analysis. (UI: uninjected.) The qPCR values were normalized to ODC. (b) The Szl(-1566) promoter was cloned into pGL3-luc+ vector. (c) Analysis of szl expression at various developmental stages in the presence of Bmp4, the reporter gene activity of the szl(-1566) promoter construct injected with bmp4 mRNA was assayed. Five sets of three, three embryos were used for each sample in each developmental stage. (a,c) Unpaired two-tailed Studentâs t-test or ANOVA were applied for statistical analysis. p â¤ 0.05 for , p â¤ 0.01 for , p â¤ 0.001 for , p â¤ 0.0001 for **, ns (non-significant) were the assignments for significance. (d) Szl(-1566) promoter was cloned into the pGL3-eGFP+ vector (luc was replaced with eGFP). (e) Embryos (30 embryos/sample) were injected with the reporter construct of szl(-1566)-eGFP+ with or without dnbr mRNA and their eGFP fluorescence was observed at stages 10.5, 12 and 24. (D: dorsal, V: ventral.). The arrows indicate the regions of eGFP fluorescence appeared with szl(-1566)-eGFP+ without dnbr mRNA and decreased with dnbr mRNA.
	Figure 2. Bmp4 signaling upregulates szl reporter gene activity via Smad1 regulatory response elements within the szl promoter: (a) Schematic diagram of serially deleted constructs of szl promoter. (b) Reporter gene activities of the serially deleted constructs of szl promoter injected with or without bmp4 and (c) smad1 mRNAs. (d) Conserved region of szl promoter consisting of a Bmp4 response element (BRE) (outlined within the dashed red box). (e) Luciferase reporter gene activity of szl(-370) wild type and mutated BRE constructs injected with or without smad1 mRNA were examined. (b,c,e) p â¤ 0.01 for , p â¤ 0.001 for , p â¤ 0.0001 for ***, ns (non-significant) were the assignments for significance. (f) Embryos were injected with 3flag-smad1 mRNA and quantitative ChIP-PCR was performed with anti-Flag immunoprecipitated genomic DNA using specific primers for the szl promoter fragment having the Smad1 binding site. Fold enrichment method used to normalize ChIP-qPCR. Smad1 binding scheme is given in the upper part of figure. (SRE: Smad1 response elements).
	Figure 3. Ventx1.1 and Ventx2.1 mediated szl transcriptional modulation acts as a secondary Bmp4 signaling response: (a) Green fluorescence of embryos injected with szl(-1566)-eGFP+ in combination of ventx1.1 and/or ventx2.1 mRNAs at the one-cell stage was observed at stage 11. (25 embryos were injected per sample) (b) Embryos were injected with, without or with some combination of ventx1.1/ventx2.1 mRNAs. Animal cap explants (10 AC/sample) were dissected at stage eight and relative expression of ventral specific marker genes, including szl at gastrula stage, through quantitative RT-PCR was performed. Ct values were normalized to ODC, a housekeeping gene. (c) Serially deleted constructs of the szl promoter were injected with or without ventx1.1 and (d) ventx2.1 mRNAs. Reporter gene activities were analyzed. (e) Szl (-1566)-expressing embryos were injected with, without or with some combination of ventx1.1/ventx2.1 mRNAs and analyzed for luciferase activity. (bâe) p â¤ 0.05 for , p â¤ 0.01 for , p â¤ 0.001 for , p â¤ 0.0001 for **, ns (non-significant) were the assignments for significance. (f) Fragment of szl promoter having the putative Ventx response elements (VRE, highlighted within the dashed red box).
	Figure 4. One VRE (ventx cis-acting response element) site for Ventx1.1/2.1 binding in szl promoter is required for szl transcription: (a) Szl(-370) wild type or szl(-370)mVRE were injected with the indicated mRNAs and their combination. Luciferase activities of the samples were analyzed. (b) Embryos (300 embryos/sample) were injected alone or with the combination of 3flag-ventx1.1 and myc-ventx2.1 mRNAs (1 ng/embryo) and cultured to the gastrula stage. ChIP-PCR analysis using specific primers for szl promoter region having the VRE site was performed. (c) Graph represents the relative band intensities of ChIP-PCR bands. (a,c) p â¤ 0.05 for , p â¤ 0.001 for , p â¤ 0.0001 for **, ns (non-significant) were the assignments for significance. (d) Immunoprecipitation assay were performed for 3F.Ventx1.1 and Myc.Ventx2.1 protein-protein interaction during szl promoter binding. (30 embryos/sample) (e) A model of Ventx1.1/2.1 alone and combination mediated regulation of szl transcription. The numbers indicate the number of lane shown on (e), indicating that each schematic model diagram is related with and based on the result(s) of lane(s) shown on (b).
	Figure 5. Smad1 enhances Ventx2.1-mediated szl activation and rescues Ventx1.1 mediated szl suppression. (a) Embryos were injected with smad1, ventx1.1 and ventx2.1 mRNAs individually and in different combinations. Animal cap explants (10 AC/sample) were dissected at stage eight and quantitative RT-PCR of ventral specific marker genes including szl at gastrula stage were analyzed. V1.1 = Ventx1.1, V2.1 = Ventx2.1 (b) Szl(-1566) promoter construct were injected with smad1, ventx1.1 and ventx2.1 mRNA alone and possible combinations and reporter gene activities were analyzed. (c) Xenopus leavis embryos (300 embryos/sample) were injected with ha.smad1, 3flag.ventx1.1, myc.ventx2.1 mRNAs alone and their possible combinations (0.5 ng/embryo each) and cultured to the gastrula stage. ChIP-PCR analysis using specific primers for the szl promoter region having the VRE site was performed. The asterisks of different colors indicate the difference of antibodies used for ChIP; red () for Myc.Ventx2.1, green () for Flag.Ventx1.1 and blue () for HA.Smad1. (d) Quantitative PCR analysis was performed with fold enrichment method and the experiment was repeated three times. (a,b,d) p â¤ 0.05 for , p â¤ 0.01 for , p â¤ 0.001 for , p â¤ 0.0001 for ***, ns (non-significant) were the assignments for significance. (e) A model of Smad1 and Ventx1.1/2.1 alone and in combinations mediated regulation of szl transcription. The numbers indicate the number of lane shown on (c), indicating that each schematic model diagram is related with and based on the result(s) of lane(s) shown on (c).
	Figure 6. Proposed model of the szl transcriptional regulation by Bmp4 signaling via Smad1/Ventx1.1/Ventx2.1 axis. This schematic overview of szl transcriptional regulation during Bmp4 synexpression fashion is based on our findings above. Szl promoter has two types of Bmp4 response elements required for synexpression; they are the Smad1 binding response element (SRE) and the Ventx binding response element (VRE). Smad1 binds to SRE (reported here as TCTG) while Ventx1.1 and Ventx2.1 bind to one VRE (described herein as TAAATT) to regulate szl expression. Ventx1.1 reduces szl expression, while Ventx2.1 induces it, and its combination further enhances it, compared to Ventx2.1 presence alone.
	Figure S1. Spatio-temporal expression pattern of szl and Bmp4 modulated promoter activities of synexpression genes. (a) Developmental expression of szl and ventx family transcription factors. (b) Dorso-ventral distribution of representative marker genes. (c and d) Bmp7.1.L (-3145).luc+ and bambi.S (-3073).luc+ promoters respectively were injected with and without dnbr mRNA at 1 cell stage of Xenopus leavis embryos to measure relative reporter gene activities.(c and d) Unpaired two-tailed Studentâs t-test or ANOVA were applied for statistical analysis. p â¤ 0.0001 for **, ns(non-significant) were the assignments for significance. a b c d e RLU bmp7.1.L(-3145) bmp7.1.L(-3145)+DNBR 0.0 0.5 1.0 1.5 bmp7.1.L (-3145) DNBR+ +- + bambi.S(-3073) bambi.S(-3073)+DN-BR 0.0 0.5 1.0 1.5 RLU (Fold) bambi.S (-3073) DNBR + + - + ** (e) The bambi(-3073).eGFP promoter were injected with and without dnbr mRNAs to visualize fluorescence at developmental stages 11, 15 and 24 and the number of embryos are indicated in the down left corner.
	Figure S2. Bmp4 signaling directly targets szl gene expression during gastrulation.(a) RT-PCR of ventral specific genes including szl expression in the animal cap explants treated with bmp4 mRNA, cycloheximide and combination to check the direct and indirect targets of Bmp4 signaling. (b) Relative reporter gene assay was performed from Xenopus embryos treated with szl serially deleted constructs alone and in combination with dnbr mRNA. p â¤ 0.0001 for ****, ns(non-significant) were the assignments for significance.
	Figure S3. One VRE site (work as a secondary BRE site) is required for Ventx1.1/Ventx2.1 mediated szl transcriptional regulation. (a) Ventx1.1 and Ventx2.1 negatively and positively regulate szl transcription respectively while together ventx1.1/2.1 over expression enhances the szl transcription in whole embryo during early gastrulation. (b) Bmp4/Smad1 response elements (BRE) and Ventxs response elements (VRE) are highlighted in szl(-370) construct.