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Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

Chen H, Babino D, Schoenbichler SA, Arkhipova V, Töchterle S, Martin F, Huck CW, von Lintig J, Meyer D - PLoS ONE (2015)

Bottom Line: We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production.Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization.HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology/CMBI, University of Innsbruck, Technikerstrasse 25, 6020, Innsbruck, Austria.

ABSTRACT
Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

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rbp7a is a direct target of FoxH1.[A] Schematic drawing of the rbp7a gene highlighting 4 potential FoxH1 binding sites (triangles: S1, S2, S3, and S4). Exons are indicated by boxes, the numbers correspond to the distance form the transcriptional start site. [B] Sequence comparison of the mouse FoxH1 consensus log [37] with the four potential sites in rbp7a. [C] In vitro EMSA studies with translated FoxH1 protein with oligonucleotides containing the four potential FoxH1 binding sites (sequences and positions were shown in [A, B]). Competition experiments with unspecific (unsp.comp) and specific (self.comp) unlabeled oligonucleotides were added to verify the binding specificity. [D] In vivo chromatin immunoprecipitation (ChIP-qPCR) experiments performed with 6hpf eGFP-foxH1 mRNA injected MZsur embryos. Bars show the enrichments of DNA fragments in the regions of FoxH1 binding sites in relation to a negative control region (rhodopsin promoter region) that was lacking FoxH1 binding sites (*P<0.05; error bars indicated the SEM). [E] Induced and depleted rbp7a expression in 5hpf wild type embryos after injection of fkh-vp16 and fkh-en mRNA, respectively.
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pone.0143825.g002: rbp7a is a direct target of FoxH1.[A] Schematic drawing of the rbp7a gene highlighting 4 potential FoxH1 binding sites (triangles: S1, S2, S3, and S4). Exons are indicated by boxes, the numbers correspond to the distance form the transcriptional start site. [B] Sequence comparison of the mouse FoxH1 consensus log [37] with the four potential sites in rbp7a. [C] In vitro EMSA studies with translated FoxH1 protein with oligonucleotides containing the four potential FoxH1 binding sites (sequences and positions were shown in [A, B]). Competition experiments with unspecific (unsp.comp) and specific (self.comp) unlabeled oligonucleotides were added to verify the binding specificity. [D] In vivo chromatin immunoprecipitation (ChIP-qPCR) experiments performed with 6hpf eGFP-foxH1 mRNA injected MZsur embryos. Bars show the enrichments of DNA fragments in the regions of FoxH1 binding sites in relation to a negative control region (rhodopsin promoter region) that was lacking FoxH1 binding sites (*P<0.05; error bars indicated the SEM). [E] Induced and depleted rbp7a expression in 5hpf wild type embryos after injection of fkh-vp16 and fkh-en mRNA, respectively.

Mentions: As FoxH1 is known to function mainly as a transcriptional activator, we investigated whether repression of rbp7a occurred by direct protein/DNA interaction. In silico screening of the rbp7a genomic region for the consensus motif of FoxH1 [37] revealed four putative binding sites (S1, S2, S3 and S4) (Fig 2A and 2B). Electrophoretic mobility shift assays (EMSAs) confirmed in vitro binding of the zebrafish FoxH1 protein to all four binding sites (Fig 2C). The specificity of these bindings was further confirmed by competition assays with DNA containing a mutated FoxH1 consensus sequence and with unlabeled oligonucleotides. To assess whether FoxH1 binds to these sites in vivo, we carried out chromatin immunoprecipitation (ChIP) assays with 6 hpf embryos. Analysis of the immunoprecipitated DNA fragments by quantitative PCR revealed an approximately 3.5-fold enrichment of DNA containing the S2 binding site (Fig 2D), but no enrichment for the S1, S3 or S4 binding sites. Finally, injection of mRNA encoding a transcription activating form of FoxH1 (fkh-VP16) or a constitutive repressing FoxH1 form (fkh-en) [25] caused ectopic expressions or loss of rbp7a expression, respectively (Fig 2E). These results are consistent with a potentially direct repression of rbp7a by FoxH1.


Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

Chen H, Babino D, Schoenbichler SA, Arkhipova V, Töchterle S, Martin F, Huck CW, von Lintig J, Meyer D - PLoS ONE (2015)

rbp7a is a direct target of FoxH1.[A] Schematic drawing of the rbp7a gene highlighting 4 potential FoxH1 binding sites (triangles: S1, S2, S3, and S4). Exons are indicated by boxes, the numbers correspond to the distance form the transcriptional start site. [B] Sequence comparison of the mouse FoxH1 consensus log [37] with the four potential sites in rbp7a. [C] In vitro EMSA studies with translated FoxH1 protein with oligonucleotides containing the four potential FoxH1 binding sites (sequences and positions were shown in [A, B]). Competition experiments with unspecific (unsp.comp) and specific (self.comp) unlabeled oligonucleotides were added to verify the binding specificity. [D] In vivo chromatin immunoprecipitation (ChIP-qPCR) experiments performed with 6hpf eGFP-foxH1 mRNA injected MZsur embryos. Bars show the enrichments of DNA fragments in the regions of FoxH1 binding sites in relation to a negative control region (rhodopsin promoter region) that was lacking FoxH1 binding sites (*P<0.05; error bars indicated the SEM). [E] Induced and depleted rbp7a expression in 5hpf wild type embryos after injection of fkh-vp16 and fkh-en mRNA, respectively.
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getmorefigures.php?uid=PMC4664474&req=5

pone.0143825.g002: rbp7a is a direct target of FoxH1.[A] Schematic drawing of the rbp7a gene highlighting 4 potential FoxH1 binding sites (triangles: S1, S2, S3, and S4). Exons are indicated by boxes, the numbers correspond to the distance form the transcriptional start site. [B] Sequence comparison of the mouse FoxH1 consensus log [37] with the four potential sites in rbp7a. [C] In vitro EMSA studies with translated FoxH1 protein with oligonucleotides containing the four potential FoxH1 binding sites (sequences and positions were shown in [A, B]). Competition experiments with unspecific (unsp.comp) and specific (self.comp) unlabeled oligonucleotides were added to verify the binding specificity. [D] In vivo chromatin immunoprecipitation (ChIP-qPCR) experiments performed with 6hpf eGFP-foxH1 mRNA injected MZsur embryos. Bars show the enrichments of DNA fragments in the regions of FoxH1 binding sites in relation to a negative control region (rhodopsin promoter region) that was lacking FoxH1 binding sites (*P<0.05; error bars indicated the SEM). [E] Induced and depleted rbp7a expression in 5hpf wild type embryos after injection of fkh-vp16 and fkh-en mRNA, respectively.
Mentions: As FoxH1 is known to function mainly as a transcriptional activator, we investigated whether repression of rbp7a occurred by direct protein/DNA interaction. In silico screening of the rbp7a genomic region for the consensus motif of FoxH1 [37] revealed four putative binding sites (S1, S2, S3 and S4) (Fig 2A and 2B). Electrophoretic mobility shift assays (EMSAs) confirmed in vitro binding of the zebrafish FoxH1 protein to all four binding sites (Fig 2C). The specificity of these bindings was further confirmed by competition assays with DNA containing a mutated FoxH1 consensus sequence and with unlabeled oligonucleotides. To assess whether FoxH1 binds to these sites in vivo, we carried out chromatin immunoprecipitation (ChIP) assays with 6 hpf embryos. Analysis of the immunoprecipitated DNA fragments by quantitative PCR revealed an approximately 3.5-fold enrichment of DNA containing the S2 binding site (Fig 2D), but no enrichment for the S1, S3 or S4 binding sites. Finally, injection of mRNA encoding a transcription activating form of FoxH1 (fkh-VP16) or a constitutive repressing FoxH1 form (fkh-en) [25] caused ectopic expressions or loss of rbp7a expression, respectively (Fig 2E). These results are consistent with a potentially direct repression of rbp7a by FoxH1.

Bottom Line: We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production.Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization.HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology/CMBI, University of Innsbruck, Technikerstrasse 25, 6020, Innsbruck, Austria.

ABSTRACT
Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

Show MeSH