Slc7a11 Modulated by POU2F1 is Involved in Pigmentation in Rabbit

Solute carrier family 7 member 11 (Slc7a11) is a cystine/glutamate xCT transporter that controls the production of pheomelanin pigment to change fur and skin color in animals. Previous studies have found that skin expression levels of Slc7a11 varied significantly with fur color in Rex rabbits. However, the molecular regulation mechanism of Slc7a11 in pigmentation is unknown. Here, rabbit melanocytes were first isolated and identified. The distribution and expression pattern of Slc7a11 was confirmed in skin from rabbits with different fur colors. Slc7a11 affected the expression of pigmentation related genes and thus affected melanogenesis. Meanwhile, Slc7a11 decreased melanocyte apoptosis, but inhibition of Slc7a11 enhanced apoptosis. Furthermore, the POU2F1 protein was found to bind to the −713 to −703 bp region of Slc7a11 promoter to inhibit its activity in a dual-luciferase reporter and site-directed mutagenesis assay. This study reveals the function of the Slc7a11 in melanogenesis and provides in-depth analysis of the mechanism of fur pigmentation.

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Slc7a11 modulated by POU2F1 is involved in pigmentation in rabbit

10.1101/607978 ◽

2019 ◽

Author(s):

Yang Chen ◽

Shuaishuai Hu ◽

Lin Mu ◽

Bohao Zhao ◽

Manman Wang ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Molecular Regulation ◽

Luciferase Reporter ◽

Regulation Mechanism ◽

Solute Carrier Family ◽

Rabbit Skin ◽

Depth Analysis ◽

Control Production ◽

Solute Carrier ◽

Rex Rabbit

AbstractSolute carrier family 7 member 11 (Slc7a11) codes for a cystine/glutamate xCT transporter and can control production of pheomelanin pigment to change fur and skin colors of animals. Previous studies found that the skin expression levels of Slc7a11 varied significantly with the fur colors of the Rex Rabbit. However, it is not yet known the molecular regulation mechanism of Slc7a11 in pigmentation. Here rabbit melanocytes were first isolated and identified. The distribution and expression pattern of Slc7a11 was confirmed in rabbit skin with different fur colors. Slc7a11 could affect the expression of pigmentation related genes and thus affect melanogenesis. Meanwhile, Slc7a11 decreased melanocytes apoptosis, but inhibition of Slc7a11 enhanced apoptosis. Furthermore, it was found that POU2F1 protein bound to the -713 to -703 bp region of Slc7a11 promoter to inhibite its activity by dual-luciferase reporter and site-directed mutagenesis assay. This study uncover the function of the Slc7a11 in melanogenesis and provided in-depth analysis of the mechanism of fur pigmentation.

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Functional Dissection of a Poliovirus cis-Acting Replication Element [PV-cre(2C)]: Analysis of Single- and Dual-cre Viral Genomes and Proteins That Bind Specifically to PV-cre RNA

Journal of Virology ◽

10.1128/jvi.77.9.5152-5166.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5152-5166 ◽

Cited By ~ 58

Author(s):

Jiang Yin ◽

Aniko V. Paul ◽

Eckard Wimmer ◽

Elizabeth Rieder

Keyword(s):

Specific Binding ◽

Site Directed Mutagenesis ◽

Luciferase Reporter ◽

Directed Mutagenesis ◽

Coding Region ◽

Luciferase Reporter Gene ◽

Successful Recovery ◽

Nontranslated Region

ABSTRACT The role of the cis replication element (cre) in the 2CATPase coding region of the poliovirus (PV) genome has been studied with a series of mutants derived from either a PV1 full-length genome or a replicon (P/L) containing the firefly luciferase reporter gene in place of the capsid region. Using the P/L replicon we have inserted cre elements at three different locations in the genome including the 5′ nontranslated region and within the open reading frame. The successful recovery of replication of a nonviable P/L (A5C) mutant replicon with an artificial cre element as “rescuer,” in addition to the results of site-directed mutagenesis and experiments with truncated forms of PV-cre(2C), indicated that (i) the sequence within the upper stem and loop regions contains the minimal cre RNA required for VPg uridylylation in vitro, (ii) the location of the cre RNA in the poliovirus genome is not relevant to RNA infectivity, and (iii) specific binding of 3CDpro to PV-cre(2C) occurs within the upper stem region and probably involves several contact residues. The role of a 14-nucleotide conserved “core” sequence among known cre structures in picornaviruses was examined by site-directed mutagenesis of individual nucleotides. In addition to a conserved AAA (4472 to 4474) triplet previously shown to be the primary RNA template for VPg uridylylation by the PV RNA polymerase 3Dpol (E. Rieder, A. V. Paul, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10371-10380, 2000), we have now shown that important residues (G4468 and A4481) are contained in a predicted internal bulge at the upper stem-loop of PV-cre(2C). We have further demonstrated that the viral proteins 3CDpro and 3Cpro form stable complexes with a transcript PV-cre(2C) RNA that can be considered critical for VPg uridylylation.

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NSD2 Promotes Tumor Angiogenesis Through Methylating and Activating STAT3 Signaling

10.21203/rs.3.rs-73073/v1 ◽

2020 ◽

Author(s):

Da Song ◽

Jingqin Lan ◽

Yaqi Chen ◽

Anyi Liu ◽

Qi Wu ◽

...

Keyword(s):

Mass Spectrometry ◽

Signaling Pathway ◽

Tumor Angiogenesis ◽

Tumor Therapy ◽

Site Directed Mutagenesis ◽

Cell Counting ◽

Directed Mutagenesis ◽

Regulation Mechanism ◽

Stat3 Signaling ◽

Stat3 Signaling Pathway

Abstract Background：Tumor angiogenesis plays important roles in tumorigenesis and development, the regulation mechanism of angiogenesis is still not been fully elucidated. Nuclear receptor binding SET domain protein 2 (NSD2), a histone methyltransferase which catalyzes the di-methylation of histone H3 at lysine 36, has been proved a critical molecule in proliferation, metastasis and tumorigenesis. But its role in tumor angiogenesis remains unknown.Methods： Cell Counting Kit 8 (CCK8), scratch assays, transwell-migration assays, tube-formation and mice xenograft model assays were used to confirm the role of NSD2 in the bioprocess of angiogenesis. Bioinformatics analysis, western blot and immunofluorescence staining were used to verify the function of NSD2 in regulating STAT3 signaling pathway. Immunofluorescence co-localization, immunoprecipitation, mass spectrometry and site-directed mutagenesis were performed to determine the NSD2-dependent methylation site of STAT3.Results: Here we demonstrated that NSD2 promoted tumor angiogenesis in vitro and in vivo. Furthermore, we confirmed that the angiogenic function of NSD2 was mediated by STAT3. Momentously, we found that NSD2 promoted the methylation of STAT3 and that the inhibition of STAT3 methylation resulted in the attenuation of STAT3 signaling pathway. In addition, mass spectrometry and site-directed mutagenesis assays revealed that NSD2 methylated STAT3 at lysine 163 (K163), and K163 was of significance in the activation of STAT3 signaling pathway.Conclusion: We conclude that methylation of STAT3 catalyzed by NSD2 promotes the activation of STAT3 pathway and enhances the ability of tumor angiogenesis. Our findings investigate a NSD2 dependent methylation-phosphorylation regulation pattern of STAT3 and reveal that NSD2/STAT3/VEGFA axis might be a potential target for tumor therapy.

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