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Functional Characterization of a Single Nucleotide Polymorphism in the 3' Untranslated Region of Sheep DLX3 Gene.

Rong E, Zhang Z, Qiao S, Yang H, Yan X, Li H, Wang N - PLoS ONE (2015)

Bottom Line: We previously identified and found that four SNPs (c. *118T>C, c. *228T>C, c. *688A>G and c. *1,038_1,039 insC) in 3' untranslated region (UTR) of sheep DLX3 were in high linkage disequilibrium with each other and significantly associated with wool crimp (P<0.05), however, the underlying mechanisms by which these SNPs affect the wool crimp remains unknown.The results showed that these SNPs were significantly associated with sheep skin DLX3 mRNA expression levels.However, neither miR-188 mimc nor inhibitor could influence the allele I (insertion) reporter activity.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Chicken Genetics and Breeding at Ministry of Agriculture, Key Laboratory of Animal Genetics, Breeding and Reproduction at Education Department of Heilongjiang Province, Harbin, 150030, China; College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, China; State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, 100193, China.

ABSTRACT
The Distal-less 3 (homeobox protein DLX-3), a transcription factor, is critical for the development of hair follicle and hair formation and regeneration. We previously identified and found that four SNPs (c. *118T>C, c. *228T>C, c. *688A>G and c. *1,038_1,039 insC) in 3' untranslated region (UTR) of sheep DLX3 were in high linkage disequilibrium with each other and significantly associated with wool crimp (P<0.05), however, the underlying mechanisms by which these SNPs affect the wool crimp remains unknown. In the present study, we performed association analysis between these four identified SNPs and DLX3 gene expression in sheep skin using quantitative real-time RT-PCR. The results showed that these SNPs were significantly associated with sheep skin DLX3 mRNA expression levels. Then, we constructed DLX3 3'UTR luciferase reporters and validated the association. The reporter assays showed that the three major haplotypes, derived from the four SNPs, had significantly different effects on luciferase reporter activity and the four SNPs also had significantly different allelic effects on the luciferase reporter activity (p < 0.05). Bioinformatics analysis showed that the SNP (c. *1,038_1,039 insC) was located within a potential miR-188 binding site of the 3'UTR of sheep DLX3 mRNA. This SNP may affect miR-188-mediated DLX3 gene expression and result in phenotypic variation. To test the hypothesis, we investigated the effects of miR-188 mimic and inhibitor on the activity of the DLX3 3'UTR luciferase reporter with different SNP alleles. The results showed that in both sheep fetal fibroblasts (SFFs) and human HaCaT cells, miR-188 mimic could significantly decrease the allele D (deletion) luciferase reporter activity (p < 0.05), but miR-188 inhibitor could increased the reporter activitiy. However, neither miR-188 mimc nor inhibitor could influence the allele I (insertion) reporter activity. In addition, transfection of miR-188 mimic dramatically decreased the endogenous expression of DLX3 in SFFs (p < 0.05). Taken together, we demonstrated that DLX3 is a target gene of miR-188 and the SNP (c. *1,038_1,039 insC) is a functional SNP, and affects miR-188-mediated gene regulation of sheep DLX3. Our finding may in part explain allelic difference in gene expression and wool crimp in our tested sheep population.

No MeSH data available.


Related in: MedlinePlus

Effects of three major haplotypes and alleles of the four SNPs on DLX3 3′UTR reporter gene activity.(A) Relative luciferase activities of the three major haplotypes of sheep DLX3 3′UTR. The reporter activity was expressed as relative luciferase activity (Rluc/Fluc ratio). The psiCHECK2 empty vector was used as a control. Different letters (a, b, c and d) above columns indicate significant difference between groups (p < 0.05). (B) Strategy applied to construct novel haplotypes. Three major haplotypes (CCGI, TTAD and TCAD) were present in our tested population. In order to investigate the allelic effects of each of these four SNPs, two novel haplotypes (TCGI and TCAI) were constructed by use of a One-Tube Point Mutation Kit (TIANDZ, China). The allelic effects of SNPs 1 to 4 on reporter gene activity were measured by comparing between CCGI and TCGI for SNP1, between TTAD type and TCAD for SNP2, between TCGI and TCAI for SNP3, and between TCAI and TCAD for SNP4, respectively. (C) Relative luciferase activity of the haplotypes of sheep DLX3 3′UTR. The psiCHECK2 empty vector was used as a control. Different letters (a, b and c) above columns indicate significant difference between groups (p < 0.05). All reporter assays were performed in SFFs. Data are representative of at least three independent experiments (error bars, S.D.).
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pone.0137135.g001: Effects of three major haplotypes and alleles of the four SNPs on DLX3 3′UTR reporter gene activity.(A) Relative luciferase activities of the three major haplotypes of sheep DLX3 3′UTR. The reporter activity was expressed as relative luciferase activity (Rluc/Fluc ratio). The psiCHECK2 empty vector was used as a control. Different letters (a, b, c and d) above columns indicate significant difference between groups (p < 0.05). (B) Strategy applied to construct novel haplotypes. Three major haplotypes (CCGI, TTAD and TCAD) were present in our tested population. In order to investigate the allelic effects of each of these four SNPs, two novel haplotypes (TCGI and TCAI) were constructed by use of a One-Tube Point Mutation Kit (TIANDZ, China). The allelic effects of SNPs 1 to 4 on reporter gene activity were measured by comparing between CCGI and TCGI for SNP1, between TTAD type and TCAD for SNP2, between TCGI and TCAI for SNP3, and between TCAI and TCAD for SNP4, respectively. (C) Relative luciferase activity of the haplotypes of sheep DLX3 3′UTR. The psiCHECK2 empty vector was used as a control. Different letters (a, b and c) above columns indicate significant difference between groups (p < 0.05). All reporter assays were performed in SFFs. Data are representative of at least three independent experiments (error bars, S.D.).

Mentions: To functionally validate the association of the identified SNPs with the sheep skin DLX3 expression, we cloned the 3′UTRs of the three major haplotypes (CCGI, TTAD and TCAD) based on these four SNPs, and generated their respective 3´UTR haplotype reporters. Reporter assays showed that reporter activities were significantly different among these three major haplotypes (p < 0.05) (Fig 1A). Of these three major haplotypes, CCGI haplotype reporter had the lowest reporter activity, and TCAD haplotype reporter had the highest reporter activity in SFFs. Consistent with the association results,these reporter assay results also suggest that some of these SNPs are functional and affect DLX3 gene expression. Then we generated two more novel haplotype reporters (psiCHECK2-TCGI and psiCHECK2-TCAI) using site-directed mutagenesis (Fig 1B) and detected the allelic effects of the four SNPs on luciferase reporter activities by comparing between psiCHECK2-CCGI and psiCHECK2-TCGI for SNP1, between psiCHECK2-TTAD and psiCHECK2-TCAD for SNP2, between psiCHECK2-TCGI and psiCHECK2-TCAI for SNP3, between psiCHECK2-TCAI and psiCHECK2-TCAD for SNP4, respectively. Surprisingly, these four SNPs had significantly different allelic effects on the luciferase reporter activity (Fig 1A and 1C) (p < 0.05), suggesting all of these four SNPs may additively or synergistically affect reporter gene expression posttranscritionally.


Functional Characterization of a Single Nucleotide Polymorphism in the 3' Untranslated Region of Sheep DLX3 Gene.

Rong E, Zhang Z, Qiao S, Yang H, Yan X, Li H, Wang N - PLoS ONE (2015)

Effects of three major haplotypes and alleles of the four SNPs on DLX3 3′UTR reporter gene activity.(A) Relative luciferase activities of the three major haplotypes of sheep DLX3 3′UTR. The reporter activity was expressed as relative luciferase activity (Rluc/Fluc ratio). The psiCHECK2 empty vector was used as a control. Different letters (a, b, c and d) above columns indicate significant difference between groups (p < 0.05). (B) Strategy applied to construct novel haplotypes. Three major haplotypes (CCGI, TTAD and TCAD) were present in our tested population. In order to investigate the allelic effects of each of these four SNPs, two novel haplotypes (TCGI and TCAI) were constructed by use of a One-Tube Point Mutation Kit (TIANDZ, China). The allelic effects of SNPs 1 to 4 on reporter gene activity were measured by comparing between CCGI and TCGI for SNP1, between TTAD type and TCAD for SNP2, between TCGI and TCAI for SNP3, and between TCAI and TCAD for SNP4, respectively. (C) Relative luciferase activity of the haplotypes of sheep DLX3 3′UTR. The psiCHECK2 empty vector was used as a control. Different letters (a, b and c) above columns indicate significant difference between groups (p < 0.05). All reporter assays were performed in SFFs. Data are representative of at least three independent experiments (error bars, S.D.).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4558038&req=5

pone.0137135.g001: Effects of three major haplotypes and alleles of the four SNPs on DLX3 3′UTR reporter gene activity.(A) Relative luciferase activities of the three major haplotypes of sheep DLX3 3′UTR. The reporter activity was expressed as relative luciferase activity (Rluc/Fluc ratio). The psiCHECK2 empty vector was used as a control. Different letters (a, b, c and d) above columns indicate significant difference between groups (p < 0.05). (B) Strategy applied to construct novel haplotypes. Three major haplotypes (CCGI, TTAD and TCAD) were present in our tested population. In order to investigate the allelic effects of each of these four SNPs, two novel haplotypes (TCGI and TCAI) were constructed by use of a One-Tube Point Mutation Kit (TIANDZ, China). The allelic effects of SNPs 1 to 4 on reporter gene activity were measured by comparing between CCGI and TCGI for SNP1, between TTAD type and TCAD for SNP2, between TCGI and TCAI for SNP3, and between TCAI and TCAD for SNP4, respectively. (C) Relative luciferase activity of the haplotypes of sheep DLX3 3′UTR. The psiCHECK2 empty vector was used as a control. Different letters (a, b and c) above columns indicate significant difference between groups (p < 0.05). All reporter assays were performed in SFFs. Data are representative of at least three independent experiments (error bars, S.D.).
Mentions: To functionally validate the association of the identified SNPs with the sheep skin DLX3 expression, we cloned the 3′UTRs of the three major haplotypes (CCGI, TTAD and TCAD) based on these four SNPs, and generated their respective 3´UTR haplotype reporters. Reporter assays showed that reporter activities were significantly different among these three major haplotypes (p < 0.05) (Fig 1A). Of these three major haplotypes, CCGI haplotype reporter had the lowest reporter activity, and TCAD haplotype reporter had the highest reporter activity in SFFs. Consistent with the association results,these reporter assay results also suggest that some of these SNPs are functional and affect DLX3 gene expression. Then we generated two more novel haplotype reporters (psiCHECK2-TCGI and psiCHECK2-TCAI) using site-directed mutagenesis (Fig 1B) and detected the allelic effects of the four SNPs on luciferase reporter activities by comparing between psiCHECK2-CCGI and psiCHECK2-TCGI for SNP1, between psiCHECK2-TTAD and psiCHECK2-TCAD for SNP2, between psiCHECK2-TCGI and psiCHECK2-TCAI for SNP3, between psiCHECK2-TCAI and psiCHECK2-TCAD for SNP4, respectively. Surprisingly, these four SNPs had significantly different allelic effects on the luciferase reporter activity (Fig 1A and 1C) (p < 0.05), suggesting all of these four SNPs may additively or synergistically affect reporter gene expression posttranscritionally.

Bottom Line: We previously identified and found that four SNPs (c. *118T>C, c. *228T>C, c. *688A>G and c. *1,038_1,039 insC) in 3' untranslated region (UTR) of sheep DLX3 were in high linkage disequilibrium with each other and significantly associated with wool crimp (P<0.05), however, the underlying mechanisms by which these SNPs affect the wool crimp remains unknown.The results showed that these SNPs were significantly associated with sheep skin DLX3 mRNA expression levels.However, neither miR-188 mimc nor inhibitor could influence the allele I (insertion) reporter activity.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Chicken Genetics and Breeding at Ministry of Agriculture, Key Laboratory of Animal Genetics, Breeding and Reproduction at Education Department of Heilongjiang Province, Harbin, 150030, China; College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, China; State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, 100193, China.

ABSTRACT
The Distal-less 3 (homeobox protein DLX-3), a transcription factor, is critical for the development of hair follicle and hair formation and regeneration. We previously identified and found that four SNPs (c. *118T>C, c. *228T>C, c. *688A>G and c. *1,038_1,039 insC) in 3' untranslated region (UTR) of sheep DLX3 were in high linkage disequilibrium with each other and significantly associated with wool crimp (P<0.05), however, the underlying mechanisms by which these SNPs affect the wool crimp remains unknown. In the present study, we performed association analysis between these four identified SNPs and DLX3 gene expression in sheep skin using quantitative real-time RT-PCR. The results showed that these SNPs were significantly associated with sheep skin DLX3 mRNA expression levels. Then, we constructed DLX3 3'UTR luciferase reporters and validated the association. The reporter assays showed that the three major haplotypes, derived from the four SNPs, had significantly different effects on luciferase reporter activity and the four SNPs also had significantly different allelic effects on the luciferase reporter activity (p < 0.05). Bioinformatics analysis showed that the SNP (c. *1,038_1,039 insC) was located within a potential miR-188 binding site of the 3'UTR of sheep DLX3 mRNA. This SNP may affect miR-188-mediated DLX3 gene expression and result in phenotypic variation. To test the hypothesis, we investigated the effects of miR-188 mimic and inhibitor on the activity of the DLX3 3'UTR luciferase reporter with different SNP alleles. The results showed that in both sheep fetal fibroblasts (SFFs) and human HaCaT cells, miR-188 mimic could significantly decrease the allele D (deletion) luciferase reporter activity (p < 0.05), but miR-188 inhibitor could increased the reporter activitiy. However, neither miR-188 mimc nor inhibitor could influence the allele I (insertion) reporter activity. In addition, transfection of miR-188 mimic dramatically decreased the endogenous expression of DLX3 in SFFs (p < 0.05). Taken together, we demonstrated that DLX3 is a target gene of miR-188 and the SNP (c. *1,038_1,039 insC) is a functional SNP, and affects miR-188-mediated gene regulation of sheep DLX3. Our finding may in part explain allelic difference in gene expression and wool crimp in our tested sheep population.

No MeSH data available.


Related in: MedlinePlus