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CtBP1 overexpression in keratinocytes perturbs skin homeostasis.

Deng H, Li F, Li H, Deng Y, Liu J, Wang D, Han G, Wang XJ, Zhang Q - J. Invest. Dermatol. (2013)

Bottom Line: Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype.Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription.In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Dermatology, University of Colorado Denver, Aurora, Colorado, USA [2] Department of Pathology, University of Colorado Denver, Aurora, Colorado, USA [3] Department of Dermatology, The Sixth People's Hospital of Shanghai, Shanghai Jiaotong University, Shanghai, China.

ABSTRACT
Carboxyl-terminal-binding protein-1 (CtBP1) is a transcriptional corepressor with multiple in vitro targets, but its in vivo functions are largely unknown. We generated keratinocyte-specific CtBP1 transgenic mice with a keratin-5 promoter (K5.CtBP1) to probe the pathological roles of CtBP1. At transgene expression levels comparable to endogenous CtBP1 in acute skin wounds, the K5.CtBP1 epidermis displayed hyperproliferation, loss of E-cadherin, and failed terminal differentiation. Known CtBP1 target genes associated with these processes, e.g., p21, Brca1, and E-cadherin, were downregulated in K5.CtBP1 skin. Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype. We found that expression of the Distal-less 3 (Dlx3), a critical regulator of hair follicle differentiation and cycling, was decreased in K5.CtBP1 mice. Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription. Consistently, K5.CtBP1 mice displayed abnormal hair follicles with decreased expression of Dlx3 downstream targets Gata3, Hoxc13, and hair keratins. In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.

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CtBP1 suppresses the Dlx3 gene transcription. (A) Down-regulation of the Dlx3 gene in K5.CtBP1 skins from 9 day-old mice. (B) CtBP1 binding to the Dlx3 promoter. ChIP assay was performed in keratinocytes after transfection with FLAG-tagged CtBP1-expressing vector, either wild type or the PLDLX-binding deficient mutant. ** p<0.01 vs. the PLDLX-binding deficient mutant (mt). (C) CtBP1 transfection represses the Dlx3 reporter. The pGL4.26 Dlx3 promoter reporter was generated by cloning the −286 to 0 bp fragment of the Dlx3 promoter. (D) siCtBP1 increases activity of the Dlx3 reporter. Fadu cells were transfected with scrambled siRNA (SC) or siRNA to CtBP1 (siCtBP1) and luciferase activity was measured.
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Figure 5: CtBP1 suppresses the Dlx3 gene transcription. (A) Down-regulation of the Dlx3 gene in K5.CtBP1 skins from 9 day-old mice. (B) CtBP1 binding to the Dlx3 promoter. ChIP assay was performed in keratinocytes after transfection with FLAG-tagged CtBP1-expressing vector, either wild type or the PLDLX-binding deficient mutant. ** p<0.01 vs. the PLDLX-binding deficient mutant (mt). (C) CtBP1 transfection represses the Dlx3 reporter. The pGL4.26 Dlx3 promoter reporter was generated by cloning the −286 to 0 bp fragment of the Dlx3 promoter. (D) siCtBP1 increases activity of the Dlx3 reporter. Fadu cells were transfected with scrambled siRNA (SC) or siRNA to CtBP1 (siCtBP1) and luciferase activity was measured.

Mentions: Next, we assayed the mRNA level of Dlx3 in K5.CtBP1 skin by qRT-PCR and compared it to that in wildtype littermates. A significant decrease in Dlx3 mRNA was detected in the CtBP1 transgenic skin (Fig. 5A). To determine if CtBP1 plays a direct role in regulation of the Dlx3 gene, we performed chromatin immunoprecipitation (ChIP) to see if CtBP1 is recruited to the Dlx3 promoter in mouse keratinocytes. Mouse keratinocytes were transfected with a vector expressing CtBP1, either wildtype or the PLDLX-binding deficient mutant tagged with the FLAG epitope, and the cross-linked chromatin was immunoprecipitated with an anti-FLAG antibody. Wildtype CtBP1 bound the Dlx3 promoter region while the PLDLX-binding deficient mutant did not (Fig. 5B). This binding is limited to the promoter region, as no signal was detected with PCR amplification of the ChIP material using primers either 5′ or 3′ 2 kb to the Dlx3 promoter (Fig. 5B). Consistent with mRNA changes observed during CtBP1 knockdown, the luciferase activity of the Dlx3 promoter decreased by 60% with CtBP1 over-expression in mouse keratinocytes (Fig. 5C), indicating that CtBP1 regulates Dlx3 transcription, at least partially, via binding to its promoter. To investigate if the CtBP1-mediated repression of Dlx3 gene occurs in human cells, we knocked down CtBP1 in Fadu cells, a human SCC cell line exhibiting high endogenous CtBP1 (Deng et al., 2010), and assayed the Dlx3-luciferase reporter. CtBP1 knockdown increased the Dlx3-luc reporter activity (Fig. 5D), suggesting that CtBP1’s repressive role in Dlx3 transcription is conserved between mouse and human.


CtBP1 overexpression in keratinocytes perturbs skin homeostasis.

Deng H, Li F, Li H, Deng Y, Liu J, Wang D, Han G, Wang XJ, Zhang Q - J. Invest. Dermatol. (2013)

CtBP1 suppresses the Dlx3 gene transcription. (A) Down-regulation of the Dlx3 gene in K5.CtBP1 skins from 9 day-old mice. (B) CtBP1 binding to the Dlx3 promoter. ChIP assay was performed in keratinocytes after transfection with FLAG-tagged CtBP1-expressing vector, either wild type or the PLDLX-binding deficient mutant. ** p<0.01 vs. the PLDLX-binding deficient mutant (mt). (C) CtBP1 transfection represses the Dlx3 reporter. The pGL4.26 Dlx3 promoter reporter was generated by cloning the −286 to 0 bp fragment of the Dlx3 promoter. (D) siCtBP1 increases activity of the Dlx3 reporter. Fadu cells were transfected with scrambled siRNA (SC) or siRNA to CtBP1 (siCtBP1) and luciferase activity was measured.
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Related In: Results  -  Collection

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Figure 5: CtBP1 suppresses the Dlx3 gene transcription. (A) Down-regulation of the Dlx3 gene in K5.CtBP1 skins from 9 day-old mice. (B) CtBP1 binding to the Dlx3 promoter. ChIP assay was performed in keratinocytes after transfection with FLAG-tagged CtBP1-expressing vector, either wild type or the PLDLX-binding deficient mutant. ** p<0.01 vs. the PLDLX-binding deficient mutant (mt). (C) CtBP1 transfection represses the Dlx3 reporter. The pGL4.26 Dlx3 promoter reporter was generated by cloning the −286 to 0 bp fragment of the Dlx3 promoter. (D) siCtBP1 increases activity of the Dlx3 reporter. Fadu cells were transfected with scrambled siRNA (SC) or siRNA to CtBP1 (siCtBP1) and luciferase activity was measured.
Mentions: Next, we assayed the mRNA level of Dlx3 in K5.CtBP1 skin by qRT-PCR and compared it to that in wildtype littermates. A significant decrease in Dlx3 mRNA was detected in the CtBP1 transgenic skin (Fig. 5A). To determine if CtBP1 plays a direct role in regulation of the Dlx3 gene, we performed chromatin immunoprecipitation (ChIP) to see if CtBP1 is recruited to the Dlx3 promoter in mouse keratinocytes. Mouse keratinocytes were transfected with a vector expressing CtBP1, either wildtype or the PLDLX-binding deficient mutant tagged with the FLAG epitope, and the cross-linked chromatin was immunoprecipitated with an anti-FLAG antibody. Wildtype CtBP1 bound the Dlx3 promoter region while the PLDLX-binding deficient mutant did not (Fig. 5B). This binding is limited to the promoter region, as no signal was detected with PCR amplification of the ChIP material using primers either 5′ or 3′ 2 kb to the Dlx3 promoter (Fig. 5B). Consistent with mRNA changes observed during CtBP1 knockdown, the luciferase activity of the Dlx3 promoter decreased by 60% with CtBP1 over-expression in mouse keratinocytes (Fig. 5C), indicating that CtBP1 regulates Dlx3 transcription, at least partially, via binding to its promoter. To investigate if the CtBP1-mediated repression of Dlx3 gene occurs in human cells, we knocked down CtBP1 in Fadu cells, a human SCC cell line exhibiting high endogenous CtBP1 (Deng et al., 2010), and assayed the Dlx3-luciferase reporter. CtBP1 knockdown increased the Dlx3-luc reporter activity (Fig. 5D), suggesting that CtBP1’s repressive role in Dlx3 transcription is conserved between mouse and human.

Bottom Line: Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype.Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription.In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Dermatology, University of Colorado Denver, Aurora, Colorado, USA [2] Department of Pathology, University of Colorado Denver, Aurora, Colorado, USA [3] Department of Dermatology, The Sixth People's Hospital of Shanghai, Shanghai Jiaotong University, Shanghai, China.

ABSTRACT
Carboxyl-terminal-binding protein-1 (CtBP1) is a transcriptional corepressor with multiple in vitro targets, but its in vivo functions are largely unknown. We generated keratinocyte-specific CtBP1 transgenic mice with a keratin-5 promoter (K5.CtBP1) to probe the pathological roles of CtBP1. At transgene expression levels comparable to endogenous CtBP1 in acute skin wounds, the K5.CtBP1 epidermis displayed hyperproliferation, loss of E-cadherin, and failed terminal differentiation. Known CtBP1 target genes associated with these processes, e.g., p21, Brca1, and E-cadherin, were downregulated in K5.CtBP1 skin. Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype. We found that expression of the Distal-less 3 (Dlx3), a critical regulator of hair follicle differentiation and cycling, was decreased in K5.CtBP1 mice. Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription. Consistently, K5.CtBP1 mice displayed abnormal hair follicles with decreased expression of Dlx3 downstream targets Gata3, Hoxc13, and hair keratins. In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.

Show MeSH
Related in: MedlinePlus