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Plakoglobin as a regulator of desmocollin gene expression.

Tokonzaba E, Chen J, Cheng X, Den Z, Ganeshan R, Műller EJ, Koch PJ - J. Invest. Dermatol. (2013)

Bottom Line: Specifically, we found that Lef-1 acts as a switch activating Dsc2 and repressing Dsc3 in the presence of Pg.Interestingly, we also determined that NF-κB pathway components, the downstream effectors of the ectodysplasin-A (EDA)/ ectodysplasin-A receptor (EDAR)/NF-κB signaling cascade, can activate Dsc2 expression.It is tempting to speculate that this shift is required for the invasive growth of placode keratinocytes into the dermis, a crucial step in skin appendage formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Colorado School of Medicine, Aurora, Colorado, USA.

ABSTRACT
Desmosomes are cell adhesion junctions required for the normal development and maintenance of mammalian tissues and organs such as the skin, skin appendages, and the heart. The goal of this study was to investigate how desmocollins (DSCs), transmembrane components of desmosomes, are regulated at the transcriptional level. We hypothesized that differential expression of the Dsc2 and Dsc3 genes is a prerequisite for normal development of skin appendages. We demonstrate that plakoglobin (Pg) in conjunction with lymphoid enhancer-binding factor 1 (Lef-1) differentially regulates the proximal promoters of these two genes. Specifically, we found that Lef-1 acts as a switch activating Dsc2 and repressing Dsc3 in the presence of Pg. Interestingly, we also determined that NF-κB pathway components, the downstream effectors of the ectodysplasin-A (EDA)/ ectodysplasin-A receptor (EDAR)/NF-κB signaling cascade, can activate Dsc2 expression. We hypothesize that Lef-1 and EDA/EDAR/NF-κB signaling contribute to a shift in Dsc isoform expression from Dsc3 to Dsc2 in placode keratinocytes. It is tempting to speculate that this shift is required for the invasive growth of placode keratinocytes into the dermis, a crucial step in skin appendage formation.

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Pg co-localizes in the nucleus with Lef1 and disrupts TCF/Lef transcription factor binding to the Dsc3 promoter(a) Co-Immunoprecipitation (Co-IP) assays demonstrating an interaction between Pg and Lef-1. (b) ChIP assays demonstrating that increasing amounts of Pg (measured in g plasmid transfected) interfere with the binding of Lef-1 to the Dsc3 promoter. Input, chromatin used for immunoprecipitation; IgG, IP with unspecific IgG. (c, d) Western blot analysis of MDCK cells transfected with Pg and Lef-1 (transfection constructs shown on top; NT, not transfected). The nuclear (N) and cytoplasmic (C) distribution of the proteins is shown. Antibodies used to detect Lef-1 and the KT3-tagged plakoglobin construct are shown on the left sides of the blots. Our Lef-1 antibody does not detect endogenous Lef-1 expression in MDCK cells. Lamin B1 (nuclear fraction) and α-tubulin (cytoplasmic fraction) antibodies were used as controls. (e) Immunofluorescence microscopy of MDCK cells transfected with Pg and Lef-1. The antibodies used for staining are indicated. Note the nuclear co-localization of Pg and Lef-1 in several cells (arrows). Bar, 50 μm
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Figure 3: Pg co-localizes in the nucleus with Lef1 and disrupts TCF/Lef transcription factor binding to the Dsc3 promoter(a) Co-Immunoprecipitation (Co-IP) assays demonstrating an interaction between Pg and Lef-1. (b) ChIP assays demonstrating that increasing amounts of Pg (measured in g plasmid transfected) interfere with the binding of Lef-1 to the Dsc3 promoter. Input, chromatin used for immunoprecipitation; IgG, IP with unspecific IgG. (c, d) Western blot analysis of MDCK cells transfected with Pg and Lef-1 (transfection constructs shown on top; NT, not transfected). The nuclear (N) and cytoplasmic (C) distribution of the proteins is shown. Antibodies used to detect Lef-1 and the KT3-tagged plakoglobin construct are shown on the left sides of the blots. Our Lef-1 antibody does not detect endogenous Lef-1 expression in MDCK cells. Lamin B1 (nuclear fraction) and α-tubulin (cytoplasmic fraction) antibodies were used as controls. (e) Immunofluorescence microscopy of MDCK cells transfected with Pg and Lef-1. The antibodies used for staining are indicated. Note the nuclear co-localization of Pg and Lef-1 in several cells (arrows). Bar, 50 μm

Mentions: In order to gain further insights in the mechanisms by which Pg and Lef-1 control Dsc gene expression, we assessed whether Pg can bind to Lef-1. As shown in Figure 3a, Lef-1 and Pg can form a complex as shown by co-immunoprecipitation experiments using Lef-1 antibodies. Most interestingly, ChIP competition experiments suggested that the interaction of Pg and Lef-1 interferes with the ability of Lef-1 to bind to the Dsc3 promoter (Figure 3b). Considering that TCF/Lef-1 complexes can act as transcriptional repressors [e.g. (Hoverter and Waterman, 2008)], these results raise the possibility that Pg could activate Dsc3 expression by interfering with the binding of a TCF/Lef repressor complex to the Dsc3 promoter. Further support for this hypothesis is provided by Western blot experiments (Figure 3c-d) demonstrating that ectopically expressed Pg can interfere with the nuclear accumulation of Lef-1 and thus potentially suppress the formation of a repressor complex at the Dsc3 promoter. On the other hand, Lef-1 is required to shuttle Pg into the cell nucleus, where it activates the Dsc2 promoter as shown in Figure 3d-e. The data summarized above demonstrate that Lef-1 and Pg localize to the nucleus, which is predicted to lead to an activation of the Dsc2 gene and a suppression of the Dsc3 gene.


Plakoglobin as a regulator of desmocollin gene expression.

Tokonzaba E, Chen J, Cheng X, Den Z, Ganeshan R, Műller EJ, Koch PJ - J. Invest. Dermatol. (2013)

Pg co-localizes in the nucleus with Lef1 and disrupts TCF/Lef transcription factor binding to the Dsc3 promoter(a) Co-Immunoprecipitation (Co-IP) assays demonstrating an interaction between Pg and Lef-1. (b) ChIP assays demonstrating that increasing amounts of Pg (measured in g plasmid transfected) interfere with the binding of Lef-1 to the Dsc3 promoter. Input, chromatin used for immunoprecipitation; IgG, IP with unspecific IgG. (c, d) Western blot analysis of MDCK cells transfected with Pg and Lef-1 (transfection constructs shown on top; NT, not transfected). The nuclear (N) and cytoplasmic (C) distribution of the proteins is shown. Antibodies used to detect Lef-1 and the KT3-tagged plakoglobin construct are shown on the left sides of the blots. Our Lef-1 antibody does not detect endogenous Lef-1 expression in MDCK cells. Lamin B1 (nuclear fraction) and α-tubulin (cytoplasmic fraction) antibodies were used as controls. (e) Immunofluorescence microscopy of MDCK cells transfected with Pg and Lef-1. The antibodies used for staining are indicated. Note the nuclear co-localization of Pg and Lef-1 in several cells (arrows). Bar, 50 μm
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getmorefigures.php?uid=PMC3760975&req=5

Figure 3: Pg co-localizes in the nucleus with Lef1 and disrupts TCF/Lef transcription factor binding to the Dsc3 promoter(a) Co-Immunoprecipitation (Co-IP) assays demonstrating an interaction between Pg and Lef-1. (b) ChIP assays demonstrating that increasing amounts of Pg (measured in g plasmid transfected) interfere with the binding of Lef-1 to the Dsc3 promoter. Input, chromatin used for immunoprecipitation; IgG, IP with unspecific IgG. (c, d) Western blot analysis of MDCK cells transfected with Pg and Lef-1 (transfection constructs shown on top; NT, not transfected). The nuclear (N) and cytoplasmic (C) distribution of the proteins is shown. Antibodies used to detect Lef-1 and the KT3-tagged plakoglobin construct are shown on the left sides of the blots. Our Lef-1 antibody does not detect endogenous Lef-1 expression in MDCK cells. Lamin B1 (nuclear fraction) and α-tubulin (cytoplasmic fraction) antibodies were used as controls. (e) Immunofluorescence microscopy of MDCK cells transfected with Pg and Lef-1. The antibodies used for staining are indicated. Note the nuclear co-localization of Pg and Lef-1 in several cells (arrows). Bar, 50 μm
Mentions: In order to gain further insights in the mechanisms by which Pg and Lef-1 control Dsc gene expression, we assessed whether Pg can bind to Lef-1. As shown in Figure 3a, Lef-1 and Pg can form a complex as shown by co-immunoprecipitation experiments using Lef-1 antibodies. Most interestingly, ChIP competition experiments suggested that the interaction of Pg and Lef-1 interferes with the ability of Lef-1 to bind to the Dsc3 promoter (Figure 3b). Considering that TCF/Lef-1 complexes can act as transcriptional repressors [e.g. (Hoverter and Waterman, 2008)], these results raise the possibility that Pg could activate Dsc3 expression by interfering with the binding of a TCF/Lef repressor complex to the Dsc3 promoter. Further support for this hypothesis is provided by Western blot experiments (Figure 3c-d) demonstrating that ectopically expressed Pg can interfere with the nuclear accumulation of Lef-1 and thus potentially suppress the formation of a repressor complex at the Dsc3 promoter. On the other hand, Lef-1 is required to shuttle Pg into the cell nucleus, where it activates the Dsc2 promoter as shown in Figure 3d-e. The data summarized above demonstrate that Lef-1 and Pg localize to the nucleus, which is predicted to lead to an activation of the Dsc2 gene and a suppression of the Dsc3 gene.

Bottom Line: Specifically, we found that Lef-1 acts as a switch activating Dsc2 and repressing Dsc3 in the presence of Pg.Interestingly, we also determined that NF-κB pathway components, the downstream effectors of the ectodysplasin-A (EDA)/ ectodysplasin-A receptor (EDAR)/NF-κB signaling cascade, can activate Dsc2 expression.It is tempting to speculate that this shift is required for the invasive growth of placode keratinocytes into the dermis, a crucial step in skin appendage formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Colorado School of Medicine, Aurora, Colorado, USA.

ABSTRACT
Desmosomes are cell adhesion junctions required for the normal development and maintenance of mammalian tissues and organs such as the skin, skin appendages, and the heart. The goal of this study was to investigate how desmocollins (DSCs), transmembrane components of desmosomes, are regulated at the transcriptional level. We hypothesized that differential expression of the Dsc2 and Dsc3 genes is a prerequisite for normal development of skin appendages. We demonstrate that plakoglobin (Pg) in conjunction with lymphoid enhancer-binding factor 1 (Lef-1) differentially regulates the proximal promoters of these two genes. Specifically, we found that Lef-1 acts as a switch activating Dsc2 and repressing Dsc3 in the presence of Pg. Interestingly, we also determined that NF-κB pathway components, the downstream effectors of the ectodysplasin-A (EDA)/ ectodysplasin-A receptor (EDAR)/NF-κB signaling cascade, can activate Dsc2 expression. We hypothesize that Lef-1 and EDA/EDAR/NF-κB signaling contribute to a shift in Dsc isoform expression from Dsc3 to Dsc2 in placode keratinocytes. It is tempting to speculate that this shift is required for the invasive growth of placode keratinocytes into the dermis, a crucial step in skin appendage formation.

Show MeSH
Related in: MedlinePlus