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Ligand-dependent corepressor contributes to transcriptional repression by C2H2 zinc-finger transcription factor ZBRK1 through association with KRAB-associated protein-1.

Calderon MR, Verway M, Benslama RO, Birlea M, Bouttier M, Dimitrov V, Mader S, White JH - Nucleic Acids Res. (2014)

Bottom Line: We identified a novel interaction between ligand-dependent corepressor (LCoR) and the corepressor KRAB-associated protein-1 (KAP-1).We propose that ZBRK1, KAP-1 and LCoR form a transcriptional complex that silences gene expression, in particular FGF2, which maintains breast cell viability.Given the broad expression patterns of both LCoR and KAP-1 during development and in the adult, this complex may have several regulatory functions that extend beyond cell survival, mediated by interactions with ZBRK1 or other C2H2 zinc-finger proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, McGill University, Montreal, QC, Canada.

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Gene knockdowns increase FGF2 secretion and alter MCF-7 cell viability. (A) FGF2 secretion was measured by ELISA after 48-h or 72-h siRNA-mediated gene knockdowns and normalized to cell counts at each time point. (B) Caspase 3/7 activity was measured with a caspase 3/7 activity detection kit after 24 or 65-h siRNA-mediated gene knockdowns. (C) Confirmation of abrogation of FGF2 mRNA increase after gene knockdowns. RT-qPCR of joint (regulator+FGF2) siRNA-mediated knockdowns in MCF-7 cells. (D) Cytotoxicity of cells as measured by the LDH assay (Roche) 65 h after gene knockdowns. (E) Cytotoxicity of cells as measured by the LDH assay (Roche) 65 h after gene knockdowns and incubation with water (control) or 100-μM suramin sodium (FGF2 inhibitor). One-way ANOVA followed by the Tukey test for multiple comparisons was performed to determine statistical significance in all cases.
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Figure 6: Gene knockdowns increase FGF2 secretion and alter MCF-7 cell viability. (A) FGF2 secretion was measured by ELISA after 48-h or 72-h siRNA-mediated gene knockdowns and normalized to cell counts at each time point. (B) Caspase 3/7 activity was measured with a caspase 3/7 activity detection kit after 24 or 65-h siRNA-mediated gene knockdowns. (C) Confirmation of abrogation of FGF2 mRNA increase after gene knockdowns. RT-qPCR of joint (regulator+FGF2) siRNA-mediated knockdowns in MCF-7 cells. (D) Cytotoxicity of cells as measured by the LDH assay (Roche) 65 h after gene knockdowns. (E) Cytotoxicity of cells as measured by the LDH assay (Roche) 65 h after gene knockdowns and incubation with water (control) or 100-μM suramin sodium (FGF2 inhibitor). One-way ANOVA followed by the Tukey test for multiple comparisons was performed to determine statistical significance in all cases.

Mentions: Previous studies revealed that increased levels of the secreted form of FGF2 reduce MCF-7 cell viability (21–23). As presented above, ablation of LCoR, KAP-1 or ZBRK1 did not lead to an apoptotic response in T47D cells, which do not express FGF2. To determine if increased FGF2 secretion resulted from ablation of ZBRK1, KAP-1 or LCoR in MCF-7 cells, we performed an ELISA for FGF2 in media supernatants. FGF2 secretion was enhanced about 4-fold 48 h after ablation of ZBRK1 or KAP-1, whereas another 24 h were necessary to further elevate (3-fold) FGF2 secretion after LCoR ablation (Figure 6A), consistent with delayed loss of LCoR expression (Supplementary Figure S5B).


Ligand-dependent corepressor contributes to transcriptional repression by C2H2 zinc-finger transcription factor ZBRK1 through association with KRAB-associated protein-1.

Calderon MR, Verway M, Benslama RO, Birlea M, Bouttier M, Dimitrov V, Mader S, White JH - Nucleic Acids Res. (2014)

Gene knockdowns increase FGF2 secretion and alter MCF-7 cell viability. (A) FGF2 secretion was measured by ELISA after 48-h or 72-h siRNA-mediated gene knockdowns and normalized to cell counts at each time point. (B) Caspase 3/7 activity was measured with a caspase 3/7 activity detection kit after 24 or 65-h siRNA-mediated gene knockdowns. (C) Confirmation of abrogation of FGF2 mRNA increase after gene knockdowns. RT-qPCR of joint (regulator+FGF2) siRNA-mediated knockdowns in MCF-7 cells. (D) Cytotoxicity of cells as measured by the LDH assay (Roche) 65 h after gene knockdowns. (E) Cytotoxicity of cells as measured by the LDH assay (Roche) 65 h after gene knockdowns and incubation with water (control) or 100-μM suramin sodium (FGF2 inhibitor). One-way ANOVA followed by the Tukey test for multiple comparisons was performed to determine statistical significance in all cases.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: Gene knockdowns increase FGF2 secretion and alter MCF-7 cell viability. (A) FGF2 secretion was measured by ELISA after 48-h or 72-h siRNA-mediated gene knockdowns and normalized to cell counts at each time point. (B) Caspase 3/7 activity was measured with a caspase 3/7 activity detection kit after 24 or 65-h siRNA-mediated gene knockdowns. (C) Confirmation of abrogation of FGF2 mRNA increase after gene knockdowns. RT-qPCR of joint (regulator+FGF2) siRNA-mediated knockdowns in MCF-7 cells. (D) Cytotoxicity of cells as measured by the LDH assay (Roche) 65 h after gene knockdowns. (E) Cytotoxicity of cells as measured by the LDH assay (Roche) 65 h after gene knockdowns and incubation with water (control) or 100-μM suramin sodium (FGF2 inhibitor). One-way ANOVA followed by the Tukey test for multiple comparisons was performed to determine statistical significance in all cases.
Mentions: Previous studies revealed that increased levels of the secreted form of FGF2 reduce MCF-7 cell viability (21–23). As presented above, ablation of LCoR, KAP-1 or ZBRK1 did not lead to an apoptotic response in T47D cells, which do not express FGF2. To determine if increased FGF2 secretion resulted from ablation of ZBRK1, KAP-1 or LCoR in MCF-7 cells, we performed an ELISA for FGF2 in media supernatants. FGF2 secretion was enhanced about 4-fold 48 h after ablation of ZBRK1 or KAP-1, whereas another 24 h were necessary to further elevate (3-fold) FGF2 secretion after LCoR ablation (Figure 6A), consistent with delayed loss of LCoR expression (Supplementary Figure S5B).

Bottom Line: We identified a novel interaction between ligand-dependent corepressor (LCoR) and the corepressor KRAB-associated protein-1 (KAP-1).We propose that ZBRK1, KAP-1 and LCoR form a transcriptional complex that silences gene expression, in particular FGF2, which maintains breast cell viability.Given the broad expression patterns of both LCoR and KAP-1 during development and in the adult, this complex may have several regulatory functions that extend beyond cell survival, mediated by interactions with ZBRK1 or other C2H2 zinc-finger proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, McGill University, Montreal, QC, Canada.

Show MeSH
Related in: MedlinePlus