Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.E16-04-0249

Figure Lengend Snippet: HGF-induced MMP expression in the absence versus the presence of GRHL2.

Article Snippet: GAL4- p300 HAT (aa 1283–1674), GAL4- p300C-terminus (aa 1665–2414), and GAL4- p300 IBID (aa 2050–2096) were generated by subcloning PCR fragments ( Mlu I- No tI) into pBIND (Promega).

Techniques: Expressing

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.E16-04-0249

Figure Lengend Snippet: GRHL2 suppresses AP-1 and p300 function. (A) GRHL2 suppresses AP1 function. HT1080 and 293T cells were cotransfected with AP-1 response element-luciferase reporter construct and GRHL2 expression vector or empty vector. PMA was used to induce AP-1 signaling in 293T cells. Values represent relative luciferase activity normalized to TK-β-galactosidase control. (B) Venn diagram comparing GRHL2-repressed genes ( Farris et al. , 2016 ) vs. p300 target genes identified via E1a ( Ferrari et al. , 2014 ). (C) GRHL2 suppresses the p300 pathway. p300 effector and target genes induced by HGF in MDCK cells with GRHL2 shRNA vs. cells with constitutive GRHL2 determined by Ingenuity Pathway Analysis. Quantitation of the number of unregulated, HGF up-regulated, or HGF down-regulated genes for both cell lines. The p300 interactome diagram on which this is based is shown in Supplemental Figure S7. (D) GRHL2 suppresses p300 function. GAL4-minimal promoter-luciferase activation by a cotransfected GAL4-p300 was assayed in HT1080 cells in the presence or absence of cotransfected GRHL2 expression vector; values represent relative luciferase activity normalized to TK-β-galactosidase control.

Article Snippet: GAL4- p300 HAT (aa 1283–1674), GAL4- p300C-terminus (aa 1665–2414), and GAL4- p300 IBID (aa 2050–2096) were generated by subcloning PCR fragments ( Mlu I- No tI) into pBIND (Promega).

Techniques: Luciferase, Construct, Expressing, Plasmid Preparation, Activity Assay, shRNA, Quantitation Assay, Activation Assay

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.E16-04-0249

Figure Lengend Snippet: GRHL2 suppresses the HAT activity of p300. (A) In vitro HAT assays with recombinant H3, p300, and GRHL2 proteins. Coomassie stains to assess the quality of recombinant proteins used throughout this study are shown in Supplemental Figure S19. (B) GRHL2 inhibits the acetylation of H3 on GRHL2-repressed but not GRHL2-induced promoters in vivo (ChIP assay). Cross-linked chromatin from HT1080+ vector HT-1080+GRHL2 cells immunoprecipitated with H3K27-Ac or rabbit IgG; the indicated promoters were assayed for H3K27Ac by qPCR using the primers in Materials and Methods . * p < 0.05. GRHL2 does not affect global histone H3K18-Ac or H3K27-Ac (Western blotting of total histones). (C) GRHL2 interacts with p300. Left, cotransfection of indicated expression constructs, followed by coimmunoprecipitation/Western blotting. Middle, coimmunoprecipitation of retrovirally expressed, S-tagged GRHL2 with endogenous p300. Right, coimmunoprecipitation of endogenous GRHL2 and endogenous P300.

Article Snippet: GAL4- p300 HAT (aa 1283–1674), GAL4- p300C-terminus (aa 1665–2414), and GAL4- p300 IBID (aa 2050–2096) were generated by subcloning PCR fragments ( Mlu I- No tI) into pBIND (Promega).

Techniques: Activity Assay, In Vitro, Recombinant, In Vivo, Plasmid Preparation, Immunoprecipitation, Western Blot, Cotransfection, Expressing, Construct

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.E16-04-0249

Figure Lengend Snippet: GRHL2 inhibits the C-terminal transactivation domain of p300. (A) The p300 HAT domain stimulates transactivation by the p300 C-terminus (cotransfection in HT1080 cells). (B) GRHL2 inhibits transactivation by the p300 C-terminus. HT1080 cells were cotransfected with GAL4 response element luciferase reporter in the presence of the indicated expression constructs. (C) The IBID domain of p300 and the coactivator SRC-1 synergize to activate transcription. HT1080 cells were cotransfected with GAL4 response element luciferase reporter I in the presence of the indicated expression constructs. (D) GRHL2 inhibits transactivation by the IBID-SRC-1 complex. HT1080 cells were cotransfected with GAL4 response element luciferase reporter in the presence of the indicated expression constructs. (E) Transactivation by the IBID-SRC-1 complex is contingent upon lysines 2086 and 2091. HT1080 cells were cotransfected with GAL4 response element luciferase reporter in the presence of the indicated expression constructs. In A–E, values represent relative luciferase activity normalized to TK-β-galactosidase control. (F) Schematic of p300 domains. Bd, bromodomain; HAT, histone acetyltransferase domain; IBID, IRF-3 binding domain; KIX, CREB-binding domain.

Article Snippet: GAL4- p300 HAT (aa 1283–1674), GAL4- p300C-terminus (aa 1665–2414), and GAL4- p300 IBID (aa 2050–2096) were generated by subcloning PCR fragments ( Mlu I- No tI) into pBIND (Promega).

Techniques: Cotransfection, Luciferase, Expressing, Construct, Activity Assay, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.E16-04-0249

Figure Lengend Snippet: A small region within the DNA-binding domain of GRHL2, aa 425–437, inhibits p300. (A) Schematic of GRHL2 domains. DBD, DNA-binding domain; DD, dimerization domain; TAD, transactivation domain. (B) HAT assays using the indicated GRHL2 fragments, assayed as GST-fusion proteins. (C) GRHL2 aa 425–437 inhibits p300 HAT activity. The indicated fragments derived from GRHL2 were assayed as GST-fusions (left) or recombinant peptides (right) for inhibition of HAT activity. (D) GRHL2 aa 425–437 are required for inhibition of an AP-1 reporter, GAL4 reporter in conjunction with GAL4-p300, MMP1 reporter, or MMP14 reporter. Values represent relative luciferase activity normalized to TK-β-galactosidase control.

Article Snippet: GAL4- p300 HAT (aa 1283–1674), GAL4- p300C-terminus (aa 1665–2414), and GAL4- p300 IBID (aa 2050–2096) were generated by subcloning PCR fragments ( Mlu I- No tI) into pBIND (Promega).

Techniques: Binding Assay, Activity Assay, Derivative Assay, Recombinant, Inhibition, Luciferase

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.E16-04-0249

Figure Lengend Snippet: The p300-inhibitory domain of GRHL2 is important for the suppression of tubulogenesis and reversion of EMT. (A) MDCK cells expressing GRHL2 wild-type or GRHL2 Δ425-437 were assayed for tubulogenesis (blue, nuclei; green, actin). Scale bar, 20 μm. Middle, quantitation; right, Western blot confirmation of protein levels. (B) MSP cells expressing GRHL2 wild-type or GRHL2 Δ425-437 were assayed for reversion of EMT by Western blotting for EMT markers (left), cell morphology (middle), or qPCR (right).

Article Snippet: GAL4- p300 HAT (aa 1283–1674), GAL4- p300C-terminus (aa 1665–2414), and GAL4- p300 IBID (aa 2050–2096) were generated by subcloning PCR fragments ( Mlu I- No tI) into pBIND (Promega).

Techniques: Expressing, Quantitation Assay, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.E16-04-0249

Figure Lengend Snippet: GRHL2 suppresses tubulogenesis and EMT by inhibition of p300.

Article Snippet: GAL4- p300 HAT (aa 1283–1674), GAL4- p300C-terminus (aa 1665–2414), and GAL4- p300 IBID (aa 2050–2096) were generated by subcloning PCR fragments ( Mlu I- No tI) into pBIND (Promega).

Techniques: Inhibition