human tfap2a construct  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: TFAP2A is functionally associated with TP63-binding sites. ( A ) Effect on the IRF6 enhancer activity of the addition of exogenous TP63 in H1299 cells as measured by luciferase activity. All luciferase graphs represent the mean fold increase of at least three independent biological ± SEM; Student’s t -test * P < 0.05. ( B ) Luciferase assay measuring the effect of the addition of increasing amounts of TFAP2A on TP63-mediated activation of the IRF6 enhancer region in H1299 cells. ( C ) Western blot analysis of exogenous TP63 and TFAP2A expression in H1299 cells. ( D ) Luciferase assay measuring the effect of the addition of increasing amounts of TFAP2A on TP63-mediated activation of the PVRL1 enhancer region in H1299 cells. ( E ) Quantitative ChIP-PCR comparing TP63 and TFAP2A binding to IRF6, FGFR2, JAG2, PVRL1 and PDGFC associated binding sites, expressed as fold enrichment compared to a negative control region within the same ChIP. Data represent the mean of two independent biological replicates. ( F ) Western blot validation of TP63 and TFAP2A depletion in HFKs used in recruitment ChIPs. ( G ) Quantitative ChIP-PCR comparing TP63 and TFAP2A binding to IRF6, FGFR2, JAG2 and PVRL1 associated binding sites in HFKs depleted for TP63 or TFAP2A compared to scrambled control. Results calculated as fold enrichment compared to a negative control region within the same ChIP and normalized to scrambled control to compare between antibodies and experiments. Data represent the mean of two independent biological replicates. ( H ) Quantitative ChIP-PCR of second negative control binding site (BCL2 upstream) in HFKs depleted for TP63 or TFAP2A expressed as fold change compared to scrambled control. Data represent the mean of two independent biological replicates. ( I ) Quantitative RT-PCR quantification of expression of CL/P associated genes in TFAP2A-depleted human foreskin keratinocytes compared to scrambled control. Graph represents the mean fold increase of at least three independent biological ± SEM; Student’s t -test * P < 0.05, ** P < 0.01.

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Binding Assay, Activity Assay, Luciferase, Activation Assay, Western Blot, Expressing, Negative Control, Biomarker Discovery, Control, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: Identification of TFAP2A as a potential TP63 co-factor. ( A ) Table of results from transcription factor motif enrichment analysis of 7574 TP63-binding sites. ( B ) Cumulative frequency distribution plots comparing distance of predicted TFAP2A sites and TP63-binding motifs from the centre of TP63-binding sites/peaks. ( C ) Comparison of localization of TP63-binding sites with or without predicted AP-2 sites. ( D and E ) Quantitative PCR ChIP validation of TP63 (D) and TFAP2A (E) binding to TP63 sites associated with CL/P genes (data represent the mean of three biological replicates expressed as % input ± SEM). ( F ) Semi-quantitative PCR results for sequential re-ChIP assay for TP63 and TFAP2A for the IRF6 , FGFR2, TGFB1 and PVRL1 associated TP63-binding sites, showing both factors co-ChIP.

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Binding Assay, Comparison, Real-time Polymerase Chain Reaction, Biomarker Discovery

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: TFAP2C interacts with a subset of TP63-binding sites. ( A ) Quantitative PCR estimation of TFAP2A and TFAP2C mRNA copy number in cycling HFKs. Data represent the mean of three independent biological replicates ± SEM. ( B ) Quantitative PCR ChIP validation comparing TFAP2A (3B5) and TFAP2C (H77) interaction with a subset of TP63-binding regions associated with CL/P genes. Data represent the mean of three biological replicates expressed as % input ± SEM). ( C and D ) Luciferase assay measuring the effect of the addition of TFAP2A or TFAP2C to six TP63 isoforms on activation of the IRF6 (C) or PVRL1 (D) enhancer region in H1299 cells. ( E ) Luciferase assay measuring the effect of the addition of TFAP2A or TFAP2C on TP63-mediated activation of the IRF6 enhancer region in primary human foreskin keratinocytes (HFKs). Luciferase graphs represent the mean fold increase of at least three independent biological ± SEM; Student’s t -test * P < 0.05, ** P < 0.01.

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Biomarker Discovery, Luciferase, Activation Assay

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: TFAP2A and TFAP2C are required for efficient differentiation of organotypic raft cultures. ( A ) Western blot analysis of TFAP2A, TFAP2C, TP63 and actin loading control of the HFKs transfected with TFAP2A, TFAP2C, TP63 targeting and scrambled control siRNA. ( B ) Sections of organotypic raft cultures generated from TFAP2A-, TFAP2C- or TP63-depleted HFKs stained for haematoxylin & Eosin (H&E) and indirect immunofluorescent staining of early [keratins 1 (KRT1), intermediate (transglutaminase-1 (TGM1)] and late [Filaggrin (FLG)] markers of differentiation (scale bar = 100 µM). ( C ) Number of bromodeoxyuridine (BrDU) incorporating cells in organotypic raft culture assessed by immunofluorescent staining. Graph represents the average number of BrDU-positive cells in the basal epithelial layer from organotypic raft cultures per 1000 µM, expressed as percentage scrambled control (mean ± SE at least 10 counts each of two independent biological replicates).

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Western Blot, Control, Transfection, Generated, Staining