wwc2  (Sino Biological)

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: The identification of bona fide TEAD1 target genes. (A) Venn graph showing potential TEAD1 targets (59 genes) by cross-referencing TKO RNA-seq (955 upregulated genes) and TEAD1-ChIP-seq (392 genes) datasets. (B) Narrowing down to nine TEAD1 direct target genes (TTG) based on regulatory sequence proximity to transcription start sites (TSS) (<1000 bp). (C) TEAD1-ChIP-seq in pancreatic progenitor cells showing strong TEAD1-bound signals that are close to TSS of the candidate TTGs. (D) TEAD1-ChIP-seq in wild-type mouse islets showing TEAD1-bound signals that are close to TSS on WWC2, NR4A3, Amotl2, and LATS2 genes. (E) Quantitative PCR showing the expression of TTGs in TKO and control islets isolated from 12-week-old male mice. (F) Illustration of human CTGF promoter (hCTGF) driven luciferase reporter and mutant human CTGF promoter (ΔhCTGF) driven luciferase reporter on which the MCAT motif was moved to the reverse strand. A luciferase assay showing no difference in activity between ΔhCTGF and hCTGF promoters with co-transfection of YAP5SA and TEAD1. (G) Luciferase assays using mouse YAP1 promoter reporter (mYAP1r) show that TEAD1 and TEAD1 + VGLL4 repress YAP1 transcription, while YAP5SA promotes YAP1 transcription. (H) Split-GFP system showing no binding as indicated by GFP signal between ΔTEAD1 (truncated TEAD1) and YAP1/TAZ/VGLL4. mCherry signal indicate transfection efficiency. Nuclei were counterstained with diamidino-2-phenylindole (DAPI) (blue). (I) mYAP1r-promoter luciferase assays show both TEAD1 and ΔTEAD1 repress YAP1 transcription. (J) ΔmYAP1r (MCATs mutant)-promoter luciferase assays show absent repression of YAP1 transcription by TEAD1 or ΔTEAD1 whereas YAP5SA transcriptional activation of YAP1 is impaired. (K) Human YAP1 promoter and (L) Human TEAD3 promoter luciferase assays show TEAD1 and ΔTEAD1 repression and YAP5SA activation of transcription. (M) ΔTEAD1 inhibit HeLa cell growth. GFP positivity demonstrate ΔTEAD1 or backbone (empty vector) lentiviral transduction. (N) YAP1 protein expression by Western blotting after TEAD1 and ΔTEAD1 overexpression in Hela cells. (O) Human TTGs promoter luciferase assay show TEAD1 and ΔTEAD1 repress the transcription of most TTGs except KNTC1, while YAP5SA promotes the transcription of all TTGs. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM.

Article Snippet: Human WWC2 cDNA ORF clone was purchased from Sino Biological Inc. (#HG19486-U).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Sequencing, Real-time Polymerase Chain Reaction, Expressing, Isolation, Luciferase, Mutagenesis, Activity Assay, Cotransfection, Binding Assay, Transfection, Activation Assay, Plasmid Preparation, Transduction, Western Blot, Over Expression

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: Function validation of TTGs. (A) HOPFLASH reporter luciferase assay show WWC2, AMOTL2, WTIP and YAP5SA regulation of Hippo signaling activity. (B) Human Ki67-promoter reporter (hKi67r) construct. (C) hKi67r reporter activities suggest differential regulatory effects of proliferation by TTGs. (D) Flow cytometry analysis of proliferative (EDU+) HeLa cells after TTGs overexpression (GFP+); double positives are indicated by grey dots. (E) Quantification of flow cytometry analysis in TTGs overexpressed INS2 cells. (F) Schematic representation of NR4A3 isoforms: NR4A3L, full-length NR4A3; NR4A3S, short form of NR4A3. (G) Quantitative PCR showing NR4A3L and NR4A3S mRNA expression in TKO islets. (H) Quantitative PCR showing Ins1, Ins2, Ki67, NR4A3L and NR4A3S mRNA expression in INS2 cells after TEAD1 overexpression (TEAD1OV). (I) Immunostaining and quantification of Ki67 + Insulin + cells in NR4A3L- and NR4A3S-overexpressing mouse islets. (J) Quantitative PCR showing Ki67, Ins1, Ins2, PDX1, MAFA and UCN3 mRNA expression in NR4A3L- and NR4A3S-overexpressed INS2 cells. (K) hKi67r reporter activity show WTIP, WWC2, AMTOL2, PKCiota and NR4A3 antagonize the proliferation effect of YAP5SA while co-transfecting with YAP5SA at a 1:1 molar ratio in HeLa cells. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM.

Article Snippet: Human WWC2 cDNA ORF clone was purchased from Sino Biological Inc. (#HG19486-U).

Techniques: Luciferase, Activity Assay, Construct, Flow Cytometry, Over Expression, Real-time Polymerase Chain Reaction, Expressing, Immunostaining