gtpγs  (Cytoskeleton Inc)

Journal: Science Advances

Article Title: YAP/TAZ-VGLL3 governs adipocyte fate via epigenetic reprogramming of PPARγ and its target enhancers

doi: 10.1126/sciadv.aea7235

Figure Lengend Snippet: ( A ) The 3D structure of human TAZ (UniProt accession Q9GZV5 ) predicted by AlphaFold is shown on the left. The domain organization of human TAZ is shown on the right: TEAD binding domain (TBD), WW domain, coiled-coil (CC) domain, transactivation domain (TAD), and PDZ binding motif. Serine-to-alanine (SA) mutation sites for constitutively active mutants (red circles) and a TEAD-binding–deficient mutant (blue circle). ( B and C ) Streptavidin pull-down assay for human embryonic kidney (HEK) 293T cells showing the interaction of green fluorescent protein (GFP)–tagged PPARγ2 (B) or endogenous TEADs and LATS1 (C) with SFB (S protein–FLAG–streptavidin binding peptide)–tagged TAZ mutants. ( D to F ) C3H10T1/2 cells stably expressing doxycycline-inducible 2xHA-tagged TAZ mutants were subjected to immunoblot analysis (D), RT-qPCR analysis ( n = 3) (E), and lipid staining with oil red O (F). Scale bars, 200 μm (F). ( G ) MA plot showing differential H3K27ac ChIP-seq signals between control and TAZ2SA-expressing C3H10T1/2 cells (left) and heatmaps showing H3K27ac ChIP-seq signals for cells expressing the indicated TAZ2SA mutants (right). Significantly changed regions (|fold change| > 2 and adj. P value of <0.05) in TAZ2SA-expressing versus control cells are colored red (fold change > 2) or blue (fold change < −2). ( H ) Genome browser views of the indicated loci showing the coverage of the H3K27ac ChIP-seq signal in differentiated C3H10T1/2 cells expressing the TAZ mutants. ( I ) Heatmap of the H3K27ac ChIP-seq signals from C3H10T1/2 cells expressing indicated TAZ mutants, aligned with PPARγ ChIP-seq peaks (D6). Data in bar graph (E) are means ± SEM and analyzed by one-way ANOVA with Tukey’s post hoc test. ** P < 0.01 and *** P < 0.001.

Article Snippet: For streptavidin-mediated pull-down assay of S protein–FLAG–streptavidin binding peptide (SFB)–tagged proteins, cleared cell lysates (1 mg of protein in 1 ml) were incubated for 2 hours at 4°C with 20 μl of Pierce High Capacity Streptavidin Agarose (20359, Thermo Fisher Scientific), and the beads were then washed three times with lysis buffer and boiled with Laemmli sample buffer for immunoblot analysis.

Techniques: Binding Assay, Mutagenesis, Pull Down Assay, Stable Transfection, Expressing, Western Blot, Quantitative RT-PCR, Staining, ChIP-sequencing, Control

Journal: Science Advances

Article Title: YAP/TAZ-VGLL3 governs adipocyte fate via epigenetic reprogramming of PPARγ and its target enhancers

doi: 10.1126/sciadv.aea7235

Figure Lengend Snippet: ( A ) Scatter plot showing correlations between RNA fold change and ATAC gene activity change in adipocyte-related cells (LAKO versus control; sn sequencing). Color indicates the maximum percentage of cells expressing each gene. ( B ) Vgll3 expression in iWAT snRNA-seq. Dediff., Dedifferentiated adipocytes. ( C ) Genomic browser view of Vgll3 locus with the indicated sequencing data. ( D ) Mouse adipose tissue RNA-seq data ( GSE138911 ) showing Vgll3 expression [fragments per million mapped reads (FPM)] in high-fat diet (HFD) or normal chow (NC)–fed adipocyte-specific YAP/TAZ KO (YTKO) mice ( Yap1 fl/fl ; Wwtr1 fl/fl ; Adipoq-Cre ). ( E ) Human visceral adipose tissue RNA data from the GTEx consortium showing the correlation between WWTR1 and VGLL3 expression (right). ( F ) C3H10T1/2 cells expressing doxycycline (Dox)–inducible HA-TAZ2SA were treated with 2 μM VT-104 for 36 hours and subjected to RT-qPCR. ( G ) C3H10T1/2 cells (D6) expressing Dox-inducible Myc-Vgll3 were analyzed by RT-qPCR ( n = 3) and immunoblot. ( H ) Oil red O staining of Dox-inducible Vgll3 or Vgll3ΔTDU-expressing cells. Scale bars, 200 μm. ( I ) Vgll3 KO C3H10T1/2 cells expressing Dox-inducible HA-TAZ2SA and its parental cells (Con) were analyzed by RT-qPCR and immunoblot. ( J ) Genomic view of Pparg (left) and Fabp4 (right) regions with the indicated ChIP-seq data from TAZ mutant–expressing cells. ( K ) H3K27ac peak heatmap aligned with PPARγ ChIP-seq peaks (D6). ( L ) Streptavidin pull-down assay of HEK293T cells cotransfected with HA-tagged histone deacetylase 3 (HDAC3) and SFB-tagged VGLL3. ( M ) C3H10T1/2 cells with inducible Vgll3 expression (TRE-Vgll3) and Hdac3 [short hairpin Hdac3 (shHdac3)] or Ncor1 (shNcor1) knockdown or parental control (−) were treated with adipogenic cocktails and Dox or vehicle control (48 hours) and subjected to RT-qPCR. ( N ) A proposed model for the role of TAZ in adipocyte differentiation and dedifferentiation. Data in bar graphs (D, E, and G) are means ± SEM and analyzed by the unpaired t test. ** P < 0.01 and *** P < 0.001.

Article Snippet: For streptavidin-mediated pull-down assay of S protein–FLAG–streptavidin binding peptide (SFB)–tagged proteins, cleared cell lysates (1 mg of protein in 1 ml) were incubated for 2 hours at 4°C with 20 μl of Pierce High Capacity Streptavidin Agarose (20359, Thermo Fisher Scientific), and the beads were then washed three times with lysis buffer and boiled with Laemmli sample buffer for immunoblot analysis.

Techniques: Activity Assay, Control, Sequencing, Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Staining, ChIP-sequencing, Mutagenesis, Pull Down Assay, Histone Deacetylase Assay, Knockdown