Journal: Human Molecular Genetics

Article Title: Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation

doi: 10.1093/hmg/ddu111

Figure Lengend Snippet: Interaction of p53 and WT1 proteins. Detection of WT1-p53 protein interactions by immunoprecipitation and western blot analysis. HeLa cells were transfected with expression plasmids encoding the WT1-GFP-fusion proteins, wild-type WT1, mutant WT1 Wilms3 and mutant WT1 Wilms2 . As control, cells were transfected with a GFP-expression plasmid alone. Lysates from transfected cells were incubated with control antibodies (lanes, αCtrl) or p53-specific antibodies (lanes, αP53). Immunoprecipitates were resolved by SDS–PAGE and immunoblotted using antibodies specific for the N-terminus of the WT1 protein (αF6WT1). As input controls, 5% of the respective lysates from transfected HeLa cells were loaded on the gel (lanes, In). Asterisk indicates a non-specific band. Wild-type and mutant WT1-GFP-fusion proteins in the input and immunoprecipitated fractions are marked by black boxes.

Article Snippet: Endogenous p53 activity in Wilms3 cells was measured with the p53-responsive reporter pG13- Luc [13 p53 binding sites, Addgene, 16 442 ( )] and the non-responsive reporter pG15- Luc [15 mutant p53 binding sites, Addgene, 16 443 ( )].

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Mutagenesis, Control, Plasmid Preparation, Incubation, SDS Page

Journal: Human Molecular Genetics

Article Title: Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation

doi: 10.1093/hmg/ddu111

Figure Lengend Snippet: Identification of p53 networks in cohorts of WT1 Wilms3 target genes. The MetaCore program ‘What are the key transcription factors and target genes in my data’ was used to identify p53 networks in subsets of genes whose expressions are either down-regulated ( A ) or up-regulated ( B ) by sh WT1 knockdown in Wilms3 cells. Blue circles mark down-regulated genes ( A ), and red circles mark up-regulated genes ( B ). The properties of individual genes are indicated by coloured symbols. These symbols are explained in Supplementary Material, Table S15 . Green arrows point to genes that are activated by p53 and red arrows to genes that are repressed by p53.

Article Snippet: Endogenous p53 activity in Wilms3 cells was measured with the p53-responsive reporter pG13- Luc [13 p53 binding sites, Addgene, 16 442 ( )] and the non-responsive reporter pG15- Luc [15 mutant p53 binding sites, Addgene, 16 443 ( )].

Techniques: Knockdown

Journal: Human Molecular Genetics

Article Title: Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation

doi: 10.1093/hmg/ddu111

Figure Lengend Snippet: Interaction of p53 and WT1 proteins. Detection of WT1-p53 protein interactions by immunoprecipitation and western blot analysis. HeLa cells were transfected with expression plasmids encoding the WT1-GFP-fusion proteins, wild-type WT1, mutant WT1 Wilms3 and mutant WT1 Wilms2 . As control, cells were transfected with a GFP-expression plasmid alone. Lysates from transfected cells were incubated with control antibodies (lanes, αCtrl) or p53-specific antibodies (lanes, αP53). Immunoprecipitates were resolved by SDS–PAGE and immunoblotted using antibodies specific for the N-terminus of the WT1 protein (αF6WT1). As input controls, 5% of the respective lysates from transfected HeLa cells were loaded on the gel (lanes, In). Asterisk indicates a non-specific band. Wild-type and mutant WT1-GFP-fusion proteins in the input and immunoprecipitated fractions are marked by black boxes.

Article Snippet: Endogenous p53 activity in Wilms3 cells was measured with the p53-responsive reporter pG13- Luc [13 p53 binding sites, Addgene, 16 442 ( )] and the non-responsive reporter pG15- Luc [15 mutant p53 binding sites, Addgene, 16 443 ( )].

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Mutagenesis, Control, Plasmid Preparation, Incubation, SDS Page

Journal: Human Molecular Genetics

Article Title: Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation

doi: 10.1093/hmg/ddu111

Figure Lengend Snippet: Identification of p53 networks in cohorts of WT1 Wilms3 target genes. The MetaCore program ‘What are the key transcription factors and target genes in my data’ was used to identify p53 networks in subsets of genes whose expressions are either down-regulated ( A ) or up-regulated ( B ) by sh WT1 knockdown in Wilms3 cells. Blue circles mark down-regulated genes ( A ), and red circles mark up-regulated genes ( B ). The properties of individual genes are indicated by coloured symbols. These symbols are explained in Supplementary Material, Table S15 . Green arrows point to genes that are activated by p53 and red arrows to genes that are repressed by p53.

Article Snippet: Endogenous p53 activity in Wilms3 cells was measured with the p53-responsive reporter pG13- Luc [13 p53 binding sites, Addgene, 16 442 ( )] and the non-responsive reporter pG15- Luc [15 mutant p53 binding sites, Addgene, 16 443 ( )].

Techniques: Knockdown

Journal: EBioMedicine

Article Title: LRH1-driven transcription factor circuitry for hepatocyte identity: Super-enhancer cistromic analysis

doi: 10.1016/j.ebiom.2018.12.056

Figure Lengend Snippet: The core TFs identified in the liver. (A) H3K27Ac ChIP-seq and DNase-seq profiles at the Hnf4a (left) and the Sec62 (right) loci in mouse liver. Gray bars indicate enhancer regions. (B) Distribution of H3K27Ac ChIP-seq signal intensities across 9891 enhancers in the liver. H3K27Ac occupancy was not evenly distributed across the enhancer regions, with a subset of 460 enhancers containing exceptionally high amounts of H3K27Ac (i.e., super-enhancers) (left). A box plot of H3K27Ac ChIP-seq densities at constituent enhancers within 9431 typical enhancers or 460 super-enhancers (right). (C) Gene ontology (GO) functional categories regarding molecular functions for super-enhancer-associated genes. Genes encoding for the factors controlling transcription were enriched. (D) A protein-protein interaction network of super-enhancer-associated transcription factors (TFs) according to STRING database. LRH1 (also known as Nr5a2 ), HNF4α, PPARα, and RXRα make a core network. The TFs were divided into two groups (multiple interactions with quadruple evidences and multiple interactions with double or triple evidences) according to the number of evidences in the above network. The red dotted line designates the cutoff dividing core and second-tier TFs. (E) A network displaying interactions between GO categories. Each node indicates GO term. The thickness of node colour represents the degree of statistical significance for enrichment. Node sizes show the number of gene counts assigned to each GO term. The network was generated by analysis of Cytoscape plugin BiNGO. (F) H3K27Ac ChIP-seq data at the loci of Nr5a2 (LRH1), Hnf4a , Ppara, and Rxra . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The plasmids encoding mouse LRH1 (#16342, RRID: Addgene_16,342) and mouse PPARα (#22751, RRID: Addgene_22,751) were supplied from Addgene (Cambridge, MA).

Techniques: ChIP-sequencing, Functional Assay, Generated

Journal: EBioMedicine

Article Title: LRH1-driven transcription factor circuitry for hepatocyte identity: Super-enhancer cistromic analysis

doi: 10.1016/j.ebiom.2018.12.056

Figure Lengend Snippet: Correlation between identified core TF and each gene transcript levels. (A) Correlation analyses in a large cohort of cirrhosis patients (GSE25097) ( N = 46). Pearson's r correlation coefficients with corresponding P -values for co-variation between each core TF mRNA levels (x axis) and hepatocyte identity gene transcripts (y axis) show robust correlations. (B) Pearson correlation analyses in mice treated as in C. For single and multiple CCl 4 treatment models, group sizes (N) are 12 and 7, respectively. (C) Results of KEGG pathway analysis of the up- or down-regulated genes after hepatocyte-specific deletion of LRH1 (GSE68718). Enriched signaling pathways of each gene cluster were analyzed using DAVID.

Article Snippet: The plasmids encoding mouse LRH1 (#16342, RRID: Addgene_16,342) and mouse PPARα (#22751, RRID: Addgene_22,751) were supplied from Addgene (Cambridge, MA).

Techniques: Protein-Protein interactions

Journal: EBioMedicine

Article Title: LRH1-driven transcription factor circuitry for hepatocyte identity: Super-enhancer cistromic analysis

doi: 10.1016/j.ebiom.2018.12.056

Figure Lengend Snippet: LRH1 protection of the liver from toxicant-induced injury. (A) LRH1 protection of the liver from APAP-induced injury. H&E staining (upper left). At four days after a hydrodynamic injection of the plasmid encoding LRH1 or mock vector (pcDNA3.1), mice were fasted overnight and subjected to a single dose of APAP (300 mg/kg), and the liver tissues were obtained six h afterward. TUNEL staining (upper right). The scale bars represent 100 μm. Serum ALT and AST activities (lower left). Liver weight per body weight ratio (middle). Correlation between serum ALT activities and LRH1 transcript levels in the liver (lower right). (B) LRH1 protection of the liver from CCl 4 -induced injury. H&E staining (upper left). Mice were subjected to a single dose of CCl 4 (0.6 mL/kg) four days after a hydrodynamic injection of the plasmid encoding LRH1 or mock vector (pcDNA3.1), and the liver tissues were obtained two days afterward. TUNEL staining (upper right). TUNEL-stained tissues were separated to non-tissue, normal and apoptotic areas by blue, green and red colors, respectively. Insets showed true-colour images. The scale bars represent 100 μm. Serum ALT and AST activities (lower left). Correlation between serum ALT activities and LRH1 ( Nr5a2 ) mRNA levels in the liver (lower right). (C) Immunoblottings for apoptosis or liver regeneration markers (left). Values were obtained using scanning densitometry (right). Data information: For A, data represent the means ± SEM (Mock+Veh, n = 7; Mock+APAP, n = 8; LRH1 + APAP, n = 13; and non-injected control (Con), n = 5, significantly different as compared to vehicle control, ** P < .01; or APAP-treated control, # P < .05; ## P < .01). For B, data represent the means ± SEM (Mock+Veh, n = 6; Mock+CCl 4 , n = 14; LRH1 + CCl 4 , n = 4; and non-injected control (Con), n = 4, significantly different as compared to vehicle control, ** P < .01; or CCl 4 -treated control: # P < .05; ## P < .01). For C, data represent the means ± SEM (n = 4 each, significantly different as compared to vehicle control, * P < .05; ** P < .01; or CCl 4 -treated control: # P < .05; ## P < .01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The plasmids encoding mouse LRH1 (#16342, RRID: Addgene_16,342) and mouse PPARα (#22751, RRID: Addgene_22,751) were supplied from Addgene (Cambridge, MA).

Techniques: Staining, Injection, Plasmid Preparation, TUNEL Assay, Control

Journal: EBioMedicine

Article Title: LRH1-driven transcription factor circuitry for hepatocyte identity: Super-enhancer cistromic analysis

doi: 10.1016/j.ebiom.2018.12.056

Figure Lengend Snippet: LRH1-dependent recovery of hepatocyte identity genes. (A) Principal component analysis of RNA-seq data. RNA-seq data was generated using liver of mice treated with APAP alone or APAP+LRH1 overexpression vector as in A. RNA-seq data are deposited in the GEO under accession number GSE104302. (B) Pie graphs showing the enhancer composition. Genes having P -values lower than 0.05 between APAP and LRH1 + APAP groups were defined as LRH1-dependent genes. (C) Heatmaps and hierarchical correlation analyses of differentially expressed genes (DEGs). DEGs were selected as the genes with independent t -test ( P -values < .05 with a fold-change of >1.5). The DEGs were hierarchically clustered and presented as heatmaps according to the row Z score. Super-enhancer- or typical enhancer (SE or TE)-associated DEGs represent significantly altered genes in the APAP group as compared to the vehicle group among the SE-associated or TE-associated genes. Not assigned DEGs are DEGs which are assigned neither to SE nor TE. Heatmaps of total DEGs (a), SE-associated DEGs (b), TE-associated DEGs (c), and not assigned DEGs (d) are presented in left. The proportions of the gene clusters depicted in the heatmaps (left) were shown as a graph (right). (D) Results of KEGG pathway analysis of the clustered DEGs. A schematic description of the gene clusters (a). Enriched signaling pathways of each gene cluster were analyzed using DAVID (b-d).

Article Snippet: The plasmids encoding mouse LRH1 (#16342, RRID: Addgene_16,342) and mouse PPARα (#22751, RRID: Addgene_22,751) were supplied from Addgene (Cambridge, MA).

Techniques: RNA Sequencing, Generated, Over Expression, Plasmid Preparation, Protein-Protein interactions

Journal: EBioMedicine

Article Title: LRH1-driven transcription factor circuitry for hepatocyte identity: Super-enhancer cistromic analysis

doi: 10.1016/j.ebiom.2018.12.056

Figure Lengend Snippet: LRH1 as a driver gene for the core TF circuitry. (A) Hepatic super-enhancer-associated TF network with gene expression changes in mice treated with APAP alone or APAP+LRH1 overexpression vector. The node colors reflect log2 gene expression ratio in mice treated with APAP alone (left) or APAP+LRH1 overexpression (right) as compared to vehicle treatment (red, upregulation; blue, downregulation). Log2 fold changes of the core TFs are presented as an inset table. (B) The core TF mRNA levels from the APAP model. (C) The core TF mRNA levels from the CCl 4 model. (D) The effect of each core TF overexpression on other core TFs. qRT-PCR assays were done on AML12 cells transfected with pcDNA3.1, LRH1, HNF4α, PPARα or RXRα for 48 h. The first lane of each graph is transfection reagent-treated control. Heatmap presents averages of core TF mRNA levels. O/E, overexpression. (E) Super-enhancer (SE)-luciferase reporter assays. Luciferase assays were done on AML12 cells co-transfected with each SE-luciferase reporter, and pcDNA3.1, LRH1, HNF4α, PPARα or RXRα overexpression vector for 24 h. Relative luciferase activities represent arbitrary units of luminescence normalized to the pcDNA3.1 group. The schematic illustrations showing each SE-luciferase construct are presented in the upper panel. The ChIP-seq signal peaks in the scheme are also shown in F. Red bars indicate the peaks excised for cloning of each SE-luciferase reporter construct. O/E, overexpression. (F) A proposed scheme showing auto-regulatory loops for the core TFs. In healthy liver, the core TFs form an interconnected feedback loop for gene expression. Upon injury, the signal circuitry loses its integrity with decrease of hepatocyte identity. LRH1 serves a driver for reconstitution of the signal circuitry. Data information: For B, data represent the means ± SEM (Mock+Veh, n = 7; Mock+APAP, n = 8; and LRH1 + APAP, n = 13, significantly different as compared to vehicle control, ** P < .01; or APAP-treated control, # P < .05; ## P < .01). For C, data represent the means ± SEM (Mock+Veh, n = 6; Mock+CCl 4 , n = 14; and LRH1 + CCl 4 , n = 4; significantly different as compared to vehicle control, * P < .05; ** P < .01; or CCl 4 -treated control: # P < .05; ## P < .01). For D and E, data represent the means ± SEM ( n = 3 each, significantly different as compared to pcDNA3.1 group, * P < .05; ** P < .01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The plasmids encoding mouse LRH1 (#16342, RRID: Addgene_16,342) and mouse PPARα (#22751, RRID: Addgene_22,751) were supplied from Addgene (Cambridge, MA).

Techniques: Gene Expression, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Transfection, Control, Luciferase, Construct, ChIP-sequencing, Cloning

Journal: Hearing research

Article Title: Mesenchymal ETV transcription factors regulate cochlear length.

doi: 10.1016/j.heares.2020.108039

Figure Lengend Snippet: Fig. 7. Mesenchymal Etv1 is dispensable for cochlear development. Whole mount immunostaining of P0 cochlea from control (Etv1fl/þ; Etv4/þ; Etv5fl/þ;Twist2Cre/þ), Etv4/5 double mutant (Etv1fl/þ;Etv4/;Etv5fl/fl;Twist2Cre/þ) and Etv1/4/5 triple mutant (Etv1fl/fl;Etv4/;Etv5fl/fl;Twist2Cre/þ) showing whole cochlea stained with Myosin 6 antibody (green) (A), and representative regions from each cochlear turn stained with F-actin (green) (B), and Prox1 staining (green) (C) as a marker for supporting cells. Scale bar ¼ 100 mm. White arrowheads mark the beginning and the end of the cochlear length measurement. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: In situ hybridization antisense riboprobes were prepared from the following mouse cDNA plasmids: Etv1 (pBluescript SKEtv1, Addgene 16,282), Etv4 (pGEM-Etv4, gift of G. Martin), Etv5 (pBluescriptSK-Etv5, gift of G. Martin), Ebf1 (pCR4-TOPO-Ebf1, Transomic BC13936), and Tgfbi (pCR-BluntII-TOPO-Tgfbi, Transomic BC129900).

Techniques: Immunostaining, Control, Mutagenesis, Staining, Marker

Journal: Hearing research

Article Title: Mesenchymal ETV transcription factors regulate cochlear length.

doi: 10.1016/j.heares.2020.108039

Figure Lengend Snippet: Fig. 8. Quantification of cochlear length, hair cell and supporting cell density in Etv1/4/ 5 compound mutants. (A) Quantification of cochlear duct length at P0 from control (Etv1fl/þ;Etv4/þ;Etv5fl/

Article Snippet: In situ hybridization antisense riboprobes were prepared from the following mouse cDNA plasmids: Etv1 (pBluescript SKEtv1, Addgene 16,282), Etv4 (pGEM-Etv4, gift of G. Martin), Etv5 (pBluescriptSK-Etv5, gift of G. Martin), Ebf1 (pCR4-TOPO-Ebf1, Transomic BC13936), and Tgfbi (pCR-BluntII-TOPO-Tgfbi, Transomic BC129900).

Techniques: Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PWWP2B Fine-Tunes Adipose Thermogenesis by Stabilizing HDACs in a NuRD Subcomplex.

doi: 10.1002/advs.202102060

Figure Lengend Snippet: Figure 4. PWWP2B suppresses thermogenic program in a cell-autonomous manner. a) Lentiviral shRNAs are transduced to immortalized brown preadipocytes and induced differentiation in vitro. PWWP2B level is analyzed by western blot in mature brown adipocytes (day 6). n = 3. b) Real-time QPCR analyzed gene expression in cells of (a). n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 versus the scrambled control by unpaired two-tailed Student’s t-test. The data shown are mean ± SEM. c) Cells generated as in (a) are treated by 10 µM FSK for 6 h before the gene expression analysis. n = 3. *P < 0.05, **P < 0.01 versus the scrambled control by unpaired two-tailed Student’s t-test. The data shown are mean ± SEM. d) Primary SVF cells are isolated from BAT of Pwwp2bflox/flox mice. Lentivirus expressing Cre infected SVFs to delete Pwwp2b. Cells are differentiated to mature adipocytes following western blot analysis. n = 3. e) Primary SVF cells isolated from iWAT of C57BL/6 mice are differentiated into beige cells. Lentiviral shRNA infected beige cells on differentiation days 2 and 4, and gene expression analysis is performed on day 6. n = 3. *P < 0.05 by unpaired two-tailed Student’s t-test. The data shown are mean ± SEM. f) Primary SVF cells isolated from iWAT of Floxed and knockout mice are differentiated into beige cells. Western blot assay for PWWP2B and UCP1 proteins. n = 3. g,h) Adenovirus carrying Pwwp2b infected immortalized brown adipocytes on differentiation day 3. Gene expression analysis is performed on day 6 with or without 10µM FSK treatment for 12 h as indicated. h) n = 3. *P < 0.05, ***P < 0.001 by unpaired two-tailed Student’s t-test. The data shown are mean ± SEM. i,j) OCR is measured by Clark oxygen electrode in mature adipocytes (day 6) that are knocked down ((i), n = 3) or overexpressed Pwwp2b ((j,n) = 3). Knockdown or overexpressing lentivirus infected brown preadipocytes following standard induction to mature adipocytes. **P < 0.01 versus the control by unpaired two-tailed Student’s t-test. The data shown are mean ± SEM.

Article Snippet: Construction and package of knockdown (Addgene, pSP-108 vector), overexpressing lentivirus (Addgene, 17 398), and adenovirus (Addgene, 16 404) was followed the standard protocol.

Techniques: In Vitro, Western Blot, Gene Expression, Control, Two Tailed Test, Generated, Isolation, Expressing, Infection, shRNA, Knock-Out, Knockdown