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: Structure (London, England : 1993)

Article Title: Integrated Structural Modeling of Full Length LRH-1 Reveals Inter-domain Interactions Contribute to Receptor Structure and Function

doi: 10.1016/j.str.2020.04.020

Figure Lengend Snippet: Key Resources Table

Article Snippet: ​ Reagent or resource Source Identifiers Bacterial and Virus Strains BL21 dE3 Chemically Competent E. coli Novagen 69450 BL21 dE3 AI Chemically Competent E. coli ThermoFisher Scientific C607003 Origami 2 dE3 Chemically Competent E. coli Novagen 71345 Chemicals, Peptides, and Recombinant Proteins cOmplete™, EDTA-free Protease Inhibitor Cocktail Sigma-Alrich 4693132001 Adenosine 5’-triphosphate disodium salt hydrate Sigma-Aldrich A2383 BS3 (bis(sulfosuccinimidyl)suberate) ThermoFisher Scientific A39266 Mouse anti-LRH-1 primary R&D Systems PP-H2325-00 Goat anti-mouse HRP conjugated secondary Promega W4021 β-actin primary Millipore Sigma A3854 PGC1α peptide ( 740 EEPSLLKKLLLAPA 753 ) New England Peptides N/A N-terminally FAM-labeled PGC1α peptide ( 740 EEPSLLKKLLLAPA 753 ) New England Peptides N/A Gene NR5A2 (LRH-1), Homo sapiens UniProt database {"type":"entrez-protein","attrs":{"text":"O00482","term_id":"7676156","term_text":"O00482"}} O00482 Deposited Data LRH-1 FL Integrated Model This study PDBDev_00000035 LRH-1 DBD:CYP7A1 Promoter Complex Solomon et al, 2005 PDB: 2A66 LRH-1 LBD: NCoA2 Peptide Complex Krylova, et al 2005 PDB: 1YOK LRH-1 LBD: RJW100: PGC1α Peptide Complex Mays et al, 2017 PDB: 5UNJ LRH-1 LBD: DLPC: NCoA2 Peptide Complex Musille et al, 2012 PDB: 4DOS mSF-1 DBD: Inhibinα Promoter Complex Little et al, 2006 PDB: 2FF0 Experimental Models: Cell Lines HEK 293T, Human embryonic kidney ATCC CRL-3216 Recombinant DNA pRSF2-LRH-1 WT Suzawa, et al., 2015 .

Techniques: Recombinant, Protease Inhibitor, Software, Reporter Assay

Journal: Thoracic Cancer

Article Title: LRH1 as a promising prognostic biomarker and predictor of metastasis in patients with non‐small cell lung cancer

doi: 10.1111/1759-7714.12887

Figure Lengend Snippet: Association between LRH1 expression level and clinicopathological characteristics of NSCLC

Article Snippet: The antibodies included mouse monoclonal antibody against β‐actin (TA‐09, 15V70208, Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China) and rabbit antibody against LRH1 (ab153944, Abcam).

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: LRH1 as a promising prognostic biomarker and predictor of metastasis in patients with non‐small cell lung cancer

doi: 10.1111/1759-7714.12887

Figure Lengend Snippet: LRH1 expression in non‐small cell lung cancer (NSCLC) tissue specimens. Representative ( a ) quantitative real‐time PCR and ( b ) Western blot analysis of LRH1 expression in 16 paired NSCLC clinical tissue specimens. Histograms of LRH1 messenger RNA (mRNA) expression and pooled data in adjacent normal lung (N, n = 16) and NSCLC (T, n = 16) tissues. The relative expression of LRH1 mRNA and LRH1 were highly expressed in NSCLC tumor tissues compared to normal lung tissues. Data are expressed as mean ± standard deviation (SD). * P < 0.05. ( c ) Representative immunohistochemistry staining images of samples from an NSCLC patient (T) and adjacent normal lung (N) tissues showed LRH1 expression in NSCLC tissues. The expression of LRH1 protein significantly increased in tumor tissues compared to adjacent normal lung tissues. A histogram of pooled data (right) of NSCLC (T, n = 156) and normal lung (N, n = 30) tissues. The percentage of NSCLC tissues with high LRH1 expression significantly increased compared to that of normal lung tissues. * P < 0.05.

Article Snippet: The antibodies included mouse monoclonal antibody against β‐actin (TA‐09, 15V70208, Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China) and rabbit antibody against LRH1 (ab153944, Abcam).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation, Immunohistochemistry, Staining

Journal: Thoracic Cancer

Article Title: LRH1 as a promising prognostic biomarker and predictor of metastasis in patients with non‐small cell lung cancer

doi: 10.1111/1759-7714.12887

Figure Lengend Snippet: Representative photomicrographs of LRH1 immunohistochemical staining. ( a ) High and ( b ) low expression in adenocarcinoma (ADC). ( c ) High and ( d ) low expression in squamous cell carcinoma (SCC) (×100 original magnification).

Article Snippet: The antibodies included mouse monoclonal antibody against β‐actin (TA‐09, 15V70208, Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China) and rabbit antibody against LRH1 (ab153944, Abcam).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Thoracic Cancer

Article Title: LRH1 as a promising prognostic biomarker and predictor of metastasis in patients with non‐small cell lung cancer

doi: 10.1111/1759-7714.12887

Figure Lengend Snippet: Univariate and multivariate analyses of OS and DFS

Article Snippet: The antibodies included mouse monoclonal antibody against β‐actin (TA‐09, 15V70208, Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China) and rabbit antibody against LRH1 (ab153944, Abcam).

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: LRH1 as a promising prognostic biomarker and predictor of metastasis in patients with non‐small cell lung cancer

doi: 10.1111/1759-7714.12887

Figure Lengend Snippet: High LRH1 levels were associated with shorter survival in non‐small cell lung cancer (NSCLC) patients. ( a ) Overall survival (OS) and ( b ) disease‐free survival (DFS) were statistically shorter in patients with high LRH1 protein expression than in those with low LRH1 protein expression. Kaplan–Meier curves of OS and DFS for high and low LRH1 expression. * P < 0.05 was considered statistically significant.

Article Snippet: The antibodies included mouse monoclonal antibody against β‐actin (TA‐09, 15V70208, Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China) and rabbit antibody against LRH1 (ab153944, Abcam).

Techniques: Expressing