wild type human adenovirus serotype 5 ad  (ATCC)

Journal: Cell

Article Title: Transcription factor protein interactomes reveal genetic determinants in heart disease.

doi: 10.1016/j.cell.2022.01.021

Figure Lengend Snippet: Figure 5. Functional impact of a highly scored CHD variant in GLYR1 (A) Simplified protein schematic depicting the domain organization of human GLYR1. Black rectangle indicates zoomed-in protein region in (B). (B) Protein sequence conservation across vertebrate species for the GLYR1 rigid loop region containing the CHD-associated P496L DNV. (C) GLYR1 dehydrogenase domains: Rossman-fold globular domain (green), the linking a9-helix (red), and the a-helical bundle (dark blue). Right panels: zoom into the WT and mutant forms of the rigid loop with aromatic residues in beige and Proline 496 in orange. (D) Distribution of the root mean square deviation (RMSD) of frames visited during the trajectories from the reference state represented by the starting structure of the WT (blue) and the P496L mutant (green) GLYR1 dehydrogenase domains within the measured time. (E) Residue flexibility analysis based on the standard deviations of the atomic positions in the simulations (RMSF) after fitting to the starting structure of the WT form (blue) and the mutant (green) GLYR1 dehydrogenase domains. F-statistic shows lower flexibility of the mutant compared to the Rossman-fold domain (residues 262–437). (F) The ability of GLYR1 WT or P496L mutant to interact with GATA4 by immunoprecipitation (IP) of GLYR1-MYC and immunoblotting with indicated antibodies. (G) Luciferase reporter assay in HeLa cells showing activation of the GATA4-dependent Nppa luciferase reporter upon addition of plasmids encoding indicated proteins. (n = 3 independent experiments). One-way ANOVA coupled with Tukey post hoc test: ***p value < 0.001. See also Figure S4 and Table S1N.

Article Snippet: Adenoviral – Human Type 5 (dE1/E3) viral particles expressing GLYR1 wild-type (Ad-GFP-EF1-h-GLYR1; PFU titer: 1x1010 PFU/mL), GLYR1 P496L mutant (Ad-GFP-EF1-h-GLYR1 P496L PFU titer: 2x109 PFU/mL) or negative control (Ad-EF1a-eGFP; PFU titer: 1.2x1010 PFU/mL) viral particles, were obtained from Vector Biolabs.

Techniques: Functional Assay, Variant Assay, Sequencing, Mutagenesis, Residue, Immunoprecipitation, Western Blot, Luciferase, Reporter Assay, Activation Assay

Journal: Cell

Article Title: Transcription factor protein interactomes reveal genetic determinants in heart disease.

doi: 10.1016/j.cell.2022.01.021

Figure Lengend Snippet: Figure 6. GATA4-associated roles for GLYR1 in transcription regulation during cardiomyocyte differentiation (A) Heatmap of genes differentially bound by GLYR1 (FDR < 0.1) between hiPSCs and CPs subjected to k-means clustering based on GLYR1 ChIP-seq signal (3 representative replicates plotted; n = 5), H3K36me3 ChIP-seq signal (n = 2), and gene expression levels (GSE137920; n = 3). Statistically enriched GO Biological Process terms and example genes per cluster on the right panel. (B) Overlap of genes bound by GLYR1 in CPs from clusters 2 and 3 (FDR < 0.1, LogFC > 0.5) with genes occupied by GATA4 within the gene body (1st intron-TES). The odds of GATA4 binding to gene bodies enriched GLYR1 signal versus no GATA4 binding is 2.38 (***p value < 2.2e16, Fisher’s exact test). (C) Gene Ontology enrichment analysis of biological process for genes up- or downregulated in CPs compared to hiPSCs (FDR < 0.05) and bound by GATA4:GLYR1, GLYR1-only, and GATA4-only. Prot., protein; dev., development.

Article Snippet: Adenoviral – Human Type 5 (dE1/E3) viral particles expressing GLYR1 wild-type (Ad-GFP-EF1-h-GLYR1; PFU titer: 1x1010 PFU/mL), GLYR1 P496L mutant (Ad-GFP-EF1-h-GLYR1 P496L PFU titer: 2x109 PFU/mL) or negative control (Ad-EF1a-eGFP; PFU titer: 1.2x1010 PFU/mL) viral particles, were obtained from Vector Biolabs.

Techniques: ChIP-sequencing, Gene Expression, Binding Assay