Journal: Cell reports

Article Title: Site-Dependent Cysteine Lipidation Potentiates the Activation of Proapoptotic BAX

doi: 10.1016/j.celrep.2020.02.057

Figure Lengend Snippet:

Article Snippet: Q5 Site Directed Mutagenesis Kit , New England Biolabs , Cat# E0554S.

Techniques: Virus, Recombinant, Mutagenesis, Cell Viability Assay, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: PLoS Genetics

Article Title: Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model

doi: 10.1371/journal.pgen.1007009

Figure Lengend Snippet: (A) Zebrafish irf6 gene structure composed of eight exons, a helix-turn-helix DNA-binding domain (yellow), and a SMIR/IAD protein-binding domain (green). The CRISPR gRNA target site was located in exon 6 at the start of the protein-binding domain. Sanger sequencing of the target site revealed a -8bp deletion (Δ8bp) that created a frameshift and premature stop codon, truncating the protein to 29 kD as predicted by in silico translation. Another +5bp insertion was also identified. (B) Breeding pedigree revealed the irf6 mutant phenotype in F3 and the importance of maternal transcripts. (C) Top: western blot at the sphere stage (4 hpf) revealed a lack of Irf6 full-length (55 kD) or truncated (29 kD) protein in all maternal irf6 Δ8bp/Δ8bp embryos but not paternal irf6 Δ8bp/Δ8bp embryos or wild type embryos. Bottom: relative gene expression by RT-qPCR revealed a lack of irf6 mRNA transcripts in all maternal irf6 Δ8bp/Δ8bp embryos but comparable levels between wild type and paternal irf6 Δ8bp/Δ8bp embryos. Error bar = 2xSEM, n = 3. (D-D’) Wild type embryos at the sphere stage (4 hpf) (D) and at the 30% epiboly stage (5 hpf) (D’). (E-E’) Maternal irf6 Δ8bp/Δ8bp embryos at the sphere stage (4 hpf) (E) and displaying the periderm rupture phenotype at 5 hpf (E’). Scale bar = 250 μm. (F) Cross-sectional schematic through the embryonic midline illustrating the zebrafish embryo epiboly process. Arrows represent cell and yolk directional movements. Wild type embryos experience rapid cellular lamination and yolk doming between 4–5 hpf, while maternal-zygotic irf6 Δ8bp/Δ8bp embryos experience incomplete periderm differentiation and animal pole/yolk separation.

Article Snippet: PCR-based site-directed mutagenesis was performed to generate pCS2+8 vectors containing irf6 missense gene variants using the Q5 site-directed mutagenesis kit (New England Biolabs) with amplification primers designed by the NEBaseChanger online tool ( http://nebasechanger.neb.com ).

Techniques: Binding Assay, Protein Binding, CRISPR, Sequencing, In Silico, Mutagenesis, Western Blot, Gene Expression, Quantitative RT-PCR

Journal: PLoS Genetics

Article Title: Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model

doi: 10.1371/journal.pgen.1007009

Figure Lengend Snippet: (A-L) Zebrafish embryos at 96 hpf stained with alcian blue for cartilaginous craniofacial elements. Maternal/zygotic-null irf6 -/- embryos were rescued by microinjection of either zebrafish irf6 mRNA (E-H) or human IRF6 mRNA (I-L) at the one-cell stage, preventing the periderm rupture phenotype and restoring normal craniofacial development compared to wild type embryos (A-D). Scale bar = 150 μm. (M-N) Dimensional measurements of dissected ethmoid plates at 96 hpf, with length ( l ) and width ( w ) denoted by dashed lines on panel (D). The length (M) and width (N) of ethmoid plates from zebrafish and human IRF6 mRNA rescued maternal/zygotic-null irf6 -/- embryos are statistically indistinguishable in dimensions compared those of wild type embryos. Error bar = 2xSEM, n = 12.

Article Snippet: PCR-based site-directed mutagenesis was performed to generate pCS2+8 vectors containing irf6 missense gene variants using the Q5 site-directed mutagenesis kit (New England Biolabs) with amplification primers designed by the NEBaseChanger online tool ( http://nebasechanger.neb.com ).

Techniques: Staining, Microinjection

Journal: PLoS Genetics

Article Title: Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model

doi: 10.1371/journal.pgen.1007009

Figure Lengend Snippet: (A-H) Whole-mount in situ hybridization analysis of wild type embryos (A-D) compared to maternal/zygotic-null irf6 -/- embryos (E-H) at the sphere stage revealed strong down-regulation of critical irf6 downstream genes such as krt4 and grhl3 . Scale bar = 150 μm. (I) Relative gene expression of wild type embryos, maternal/zygotic-null irf6 -/- embryos, and maternal/zygotic-null irf6 -/- embryos rescued with either wild type zebrafish or human IRF6 mRNA (100 pg) microinjections, for a panel of genes with crucial roles in the irf6 gene regulatory network. Error bar = 2xSEM, n = 3.

Article Snippet: PCR-based site-directed mutagenesis was performed to generate pCS2+8 vectors containing irf6 missense gene variants using the Q5 site-directed mutagenesis kit (New England Biolabs) with amplification primers designed by the NEBaseChanger online tool ( http://nebasechanger.neb.com ).

Techniques: In Situ Hybridization, Gene Expression

Journal: PLoS Genetics

Article Title: Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model

doi: 10.1371/journal.pgen.1007009

Figure Lengend Snippet: (A) Experimental approach for characterizing protein functions of human IRF6 missense gene variants. Variant mRNAs were synthesized and microinjected into maternal-null irf6 -/- embryos at the one-cell stage and assessed for phenotypic rescue at 24 hpf. (B-C) Protein modeling of the protein-binding domain and C-terminus of IRF6 using ExPASy with crystalline structures of IRF1. (B) is mapped with missense variant amino acid residues (green) whose mRNA rescued the periderm rupture phenotype, while (C) is mapped with missense variant amino acid residues (red) whose mRNA failed to rescue. (D-F) Results for functional rescue of periderm rupture with maternal-null irf6 -/- embryos for representative human IRF6 missense gene variants. Results were classified as rescued if any maternal-null irf6 -/- embryos injected with variant mRNA remained alive and phenotypically wild type at 24 hpf (50 embryos/round, n = 3). Missense gene variants were categorized by location within the IRF6 protein, and by computational results from PolyPhen-2 and SIFT on whether the in silico predictions agreed on the deleterious effects of the missense gene variants on protein function. Further shown are ACMG guideline pathogenicity predictions (pathogenic, likely pathogenic, uncertain, and benign), and the number of families identified for each variant (all gene variant annotations were based on NM_006147.3).

Article Snippet: PCR-based site-directed mutagenesis was performed to generate pCS2+8 vectors containing irf6 missense gene variants using the Q5 site-directed mutagenesis kit (New England Biolabs) with amplification primers designed by the NEBaseChanger online tool ( http://nebasechanger.neb.com ).

Techniques: Variant Assay, Synthesized, Protein Binding, Functional Assay, Injection, In Silico

Journal: PLoS Genetics

Article Title: Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model

doi: 10.1371/journal.pgen.1007009

Figure Lengend Snippet: (A) Identification and characterization of IRF6 missense gene variants in the ExAC and gnomAD databases. p.V274I alleles were identified in all populations. (B) mRNA dosage titration experiement results for a subset of missense gene variants correlating amount of variant mRNA microinjected to the percent of maternal-null irf6 -/- embryos rescued from rupture and undergoing normal embryonic development at 24 hpf. The missense variants were classified into three categories based on levels of protein function. No variant was identified in ExAC/gnomAD that could not rescue the periderm rupture phenotype. Error bar = 2xSEM, 50 embryos/round, n = 3.

Article Snippet: PCR-based site-directed mutagenesis was performed to generate pCS2+8 vectors containing irf6 missense gene variants using the Q5 site-directed mutagenesis kit (New England Biolabs) with amplification primers designed by the NEBaseChanger online tool ( http://nebasechanger.neb.com ).

Techniques: Titration, Variant Assay

Journal: PLoS Genetics

Article Title: Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model

doi: 10.1371/journal.pgen.1007009

Figure Lengend Snippet: (A-T) Craniofacial morphologies of maternal-null irf6 -/- embryos rescued by human IRF6 missense gene variant mRNA microinjections (100 pg/embryo) at 96 hpf stained with alcian blue. (A-D) Uninjected wild type control. (E-H) p.P12L. (I-L) p.P76L. (M-P) p.T100A. (Q-T) p.P222L. Scale bars = 150 μm, n = 3.

Article Snippet: PCR-based site-directed mutagenesis was performed to generate pCS2+8 vectors containing irf6 missense gene variants using the Q5 site-directed mutagenesis kit (New England Biolabs) with amplification primers designed by the NEBaseChanger online tool ( http://nebasechanger.neb.com ).

Techniques: Variant Assay, Staining, Control

Journal: Molecular cell

Article Title: Partitioning of MLX-Family Transcription Factors to Lipid Droplets Regulates Metabolic Gene Expression

doi: 10.1016/j.molcel.2020.01.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid construction Genomic DNA was isolated using GenElute™ Mammalian Genomic DNA Miniprep Kit (G1N350, Sigma-Aldrich), PCRs were performed using Q5® High-Fidelity PCR Kit or Phusion® High-Fidelity PCR Kit (M0493L and M0530L, New England Biolabs), and restriction enzymes were from New England Biolabs.

Techniques: Binding Assay, Recombinant, Transfection, Sequencing, Staining, Chromatin Immunoprecipitation, PCR Cloning, Bicinchoninic Acid Protein Assay, SYBR Green Assay, RNA Sequencing Assay, Clone Assay, Plasmid Preparation, Software