Journal: eLife

Article Title: Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract

doi: 10.7554/eLife.18065

Figure Lengend Snippet: ( A ) General schematic of trans N17 addition experiments to ∆N aggregation reaction to determine how N17 impacts the balance between stable, ∆N oligomers and amyloid fibrillar aggregates. ( B ) 12.5x excess of N17 peptide was added when ∆N aggregation was initiated. Then 60 hr after initiation of aggregation, the fibers were analyzed by cryo-EM. N17-seeded ∆N aggregates become more bundled and more resemble the morphology of Ex1 aggregates . ( C ) 12.5x or 2.5x excess of N17 peptide was added in trans when ∆N aggregation was initiated. The impact of N17 peptide on ∆N oligomers was analyzed by 0.1% SDS-AGE gels and immunoprobed for the C-terminal S-tag. Trans addition of N17 promotes disappearance of these stable ∆N oligomers from the AGE gel. ( D ) 12.5x excess of N17 peptide was added in trans 7 hr after initiation of ∆N aggregation, allowing for the formation of ∆N oligomers before N17 addition. Impact of N17 peptide on ∆N oligomers was analyzed by 0.1% SDS-AGE gels and immunoprobed for the C-terminal S-tag. ( E ) Schematic of in vivo N17 addition experiment: ST14a striatal-derived neurons were transfected with the C-terminally GFP tagged mHtt-Ex1 (mHtt-Ex1-GFP). 10 hr after transfection, when expressed mHtt protein is still completely soluble, cells are protein transfected using the Xfect kit (Clontech) with N17 or a mutant N17 peptide (NA) that inhibits aggregation. After 10 hr, resulting cells containing GFP fluorescent puncta are counted. ( F ) Fluorescence microscopy of ST14a striatal neurons transfected with mHtt-Ex1-GFP and N17 variant peptides (left) and quantification of cells containing puncta (right). Data are mean ± SEM of three independent experiments with at least 200 cells counted in each condition. Scale bar is 20 µm. *p<0.05. DOI: http://dx.doi.org/10.7554/eLife.18065.013

Article Snippet: 10 hr after transfection, when expressed mHtt protein is still completely soluble, cells are protein transfected using the Xfect kit (Clontech) with N17 or a mutant N17 peptide (NA) that inhibits aggregation.

Techniques: Cryo-EM Sample Prep, In Vivo, Derivative Assay, Transfection, Mutagenesis, Fluorescence, Microscopy, Variant Assay

Journal: Cell Research

Article Title: Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

doi: 10.1038/cr.2013.122

Figure Lengend Snippet: dCas9VP160 activates endogenous genes. (A) Protein architecture of dCas9VP160 compared to VP48. (B) Schematic of the human IL1RN promoter region. Locations of transcription start site (TSS) and start codon (ATG) are indicated. Short lines with number indicate targeting sites of the sgRNAs. (C) Activation of human IL1RN expression in HEK293T cells. Cells were analyzed by qRT-PCR 2 days after transfection with dCas9VP160 and the indicated sgRNAs. (D) Schematic of the human SOX2 promoter region. Locations of TSS and start codon (ATG) are indicated. Short lines with number indicate targeting sites of sgRNAs. (E) Activation of SOX2 . Cells were analyzed by qRT-PCR 2 days after transfection with dCas9VP160 and the indicated sgRNAs. (F) Schematic of the human OCT4 promoter region. Locations of transcription start site (TSS) and start codon (ATG) are indicated. Short lines with number indicate targeting sites of sgRNAs. (G) Activation of OCT4 . Cells transfected with dCas9VP160 and the indicated sgRNAs were analyzed by qRT-PCR 2 days later. sgTetO-mut, negative control sgRNA. Error bars show SD among triplicates.

Article Snippet: mESCs from mice carrying a Dox-inducible Musashi-1 (MSI1) allele in the Col1A1 locus were transfected with dCas9VP48 using Xfect mESC transfection reagent (Clontech) or were cultured in mouse ES medium with 2 μg/ml Doxycycline.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Negative Control

Journal: Cell Research

Article Title: Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

doi: 10.1038/cr.2013.122

Figure Lengend Snippet: Multiple exogenous and endogenous genes were simultaneously activated by CRISPR-on. (A) One exogenous and two endogenous genes were simultaneously activated by CRISPR-on. Cells were analyzed by qRT-PCR 2 days after transfection with dCas9VP160 and the indicated sgRNAs. (B) Three endogenous genes, SOX2 , IL1RN and OCT4 , can be simultaneously activated by dCas9VP160/sgRNAs. Cells were transfected with dCas9VP160 and the indicated sgRNAs and were analyzed by qRT-PCR 2 days after transfection. The Last three sets of bars represent triple activation experiments using sgSOX2, sgOCT4 and sgIL1RN with three different ratios of sgSOX2:sgIL1RN, keeping the amount of sgOCT4 constant, as indicated by numbers above line. sgTetO-mut, negative control sgRNA. Error bars show SD among triplicates.

Article Snippet: mESCs from mice carrying a Dox-inducible Musashi-1 (MSI1) allele in the Col1A1 locus were transfected with dCas9VP48 using Xfect mESC transfection reagent (Clontech) or were cultured in mouse ES medium with 2 μg/ml Doxycycline.

Techniques: CRISPR, Quantitative RT-PCR, Transfection, Activation Assay, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases

doi: 10.3390/ijms22073296

Figure Lengend Snippet: The CNS and reference gene (RG) promoters are selectively activated by specific single guide RNAs (sgRNAs) designed to target them. ( a ) Schematic representation of the PPARGC1A locus showing the CNS and RG promoters (top), locations of sgRNAs used for transfections; CNS-specific exons B1 , B4 and B5 in color; the structure of the RG is displayed on the right; CNS-specific transcripts and RG transcripts are shown below; * pink or red lines refer to alternatively spliced transcripts encoding stop codons in exon 7A or in an extension of exon 3, respectively. ( b , c ) Selective effects of individual sgRNAs targeting the RG or CNS promoters on transcription initiation; individual sgRNAs or their mixtures were transfected into clonal SH-SY5Y cells expressing CRISPR-associated deactivated (dCas9) protein fused to the tripartite transcriptional activator VPR and levels of E1E2 and B1B4 transcripts selective for RG or CNS promoter activation were measured by qRT-PCR 36 h after transfection. Log-fold levels are expressed relative to transcript levels of the clonal cells transfected with scrambled sgRNA. Interactions of all sgRNA mixtures shown were significant ( p < 0.001) when the use of three, two, or one sgRNA was compared. ( d ) Effects of sgRNA transfections on CNS-specific and RG transcript levels and levels of transcripts encoding truncated isoforms and initiated at either promoter.

Article Snippet: For the stable integration of dCas9-VPR into the genome of cell lines, pLenti-EF1a-dCas9-SAM plasmids (ABM) along with the trans-complementation plasmids pMD2.G expressing VSV-G envelope and phCMV-8.91 expressing GAG/POL were transfected into HEK293T cells using jetPEI or Xfect transfection reagents (Polyplus Transfection T or Clontech, Mountain View, CA, USA) in serum and antibiotic free medium.

Techniques: Transfection, Expressing, CRISPR, Activation Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases

doi: 10.3390/ijms22073296

Figure Lengend Snippet: RNA sequencing confirms the predicted structure of transcripts generated by activation of the RG or CNS promoters and reveals differentially expressed genes (DEGs) for either promoter activation. ( a ) Sashimi plots of PPARGC1A transcripts generated by transfection of clonal SH-SY5Y cells expressing dCas9-VPR with sgRNAs activating the RG or the CNS promoters or with scrambled sgRNAs, each merged from three biological replicates; read densities across exons are normalized to obtain comparable measures of expression above the x -axis and normalized single-end junction reads are shown as arcs below the x -axis; structure of visualized exons is shown at the bottom. ( b , c ) Volcano plots of DEGs in cells with RG or CNS activated promoters, respectively, in comparison to cells transfected with scrambled sgRNAs. The top 20 most DEGS are highlighted.

Article Snippet: For the stable integration of dCas9-VPR into the genome of cell lines, pLenti-EF1a-dCas9-SAM plasmids (ABM) along with the trans-complementation plasmids pMD2.G expressing VSV-G envelope and phCMV-8.91 expressing GAG/POL were transfected into HEK293T cells using jetPEI or Xfect transfection reagents (Polyplus Transfection T or Clontech, Mountain View, CA, USA) in serum and antibiotic free medium.

Techniques: RNA Sequencing Assay, Generated, Activation Assay, Transfection, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases

doi: 10.3390/ijms22073296

Figure Lengend Snippet: Differentially expressed genes resulting from activation of the RG or CNS promoters reveal promoter selectivity with partial overlap. ( a ) Venn diagram of differentially expressed genes (DEGS) produced by transfection of clonal SH-SY5Y cells expressing dCas9-VPR with sgRNAs activating the RG or the CNS promoters are compared with cells transfected with scrambled sgRNAs. ( b , c ) Top canonical signaling pathways for DEGs after RG or CNS promoter activation, respectively. ( d ) Top nervous system signaling pathways for DEGs after CNS promoter activation; red lines refer to adjusted p -values of 0.05; z -scores > 2.0 or <−2.0 are significant and indicate the direction for the expected entity; n.a, not available; B-H, Bernini-Hochberg adjusted p -values for multiple testing.

Article Snippet: For the stable integration of dCas9-VPR into the genome of cell lines, pLenti-EF1a-dCas9-SAM plasmids (ABM) along with the trans-complementation plasmids pMD2.G expressing VSV-G envelope and phCMV-8.91 expressing GAG/POL were transfected into HEK293T cells using jetPEI or Xfect transfection reagents (Polyplus Transfection T or Clontech, Mountain View, CA, USA) in serum and antibiotic free medium.

Techniques: Activation Assay, Produced, Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases

doi: 10.3390/ijms22073296

Figure Lengend Snippet: CNS-specific and reference PGC-1α proteins affect exon usage via distinct and similar mechanisms. ( a ) Venn diagram showing the number of genes (left) or exons (right) alternatively spliced after activation of the RG or the CNS promoters in comparison to cells transfected with scrambled sgRNAs. Exon usage of SQSTM1 after RG and CNS promoter activation reveals similar changes to transfections of scrambled sgRNAs (control) and additional differences between each other; comparison between ( b ) CNS and RG promoter activation, ( c ) RG promoter activation and control and ( d ) CNS promoter activation and control; different exon usage between pairwise comparisons is indicated by purple boxes representing the affected exon bins; significant differences ( p < 0.0001) are highlighted by stars; * and **, regions different between RG or CNS vs. scrambled; ***, region, different between CNS vs. RG or scrambled. The black lines refer to differences in any comparison.

Article Snippet: For the stable integration of dCas9-VPR into the genome of cell lines, pLenti-EF1a-dCas9-SAM plasmids (ABM) along with the trans-complementation plasmids pMD2.G expressing VSV-G envelope and phCMV-8.91 expressing GAG/POL were transfected into HEK293T cells using jetPEI or Xfect transfection reagents (Polyplus Transfection T or Clontech, Mountain View, CA, USA) in serum and antibiotic free medium.

Techniques: Activation Assay, Comparison, Transfection