Journal: Cell reports

Article Title: A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability

doi: 10.1016/j.celrep.2018.09.045

Figure Lengend Snippet: (A) WNT7A binds to RECK and FZD8. HEK293 RECK −/− WNT7A cells were cultured in medium supplemented with indicated purified proteins (25 nM) for 72 hr and CM subjected to Protein A pull-down. (B) WNT3A binds to FZD4 and FZD8, but not to GPR124 and RECK. HEK293 RECK −/− WNT3A cells were cultured in medium supplemented with indicated purified proteins (25 nM) for 72 hr and CM subjected to Protein A pull-down. (C) Super TOP-Flash (STF) canonical Wnt/β-catenin reporter gene assay. HEK293 RECK +/+ or RECK −/− cells were co-transfected with the indicated expression constructs and STF/RLuc reporters. 24 hr after transfection sRECK-Fc protein was added at 0, 10, 100, or 1,000 nM (wedges). 24 hr later STF activity was normalized with RLuc activity. Mean (n = 3) ± SD. *p < 0.05 versus no protein. (D) WNT7A binding to RECK is mediated by RECK cystine knot motif (CK) and cysteine-rich domains (CRD). HEK293 RECK −/− WNT7A cell culture medium was supplemented with the indicated proteins (3 nM) and conditioned for 96 hr. CM were subjected to Protein A pull-down. (E and F) Cell surface RECK binds to WNT7A, but not to WNT3A. HEK293 RECK −/− WNT7A (E) or WNT3A (F) cells were transfected with the indicated expression constructs. 48 hr after transfection, SNAP tags were specifically biotinylated (bSNAP) and cell surface proteins cross-linked in situ using DTSSP (100 μM). Cells were lysed, subjected to streptavidin pull-down, and cross-links reversed. WCL, whole cell lysate. All western blots (WB) were performed under reducing conditions. All data are representative of at least two independent experiments with similar results. CM, conditioned medium; s, soluble ECD; Fc, IgG Fc fragment. See also and .

Article Snippet: pcDNA-WNT7A (human) , , Addgene plasmid #35914.

Techniques: Cell Culture, Purification, Reporter Gene Assay, Transfection, Expressing, Construct, Activity Assay, Binding Assay, In Situ, Western Blot

Journal: Cell reports

Article Title: A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability

doi: 10.1016/j.celrep.2018.09.045

Figure Lengend Snippet: (A) RECK and FZD8 only bind newly secreted WNT7A. Culture media of HEK293 RECK −/− WNT7A cells were supplemented with the indicated proteins (100 nM) and conditioned for 72 hr (“during conditioning”). Alternatively, the indicated proteins were added to the CM after harvesting after 72h (“after conditioning”). CM were subjected to Protein A pull-down. (B and C) CM from HEK293 RECK −/− WNT7A (B) or WNT3A (C) cells were analyzed by reducing (+DTT) or non-reducing (−DTT) SDS-PAGE/WB. Where indicated CM were subjected to Triton X-114 phase separation. (D) sRECK-Fc protein stabilizes newly secreted, monomeric, hydrophobic WNT7A. Culture medium of HEK293 RECK −/− WNT7A cells was supplemented with indicated proteins (100 nM) and conditioned for 72 hr (“during conditioning”). Alternatively, indicated proteins were added to CM after harvesting after 72 hr (“after conditioning”). CM aliquots were subjected to Triton X-114 phase separation and remaining CM to Protein A pull-down followed by Triton X-114 phase separation of pull-downs and unbound supernatants. Irrelevant lanes (between lanes 4 and 5) have been removed from the WB. (E) RECK and WNT7A form a 1:1 complex. Expi293F cells were co-transfected with sRECK-His and WNT7A. 96 hr after transfection CM was harvested and sRECK-His:WNT7A complex purified by combined chemical cross-linking (BS 3 )/tandem affinity purification. Purified complex was analyzed by reducing SDS-PAGE/WB. (F) Free, hydrophobic WNT7A is highly unstable. sRECK-Fc:WNT7A, sFZD8-Fc-Biotin:WNT7A, and sFZD8-Fc-Biotin:WNT3A complexes were isolated from the corresponding HEK293 RECK −/− WNT7A/WNT3A CM (96 hr, 100 nM recombinant protein) using Protein A or streptavidin agarose. Bound Wnt proteins were eluted by low pH, neutralized, diluted into PBS/10% FBS and incubated at 37°C. At indicated time points Wnt eluate was subjected to Triton X-114 phase separation. All data are representative of at least two independent experiments with similar results. CM, conditioned medium; WB, western blot; T, total; De, detergent phase; Aq, aqueous phase; s, soluble ECD; Fc, IgG Fc fragment. See also Figures and .

Article Snippet: pcDNA-WNT7A (human) , , Addgene plasmid #35914.

Techniques: SDS Page, Transfection, Purification, Affinity Purification, Isolation, Recombinant, Incubation, Western Blot

Journal: Cell reports

Article Title: A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability

doi: 10.1016/j.celrep.2018.09.045

Figure Lengend Snippet: (A) GPR124, RECK, and WNT7A form a ternary complex. HEK293 RECK −/− (control) or HEK293 RECK −/− WNT7A cells were cultured in medium supplemented with the indicated proteins (50 nM) for 72 hr. CM were harvested and subjected to Protein A pull-down. (B) GPR124 does not regulate cell surface RECK:WNT7A complex formation. Cells were transfected with indicated expression constructs for 48 hr. SNAP tags were specifically biotinylated (bSNAP) and cell surface proteins cross-linked in situ by DTSSP (100 μM). Cells were lysed, subjected to streptavidin pull-down, and cross-links reversed. WCL, whole cell lysate. β2AR, β 2 -adrenergic receptor. (C) FZD/GPR124/RECK/WNT7A quaternary complex formation is not detectable in vitro . sRECK-His:WNT7A complex was purified from HEK293 RECK −/− WNT7A CM (72 hr, 100 nM sRECK-His) using Ni-NTA agarose. Indicated purified proteins/sRECK:WNT7A complex (1 μM each) were allowed to form complexes and subjected to Protein A pull-down. Faint non-specific sRECK binding in all lanes. (D) FZD8 elutes WNT7A from RECK. sRECK-Fc:WNT7A complex was isolated from HEK293 RECK −/− WNT7A CM (72 hr, 100 nM sRECK-Fc) by Protein A agarose. Beads were incubated with 0.1, 1, or 10 μM sFZD4/sFZD8 protein (wedges) or glycine, pH 2.9 for 1 hr (eluate 1) and then with 1% SDS (eluate 2). (E) WNT7A binding to RECK and FZD8 is mutually exclusive. HEK293 RECK −/− WNT7A cells were cultured in medium supplemented with indicated proteins (25 nM) for 72 hr. CM were subjected to sequential Ni-NTA agarose (1 st ) and Protein A agarose (2 nd ) pull-downs. (F) RECK dose-dependently enhances FZD8:WNT7A complex formation. HEK293 RECK −/− WNT7A cells were cultured in medium supplemented with sFZD8-His and sRECK-Fc proteins as indicated for 72 hr. CM were subjected to Ni-NTA pull-down. (G) Model of RECK/GPR124-regulated canonical WNT7 signaling. GPR124 ECD binding to RECK strongly enhances RECK/WNT7-induced canonical Wnt signaling by an extracellular mechanism that does not involve intrinsic GPR124 signal transduction or regulation of RECK:WNT7 complex formation. The RECK cystine knot motifs (blue) and cysteine-rich domain (purple) mediate binding of RECK:GPR124 to WNT7, stabilizing WNT7 in its active, monomeric, hydrophobic form. Free WNT7 rapidly converts into an inactive, aggregated, hydrophilic state. The GPR124:RECK complex acts as a stabilizing receptor for WNT7 increasing bioavailability of active, monomeric cell surface WNT7. GPR124:RECK-bound WNT7 is eventually transferred to FZD and LRP5/6 co-receptors by transient direct or indirect interactions. Alternatively, GPR124/RECK/WNT7 could form a stable multi-protein receptor complex with FZD/LRP5/6. All western blots (WB) were performed under reducing conditions. All data are representative of at least two independent experiments with similar results. CM, conditioned medium; s, soluble ECD; Fc, IgG Fc fragment. See also .

Article Snippet: pcDNA-WNT7A (human) , , Addgene plasmid #35914.

Techniques: Control, Cell Culture, Transfection, Expressing, Construct, In Situ, In Vitro, Purification, Binding Assay, Isolation, Incubation, Transduction, Western Blot

Journal: Cell reports

Article Title: A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability

doi: 10.1016/j.celrep.2018.09.045

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pcDNA-WNT7A (human) , , Addgene plasmid #35914.

Techniques: Virus, Recombinant, Reporter Assay, Plasmid Preparation, Luciferase, Software, CRISPR

Journal: The FASEB Journal

Article Title: Sirtuin 6 protects against hepatic fibrogenesis by suppressing the YAP and TAZ function

doi: 10.1096/fj.202200522r

Figure Lengend Snippet: FIGURE 9 SIRT6 deacetylates YAP1 at multiple lysine residues. (A) A diagram of human YAP1 domain structure and several known acetylated lysine residues. (B) Real-time PCR analysis of CYR61, CTGF, and ANKRD1 mRNAs in the LX-2 cells transfected with either WT or mutant human YAP1 plasmids in the presence of 5 ng/ml TGF-β1 (n = 3). (C) YAP1 acetylation analysis in LX-2 cells transfected with WT or mutant human YAP1 plasmids together with vector or SIRT6 plasmids in the presence of 5 ng/ml TGF-β1. Data are presented as mean ± SEM. *p < .05, **p < .01, ***p < .001 versus vector control, and #p < .05, ##p < .001, ###p < .001 versus WT YAP.

Article Snippet: Human YAP1 was a gift from Yosef Shaul (Addgene, Cambridge, MA; plasmid # 18881; http:// n2t.net/addge ne:18881; RRID:Addgene_18881).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation, Control

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Genes targeted in vivo by dCas9-effectors in this study.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: In Vivo, Binding Assay

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Rb paralogs have similar transcriptional effects on E2F2 and Mpp6 reporters in S2 cells. a) For all experiments described here, S2 cells were transfected with actin -GAL4, one of the dCas9 effectors, a single gRNA, and a luciferase reporter. b) Schematic of the Mpp6 -luciferase reporter that was designed to be regulated by the Mpp6 promoter. gRNAs are identical to those used in vivo, but lack gRNA 6 and 7, as they bind within the protein coding sequence not present in the reporter. c) dCas9-Rbf1 recruitment to the Mpp6 reporter significantly represses luciferase expression from position 2 and 3 (50%). Position 5 repression appears to represent a nonspecific steric hindrance, as it is seen with dCas9 alone e). d) dCas9-Rbf2 has a similar pattern and degree of repression of this reporter as dCas9-Rbf1. e) dCas9 has little effect on this promoter aside from position 5, which suggests steric hindrance from downstream of the TSS. f) Schematic of the E2F2 -luciferase reporter that was designed to be regulated by the E2F2 promoter. Here, the gRNAs are in opposite orientation from what is shown in a), as this is the same genomic region, but on the opposite strand. g) dCas9-Rbf1 recruitment to the E2F2 reporter significantly represses luciferase expression from position A (25%) and position B (60%). Recruitment to position 2 causes the same level of repression as dCas9 alone, suggesting steric hindrance from this site, as seen in i). h) dCas9-Rbf2 has a similar pattern and degree of repression of this reporter as dCas9-Rbf1. i) dCas9 has little effect on this promoter, aside from position 2 and 1, which suggests that the E2F2 promoter is more sensitive than the Mpp6 promoter to recruitment of dCas9. Error bars indicate SEM, and * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Expression is normalized to the empty gRNA vector control.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: Transfection, Luciferase, In Vivo, Sequencing, Expressing, Plasmid Preparation, Control

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Creation of an in vivo CRISPRi system in Drosophila to target Rb paralogs to endogenous gene promoters. a) The fly Rbf1 and Rbf2 FLAG-tagged coding sequences were fused to the C-terminus of the S. pyogenes nuclease dead Cas9 (dCas9; asterisks denote inactivating mutations), and placed under UAS expression. FLAG-tagged dCas9 was created as a negative control. dCas9-VPR was obtained as a fly line (#67055 from BDSC); VPR is a tripartite activator that was previously characterized as a Drosophila transcriptional activator ( Lin et al . 2015 ). b) Here, we generated homozygous flies expressing UAS:dCas9-Rb chimeras under the expression of nubbin -GAL4 (expression predominantly in the wing pouch of L3 wing discs; ; ). We crossed these fly lines to homozygous flies expressing 2 gRNAs for a single gene's promoter (from the DRSC). These tandem gRNAs bind within 400 bp of a gene's TSS, and are ubiquitously expressed. c) Progeny from the cross indicated in b) express 3 transgenes in order to target one of the dCas9 chimeras to a single gene's promoter in the L3 wing disc; these flies were used for most of the in vivo experiments described in this study. d) Generation of flies expressing UAS:dCas9-Rb effectors in the follicle cells of the adult female ovary using a traffic jam -GAL4 driver (#104055 from the DGRC). Fly crossing scheme is as depicted in b).

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: In Vivo, Expressing, Negative Control, Generated

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Targeting dCas9-Rb chimeras to endogenous promoters leads to gene-specific effects in the wing. a–c) Wings ( n = 100) were dissected from adult flies for phenotype analysis after dCas9-effectors were targeted to endogenous gene promoters in L3 wing discs. Representative images from each cross are shown. a) A non-targeting gRNA control (QUAS) demonstrates that expression of effectors not targeted to any locus on the genome do not cause adult wing phenotypes. b) Targeting the wg promoter with VPR leads to severe developmental defects, but Rb effects on this promoter are mild to non-existent. c) Targeting the E2F2 promoter with Rbf1 and Rbf2 leads to severe morphological defects that are Rb-specific, confirming the feasibility of the dCas9-Rb fusions in vivo in Drosophila. d–f) Quantification of the wings analyzed in a–c). Values are indicated as a proportion of the total number of wings. d) Quantification of images in a). Background effects are essentially non-existent when expressing the dCas9-effectors with a non-targeting gRNA. e) Quantification of images from crosses depicted in b). dCas9-VPR has a severe phenotypic effect on wg , due to wg overexpression, as previously reported ( Ewen-Campen et al . 2017 ). Minor phenotypes are observed in ∼20% of wings with dCas9-Rbf2. f) Quantification of images from crosses depicted in c). Effects of targeting the E2F2 promoter are severe with both Rb paralogs, and the phenotype is fully penetrant. Legend in f) is shown for all graphs.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: Control, Expressing, In Vivo, Over Expression

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Rbf1 and Rbf2 recruitment to the E2F2/ Mpp6 promoter leads to Rb-specific repression. a) Diagram of the E2F2 gene, which shares its promoter with Mpp6 as a divergently paired gene. gRNA binding sites on the promoter of E2F2 , which overlaps with the promoter of Mpp6 . Horizontal lines below the genes are the locations of the E2F2 gRNAs, with approximate distance from the indicated E2F2 TSS. b, c) Expression level of E2F2 and Mpp6 measured in L3 wing discs after dCas9-effector recruitment, using RT-qPCR. Control samples are dCas9 flies crossed to a non-targeting gRNA fly line (QUAS). b) Rbf1 and Rbf2 significantly repress E2F2 expression. dCas9 alone leads to some modest repression, while the VPR activator has no significant effect on expression of E2F2 or Mpp6 . c) Rbf1 and Rbf2 significantly repress Mpp6 expression, to a greater degree than observed for E2F2 . Effects are significantly greater than by dCas9 alone. d, e) Expression level of E2F2 and Mpp6 measured in ovaries after dCas9-effector recruitment, using RT-qPCR. d) E2F2 is significantly repressed by dCas9-Rbf1, which is greater than dCas9-Rbf2 and dCas9 effects. e) Mpp6 is significantly repressed by dCas9-Rbf1, and dCas9-Rbf1 appears to be a better repressor than Rbf2, although the difference is not statistically significant. For b–e), values in the bars indicate the average expression, error bars are SEM, and asterisks denote statistically significant differences: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Control

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Position-sensitive Rb paralog effects on the E2F2/ Mpp6 bidirectional promoter. a) Schematic of single gRNAs targeting the 1 kb promoter between the E2F2 and Mpp6 transcribed regions. gRNA 4 is what is indicated as −577 in , and gRNA 5 is what is indicated as −672. 4 + 5 are the 2 tandem gRNAs used in . dCas9 effectors were recruited to these sites using one gRNA at a time. b) Adult wings ( n = 50) were dissected after targeting by dCas9, dCas9-Rbf1, and dCas9-Rbf2. Images are representative wings. The severe morphological defect which is clear with 4 + 5 is only observed with gRNA 4 or 5 alone with Rbf2. Rbf1 targeting with the single gRNA 4 or gRNA 5 does not produce this severe effect. c) Quantification of the percentage of wings with particular phenotypes as indicated in the legend on the bottom right. Milder effects were observed from most sites, with supernumerary bristles or ectopic veins forming in a small proportion of wings.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques:

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Repression of E2F2 and Mpp6 in a distance-dependent manner. a) Schematic of gRNAs designed here to target the E2F2/ Mpp6 promoter. dCas9 effectors were recruited to these sites using one gRNA at a time, and L3 wing discs were dissected for RT-qPCR analysis. b) Expression of E2F2 after recruitment of dCas9-Rbf1 to each gRNA site. Position 2 is highlighted, where the greatest repression was measured (∼40%). c) Expression of Mpp6 after recruitment of dCas9-Rbf1 to each gRNA site. Positions 4 and 5 are highlighted, where the greatest repression was measured. Rbf1 is a better repressor when 2 molecules are recruited to the Mpp6 TSS. d) Expression of E2F2 after recruitment of dCas9-Rbf2 to each gRNA site. Position 2 is highlighted, where the greatest repression was measured (∼40%), identical to what was measured by dCas9-Rbf1. e) Expression of Mpp6 after recruitment of dCas9-Rbf2 to each gRNA site. Positions 4 and 5 are highlighted, where the greatest repression was measured. Whether a single molecule or 2 molecules of Rbf2 are recruited does not make a big difference in magnitude of repression (∼50%). f) Expression of E2F2 after recruitment of dCas9 to each gRNA site. dCas9 is not able to repress this gene from most sites. g) Expression of Mpp6 after recruitment of dCas9 to each gRNA site. dCas9 is not able to repress this gene from most sites, aside from a few. Error bars indicate SEM, and * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: Quantitative RT-PCR, Expressing

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: p300 is a regulator of both transcription and alternative splicing. MCF7 cells were transfected with nontargeting siRNA (siNT) and sip300 for 72 h. RNA was extracted, libraries prepared, and RNA-seq was conducted. ( A ) Venn diagram representing differentially expressed genes (light green) and alternatively spliced genes (light orange). ( B , C ) Functional analysis was conducted using the DAVID functional annotation tool (DAVID, https://david.ncifcrf.gov/ ) for ( B ) differentially expressed genes and ( C ) alternative splicing genes. ( D ) Real-time PCR analysis of total mRNA amount of CD44 relative to CycloA reference gene. ( E ) Real-time PCR analysis of CD44s and CD44v5-6 relative to CD44 total mRNA amount. PSI was calculated by CD44v5-6/CD44s + CD44v5-6. Values represent averages of three independent experiments ± SE; (**) P < 0.01 (paired Student's t -test).

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Alternative Splicing, Transfection, RNA Sequencing, Functional Assay, Real-time Polymerase Chain Reaction

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: Tethering p300 to CD44 promoter up-regulates its expression and promotes CD44v inclusion. ( A – C ) HEK293T cells were transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the CD44 promoter, Intron 8 (In8) or 3′ UTR for 30 h. Total RNA was extracted and analyzed by real-time PCR for total mRNA amount of CD44 relative to CycloA reference gene ( A ) and for total mRNA amount of CD44s and CD44v6-7 relative to CD44 total mRNA amount ( B ). PSI was calculated by CD44v6-7/CD44s + CD44v6-7. ChIP was performed of H3 pan-acetylated, along the CD44 gene ( C ). Values represent averages of three independent experiments ± SE and are expressed as dCas9-p300 core (WT) relative to dCas9-p300 core (mut) (*) P < 0.05, (**) P < 0.01, for gRNA to promoter; (#) P < 0.05 gRNA to In8; (†††) P < 0.005 gRNA to 3′ UTR; Student's paired t -test. ( D , E ) HEK293T cells were transfected with either WT RNAPII or Slow RNAPII and after 24 h treated with α-amanitin for 24 h ( D ); or cells were treated with 6 µM CPT for 6 h ( E ). Total RNA was extracted and analyzed by real-time PCR for CD44s and CD44v6-7 relative to CD44 total mRNA amount. PSI was calculated by CD44v6-7/CD44s + CD44v6-7. Values represent mean ± SE of seven ( D ) or three ( E ) independent experiments.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: p300 acetylates Sam68 and hnRNP M. ( A ) Enrichment of binding sites of six of CD44's known splicing factors in p300 regulated exons: SAM68, hnRNP M, hnRNP L, TRA2β, hnRNP F, hnRNP A1. Shown are the −lg ( P -values) enrichment of binding sites in skipped exons versus included ( right bars) and included versus skipped ( left bars). Dashed red line denotes significant P -value (corrected for multiple testing, Bonferroni correction). As can be seen, the binding sites of SAM68 and hnRNP M were found to be significantly enriched in the skipped exons. ( B , C ) HEK293T cells were transfected with nontargeting siRNA (siNT) and sip300 for 72 h. Sam68 was immunoprecipitated and acetylated lysine (AK) was detected ( B ). (*) Heavy chain of Sam68 Ab. hnRNP M was immunoprecipitated and acetylated lysine (KA) was detected ( C ). ( D ) MCF7 cells were transfected with nontargeting siRNA (siNT) or siRNA against p300 (sip300) or against Sam68 (siSam68) or hnRNP M (sihnRNP M) for 72 h. Total RNA was extracted and analyzed by real-time PCR for CD44s and CD44v5-6 relative to CD44 total mRNA amount. PSI was calculated by CD44v5-6/CD44s + CD44v5-6. Values represent averages of three independent experiments ± SE. (*) P < 0.05; (***) P < 0.001; Student's paired t -test. ( E ) HEK293T transfected with nontargeting siRNA (siNT) or siRNA against p300 (sip300) and transfected again after 48 h with either pcDNA3-empty vector or hnRNP M for 24 h. Total RNA was extracted and analyzed by real-time PCR for total mRNA amount of CD44s and CD44v6-7 relative to CD44 total mRNA amount. PSI was calculated by CD44v6-7/CD44s + CD44v6-7. Values represent averages of three independent experiments ± SE.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: p300 excludes hnRNP M from CD44 pre-mRNA and promotes cell motility. ( A ) RNA-IP of hnRNP M in HEK293T cells transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the CD44 promoter. Real-time PCR analysis of CD44 intron 8. Values represent averages of three independent experiments ± SE. ( B ) hnRNP M was immunoprecipitated from HEK293T cells and interacting proteins were detected with indicated antibodies. ( C ) HDAC1 was immunoprecipitated from HEK293T cells and interacting proteins were detected with indicated antibodies. (*) Long exposure. ( D ) HEK293T cells were transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the CD44 promoter for 30 h. ChIP was performed on HDAC1, along the CD44 gene. ( E ) HEK293T cells were treated with 10 µM TSA for 2 h. Real-time PCR analysis of CD44s and CD44v5-6 relative to CD44 total mRNA amount. PSI was calculated by CD44v5-6/CD44s + CD44v5-6. ( F , G ) MCF7 cells stable for dCas9-p300 core (mut) or dCas9-p300 core (WT) were transfected with four gRNAs targeted to CD44 promoter or 3′ UTR for 30 h. Cells were seeded in 96-well plates and a scratch assay was conducted. Results were calculated for gRNA to promoter relative to 3′ UTR. Values represent averages of three independent experiments ± SE. ( G ) Western blot was conducted using the indicated antibodies.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation, Wound Healing Assay, Western Blot

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: Schematic representation of the proposed mechanism. The chromatin factor p300 acetylates histones as well as splicing factors to regulate, respectively, transcription and alternative splicing.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Alternative Splicing