Journal: OncoTargets and therapy

Article Title: Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells

doi: 10.2147/OTT.S124595

Figure Lengend Snippet: Expression levels of TUG1 and ZEB2 in bladder cancer tissues (n=36) and corresponding nontumor tissues (n=36). Notes: ( A ) TUG1 was significantly upregulated in bladder cancer tissues. ( B and C ) The mRNA and protein expression levels of ZEB2 were significantly upregulated in bladder cancer tissues. ( D ) The positive correlation between TUG1 and ZEB2 mRNA in bladder cancer tissues ( r =0.5707, P =0.0029). * P <0.05. Abbreviations: mRNA, messenger RNA; N, adjacent normal tissues; T, tumor tissues; TUG1, taurine upregulated gene 1; ZEB2, zinc finger E-box binding homeobox 2.

Article Snippet: miR-124 mimics (miR-142), small interfering RNA (siRNA) against TUG1 (si-TUG1), TUG1 expression plasmid (pcDNA-TUG1), siRNA against ZEB2 (si-ZEB2) and ZEB2 expression plasmid (pcDNA-ZEB2) were purchased from Ribobio Co. (Guangzhou, China). siRNA sequences for TUG1 were 5′-GCU UGG CUU CUA UUC UGA AUC CUU U-3′ (sense) and 5′-AAA GGA UUC AGA AUA GAA GCC AAG C-3′ (antisense). siRNA sequences for ZEB2 were 5′-CAU CAG ACU UUG AGG AAU A-3′ (sense) and 5′-UAU UCC UCA AAG UCU GAU G-3′ (antisense).

Techniques: Expressing, Binding Assay

Journal: OncoTargets and therapy

Article Title: Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells

doi: 10.2147/OTT.S124595

Figure Lengend Snippet: Knockdown of TUG1 suppressed bladder cancer cell proliferation and induced apoptosis in vitro. Notes: ( A ) The expression levels of TUG1 in HCV-29, T24, and BIU-87 cells were detected by qRT-PCR. ( B and C ) MTT assay was used to detected cell viability of T24 and BIU-87 cells transfected with si-TUG1 at the indicated time points. ( D – G ) Flow cytometry was used to detect apoptosis of T24 and BIU-87 cells transfected with si-TUG1 at 48 h. * P <0.05. Abbreviations: FITC, fluorescein isothiocyanate; NC, negative control; OD, optical density; PI, propidium iodide; si, small interfering; TUG1, taurine upregulated gene 1.

Article Snippet: miR-124 mimics (miR-142), small interfering RNA (siRNA) against TUG1 (si-TUG1), TUG1 expression plasmid (pcDNA-TUG1), siRNA against ZEB2 (si-ZEB2) and ZEB2 expression plasmid (pcDNA-ZEB2) were purchased from Ribobio Co. (Guangzhou, China). siRNA sequences for TUG1 were 5′-GCU UGG CUU CUA UUC UGA AUC CUU U-3′ (sense) and 5′-AAA GGA UUC AGA AUA GAA GCC AAG C-3′ (antisense). siRNA sequences for ZEB2 were 5′-CAU CAG ACU UUG AGG AAU A-3′ (sense) and 5′-UAU UCC UCA AAG UCU GAU G-3′ (antisense).

Techniques: Knockdown, In Vitro, Expressing, Quantitative RT-PCR, MTT Assay, Transfection, Flow Cytometry, Negative Control

Journal: OncoTargets and therapy

Article Title: Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells

doi: 10.2147/OTT.S124595

Figure Lengend Snippet: Knockdown of ZEB2 suppressed bladder cancer cell proliferation and induced apoptosis in vitro. Notes: ( A , B ) The mRNA and protein expression levels of ZEB2 in HCV-29, T24, and BIU-87 cells were detected by qRT-PCR and Western blot, respectively. ( C ) The expression levels of ZEB2 in T24 and BIU-87 cells 48 h after transfection with si-ZEB2 or pcDNA-ZEB2. ( D – G )1 MTT assay was used to detect cell viability of T24 and BIU-87 cells transfected with si-ZEB2 or pcDNA-ZEB2 at the indicated time points. ( H and I ) Flow cytometry was used to evaluate apoptosis of T24 and BIU-87 cells transfected with si-ZEB2 at 48 h. * P <0.05. Abbreviations: mRNA, messenger RNA; NC, negative control; OD, optical density; si, small interfering; ZEB2, zinc finger E-box binding homeobox 2.

Article Snippet: miR-124 mimics (miR-142), small interfering RNA (siRNA) against TUG1 (si-TUG1), TUG1 expression plasmid (pcDNA-TUG1), siRNA against ZEB2 (si-ZEB2) and ZEB2 expression plasmid (pcDNA-ZEB2) were purchased from Ribobio Co. (Guangzhou, China). siRNA sequences for TUG1 were 5′-GCU UGG CUU CUA UUC UGA AUC CUU U-3′ (sense) and 5′-AAA GGA UUC AGA AUA GAA GCC AAG C-3′ (antisense). siRNA sequences for ZEB2 were 5′-CAU CAG ACU UUG AGG AAU A-3′ (sense) and 5′-UAU UCC UCA AAG UCU GAU G-3′ (antisense).

Techniques: Knockdown, In Vitro, Expressing, Quantitative RT-PCR, Western Blot, Transfection, MTT Assay, Flow Cytometry, Negative Control, Binding Assay

Journal: OncoTargets and therapy

Article Title: Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells

doi: 10.2147/OTT.S124595

Figure Lengend Snippet: ZEB2 overexpression reversed effects of TUG1 knockdown on proliferation and apoptosis of bladder cancer cells. Notes: ( A and C ) Western blot analyses showed the expression of ZEB2 was effectively upregulated by pcDNA-TUG1 in T24 and BIU-87 cells. ( B and D ) Western blot analyses indicated that the expression of ZEB2 was effectively downregulated by si-TUG1 in T24 and BIU-87 cells. ( E and F ) The viability of T24 and BIU-87 cells transfected with si-TUG1 or si-TUG1 + pcDNA-ZEB2 was detected at the indicated time points by MTT. ( G and H ) Flow cytometry was used to measure apoptosis of T24 and BIU-87 cells transfected with si-TUG1 or si-TUG1 + pcDNA-ZEB2 at 48 h. * P <0.05. Abbreviations: NC, negative control; OD, optical density; si, small interfering; TUG1, taurine upregulated gene 1; ZEB2, zinc finger E-box binding homeobox 2.

Article Snippet: miR-124 mimics (miR-142), small interfering RNA (siRNA) against TUG1 (si-TUG1), TUG1 expression plasmid (pcDNA-TUG1), siRNA against ZEB2 (si-ZEB2) and ZEB2 expression plasmid (pcDNA-ZEB2) were purchased from Ribobio Co. (Guangzhou, China). siRNA sequences for TUG1 were 5′-GCU UGG CUU CUA UUC UGA AUC CUU U-3′ (sense) and 5′-AAA GGA UUC AGA AUA GAA GCC AAG C-3′ (antisense). siRNA sequences for ZEB2 were 5′-CAU CAG ACU UUG AGG AAU A-3′ (sense) and 5′-UAU UCC UCA AAG UCU GAU G-3′ (antisense).

Techniques: Over Expression, Knockdown, Western Blot, Expressing, Transfection, Flow Cytometry, Negative Control, Binding Assay

Journal: OncoTargets and therapy

Article Title: Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells

doi: 10.2147/OTT.S124595

Figure Lengend Snippet: TUG1 targeted miR-142 to regulate ZEB2 expression. Notes: ( A ) The predicted binding sites of miR-142 in TUG1. ( B and C ) The luciferase activity in T24 and BIU-87 cells simultaneously transfected with miR-142 and TUG1-WT was significantly reduced, whereas cotransfection of miR-142 and TUG1-MUT did not change the luciferase activity. ( D and E ) The expression of miR-142 in T24 and BIU-87 cells treated with si-TUG1 or pcDNA-TUG1 was detected by qRT-PCR. ( F ) The predicted binding sites of miR-142 in 3′-UTR of ZEB2. ( G and H ) The 3′-UTR of wild type ZEB2 (WT) or 3′-UTR of mutant ZEB2 (MUT) reporter plasmids was co-transfected into T24 or BIU-87 cells with miR-control, miR-142, miR-142 + pcDNA-control, or miR-142 + pcDNA-TUG1. The luciferase activity was detected 36 h posttransfection. ( I and J ) The expression of ZEB2 in T24 and BIU-87 cells treated with miR-142 or miR-142 + pcDNA-TUG1 was detected by Western blot analysis. * P <0.05. Abbreviations: MUT, mutant; NC, negative control; qRT-PCR, quantitative real time polymerase chain reaction; si, small interfering; TUG1, taurine upregulated gene 1; ZEB2, zinc finger E-box binding homeobox 2; WT, wild type.

Article Snippet: miR-124 mimics (miR-142), small interfering RNA (siRNA) against TUG1 (si-TUG1), TUG1 expression plasmid (pcDNA-TUG1), siRNA against ZEB2 (si-ZEB2) and ZEB2 expression plasmid (pcDNA-ZEB2) were purchased from Ribobio Co. (Guangzhou, China). siRNA sequences for TUG1 were 5′-GCU UGG CUU CUA UUC UGA AUC CUU U-3′ (sense) and 5′-AAA GGA UUC AGA AUA GAA GCC AAG C-3′ (antisense). siRNA sequences for ZEB2 were 5′-CAU CAG ACU UUG AGG AAU A-3′ (sense) and 5′-UAU UCC UCA AAG UCU GAU G-3′ (antisense).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Cotransfection, Quantitative RT-PCR, Mutagenesis, Control, Western Blot, Negative Control, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells

doi: 10.2147/OTT.S124595

Figure Lengend Snippet: TUG1 regulated the Wnt/β-catenin pathway by affecting ZEB2. Notes: ( A and B ) Protein expressions of β-catenin, c-Myc, and cyclinD1 were evaluated at 48 h in T24 cells transfected with pcDNA-TUG1 or pcDNA-TUG1 + si-ZEB2 by Western blot analysis. ( C and D ) Protein expressions of β-catenin, c-myc, and cyclinD1 were measured at 48 h in T24 cells transfected with si-TUG1 and si-TUG1 + pcDNA-ZEB2 by Western blot analysis. * P <0.05. Abbreviations: si, small interfering; TUG1, taurine upregulated gene 1; ZEB2, zinc finger E-box binding homeobox 2.

Article Snippet: miR-124 mimics (miR-142), small interfering RNA (siRNA) against TUG1 (si-TUG1), TUG1 expression plasmid (pcDNA-TUG1), siRNA against ZEB2 (si-ZEB2) and ZEB2 expression plasmid (pcDNA-ZEB2) were purchased from Ribobio Co. (Guangzhou, China). siRNA sequences for TUG1 were 5′-GCU UGG CUU CUA UUC UGA AUC CUU U-3′ (sense) and 5′-AAA GGA UUC AGA AUA GAA GCC AAG C-3′ (antisense). siRNA sequences for ZEB2 were 5′-CAU CAG ACU UUG AGG AAU A-3′ (sense) and 5′-UAU UCC UCA AAG UCU GAU G-3′ (antisense).

Techniques: Transfection, Western Blot, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: Primers for qRT-PCR.

Article Snippet: The Klf4 over-expression plasmid (pcDNA- Klf4 ) and empty pcDNA3.1 plasmid (control) were purchased from RIBOBIO (Guangzhou, China).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: Antihypertrophic effects of OT in vitro . Neonatal rat cardiomyocytes stimulated with ISO for 24 h in the presence or absence of OT. (A) The cell morphology was evaluated by H&E staining. (B) Statistical results of measurement of cell surface areas. (C,D) Effects of OT on the protein expressions of BNP and β-MHC. (E) Effects of OT on the expression of lncRNA GAS5. (F) Effects of OT on the expression of miR-375-3p. (G,H) Effects of OT on the mRNA and protein expressions of KLF4. (I) Effects of OT on the p-PI3K/PI3K ratio. (J) Effects of OT on the p-AKT1/AKT1 ratio. (K) Western blot images of BNP, β-MHC, KLF4, p-PI3K, PI3K, AKT1, p-AKT1, and β-actin levels. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The Klf4 over-expression plasmid (pcDNA- Klf4 ) and empty pcDNA3.1 plasmid (control) were purchased from RIBOBIO (Guangzhou, China).

Techniques: In Vitro, Staining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: The interactions of miR-375-3p with lncRNA GAS5 and KLF4. (A,C) Predicted duplex formation between lncRNA GAS5 and miR-375-3p (A) , and KLF4 3′-UTR and miR-375-3p (C) . (B) The WT (MUT) GAS5 vector and miR-375-3p mimics or mimics NC were co-transfected into 293T cells. Dual luciferase gene reporter assay was performed to indicate that miR-375-3p could directly bind with the WT GAS5. (D) The WT (MUT) KLF4 3′-UTR vector and miR-375-3p mimics or mimics NC were co-transfected into 293T cells. Dual luciferase gene reporter assay was performed to indicate that miR-375-3p could directly bind with the WT KLF4. All the experiments were repeated three times. ** p < 0.01. WT, wild-type; MUT, mutant type; NC, negative control.

Article Snippet: The Klf4 over-expression plasmid (pcDNA- Klf4 ) and empty pcDNA3.1 plasmid (control) were purchased from RIBOBIO (Guangzhou, China).

Techniques: Plasmid Preparation, Transfection, Luciferase, Reporter Assay, Mutagenesis, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: Down-regulation of lncRNA GAS5 inhibits the anti-hypertrophic effects of OT via miR-375-3p in ISO-treated primary cardiomyocytes. (A) qRT-PCR was performed to quantitatively measure lncRNA GAS5 expression after transfection of shRNA-GAS5#1, shRNA-GAS5#2, shRNA-GAS5#3 in cardiomyocytes. (B) Expression of lncRNA GAS5 was determined by qRT-PCR. (C,D) Expressions of BNP and β-MHC proteins were measured by Western blot analysis. (E) Expression of miR-375-3p was determined by qRT-PCR. (F) Expression of KLF4 mRNA was determined by qRT-PCR. (G) Expression of KLF4 protein was determined by Western blot analysis. (H) Statistical results of measurement of cell surface areas. (I) Cardiomyocyte surface areas were measured by immunofluorescent staining. Scale bars represent 20 µm. Images were captured at ×400 magnification (J) Representative Western blot images of KLF4, BNP, β-MHC and GAPDH. (a) ISO+OT+shRNA-scramble group. (b) ISO+OT+shRNA-GAS5 group. (c) ISO+OT+shRNA-GAS5+miR-375-3p inhibitor NC group. (d) ISO+OT+shRNA-GAS5+miR-375-3p inhibitor group. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The Klf4 over-expression plasmid (pcDNA- Klf4 ) and empty pcDNA3.1 plasmid (control) were purchased from RIBOBIO (Guangzhou, China).

Techniques: Quantitative RT-PCR, Expressing, Transfection, shRNA, Western Blot, Staining

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: Over-expression of miR-375-3p blunted anti-hypertrophic effects of oxytocin via KLF4 and modulated the PI3K/AKT signaling pathway. (A) Expression of miR-375-3p in cardiomyocytes following transfection of miR-375-3p mimics and miR-375-3p mimics NC. (B) Expression of miR-375-3p was detected by qRT-PCR. (C,D) Expressions of BNP and β-MHC proteins were measured by Western blot analysis. (E) Expression of KLF4 mRNA was detected by qRT-PCR. (F) Expression of KLF4 protein was measured by Western blot analysis. (G) p-PI3K/PI3K ratio. (H) p-AKT1/AKT1 ratio. (I) Statistical results of measurement of cell surface areas. (J) Representative western blot images of KLF4, PI3K, p-PI3K, AKT1, p-AKT1, BNP, β-MHC and GAPDH levels. (K) Cardiomyocyte surface areas were measured by immunofluorescent staining. Scale bars represent 20 µm. Images were captured at ×400 magnification. (a) ISO+OT+miR-375-3p mimics NC group. (b) ISO+OT+miR-375-3p mimics group. (c) ISO+OT+miR-375-3p mimics+pcDNA-NC group. (d) ISO+OT+miR-375-3p mimics+pcDNA-KLF4 group. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The Klf4 over-expression plasmid (pcDNA- Klf4 ) and empty pcDNA3.1 plasmid (control) were purchased from RIBOBIO (Guangzhou, China).

Techniques: Over Expression, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Staining

Journal: Frontiers in Pharmacology

Article Title: Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism

doi: 10.3389/fphar.2021.766024

Figure Lengend Snippet: knock-down of KLF4 blunted anti-hypertrophic effects of oxytocin via PI3K/AKT pathway. (A–C) Detection of relative shRNA-KLF4 interference effects. The changes in KLF4 mRNA and protein levels were detected by qRT-PCR and Western blotting after transfection of shRNA-KLF4 #1, shRNA-KLF4 #2, shRNA-KLF4 #3 in primary cardiomyocytes. (D) Expression of KLF4 mRNA was determined by qRT-PCR. (E) Expression of KLF4 protein was measured by Western blot analysis. (F,G) Expressions of BNP, β-MHC proteins were measured by Western blot analysis. (H) p-PI3K/PI3K ratio. (I) p-AKT1/AKT1 ratio. (J) Statistical results of measurement of cell surface areas. (K) Cardiomyocyte surface areas were measured by immunofluorescent staining. Scale bars represent 20 µm. Images were captured at ×400 magnification. (L) Western blot images of KLF4, PI3K, p-PI3K, AKT1, p-AKT1, BNP, β-MHC and GAPDH levels. (a) ISO+OT+shRNA-NC group. (b) ISO+OT+shRNA-KLF4 group. (c) ISO+OT+shRNA-KLF4+LY194002 group. Data are shown as the mean ± sd of three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The Klf4 over-expression plasmid (pcDNA- Klf4 ) and empty pcDNA3.1 plasmid (control) were purchased from RIBOBIO (Guangzhou, China).

Techniques: shRNA, Quantitative RT-PCR, Western Blot, Transfection, Expressing, Staining

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Schematic of enCRISPRa containing three components: a dCas9-p300 fusion protein, the sgRNA with two MS2 hairpins, and the MCP-VP64 fusion protein. b Expression of MYOD upon dCas9 alone, dCas9-VP64 (V), dCas9-p300 (P), dCas9-VP64 + MCP-p300 (enCRISPRa-VP) or dCas9-p300 + MCP-VP64 (enCRISPRa-PV)-mediated enhancer activation in HEK293T cells. mRNA expression relative to nontransduced cells (control) is shown as mean ± SEM ( n = 4 experiments). The differences between control and dCas9 activators were analyzed by a one-way ANOVA. # P < 0.05, ### P < 0.001. The difference between different dCas9 activators were analyzed by a one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant. c Expression of HBE1 , HBG1/2 and HBB upon dCas9 alone, dCas9-VP64 (V), dCas9-p300 (P), or enCRISPRa (VP and PV)-mediated activation of the HS2 enhancer in HEK293T cells. mRNA expression relative to nontransduced cells is shown as mean ± SEM ( n = 4 experiments) and analyzed by a one-way ANOVA. d Expression of MYOD upon dxCas9-VPR, SunTag, SAM or enCRISPRa-mediated enhancer activation in HEK293T cells. mRNA expression relative to nontransduced cells is shown as mean ± SEM ( n = 4 experiments). The differences between control and dCas9 activators were analyzed by a one-way ANOVA. # P < 0.05, ### P < 0.001. The difference between different dCas9 activators were analyzed by a one-way ANOVA. *** P < 0.001. e Expression of HBE1 , HBG1/2 and HBB upon dxCas9-VPR, SunTag, SAM or enCRISPRa-mediated activation of HS2 in HEK293T cells. mRNA expression relative to nontransduced cells is shown as mean ± SEM ( n = 4 experiments) and analyzed by a one-way ANOVA. f Genome-wide analysis of dCas9 binding in HEK293T cells expressing HS2-specific sgRNA (two replicates sgHS2-rep1 and sgHS2-rep2) or nontargeting sgGal4. Data points for the sgRNA target regions and the predicted off-targets are shown as green and red, respectively. The x - and y axis denote the normalized read counts (left) or mean normalized read counts from n = 2 experiments (right). Source data are provided as a Source Data file.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: Expressing, Activation Assay, Genome Wide, Binding Assay

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Schematic of enCRISPRi containing a dCas9-LSD1 fusion protein, the sgRNA with two MS2 hairpins, and the MCP-KRAB fusion protein. b Expression of β-globin genes in K562 cells upon dCas9-KRAB (K), dCas9-LSD1 (L) or enCRISPRi (LK and KL)-mediated repression of the HS2 enhancer using four HS2-targeting sgRNAs individually (sgHS2-1 to sgHS2-4) or combined (sgHS2-all). The nontargeting sgGal4 was analyzed as the control. mRNA expression relative to nontransduced cells is shown as mean ± SEM ( n = 4 experiments) and analyzed by a two-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant. c RNA-seq profiles in K562 cells upon dCas9-KRAB, dCas9-LSD1 or enCRISPRi-mediated repression of the HS2 enhancer using four sgRNAs (sgHS2-all). Scatter plot is shown for each gene by the mean of log2 normalized RNA-seq signals as transcripts per million or TPM ( n = 2 experiments) ( x axis) and log2 fold changes of mean TPM in cells expressing sgHS2 and nontransduced cells ( y axis). β-globin genes are indicated by red arrowheads. d Genome-wide analysis of dCas9 binding in K562 cells expressing HS2-specific sgRNA (sgHS2-rep1 and sgHS2-rep2) or nontargeting sgGal4. Data points for the sgRNA target regions and the predicted off-targets are shown as green and red, respectively. e Density maps are shown for DHS, ChIP-seq of H3K27ac, H3K4me1, H3K4me2, CTCF, and RNA-seq at the β-globin cluster (chr11: 5,222,500–5,323,700; hg19). The zoom-in view of the HS2 proximity region is shown on the top. Dashed lines denote the positions of sgRNAs. f Expression of β-globin genes in K562 cells coexpressing enCRISPRi and target-specific sgRNAs at various positions within the β-globin cluster, control sgRNAs (sgCtrl, sgTAD1, sgTAD2, sgCTCF1 and sgCTCF2) or nontargeting sgGal4. mRNA expression relative to nontransduced cells is shown as mean ± SEM. The differences between control sgGal4 and other sgRNAs were analyzed by a one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant. The differences between sgHS2 and other sgRNAs were analyzed by a one-way ANOVA. ## P < 0.01, ### P < 0.001. Source data are provided as a Source Data file.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: Expressing, RNA Sequencing Assay, Genome Wide, Binding Assay, ChIP-sequencing

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Density maps are shown for ATAC-seq and H3K27ac ChIP-seq at TAL1 (chr1:47,679,000–47,722,000; hg19) in Jurkat cells. The TAL1 oncogenic SE is shown as blue shaded lines. The positions of sgRNAs and PAM sequences are shown as colored lines and boxes, respectively. b Chromatin occupancy of dCas9-p300 by target-specific (sgMut1, sgMut2, sgWT1 and sgWT2) or nontargeting sgGal4 in Jurkat or K562 cells. ChIP signals (% of input) are shown as mean ± SEM ( n = 4 experiments). c Expression of TAL1 mRNA in Jurkat cells upon enCRISPRa-mediated enhancer activation. Results are mean ± SEM ( n = 4 experiments). d Expression of TAL1 protein upon enCRISPRa-mediated enhancer activation. The quantified TAL1 expression is shown. e Activation of TAL1 SE promoted T-ALL growth in vitro. Relative absorbance by cell viability assays ( y axis) at different days of culture ( x axis) is shown ( n = 3 experiments). f Expression of TAL1 upon enCRISPRi-mediated enhancer repression in Jurkat cells. Results are mean ± SEM ( n = 4 experiments). g Expression of TAL1 protein upon enCRISPRi-mediated enhancer repression. h Repression of TAL1 SE impaired T-ALL growth in vitro ( n = 3 experiments). i Activation of TAL1 SE promoted T-ALL growth in NSG mice xenografted with Jurkat cells transduced with sgGal4, sgMut2 or sgWT2, respectively. Bioluminescence intensity is shown at 4 h, 2 and 4 weeks post transplantation. j Quantification of bioluminescence intensity is shown. Results are mean ± SEM ( n = 5 recipients per group). k Frequencies of leukemia cells in BM and PB of xenografted NSG mice 4 weeks post transplantation. Results are mean ± SEM ( n = 5, 5, and 4 recipients for sgGal4, sgMut2 and sgWT2, respectively). l Representative bloodsmear images of NSG mice 4 weeks post transplantation. The inset images indicate the zoom-in view. Representative leukemia cells are indicated by arrowheads. Scale bars, 200 and 20 µm for full and insert images, respectively. Results are mean ± SEM and analyzed by a one-way or two-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, ### P < 0.001, n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: ChIP-sequencing, Expressing, Activation Assay, In Vitro, Transduction, Transplantation Assay

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Schematic of site-specific KI of tetracycline-inducible dCas9-KRAB into the Col1a1 locus. Coexpression of dCas9-KRAB, sgRNA-MS2 and MCP-LSD1 leads to assembly of enCRISPRi complex in vivo. b Validation of dCas9-KRAB and rtTA KI or WT alleles by genotyping PCR. C57BL/6 WT mouse and targeted KH2-ESC were used as controls. Two independent heterozygous (Het) and homozygous (Hom) KI mice were analyzed. c Dox-inducible expression of dCas9-KRAB fusion protein was confirmed by Western blot in the targeted KH2-ESC and two independent dCas9-KRAB KI mice. β-tubulin was analyzed as the loading control. d Schematic of in vivo perturbation of lineage-specific enhancers in dCas9-KRAB KI mice. e In vivo enCRISPRi perturbation of Cebpa CREs revealed lineage-specific requirement of Cebpa enhancers during hematopoiesis. Waterfall plots are shown for target-specific sgRNAs (green and red dots) and nontargeting control sgRNAs (gray dots) by the mean normalized log2 fold changes in HSPCs, myeloid, T or B cells 16 weeks post BMT (T2) relative to pooled sgRNA-transduced HSPCs (T1) from two independent replicate screens ( n = 3 recipient mice per screen). Density maps are shown for ATAC-seq and H3K27ac ChIP-seq at the Cebpa locus (chr7:35,877,000–35,951,000; mm9) in bone marrow HSC, granulocytes (GN), monocytes (Mono), B, CD4+ and CD8+ T cells, respectively. The annotated Cebpa promoter (P) and enhancers (E1 to E4) are indicated by green and blue shaded lines. Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. . f In vivo enCRISPRi perturbation of Spi1 CREs during hematopoiesis. Density maps are shown for ATAC-seq and H3K27ac ChIP-seq at the Spi1 locus (chr2:90,911,000–90,957,000; mm9) in bone marrow HSC, GN, Mono, B, CD4+ and CD8+ T cells, respectively. The annotated Spi1 promoter (P) and enhancer (E) are indicated by green and blue shaded lines. Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: In Vivo, Expressing, Western Blot, ChIP-sequencing

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Schematic of in vivo pooled sgRNA-based multiloci perturbations of developmentally regulated enhancers in dCas9-KRAB KI mice. b In vivo perturbation of annotated CREs for five key hematopoietic TFs revealed the functional requirement of lineage-specific enhancers for HSC differentiation to one or multiple hematopoietic lineages. Waterfall plots are shown for target-specific sgRNAs (green and red dots) and nontargeting control sgRNAs (gray dots) by the mean normalized log2 fold changes in HSPCs, myeloid, T or B cells 16 weeks post BMT (T2) relative to pooled sgRNA-transduced HSPCs (T1) from two independent replicate screens ( n = 15 recipient mice per replicate screen). Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. . c In vivo enCRISPRi of Runx1 CREs during hematopoiesis by single locus-based perturbation. Density maps are shown for ATAC-seq and H3K27ac ChIP-seq at the Runx1 locus (chr16:92,579,000–93,050,000; mm9) in bone marrow HSC, GN, Mono, B, CD4+ and CD8+ T cells, respectively. The annotated Runx1 promoters (P1 and P2) and enhancers (E1–E3) are indicated by green and blue shaded lines. Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. . d In vivo enCRISPRi of Runx1 CREs during hematopoiesis by multiplexed perturbation. Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. .

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: In Vivo, Functional Assay, ChIP-sequencing

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: Density maps are shown for ChIP-seq of dCas9, active histone marks (H3K4me1, H3K4me2, and H3K27ac), repressive H3K9me3 and H3K27me3, GATA1, TAL1, and CTCF at the β-globin cluster (chr11: 5,222,500−5,323,700; hg19) in K562 cells coexpressing nontargeting sgGal4 (control or C) or sgHS2 with dCas9-KRAB (K), dCas9-LSD1 (L) or enCRISPRi (LK and KL). Regions showing increased or decreased ChIP-seq signals in enCRISPRi (LK) relative to control, dCas9-KRAB, dCas9-LSD1 or enCRISPRi (KL) (enCRISPRi—C, enCRISPRi—K, enCRISPRi—L, or enCRISPRi—KL) are depicted in green and red, respectively. Blue bars denote the sgRNA-targeted HS2 enhancer. Green bars denote the β-globin genes. Independent replicate experiments are shown as rep1 and rep2 in Supplementary Figs. and , respectively.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: ChIP-sequencing