Journal: Molecular cell

Article Title: Dismissal of RNA Polymerase II Underlies a Large Ligand-induced Enhancer Decommissioning Program

doi: 10.1016/j.molcel.2018.07.039

Figure Lengend Snippet: A, The enrichment of CBP (E-TABM-785), p300 (E-TABM-785) and SRC3 (E-TABM-785) spanning ±3kb region from the center of BAER enhancer under Veh and E2 conditions, represented as heatmaps. B, The enrichment of Pol II spanning ±3kb region from the center of E2 active and BAER enhancer under Veh and E2 conditions were represented as heatmaps. C, The enrichment of Pol II at selected enhancers was measured by RT-qPCR when endogenous ERα was knocked-down using two different siRNAs targeting the 3’UTR of ERα in HA-tagged P-box mutant ERα stable cells. Data are presented as mean ± s.d. (N = 3; unpaired t test). D, Plekhf2 and Sytl2 eRNAs were knocked-down using two different ASOs. The expression level of Plekhf2 and Sytl2 mRNA and eRNAs are measured by RT-qPCR. Data are presented as mean ± s.d. P values denoting differences between between control sgRNA and sgRNAs target to Plekhf2- or Sytl2-enhancers. (N = 3; unpaired t test). E, Plekhf2 and Sytl2 enhancers were knockeddown by dCas9-KRAB with 2 different pairs of sgRNAs. The expression level of Plekhf2 and Sytl2 mRNA and eRNAs were measured by RT-qPCR. Data are presented as mean ± s.d. P values denoting differences between control sgRNA and sgRNAs target to Plekhf2- or Sytl2-enhancers. (N = 3; unpaired t test). F, Insertion of Pol II transcription terminators to Plekhf2 and Sytl2 enhancers could decrease downstream target reporter gene (luciferase) expression in HEK-293T cells. Plekhf2 and Sytl2 enhancers with ~500bp Pol II transcription terminators were cloned into pGL4.23 and transfected into HEK-293T cells, the luciferase value was detected by Dual-Luciferase® Reporter Assay System (Promega). G, 4C-seq under vehicle or 1hr E2 treatment in MCF7 cells is shown. The black line represents the interaction frequency based on liner mean in 200kb window. Gray cloud indicates 20th and 80th percentiles of confidence intervals of the main trend on the curve (black line). Domainogram (heatmap) represents the normalized contact profile, which resolution was ranked from 2kb on the top to 50kb on the bottom. Coordinate of enhancers are indicated with red box, while the promoters are indicated with blue box. H, 3CPCR assays showing the changes of the interaction between enhancer and promoter at Plekhf2 and Sytl2 loci upon vehicle or E2 treatment.

Article Snippet: The dCas9-KRAB lenti-virus was prepared by co-transfection of pVSVg (Addgene, Cat#8454) and psPAX2 (Addgene Cat#12260), and pHAGE-TRE-dCas9-KRAB vector (Addgene, Plasmid #50917) into HEK-293T cell using Lipofectamine2000 and harvest the lenti-virus in 48 h and 72 h after transfection.

Techniques: Quantitative RT-PCR, Mutagenesis, Expressing, Luciferase, Clone Assay, Transfection, Reporter Assay

Journal: Molecular cell

Article Title: Dismissal of RNA Polymerase II Underlies a Large Ligand-induced Enhancer Decommissioning Program

doi: 10.1016/j.molcel.2018.07.039

Figure Lengend Snippet: A, E2-mediated decrease of Pol II at the Plekhf2 and Sytl2 enhancers is abolished by 10 M MG132 treatment for 3 h, as shown by Pol II ChIP-qPCR. ChIP signals are presented as percentage of input. Data are mean ± s.d. P values denoting differences between Veh and E2 condition are shown. B, RT-qPCR showing gene expression levels of E2 repressed gene-Sytl2 and Plekhf2 in response of E2 when MCF7 cells was treated with 10 M MG132. Data are presented as mean ± s.d. P values denote differences between +/− E2 treatment are shown (N = 3; unpaired t test). C, The enrichment of NEDD4 spanning ±3kb region from the center of BAER or activated enhancers in Veh and E2 conditions is represented as heatmaps. D, Box-and-whisker plots of NEDD4 ChIP-seq signals (tag counts) in siCTL or Kdm2a knockdown MCF7 cells before and after E2 treatment at BAER enhancers are shown. P values denote statistical differences between treatment conditions. E, Box-and-whisker plots of Pol II ChIP-seq signals (tag counts) in siCTL or Nedd4 knockdown MCF7 cells before and after E2 treatment at BAER enhancers, but not at E2 activated enhancers, are shown. P values denote statistical differences between treatment conditions. Centerlines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles. F, Plekhf2 and Sytl2 enhancers were repressed by dCas9 fused with KDM2A or truncated NEDD4 (containing C terminal HECT domain) proteins generated using two different groups of sgRNAs. The expression level of Plekhf2 and Sytl2 mRNA and eRNAs are measured by RT-qPCR. Data are presented as mean ± s.d. P values denoting differences between control sgRNA and sgRNAs target to Plekhf2- or Sytl2-enhancers. (N = 3; unpaired t test; * = p< 0.05, ** = p< 0.01, *** =p< 0.001.). G, Endogenous ERα and Kdm2a or Nedd4 were knocked-down using siRNAs targeting in the ERα P-box mutated stable cell line. The enrichment of Pol II at selected enhancers was measured by RT-qPCR. Data are presented as mean ± s.d. (N = 3; unpaired t test; * = p< 0.05, ** = p< 0.01, *** =p< 0.001.). H, Working model: E2 treatment causing an increased enrichment of coactivators, such as P300, CBP, SRC3 at BAER enhancers. KDM2A interacts with the DBD of ERα, recruits NEDD4 to the enhancers, and dismisses Pol II to impede enhancer activation to eliminate target gene transcription.

Article Snippet: The dCas9-KRAB lenti-virus was prepared by co-transfection of pVSVg (Addgene, Cat#8454) and psPAX2 (Addgene Cat#12260), and pHAGE-TRE-dCas9-KRAB vector (Addgene, Plasmid #50917) into HEK-293T cell using Lipofectamine2000 and harvest the lenti-virus in 48 h and 72 h after transfection.

Techniques: Quantitative RT-PCR, Expressing, Whisker Assay, ChIP-sequencing, Generated, Stable Transfection, Activation Assay

Journal: Cell reports

Article Title: Systematic evaluation of retroviral LTRs as cis -regulatory elements in mouse embryos

doi: 10.1016/j.celrep.2024.113775

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The dCas9-HA-NLS-NLS-KRAB-NLS sequence was amplified from the plasmid PB-TRE-dCas9-KRAB-MeCP2 (addgene 122267) using PrimeSTAR Max2× premix (Takara).

Techniques: Virus, Recombinant, SYBR Green Assay, Protease Inhibitor, Immunoprecipitation, Transfection, cDNA Synthesis, Expressing, Plasmid Preparation, Software