ori neg  (Addgene inc)

Journal: bioRxiv

Article Title: Selective Enhancer Dependencies in MYC -Intact and MYC -Rearranged Germinal Center B-cell Diffuse Large B-cell Lymphoma

doi: 10.1101/2023.05.02.538892

Figure Lengend Snippet: A. Heatmap showing differential acetylation of enhancer clusters with increased acetylation in GCB-DLBCL cell lines. MYC rearrangement status is coded as follows: white, no rearrangement; gray, positive for MYC::BCL6-SE rearrangement; black, positive for other MYC rearrangement. Heatmaps strips at right show significance of motif enrichment for the OCT2 and E2A ( TCF3 ) motifs in HOMER known motif analysis; black arrowheads indicate that a similar motif was identified as the most significantly enriched motif in that cluster by de novo motif analysis. See Supplementary Figures 5B-C for the corresponding data for all enhancer clusters and all cell lines. B. Western blot of nuclear extracts from GCB-DLBCL cell lines for MEF2B and CTCF (loading control). C. Genomic copy gains (log scale) for POU2AF1 in B-cell lymphoma cell lines (DepMap). GCBME-1-dependent cell lines are indicated. D. POU2F2 and POU2AF1 transcript levels in B-cell lymphoma cell lines (DepMap). GCBME-1-dependent cell lines are indicated. E. Plot of sgRNA depletion or enrichment (MAGeCK RRA log2-fold-change) for optimized gene promoter-targeting sgRNAs in lymphoma CRISPRi screens (B cell expressed transcription factors, B-cell-specific essential genes, and pan-essential genes). Target intervals are ranked along the X axis by lowest negative selection FDR p-value (left to right, intervals with log2-fold change <0) or lowest positive selection FDR p-value (right to left, intervals with log2-fold change >0). Genes with abs(log2FC) > 0.5 and fdr < 0.05 are colored blue. TF genes of interest are indicated. F. Detail of ATAC-Seq, H3K27 ChIP-Seq, and ChIP-Seq for the OCT2, OCA-B, MEF2B, and p300 in the GCBME1 enhancer cluster. CRISPRi screen NFR intervals are as indicated, with conserved subregions A and B of NFRs 195 and 196 indicated below. Inset at bottom shows further detail of the Phastcons conserved element track for sub-region 195B (see Supplementary Figure 5G for further detail), with the position of the conserved OCT2 motif indicated. G. Change in MYC expression (qRT-PCR) in GCBME1-dependent DLBCL cell lines expressing doxycycline-inducible dCas9-KRAB after transduction with indicated sgRNAs targeting MEF2B, POU2F2, and POU2AF1 (**** p<0.0001, *** p<0.001, ** p<0.01, * p<0.01, t-test of pooled replicates for both TF gene promoter-targeting sgRNAs versus both control sgRNAs, color-coded by measured transcript per legend). H. Transcriptional reporter assay in SUDHL-5 cells for sub-regions of the GCBME-1 cloned into the STARR-Seq_Ori luciferase vector. Firefly luciferase signal for each sample was normalized to internal transfection control (Renilla luciferase signal) and then to the normalized signal for the no-insert control vector (Ori_neg). I. Transcriptional reporter assay in the indicated GCBME-1-dependent cell lines for constructs bearing the NFR 195B enhancer region, with or without OCT2 motif deletion, normalized as in H.

Article Snippet: Vectors ORI_SV40 (Addgene:99309) and ORI_neg.cont (Addgene: 99315) were used as positive and negative controls.

Techniques: Western Blot, Selection, ChIP-sequencing, Expressing, Quantitative RT-PCR, Transduction, Reporter Assay, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Construct

Journal: Nucleic Acids Research

Article Title: Glucocorticoids unmask silent non-coding genetic risk variants for common diseases

doi: 10.1093/nar/gkac1045

Figure Lengend Snippet: Generation and integration of H3K27ac HiChIP before and after glucocorticoid treatment with other datasets . ( A ) Validation of drug treatment effect for HiChIP samples as measured by qRT-PCR for FKBP5 , a prototypical GR-targeted gene, using RNA extracted from the same cells. Statistical significance was evaluated with Student's t-test, achieving P < 0.005. Each dot represents a replicate. ( B ) Number of cortisol-regulated genes that were connected to a cortisol-induced GR ChIP-peak by a H3K27ac loop in different treatment conditions. ( C ) Cortisol-regulated genes that were connected to one or more cortisol-induced GR ChIP-peaks by differential cortisol-regulated H3K27ac loop(s) (fold change > 1.5 or a change from 0 that is more than 1). The X axis shows gene names (Please see for a complete list of gene names). ( D ) mRNA expression of FKBP5 before and after drug treatment as determined by RNA-seq. CPM represents counts per million. ( E ) Transcriptional activity driven by the enhancer region upstream of FKBP5 as measured by STARR-seq. ( F ) Number of HiChIP H3K27ac loops that connected GR-binding sites to the FKBP5 gene. ( G ) Integrative Genomics Viewer (IGV) plots of two different GR-dependent enhancers over a distance of 50kb, which together regulated FKBP5 . These two enhancers were predicted to be strong enhancers with looping properties by ChromHMM. ( H ) Number of PGx-eQTLs that displayed physical interactions between SNP loci (categorized by enhancer/non-enhancer states) and eQTL genes as demonstrated by H3K27ac HiChIP.

Article Snippet: To validate selected STARR-seq signals with clinical significance, we conducted luciferase reporter gene assays using the STARR-seq luciferase validation vector (Addgene #9927).

Techniques: HiChIP, Quantitative RT-PCR, Expressing, RNA Sequencing Assay, Activity Assay, Binding Assay

Journal: Nucleic Acids Research

Article Title: Glucocorticoids unmask silent non-coding genetic risk variants for common diseases

doi: 10.1093/nar/gkac1045

Figure Lengend Snippet: Functional Validation of rs1697139- MAST4 GR-dependent PGx-eQTL in Breast Cancer Cells . ( A ) SNP-dependent and drug-dependent enhancer activity of the PGx locus associated with breast cancer in Figure as measured by luciferase reporter gene assay in MDA-MB-231, a triple negative breast cancer cell line. RLU stands for relative light units and reflects normalization of luciferase signal to Renilla signal as an internal control. ( B ) ChIP-qPCR assays that tested GR binding at the PGx locus for MAST4 in MDA-MB-231 using two different primers. ChIP-qPCR targeting FKBP5 peak served as a positive control. P -values from Student's t-tests. ( C ) Dose-dependent repression of MAST4 by glucocorticoids in MDA-MB-231 cells. ( D ) Epigenomic datasets for MDA-MB-231 cells demonstrated that the SNP locus also looped to MAST4 in MDA-MB-231. GR ChIP-seq was downloaded from the database ReMap2022. ( E ) Designs of guide RNAs utilized in the CRISPR/Cas9 experiment that cut out the PGx SNP enhancer locus where GR binds and primers to test editing outcomes. ( F ) Gel electrophoresis of PCR products amplified with primers targeting the cut region demonstrated successful edits. ( G ) MAST4 gene expression as measured by qRT-PCR after CRISPR/Cas9 edits showed that the glucocorticoid-dependent repression was alleviated after the PGx SNP locus was cut out across two colonies of pure knock-out cells. Data included three biological replicates. ( H ) Expression of MAST4 in tumors from breast cancer patients in the TCGA database. **** P -values < 0.0001 by Student's t -tests. TPM stands for transcripts per million. ( I ) Kaplan–Meier curves for relapse-free survival rate in 1764 breast cancer patients predicted based on MAST4 expression. ( J ) Summary of functional insights gaining from in-depth studies of a GR-dependent eQTL and their implications for breast cancer risk.

Article Snippet: To validate selected STARR-seq signals with clinical significance, we conducted luciferase reporter gene assays using the STARR-seq luciferase validation vector (Addgene #9927).

Techniques: Functional Assay, Activity Assay, Luciferase, Reporter Gene Assay, Binding Assay, Positive Control, ChIP-sequencing, CRISPR, Nucleic Acid Electrophoresis, Amplification, Expressing, Quantitative RT-PCR, Knock-Out