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
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