Journal: Nature Communications

Article Title: Comprehensive dissection of cis- regulatory elements in a 2.8 Mb topologically associated domain in six human cancers

doi: 10.1038/s41467-025-56568-5

Figure Lengend Snippet: a CRISPR inhibition (CRISPRi) screen tiling across a 2.8 Mb topologically-associated domain containing the oncogene MYC using ~112,000 gRNAs. b MYC expression and CRISPR screen depletion (Chronos) scores from Broad DepMap ( n = 1021 cell lines). c Sliding window analysis for identification of cis -regulatory elements (CREs). d Upset plot of shared and cell-specific enhancer-like CREs across six pooled CRISPRi screens. Source data are provided as a Source Data file.

Article Snippet: To generate CRISPRi guide plasmids with mStayGold (a green fluorescent protein), we first cloned the U6-sgRNA(F + E) cassette from pCC_01 (Addgene 139086) into pLentiRNAGuide_003 (Addgene 192505) using PacI and NheI restriction sites and termed the plasmid pLentiSpCas9guide (GFP-P2A-Puro).

Techniques: CRISPR, Inhibition, Expressing

Journal: Nature Communications

Article Title: Comprehensive dissection of cis- regulatory elements in a 2.8 Mb topologically associated domain in six human cancers

doi: 10.1038/s41467-025-56568-5

Figure Lengend Snippet: a CRISPRi log 2 fold-change in A375 melanoma cells for ~112,000 gRNAs tiling the MYC TAD. Positive values indicate regions/perturbations that are highly depleted. The shaded area indicates the MYC promoter. b Detected enhancers (corresponding to depleted gRNAs over the ~4 week cell culture period) and repressors (enriched gRNAs) in each CRISPRi screen for each cell line. Repressor CREs are denoted by an asterisk after the CRE identifier. CREs from prior studies are those referenced in a review of MYC TAD CREs by Lancho et al. . and from Fulco et al. . For the CRISPRi screen CREs, those in red text match a previously MYC enhancer found in the same tissue/cancer type. Source data are provided as a Source Data file.

Article Snippet: To generate CRISPRi guide plasmids with mStayGold (a green fluorescent protein), we first cloned the U6-sgRNA(F + E) cassette from pCC_01 (Addgene 139086) into pLentiRNAGuide_003 (Addgene 192505) using PacI and NheI restriction sites and termed the plasmid pLentiSpCas9guide (GFP-P2A-Puro).

Techniques: Cell Culture

Journal: Nature Communications

Article Title: Comprehensive dissection of cis- regulatory elements in a 2.8 Mb topologically associated domain in six human cancers

doi: 10.1038/s41467-025-56568-5

Figure Lengend Snippet: a Locations of indicated CREs within the MYC TAD. b Cell proliferation (CellTiter Glo) for CRISPRi perturbations of CREs identified in the TAD-wide screen ( n = 3 biological replicates of 2–3 gRNAs per CRE or non-targeting control). Error bars represent mean ± s.e.m. The p values are determined by unpaired, one-sided t tests. * p < 0.05, ** p < 0.01. Peak p values: HT29/PC9 (HT29: 0.01, PC9: 0.028, A375: 0.84), A375 (A375: 0.004, PC9: 0.49), K562 (K562: 0.001, MDA-MB-231: 0.41). c MYC expression after CRISPRi perturbations of CREs identified in the TAD-wide screen ( n = 3 biological replicates of 2–3 gRNAs per CRE or non-targeting control). Boxplot whiskers extend from minimum to maximum, the box extends from 25th to 75th percentile and the mid-line represents the median. The p values are determined by unpaired, one-sided t tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Peak p values: HT29/PC9 (HT29: 0.000004, PC9: 0.004, A375: 0.08), A375 (A375: 0.002, PC9: 0.49), K562 (K562: 0.009, MDA-MB-231: 0.25). d Heatmap of H3K27ac and chromatin accessibility from specific cell lines ( x -axis) for CREs found in the indicated cell lines ( y -axis). Regions range from 115 to 215 kb. H3K27ac and chromatin accessibility biochemical signatures are rarely found in cell lines where the CRE is not functional. e Overlap of CREs with enhancer and promoter-associated biochemical signatures ( n = 5 association tests with 2000 samples per test). Error bars represent mean ± SD of association tests. Source data are provided as a Source Data file.

Article Snippet: To generate CRISPRi guide plasmids with mStayGold (a green fluorescent protein), we first cloned the U6-sgRNA(F + E) cassette from pCC_01 (Addgene 139086) into pLentiRNAGuide_003 (Addgene 192505) using PacI and NheI restriction sites and termed the plasmid pLentiSpCas9guide (GFP-P2A-Puro).

Techniques: Control, Expressing, Functional Assay

Journal: Nature Communications

Article Title: Comprehensive dissection of cis- regulatory elements in a 2.8 Mb topologically associated domain in six human cancers

doi: 10.1038/s41467-025-56568-5

Figure Lengend Snippet: a Schematic of H3K27ac HiChIP workflow. b ( left ) H3K27ac HiChIP contacts at 20 kb resolution between MYC and other elements in the TAD. Highlighted in color are the significant CREs identified in each CRISPRi screen and their HiChIP contacts with MYC . ( right ) The percent of CRISPRi-identified CREs that contact MYC ( first column ) and the percent of total contacts with MYC that include CRISPRi CREs ( second column ). c Pearson correlation of H3K27ac HiChIP from A549 cells with histone modifications. d Recall analysis of gRNA sequences within CREs by the indicated biochemical signature. Plot represents an average across all cell lines. e Overlap of H3K27ac HiChIP and CRISPRi perturbation screens for A549 (lung), PC-9 (lung) and HT-29 (colon). These three cell lines share a CRE (dotted box) at the promoter of CCAT1 . The single dotted line indicates MYC . Source data are provided as a Source Data file.

Article Snippet: To generate CRISPRi guide plasmids with mStayGold (a green fluorescent protein), we first cloned the U6-sgRNA(F + E) cassette from pCC_01 (Addgene 139086) into pLentiRNAGuide_003 (Addgene 192505) using PacI and NheI restriction sites and termed the plasmid pLentiSpCas9guide (GFP-P2A-Puro).

Techniques: HiChIP

Journal: Nature Communications

Article Title: Comprehensive dissection of cis- regulatory elements in a 2.8 Mb topologically associated domain in six human cancers

doi: 10.1038/s41467-025-56568-5

Figure Lengend Snippet: a Identification of transcription factor binding at CREs and summary of key transcription factors found. b Number of transcription factors significantly enriched in the CREs from 1 or more cell lines. c Fold enrichment for top 25 transcription factors in each cell line. d Transcription factor ChIP-seq for selected transcription factors from panel c . JUNB, JUN, and FOSL2 ChIP-seq are from A549, GATA2 ChIP-seq is from K562, THAP11 ChIP-seq is from HepG2 and ETS1 ChIP-seq is from SCC25. e Correlation in primary tumors from The Cancer Genome Atlas (TCGA) between MYC expression and top-ranked transcription factors from panel ( c ). TCGA tumor types are matched to the same cell/tissue type as CRISPRi cell lines in which the transcription factor is enriched ( n = 2855 TCGA primary tumors; LUAD: n = 512, COAD: n = 594, SKCM: n = 472, AML: n = 175, BRCA: n = 1102 tumors). Source data are provided as a Source Data file.

Article Snippet: To generate CRISPRi guide plasmids with mStayGold (a green fluorescent protein), we first cloned the U6-sgRNA(F + E) cassette from pCC_01 (Addgene 139086) into pLentiRNAGuide_003 (Addgene 192505) using PacI and NheI restriction sites and termed the plasmid pLentiSpCas9guide (GFP-P2A-Puro).

Techniques: Binding Assay, ChIP-sequencing, Expressing