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transcription factor crispr screen sgrna pooled library  (Addgene inc)


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    Addgene inc transcription factor crispr screen sgrna pooled library
    ( A ) SOX9 resides in the most hyper-interacting TNBC MB157 promoter-enhancer hub. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. SOX9 hyper-interacting promoter-enhancer hub is marked in red. ( B and C ) Domain-focused <t>CRISPR</t> screens of 1486 <t>transcription</t> factors identify SOX9 as an essential gene in TNBC MB157 (B) and MDA-MB-468 (C). Genes are ranked based on MAGeCK Robust Rank Aggregation (RRA) score for negative selection. A smaller RRA score indicates stronger negative selection of the corresponding gene. Essential genes residing in hyper-interacting promoter-enhancer hubs are indicated with colored dots, including SOX9 . Essentiality significance: P value < 0.05. ( D ) Fifty-seven essential transcription factors are shared between TNBC MB157 and MDA-MB-468. Upset plot of essential and expressed transcription factors from transcription factor CRISPR screen in MB157 and MDA-MB-468. ( E to G ) Loss of SOX9 significantly reduces growth of TNBC. Relative cell growth (CellTiter-Glo) was tracked for 9 days in Cas9-expressing TNBC MB157 (E) MDA-MB-468 (F) MDA-MB-231 (G) cells after transduction with control <t>sgRNA</t> (CTRL) or SOX9-targeting sgRNA [SOX9 knockout (KO)]. CellTiter-Glo day 0 are 4 days post-sgRNA transduction. N = 5 technical replicates. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 .
    Transcription Factor Crispr Screen Sgrna Pooled Library, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 39 article reviews
    transcription factor crispr screen sgrna pooled library - by Bioz Stars, 2026-09
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    1) Product Images from "Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells"

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells

    Journal: Science Advances

    doi: 10.1126/sciadv.adl4043

    ( A ) SOX9 resides in the most hyper-interacting TNBC MB157 promoter-enhancer hub. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. SOX9 hyper-interacting promoter-enhancer hub is marked in red. ( B and C ) Domain-focused CRISPR screens of 1486 transcription factors identify SOX9 as an essential gene in TNBC MB157 (B) and MDA-MB-468 (C). Genes are ranked based on MAGeCK Robust Rank Aggregation (RRA) score for negative selection. A smaller RRA score indicates stronger negative selection of the corresponding gene. Essential genes residing in hyper-interacting promoter-enhancer hubs are indicated with colored dots, including SOX9 . Essentiality significance: P value < 0.05. ( D ) Fifty-seven essential transcription factors are shared between TNBC MB157 and MDA-MB-468. Upset plot of essential and expressed transcription factors from transcription factor CRISPR screen in MB157 and MDA-MB-468. ( E to G ) Loss of SOX9 significantly reduces growth of TNBC. Relative cell growth (CellTiter-Glo) was tracked for 9 days in Cas9-expressing TNBC MB157 (E) MDA-MB-468 (F) MDA-MB-231 (G) cells after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA [SOX9 knockout (KO)]. CellTiter-Glo day 0 are 4 days post-sgRNA transduction. N = 5 technical replicates. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 .
    Figure Legend Snippet: ( A ) SOX9 resides in the most hyper-interacting TNBC MB157 promoter-enhancer hub. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. SOX9 hyper-interacting promoter-enhancer hub is marked in red. ( B and C ) Domain-focused CRISPR screens of 1486 transcription factors identify SOX9 as an essential gene in TNBC MB157 (B) and MDA-MB-468 (C). Genes are ranked based on MAGeCK Robust Rank Aggregation (RRA) score for negative selection. A smaller RRA score indicates stronger negative selection of the corresponding gene. Essential genes residing in hyper-interacting promoter-enhancer hubs are indicated with colored dots, including SOX9 . Essentiality significance: P value < 0.05. ( D ) Fifty-seven essential transcription factors are shared between TNBC MB157 and MDA-MB-468. Upset plot of essential and expressed transcription factors from transcription factor CRISPR screen in MB157 and MDA-MB-468. ( E to G ) Loss of SOX9 significantly reduces growth of TNBC. Relative cell growth (CellTiter-Glo) was tracked for 9 days in Cas9-expressing TNBC MB157 (E) MDA-MB-468 (F) MDA-MB-231 (G) cells after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA [SOX9 knockout (KO)]. CellTiter-Glo day 0 are 4 days post-sgRNA transduction. N = 5 technical replicates. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 .

    Techniques Used: HiChIP, CRISPR, Selection, Expressing, Transduction, Control, Knock-Out

    ( A ) Enrichment of active histone mark H3K27ac 3′ of SOX9 at gray box-marked genomic regions SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 in TNBC but not ER + breast cancer cells. Tracks from top to bottom: H3K27ac ChIP-seq in TNBC MB157, MDA-MB-468, and MDA-MB-231, ER + MCF7 and LTED, and TNBC cell-of-origin primary luminal progenitor cells from human donors. Blue lines represent the position of sgRNA-targeted sequences. ( B to D ) dCas9-KRAB experiments show that elements within highly acetylated SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 are functional SOX9 enhancers in TNBC but not ER + cells. Bar plots showing normalized expression of SOX9 in dCas9-KRAB–expressing TNBC MB157, MDA-MB-231, and MDA-MB-468 and ER + MCF7 after inhibition of marked elements within SOX9 .EC1 (B), SOX9 .EC2 (C), and SOX9 .EC3 (D) with noted sgRNAs relative to control sgRNA (CTRL) 6 days post-sgRNA transduction. Each condition is normalized to its corresponding CTRL sgRNA experiment. Data represent N = 4 biological replicates and presented as mean ± SEM. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 . ( E to H ) Activity of enhancers within SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 promote proliferation of TNBC but not ER + cells. Relative cell growth (CellTiter-Glo) was tracked for 9 days in dCas9-KRAB–expressing TNBC MB157 (E) and MDA-MB-231 (F) MDA-MB-468 (G) and MCF7 (H) after transduction with CTRL, SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 targeting sgRNA. N = 5 technical replicates. Data presented as mean ± SEM t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 . ns, not significant.
    Figure Legend Snippet: ( A ) Enrichment of active histone mark H3K27ac 3′ of SOX9 at gray box-marked genomic regions SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 in TNBC but not ER + breast cancer cells. Tracks from top to bottom: H3K27ac ChIP-seq in TNBC MB157, MDA-MB-468, and MDA-MB-231, ER + MCF7 and LTED, and TNBC cell-of-origin primary luminal progenitor cells from human donors. Blue lines represent the position of sgRNA-targeted sequences. ( B to D ) dCas9-KRAB experiments show that elements within highly acetylated SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 are functional SOX9 enhancers in TNBC but not ER + cells. Bar plots showing normalized expression of SOX9 in dCas9-KRAB–expressing TNBC MB157, MDA-MB-231, and MDA-MB-468 and ER + MCF7 after inhibition of marked elements within SOX9 .EC1 (B), SOX9 .EC2 (C), and SOX9 .EC3 (D) with noted sgRNAs relative to control sgRNA (CTRL) 6 days post-sgRNA transduction. Each condition is normalized to its corresponding CTRL sgRNA experiment. Data represent N = 4 biological replicates and presented as mean ± SEM. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 . ( E to H ) Activity of enhancers within SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 promote proliferation of TNBC but not ER + cells. Relative cell growth (CellTiter-Glo) was tracked for 9 days in dCas9-KRAB–expressing TNBC MB157 (E) and MDA-MB-231 (F) MDA-MB-468 (G) and MCF7 (H) after transduction with CTRL, SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 targeting sgRNA. N = 5 technical replicates. Data presented as mean ± SEM t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 . ns, not significant.

    Techniques Used: ChIP-sequencing, Functional Assay, Expressing, Inhibition, Control, Transduction, Activity Assay

    ( A and B ) SOX9 promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 cells. Left: Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both (A) and SOX9 .EC2, SOX9 .EC3, or both (B) in MB157 ( n = alleles). Right-top: SOX9 locus schematic, three-color DNA FISH 50-kb probes at SOX9 promoter (green), SOX9 .EC3 (magenta), and SOX9 .EC1 (A, red) or SOX9 .EC2 (B, yellow). Locations per fig. S3A. Right-bottom: Representative cells. Blue: 4′,6-Diamidino-2-phenylindole (DAPI). ( C and F ) SOX9 enhancers inactivation expands SOX9 -EC1-EC3 and SOX9 -EC2-EC3 hubs in individual TNBC MB157 cells. Cumulative distribution functions (CDFs) of SOX9 -EC1-EC3 (C) and SOX9 -EC2-EC3 (F) spatial perimeters in each MB157-dCas9-KRAB expressing control (CTRL), SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 sgRNA [Kolmogorov-Smirnov (KS) test, n = cells]. Mean (±SD) perimeters (micrometers): (C) Left: CTRL/ SOX9 .EC1 sgRNA: 3.78 (±2.63)/4.36 (±2.63); middle: CTRL/ SOX9 .EC2 sgRNA: 3.78 (±2.63)/4.47 (±2.64); right: CTRL/ SOX9 .EC3 sgRNA: 3.39 (±2.56)/4.28 (±2.65). (G) Left: CTRL/ SOX9 .EC1 sgRNA: 3.83 (±2.71)/4.45 (±2.72); middle: CTRL/ SOX9 .EC2 sgRNA: 3.19 (±2.44)/4.26 (±2.61); right: CTRL/ SOX9 .EC3 sgRNA: 3.83 (±2.71)/4.22 (±2.56). ( D and G ) Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both (D) and SOX9 .EC1, SOX9 .EC3, or both (G) in MB157-dCas9-KRAB expressing CTRL, SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 sgRNA ( n = alleles). ( E and H ) Representative cells of 3C and 3D (E) or 3F and 3G (H). Blue: DAPI. ( I ) SOX9 promoter inactivation decreases SOX9 -EC1-EC3 three-way interaction frequency across individual alleles in TNBC MB157. Top-left: Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both in MB157-dCas9-KRAB expressing CTRL or SOX9 promoter sgRNA ( SOX9 .P sgRNA) ( n = alleles). Bottom-left: CDFs of SOX9 -EC1-EC3 spatial perimeter in each MB157-dCas9-KRAB cell (KS test, n = cells). CTRL/ SOX9 .P sgRNA mean (±SD) perimeter: 3.90 (±2.62)/4.44 (±2.66) μm. Right: Representative cells. Blue: DAPI. Scale bars, 3 μm for nuclei and 0.5 μm for alleles.
    Figure Legend Snippet: ( A and B ) SOX9 promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 cells. Left: Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both (A) and SOX9 .EC2, SOX9 .EC3, or both (B) in MB157 ( n = alleles). Right-top: SOX9 locus schematic, three-color DNA FISH 50-kb probes at SOX9 promoter (green), SOX9 .EC3 (magenta), and SOX9 .EC1 (A, red) or SOX9 .EC2 (B, yellow). Locations per fig. S3A. Right-bottom: Representative cells. Blue: 4′,6-Diamidino-2-phenylindole (DAPI). ( C and F ) SOX9 enhancers inactivation expands SOX9 -EC1-EC3 and SOX9 -EC2-EC3 hubs in individual TNBC MB157 cells. Cumulative distribution functions (CDFs) of SOX9 -EC1-EC3 (C) and SOX9 -EC2-EC3 (F) spatial perimeters in each MB157-dCas9-KRAB expressing control (CTRL), SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 sgRNA [Kolmogorov-Smirnov (KS) test, n = cells]. Mean (±SD) perimeters (micrometers): (C) Left: CTRL/ SOX9 .EC1 sgRNA: 3.78 (±2.63)/4.36 (±2.63); middle: CTRL/ SOX9 .EC2 sgRNA: 3.78 (±2.63)/4.47 (±2.64); right: CTRL/ SOX9 .EC3 sgRNA: 3.39 (±2.56)/4.28 (±2.65). (G) Left: CTRL/ SOX9 .EC1 sgRNA: 3.83 (±2.71)/4.45 (±2.72); middle: CTRL/ SOX9 .EC2 sgRNA: 3.19 (±2.44)/4.26 (±2.61); right: CTRL/ SOX9 .EC3 sgRNA: 3.83 (±2.71)/4.22 (±2.56). ( D and G ) Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both (D) and SOX9 .EC1, SOX9 .EC3, or both (G) in MB157-dCas9-KRAB expressing CTRL, SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 sgRNA ( n = alleles). ( E and H ) Representative cells of 3C and 3D (E) or 3F and 3G (H). Blue: DAPI. ( I ) SOX9 promoter inactivation decreases SOX9 -EC1-EC3 three-way interaction frequency across individual alleles in TNBC MB157. Top-left: Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both in MB157-dCas9-KRAB expressing CTRL or SOX9 promoter sgRNA ( SOX9 .P sgRNA) ( n = alleles). Bottom-left: CDFs of SOX9 -EC1-EC3 spatial perimeter in each MB157-dCas9-KRAB cell (KS test, n = cells). CTRL/ SOX9 .P sgRNA mean (±SD) perimeter: 3.90 (±2.62)/4.44 (±2.66) μm. Right: Representative cells. Blue: DAPI. Scale bars, 3 μm for nuclei and 0.5 μm for alleles.

    Techniques Used: Expressing, Control

    ( A ) Genome tracks showing enrichment of pairwise MYC enhancer-enhancer and promoter-enhancer interactions in a population of MB157 cells. From top to bottom: Colored circles marking location of Oligopaint DNA FISH probes labeling 50-kb regions at MYC promoter (green), MYC .EC1 (magenta), MYC .EC2 (red), MYC .EC3 (yellow), and T-ALL-restricted enhancer (black), H3K27ac and SOX9 levels as measured by ChIP-seq, and normalized interaction frequency as measured by SMC1 HiChIP at the MYC locus in MB157. MYC enhancer clusters are marked by grey boxes. ( B ) A total of 80% of differentially expressed genes with SOX9-bound promoter and distal enhancer participate in ensemble hyper-interacting hubs. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. Hyper-interacting ensemble promoter-enhancer hubs containing genes that are significantly down-regulated in MB157-Cas9 cells transfected with SOX9 targeting sgRNA versus control sgRNA for 4 days and have SOX9-bound promoter and distal enhancer are marked in orange. ( C to E ) SOX9 loss significantly increases 3D distances between the MYC promoter and SOX9-bound MYC .EC2 (C) or MYC .EC3 (D) and SOX9-unbound MYC .EC1 (E) in individual cells. CDFs (left) and box and whiskers (middle) of the distances between the MYC promoter and SOX9-bound MYC .EC2 (C) and MYC .EC3 (D) and SOX9-unbound MYC .EC1 (E) in each MB157-Cas9 6 days after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA (SOX9 KO) (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) distance between MYC promoter and MYC .EC2: 0.389 (±0.358)/0.749 (±0.666) μm; MYC .EC3: 0.447 (±0.457)/0.591 (±0.551) μm; MYC .EC1: 0.494 (±0.447)/0.651 (±0.556) μm. Right: Representative cells. Scale bar per 3A. Blue: DAPI.
    Figure Legend Snippet: ( A ) Genome tracks showing enrichment of pairwise MYC enhancer-enhancer and promoter-enhancer interactions in a population of MB157 cells. From top to bottom: Colored circles marking location of Oligopaint DNA FISH probes labeling 50-kb regions at MYC promoter (green), MYC .EC1 (magenta), MYC .EC2 (red), MYC .EC3 (yellow), and T-ALL-restricted enhancer (black), H3K27ac and SOX9 levels as measured by ChIP-seq, and normalized interaction frequency as measured by SMC1 HiChIP at the MYC locus in MB157. MYC enhancer clusters are marked by grey boxes. ( B ) A total of 80% of differentially expressed genes with SOX9-bound promoter and distal enhancer participate in ensemble hyper-interacting hubs. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. Hyper-interacting ensemble promoter-enhancer hubs containing genes that are significantly down-regulated in MB157-Cas9 cells transfected with SOX9 targeting sgRNA versus control sgRNA for 4 days and have SOX9-bound promoter and distal enhancer are marked in orange. ( C to E ) SOX9 loss significantly increases 3D distances between the MYC promoter and SOX9-bound MYC .EC2 (C) or MYC .EC3 (D) and SOX9-unbound MYC .EC1 (E) in individual cells. CDFs (left) and box and whiskers (middle) of the distances between the MYC promoter and SOX9-bound MYC .EC2 (C) and MYC .EC3 (D) and SOX9-unbound MYC .EC1 (E) in each MB157-Cas9 6 days after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA (SOX9 KO) (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) distance between MYC promoter and MYC .EC2: 0.389 (±0.358)/0.749 (±0.666) μm; MYC .EC3: 0.447 (±0.457)/0.591 (±0.551) μm; MYC .EC1: 0.494 (±0.447)/0.651 (±0.556) μm. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

    Techniques Used: Labeling, ChIP-sequencing, HiChIP, Transfection, Control, Transduction

    ( A ) MYC promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 and MDA-MB-468 but not ER + MCF7. Left: Percentage of alleles with MYC promoter interacting (<350 nm) with SOX9-unbound MYC .EC1, SOX9-bound MYC .EC3, or both MYC .EC1 and MYC .EC3 in MB157, MDA-MB-468, and MCF7 as measured by three-color Oligopaint DNA FISH with probes marked in top genome track ( n = alleles). Right: Representative MB157, MDA-MB-468, and MCF7 nuclei and two magnified alleles from three-color DNA FISH. Scale bar per 3A. Blue: DAPI. ( B and C ) SOX9 loss expands MYC -EC1-EC2 (B) and MYC -EC1-EC3 (C) promoter-enhancer hubs in individual MB157 and decreases three-way interaction frequency across individual alleles. Left: CDFs of MYC -EC1-EC2 (B) and MYC -EC1-EC3 (C) spatial perimeters in each MB157-Cas9 cell expressing CTRL or SOX9 KO sgRNA (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) perimeters MYC -EC1-EC2 (B): 3.84 (±2.70)/4.53 (±2.67) μm; MYC -EC1-EC3 (C): 3.10 (±2.58)/3.90 (±2.64) μm ( n = cells). Middle: Allele percentages with MYC promoter interacting (<350 nm) with MYC .EC1, MYC .EC2, or both MYC .EC1 and MYC .EC2 (B) and MYC .EC1, MYC .EC3, or both MYC .EC1 and MYC .EC3 (C) in CTRL and SOX9 KO MB157-Cas9. Right: Representative cells. Scale bar per 3A. Blue: DAPI.
    Figure Legend Snippet: ( A ) MYC promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 and MDA-MB-468 but not ER + MCF7. Left: Percentage of alleles with MYC promoter interacting (<350 nm) with SOX9-unbound MYC .EC1, SOX9-bound MYC .EC3, or both MYC .EC1 and MYC .EC3 in MB157, MDA-MB-468, and MCF7 as measured by three-color Oligopaint DNA FISH with probes marked in top genome track ( n = alleles). Right: Representative MB157, MDA-MB-468, and MCF7 nuclei and two magnified alleles from three-color DNA FISH. Scale bar per 3A. Blue: DAPI. ( B and C ) SOX9 loss expands MYC -EC1-EC2 (B) and MYC -EC1-EC3 (C) promoter-enhancer hubs in individual MB157 and decreases three-way interaction frequency across individual alleles. Left: CDFs of MYC -EC1-EC2 (B) and MYC -EC1-EC3 (C) spatial perimeters in each MB157-Cas9 cell expressing CTRL or SOX9 KO sgRNA (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) perimeters MYC -EC1-EC2 (B): 3.84 (±2.70)/4.53 (±2.67) μm; MYC -EC1-EC3 (C): 3.10 (±2.58)/3.90 (±2.64) μm ( n = cells). Middle: Allele percentages with MYC promoter interacting (<350 nm) with MYC .EC1, MYC .EC2, or both MYC .EC1 and MYC .EC2 (B) and MYC .EC1, MYC .EC3, or both MYC .EC1 and MYC .EC3 (C) in CTRL and SOX9 KO MB157-Cas9. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

    Techniques Used: Expressing

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    CRISPR:

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.
    Article Snippet: .. For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311). .. Briefly, 10 μg of plasmid DNA, 5 μg of VSVG, 7.5 μg of psPAX2, and 26 μl of FuGene HD (Promega, catalog no. E2311) were mixed, incubated, and added to the 10- cm plate of HEK293T cells, and media was replaced 6 to 8 hours posttransfection.

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells
    Article Snippet: .. For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311). .. Briefly, 10 μg of plasmid DNA, 5 μg of VSVG, 7.5 μg of psPAX2, and 26 μl of FuGene HD (Promega, catalog no. E2311) were mixed, incubated, and added to the 10-cm plate of HEK293T cells, and media was replaced 6 to 8 hours posttransfection.

    Produced:

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.
    Article Snippet: .. For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311). .. Briefly, 10 μg of plasmid DNA, 5 μg of VSVG, 7.5 μg of psPAX2, and 26 μl of FuGene HD (Promega, catalog no. E2311) were mixed, incubated, and added to the 10- cm plate of HEK293T cells, and media was replaced 6 to 8 hours posttransfection.

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells
    Article Snippet: .. For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311). .. Briefly, 10 μg of plasmid DNA, 5 μg of VSVG, 7.5 μg of psPAX2, and 26 μl of FuGene HD (Promega, catalog no. E2311) were mixed, incubated, and added to the 10-cm plate of HEK293T cells, and media was replaced 6 to 8 hours posttransfection.



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    ( A ) SOX9 resides in the most hyper-interacting TNBC MB157 promoter-enhancer hub. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. SOX9 hyper-interacting promoter-enhancer hub is marked in red. ( B and C ) Domain-focused <t>CRISPR</t> screens of 1486 <t>transcription</t> factors identify SOX9 as an essential gene in TNBC MB157 (B) and MDA-MB-468 (C). Genes are ranked based on MAGeCK Robust Rank Aggregation (RRA) score for negative selection. A smaller RRA score indicates stronger negative selection of the corresponding gene. Essential genes residing in hyper-interacting promoter-enhancer hubs are indicated with colored dots, including SOX9 . Essentiality significance: P value < 0.05. ( D ) Fifty-seven essential transcription factors are shared between TNBC MB157 and MDA-MB-468. Upset plot of essential and expressed transcription factors from transcription factor CRISPR screen in MB157 and MDA-MB-468. ( E to G ) Loss of SOX9 significantly reduces growth of TNBC. Relative cell growth (CellTiter-Glo) was tracked for 9 days in Cas9-expressing TNBC MB157 (E) MDA-MB-468 (F) MDA-MB-231 (G) cells after transduction with control <t>sgRNA</t> (CTRL) or SOX9-targeting sgRNA [SOX9 knockout (KO)]. CellTiter-Glo day 0 are 4 days post-sgRNA transduction. N = 5 technical replicates. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 .
    Transcription Factor Crispr Screen Sgrna Pooled Library, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( A ) SOX9 resides in the most hyper-interacting TNBC MB157 promoter-enhancer hub. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. SOX9 hyper-interacting promoter-enhancer hub is marked in red. ( B and C ) Domain-focused CRISPR screens of 1486 transcription factors identify SOX9 as an essential gene in TNBC MB157 (B) and MDA-MB-468 (C). Genes are ranked based on MAGeCK Robust Rank Aggregation (RRA) score for negative selection. A smaller RRA score indicates stronger negative selection of the corresponding gene. Essential genes residing in hyper-interacting promoter-enhancer hubs are indicated with colored dots, including SOX9 . Essentiality significance: P value < 0.05. ( D ) Fifty-seven essential transcription factors are shared between TNBC MB157 and MDA-MB-468. Upset plot of essential and expressed transcription factors from transcription factor CRISPR screen in MB157 and MDA-MB-468. ( E to G ) Loss of SOX9 significantly reduces growth of TNBC. Relative cell growth (CellTiter-Glo) was tracked for 9 days in Cas9-expressing TNBC MB157 (E) MDA-MB-468 (F) MDA-MB-231 (G) cells after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA [SOX9 knockout (KO)]. CellTiter-Glo day 0 are 4 days post-sgRNA transduction. N = 5 technical replicates. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 .

    Journal: Science Advances

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells

    doi: 10.1126/sciadv.adl4043

    Figure Lengend Snippet: ( A ) SOX9 resides in the most hyper-interacting TNBC MB157 promoter-enhancer hub. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. SOX9 hyper-interacting promoter-enhancer hub is marked in red. ( B and C ) Domain-focused CRISPR screens of 1486 transcription factors identify SOX9 as an essential gene in TNBC MB157 (B) and MDA-MB-468 (C). Genes are ranked based on MAGeCK Robust Rank Aggregation (RRA) score for negative selection. A smaller RRA score indicates stronger negative selection of the corresponding gene. Essential genes residing in hyper-interacting promoter-enhancer hubs are indicated with colored dots, including SOX9 . Essentiality significance: P value < 0.05. ( D ) Fifty-seven essential transcription factors are shared between TNBC MB157 and MDA-MB-468. Upset plot of essential and expressed transcription factors from transcription factor CRISPR screen in MB157 and MDA-MB-468. ( E to G ) Loss of SOX9 significantly reduces growth of TNBC. Relative cell growth (CellTiter-Glo) was tracked for 9 days in Cas9-expressing TNBC MB157 (E) MDA-MB-468 (F) MDA-MB-231 (G) cells after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA [SOX9 knockout (KO)]. CellTiter-Glo day 0 are 4 days post-sgRNA transduction. N = 5 technical replicates. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 .

    Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

    Techniques: HiChIP, CRISPR, Selection, Expressing, Transduction, Control, Knock-Out

    ( A ) Enrichment of active histone mark H3K27ac 3′ of SOX9 at gray box-marked genomic regions SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 in TNBC but not ER + breast cancer cells. Tracks from top to bottom: H3K27ac ChIP-seq in TNBC MB157, MDA-MB-468, and MDA-MB-231, ER + MCF7 and LTED, and TNBC cell-of-origin primary luminal progenitor cells from human donors. Blue lines represent the position of sgRNA-targeted sequences. ( B to D ) dCas9-KRAB experiments show that elements within highly acetylated SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 are functional SOX9 enhancers in TNBC but not ER + cells. Bar plots showing normalized expression of SOX9 in dCas9-KRAB–expressing TNBC MB157, MDA-MB-231, and MDA-MB-468 and ER + MCF7 after inhibition of marked elements within SOX9 .EC1 (B), SOX9 .EC2 (C), and SOX9 .EC3 (D) with noted sgRNAs relative to control sgRNA (CTRL) 6 days post-sgRNA transduction. Each condition is normalized to its corresponding CTRL sgRNA experiment. Data represent N = 4 biological replicates and presented as mean ± SEM. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 . ( E to H ) Activity of enhancers within SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 promote proliferation of TNBC but not ER + cells. Relative cell growth (CellTiter-Glo) was tracked for 9 days in dCas9-KRAB–expressing TNBC MB157 (E) and MDA-MB-231 (F) MDA-MB-468 (G) and MCF7 (H) after transduction with CTRL, SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 targeting sgRNA. N = 5 technical replicates. Data presented as mean ± SEM t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 . ns, not significant.

    Journal: Science Advances

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells

    doi: 10.1126/sciadv.adl4043

    Figure Lengend Snippet: ( A ) Enrichment of active histone mark H3K27ac 3′ of SOX9 at gray box-marked genomic regions SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 in TNBC but not ER + breast cancer cells. Tracks from top to bottom: H3K27ac ChIP-seq in TNBC MB157, MDA-MB-468, and MDA-MB-231, ER + MCF7 and LTED, and TNBC cell-of-origin primary luminal progenitor cells from human donors. Blue lines represent the position of sgRNA-targeted sequences. ( B to D ) dCas9-KRAB experiments show that elements within highly acetylated SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 are functional SOX9 enhancers in TNBC but not ER + cells. Bar plots showing normalized expression of SOX9 in dCas9-KRAB–expressing TNBC MB157, MDA-MB-231, and MDA-MB-468 and ER + MCF7 after inhibition of marked elements within SOX9 .EC1 (B), SOX9 .EC2 (C), and SOX9 .EC3 (D) with noted sgRNAs relative to control sgRNA (CTRL) 6 days post-sgRNA transduction. Each condition is normalized to its corresponding CTRL sgRNA experiment. Data represent N = 4 biological replicates and presented as mean ± SEM. t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 . ( E to H ) Activity of enhancers within SOX9 .EC1, SOX9 .EC2, and SOX9 .EC3 promote proliferation of TNBC but not ER + cells. Relative cell growth (CellTiter-Glo) was tracked for 9 days in dCas9-KRAB–expressing TNBC MB157 (E) and MDA-MB-231 (F) MDA-MB-468 (G) and MCF7 (H) after transduction with CTRL, SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 targeting sgRNA. N = 5 technical replicates. Data presented as mean ± SEM t test: * P < 0.05, ** P < 0.01, *** P < 1 × 10 −3 , and **** P < 1 × 10 −4 . ns, not significant.

    Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

    Techniques: ChIP-sequencing, Functional Assay, Expressing, Inhibition, Control, Transduction, Activity Assay

    ( A and B ) SOX9 promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 cells. Left: Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both (A) and SOX9 .EC2, SOX9 .EC3, or both (B) in MB157 ( n = alleles). Right-top: SOX9 locus schematic, three-color DNA FISH 50-kb probes at SOX9 promoter (green), SOX9 .EC3 (magenta), and SOX9 .EC1 (A, red) or SOX9 .EC2 (B, yellow). Locations per fig. S3A. Right-bottom: Representative cells. Blue: 4′,6-Diamidino-2-phenylindole (DAPI). ( C and F ) SOX9 enhancers inactivation expands SOX9 -EC1-EC3 and SOX9 -EC2-EC3 hubs in individual TNBC MB157 cells. Cumulative distribution functions (CDFs) of SOX9 -EC1-EC3 (C) and SOX9 -EC2-EC3 (F) spatial perimeters in each MB157-dCas9-KRAB expressing control (CTRL), SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 sgRNA [Kolmogorov-Smirnov (KS) test, n = cells]. Mean (±SD) perimeters (micrometers): (C) Left: CTRL/ SOX9 .EC1 sgRNA: 3.78 (±2.63)/4.36 (±2.63); middle: CTRL/ SOX9 .EC2 sgRNA: 3.78 (±2.63)/4.47 (±2.64); right: CTRL/ SOX9 .EC3 sgRNA: 3.39 (±2.56)/4.28 (±2.65). (G) Left: CTRL/ SOX9 .EC1 sgRNA: 3.83 (±2.71)/4.45 (±2.72); middle: CTRL/ SOX9 .EC2 sgRNA: 3.19 (±2.44)/4.26 (±2.61); right: CTRL/ SOX9 .EC3 sgRNA: 3.83 (±2.71)/4.22 (±2.56). ( D and G ) Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both (D) and SOX9 .EC1, SOX9 .EC3, or both (G) in MB157-dCas9-KRAB expressing CTRL, SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 sgRNA ( n = alleles). ( E and H ) Representative cells of 3C and 3D (E) or 3F and 3G (H). Blue: DAPI. ( I ) SOX9 promoter inactivation decreases SOX9 -EC1-EC3 three-way interaction frequency across individual alleles in TNBC MB157. Top-left: Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both in MB157-dCas9-KRAB expressing CTRL or SOX9 promoter sgRNA ( SOX9 .P sgRNA) ( n = alleles). Bottom-left: CDFs of SOX9 -EC1-EC3 spatial perimeter in each MB157-dCas9-KRAB cell (KS test, n = cells). CTRL/ SOX9 .P sgRNA mean (±SD) perimeter: 3.90 (±2.62)/4.44 (±2.66) μm. Right: Representative cells. Blue: DAPI. Scale bars, 3 μm for nuclei and 0.5 μm for alleles.

    Journal: Science Advances

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells

    doi: 10.1126/sciadv.adl4043

    Figure Lengend Snippet: ( A and B ) SOX9 promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 cells. Left: Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both (A) and SOX9 .EC2, SOX9 .EC3, or both (B) in MB157 ( n = alleles). Right-top: SOX9 locus schematic, three-color DNA FISH 50-kb probes at SOX9 promoter (green), SOX9 .EC3 (magenta), and SOX9 .EC1 (A, red) or SOX9 .EC2 (B, yellow). Locations per fig. S3A. Right-bottom: Representative cells. Blue: 4′,6-Diamidino-2-phenylindole (DAPI). ( C and F ) SOX9 enhancers inactivation expands SOX9 -EC1-EC3 and SOX9 -EC2-EC3 hubs in individual TNBC MB157 cells. Cumulative distribution functions (CDFs) of SOX9 -EC1-EC3 (C) and SOX9 -EC2-EC3 (F) spatial perimeters in each MB157-dCas9-KRAB expressing control (CTRL), SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 sgRNA [Kolmogorov-Smirnov (KS) test, n = cells]. Mean (±SD) perimeters (micrometers): (C) Left: CTRL/ SOX9 .EC1 sgRNA: 3.78 (±2.63)/4.36 (±2.63); middle: CTRL/ SOX9 .EC2 sgRNA: 3.78 (±2.63)/4.47 (±2.64); right: CTRL/ SOX9 .EC3 sgRNA: 3.39 (±2.56)/4.28 (±2.65). (G) Left: CTRL/ SOX9 .EC1 sgRNA: 3.83 (±2.71)/4.45 (±2.72); middle: CTRL/ SOX9 .EC2 sgRNA: 3.19 (±2.44)/4.26 (±2.61); right: CTRL/ SOX9 .EC3 sgRNA: 3.83 (±2.71)/4.22 (±2.56). ( D and G ) Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both (D) and SOX9 .EC1, SOX9 .EC3, or both (G) in MB157-dCas9-KRAB expressing CTRL, SOX9 .EC1, SOX9 .EC2, or SOX9 .EC3 sgRNA ( n = alleles). ( E and H ) Representative cells of 3C and 3D (E) or 3F and 3G (H). Blue: DAPI. ( I ) SOX9 promoter inactivation decreases SOX9 -EC1-EC3 three-way interaction frequency across individual alleles in TNBC MB157. Top-left: Allele percentages with SOX9 promoter interacting with SOX9 .EC1, SOX9 .EC3, or both in MB157-dCas9-KRAB expressing CTRL or SOX9 promoter sgRNA ( SOX9 .P sgRNA) ( n = alleles). Bottom-left: CDFs of SOX9 -EC1-EC3 spatial perimeter in each MB157-dCas9-KRAB cell (KS test, n = cells). CTRL/ SOX9 .P sgRNA mean (±SD) perimeter: 3.90 (±2.62)/4.44 (±2.66) μm. Right: Representative cells. Blue: DAPI. Scale bars, 3 μm for nuclei and 0.5 μm for alleles.

    Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

    Techniques: Expressing, Control

    ( A ) Genome tracks showing enrichment of pairwise MYC enhancer-enhancer and promoter-enhancer interactions in a population of MB157 cells. From top to bottom: Colored circles marking location of Oligopaint DNA FISH probes labeling 50-kb regions at MYC promoter (green), MYC .EC1 (magenta), MYC .EC2 (red), MYC .EC3 (yellow), and T-ALL-restricted enhancer (black), H3K27ac and SOX9 levels as measured by ChIP-seq, and normalized interaction frequency as measured by SMC1 HiChIP at the MYC locus in MB157. MYC enhancer clusters are marked by grey boxes. ( B ) A total of 80% of differentially expressed genes with SOX9-bound promoter and distal enhancer participate in ensemble hyper-interacting hubs. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. Hyper-interacting ensemble promoter-enhancer hubs containing genes that are significantly down-regulated in MB157-Cas9 cells transfected with SOX9 targeting sgRNA versus control sgRNA for 4 days and have SOX9-bound promoter and distal enhancer are marked in orange. ( C to E ) SOX9 loss significantly increases 3D distances between the MYC promoter and SOX9-bound MYC .EC2 (C) or MYC .EC3 (D) and SOX9-unbound MYC .EC1 (E) in individual cells. CDFs (left) and box and whiskers (middle) of the distances between the MYC promoter and SOX9-bound MYC .EC2 (C) and MYC .EC3 (D) and SOX9-unbound MYC .EC1 (E) in each MB157-Cas9 6 days after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA (SOX9 KO) (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) distance between MYC promoter and MYC .EC2: 0.389 (±0.358)/0.749 (±0.666) μm; MYC .EC3: 0.447 (±0.457)/0.591 (±0.551) μm; MYC .EC1: 0.494 (±0.447)/0.651 (±0.556) μm. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

    Journal: Science Advances

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells

    doi: 10.1126/sciadv.adl4043

    Figure Lengend Snippet: ( A ) Genome tracks showing enrichment of pairwise MYC enhancer-enhancer and promoter-enhancer interactions in a population of MB157 cells. From top to bottom: Colored circles marking location of Oligopaint DNA FISH probes labeling 50-kb regions at MYC promoter (green), MYC .EC1 (magenta), MYC .EC2 (red), MYC .EC3 (yellow), and T-ALL-restricted enhancer (black), H3K27ac and SOX9 levels as measured by ChIP-seq, and normalized interaction frequency as measured by SMC1 HiChIP at the MYC locus in MB157. MYC enhancer clusters are marked by grey boxes. ( B ) A total of 80% of differentially expressed genes with SOX9-bound promoter and distal enhancer participate in ensemble hyper-interacting hubs. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. Hyper-interacting ensemble promoter-enhancer hubs containing genes that are significantly down-regulated in MB157-Cas9 cells transfected with SOX9 targeting sgRNA versus control sgRNA for 4 days and have SOX9-bound promoter and distal enhancer are marked in orange. ( C to E ) SOX9 loss significantly increases 3D distances between the MYC promoter and SOX9-bound MYC .EC2 (C) or MYC .EC3 (D) and SOX9-unbound MYC .EC1 (E) in individual cells. CDFs (left) and box and whiskers (middle) of the distances between the MYC promoter and SOX9-bound MYC .EC2 (C) and MYC .EC3 (D) and SOX9-unbound MYC .EC1 (E) in each MB157-Cas9 6 days after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA (SOX9 KO) (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) distance between MYC promoter and MYC .EC2: 0.389 (±0.358)/0.749 (±0.666) μm; MYC .EC3: 0.447 (±0.457)/0.591 (±0.551) μm; MYC .EC1: 0.494 (±0.447)/0.651 (±0.556) μm. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

    Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

    Techniques: Labeling, ChIP-sequencing, HiChIP, Transfection, Control, Transduction

    ( A ) MYC promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 and MDA-MB-468 but not ER + MCF7. Left: Percentage of alleles with MYC promoter interacting (<350 nm) with SOX9-unbound MYC .EC1, SOX9-bound MYC .EC3, or both MYC .EC1 and MYC .EC3 in MB157, MDA-MB-468, and MCF7 as measured by three-color Oligopaint DNA FISH with probes marked in top genome track ( n = alleles). Right: Representative MB157, MDA-MB-468, and MCF7 nuclei and two magnified alleles from three-color DNA FISH. Scale bar per 3A. Blue: DAPI. ( B and C ) SOX9 loss expands MYC -EC1-EC2 (B) and MYC -EC1-EC3 (C) promoter-enhancer hubs in individual MB157 and decreases three-way interaction frequency across individual alleles. Left: CDFs of MYC -EC1-EC2 (B) and MYC -EC1-EC3 (C) spatial perimeters in each MB157-Cas9 cell expressing CTRL or SOX9 KO sgRNA (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) perimeters MYC -EC1-EC2 (B): 3.84 (±2.70)/4.53 (±2.67) μm; MYC -EC1-EC3 (C): 3.10 (±2.58)/3.90 (±2.64) μm ( n = cells). Middle: Allele percentages with MYC promoter interacting (<350 nm) with MYC .EC1, MYC .EC2, or both MYC .EC1 and MYC .EC2 (B) and MYC .EC1, MYC .EC3, or both MYC .EC1 and MYC .EC3 (C) in CTRL and SOX9 KO MB157-Cas9. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

    Journal: Science Advances

    Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells

    doi: 10.1126/sciadv.adl4043

    Figure Lengend Snippet: ( A ) MYC promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 and MDA-MB-468 but not ER + MCF7. Left: Percentage of alleles with MYC promoter interacting (<350 nm) with SOX9-unbound MYC .EC1, SOX9-bound MYC .EC3, or both MYC .EC1 and MYC .EC3 in MB157, MDA-MB-468, and MCF7 as measured by three-color Oligopaint DNA FISH with probes marked in top genome track ( n = alleles). Right: Representative MB157, MDA-MB-468, and MCF7 nuclei and two magnified alleles from three-color DNA FISH. Scale bar per 3A. Blue: DAPI. ( B and C ) SOX9 loss expands MYC -EC1-EC2 (B) and MYC -EC1-EC3 (C) promoter-enhancer hubs in individual MB157 and decreases three-way interaction frequency across individual alleles. Left: CDFs of MYC -EC1-EC2 (B) and MYC -EC1-EC3 (C) spatial perimeters in each MB157-Cas9 cell expressing CTRL or SOX9 KO sgRNA (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) perimeters MYC -EC1-EC2 (B): 3.84 (±2.70)/4.53 (±2.67) μm; MYC -EC1-EC3 (C): 3.10 (±2.58)/3.90 (±2.64) μm ( n = cells). Middle: Allele percentages with MYC promoter interacting (<350 nm) with MYC .EC1, MYC .EC2, or both MYC .EC1 and MYC .EC2 (B) and MYC .EC1, MYC .EC3, or both MYC .EC1 and MYC .EC3 (C) in CTRL and SOX9 KO MB157-Cas9. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

    Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

    Techniques: Expressing