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A genetically engineered mouse model of NUT carcinoma (CUT&RUN)

ABSTRACT: A genetically engineered mouse model of NUT carcinoma (CUT&RUN)

PROVIDER: PRJNA1008244 | ENA |

REPOSITORIES: ENA

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A genetically engineered mouse model of NUT carcinoma (CUT&RUN)

Project description:We created a genetically engineered mouse model (GEMM) of NC that forms a Brd4-NUTM1 fusion gene upon tamoxifen-induction of Sox2-driven Cre. Two GEMM-derived cell lines were developed whose transcriptomic and epigenetic landscapes, characterized by RNAseq and CUT&RUN, show striking overlap with those of primary GEMM tumors. GEMM primary tumor and cell lines form very large H3K27ac-enriched super-enhancers that are unique to hNC, termed megadomains, that are invariably associated with key hNC-defining transcriptional oncogenic targets, Myc and Trp63.

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A genetically engineered mouse model of NUT carcinoma

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Project description:NUT carcinoma (NC) is a highly aggressive subtype of squamous carcinoma driven by the BRD4-NUT fusion oncoprotein. Closely resembling human NC (hNC), GEMM tumors (mNC) are poorly differentiated squamous carcinomas that express high levels of MYC and metastasize to organs (liver, lung) and regional lymph nodes. Two GEMM-derived cell lines were developed whose transcriptomic and epigenetic landscapes, characterized by RNAseq and CUT&RUN, show striking overlap with those of primary GEMM tumors. As in hNC, BRD4-NUT functions to block differentiation and maintain growth of mNC, as evidenced by BRD4-NUT knockdown and treatment of mNC cell lines with BET bromodomain inhibitors (BETi). Mechanistically, GEMM primary tumor and cell lines form very large H3K27ac-enriched super-enhancers that are unique to hNC, termed megadomains, that are invariably associated with key hNC-defining transcriptional oncogenic targets, Myc and Trp63.

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Project description:SOX6 CUT&RUN on HUDEP1 over expressing SOX6-Flag. The experiment is done using and anti Flag Ab to assist the genome wide binding profile of SOX6 in HUDEP1 (Human Umbilical cord blood-Derived Erythroid Progenitor-1).

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Identification of chromatin states during zebrafish gastrulation using CUT&RUN and CUT&Tag

Project description:Here we describe successful implementation of CUT&RUN for profiling protein-DNA interactions in zebrafish embryos. We apply modified a CUT&RUN method to generate high resolution maps of enrichment for H3K4me3, H3K27me3, H3K9me3, and RNA polymerase II during zebrafish gastrulation. Using this data, we identify a conserved subset of developmental genes that are enriched in both H3K4me3 and H3K27me3 during gastrulation, and we demonstrate the increased effectiveness of CUT&RUN for detecting protein enrichment at repetitive sequences with reduced mappability. Our work demonstrates the power of combining CUT&RUN with the strengths of the zebrafish system to better understand the changing embryonic chromatin landscape and its roles in shaping development.

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CUT&RUN sequencing of human kidney tubuloid to study genome-wide binding site in tRCC patients

Project description:Translocation renal cell carcinoma (tRCC) is a rare, aggressive kidney cancer primarily occurring in children. They are genetically defined by translocations involving MiT/TFE gene family members, TFE3. We utilized human kidney organoids, or tubuloids, to engineer a tRCC model by expressing of one of the most common MiT/TFE fusions, SFPQ-TFE3. Lentiviral transductions were performed as previously described with lentiviruses encoding either pLKO.1-UbC-luciferase-blast (TubCtrl), pLKO.1-UbC-TFE3-blast (TubTFE3), or pLKO.1-UbC-SFPQ-TFE3-blast (TubFus). Two days post transduction, 5 µg/ml blasticidin was added to the culture medium to select for successfully transduced cells. To study the genome-wide binding sites of the fusion, we conducted CUT&RUN sequencing. CUT&RUN experiments were performed using a modified protocol for low cell numbers using the following antibodies: anti-TFE3 (ab93808, Abcam, 1:2000). Libraries were sequenced using an Illumina NextSeq2000 (2x100bp).

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