Project description:Enhancers are critical regulatory elements in the genome that help orchestrate spatiotemporal patterns of gene expression during development and normal physiology. In cancer, enhancers are often rewired by various genetic and epigenetic mechanisms for the activation of oncogenes that lead to initiation and progression. A key feature of active enhancers is the production of non-coding RNA molecules called enhancer RNAs, whose functions remain unknown but can be used to specify active enhancers de novo. Using a combination of eRNA transcription and chromatin modifications, we have identified a novel enhancer located 30 kb upstream of Colony Stimulating Factor 1 (CSF1). Notably, CSF1 is implicated in the progression of breast cancer, is overexpressed in triple-negative breast cancer (TNBC) cell lines, and its enhancer is primarily active in TNBC patient tumors. Genomic deletion of the enhancer (via CRISPR/Cas9) enabled us to validate this regulatory element as a bona fide enhancer of CSF1 and subsequent cell-based assays revealed profound effects on cancer cell proliferation, colony formation, and migration. Epigenetic silencing of the enhancer via CRISPR-interference assays (dCas9-KRAB) coupled to RNA-sequencing, enabled unbiased identification of additional target genes, such as RSAD2, that are predictive of clinical outcome. Additionally, we repurposed the RNA-guided RNA-targeting CRISPR-Cas13 machinery to specifically degrade the eRNAs transcripts produced at this enhancer to determine the consequences on CSF1 mRNA expression, suggesting a post-transcriptional role for these non-coding transcripts. Finally, we test our eRNA-dependent model of CSF1 enhancer function and demonstrate that our results are extensible to other forms of cancer. Collectively, this work describes a novel enhancer that is active in the TNBC subtype, which is associated with cellular growth, and requires eRNA transcripts for proper enhancer function. These results demonstrate the significant impact of enhancers in cancer biology and highlight their potential as tractable targets for therapeutic intervention.

Project description:Triple-negative breast cancer (TNBC) is the most therapeutically recalcitrant form of breast cancer. Pioneering genomic studies have identified the repertoire of expressed genes in the distinct molecular subtypes of breast cancer and have been instrumental in providing diagnostic, prognostic, and therapeutic guidance. Yet breast cancer remains the second leading cause of cancer mortality in women. This is due, in part, to the paucity of targeted therapies for triple-negative breast cancer (TNBC). We posit that a systematic analysis of regulatory elements that extend beyond protein coding genes will lead to novel avenues for therapeutic intervention. To this end, we analyzed the regulatory mechanisms of TNBC-specific transcriptional enhancers together with their non-coding enhancer RNA (eRNA) transcripts. We systematically probed the functions of the top 30 eRNA-producing super-enhancers using a high-throughput CRISPR-interference (dCas9-KRAB) assay coupled to RNA-seq that enabled unbiased detection of target genes genome-wide. Utilizing Hi-C, we constructed 3D chromatin interaction maps to annotate direct target genes for each super-enhancer and highlight their proclivity for genes that portend worse clinical outcomes in TNBC patients. To illustrate the utility of this dataset, we selected a novel super-enhancer controlling the nearby PODXL gene and demonstrate that deletion of the super-enhancer (via Cas9 deletion) or specific degradation of its enhancer RNAs (via Cas13 RNA cleavage) leads to profound effects on target gene expression, cell proliferation, and migration. Furthermore, complete knockout of the PODXL super-enhancer leads to a significant decrease in tumor growth and metastatic potential in mouse xenograft experiments. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of triple-negative breast cancer.

			Action	DRS
	SRR16203123_1.fastq.gz	Fastqsanger.gz
	SRR16203123_2.fastq.gz	Fastqsanger.gz
	SRR16203124_1.fastq.gz	Fastqsanger.gz
	SRR16203124_2.fastq.gz	Fastqsanger.gz
	SRR16203125_1.fastq.gz	Fastqsanger.gz

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Enhancer RNA transcription defines novel CSF1 enhancer in triple-negative breast cancer

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