Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer
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 initiat...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-04-01
|
| Series: | Cancers |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2072-6694/14/7/1852 |
| _version_ | 1797439998100766720 |
|---|---|
| author | Michael W. Lewis Kamila Wisniewska Caitlin M. King Shen Li Alisha Coffey Michael R. Kelly Matthew J. Regner Hector L. Franco |
| author_facet | Michael W. Lewis Kamila Wisniewska Caitlin M. King Shen Li Alisha Coffey Michael R. Kelly Matthew J. Regner Hector L. Franco |
| author_sort | Michael W. Lewis |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-09T12:01:45Z |
| format | Article |
| id | doaj.art-7a06293b4d9444d790fc53dcf9e4a12c |
| institution | Directory Open Access Journal |
| issn | 2072-6694 |
| language | English |
| last_indexed | 2024-03-09T12:01:45Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cancers |
| spelling | doaj.art-7a06293b4d9444d790fc53dcf9e4a12c2023-11-30T23:02:56ZengMDPI AGCancers2072-66942022-04-01147185210.3390/cancers14071852Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast CancerMichael W. Lewis0Kamila Wisniewska1Caitlin M. King2Shen Li3Alisha Coffey4Michael R. Kelly5Matthew J. Regner6Hector L. Franco7The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAThe Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAThe Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAThe Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAThe Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAThe Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAThe Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAThe Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAEnhancers 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.https://www.mdpi.com/2072-6694/14/7/1852breast cancerovarian cancerenhancereRNACRISPR-Cas9dCas9-KRAB |
| spellingShingle | Michael W. Lewis Kamila Wisniewska Caitlin M. King Shen Li Alisha Coffey Michael R. Kelly Matthew J. Regner Hector L. Franco Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer Cancers breast cancer ovarian cancer enhancer eRNA CRISPR-Cas9 dCas9-KRAB |
| title | Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer |
| title_full | Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer |
| title_fullStr | Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer |
| title_full_unstemmed | Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer |
| title_short | Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer |
| title_sort | enhancer rna transcription is essential for a novel csf1 enhancer in triple negative breast cancer |
| topic | breast cancer ovarian cancer enhancer eRNA CRISPR-Cas9 dCas9-KRAB |
| url | https://www.mdpi.com/2072-6694/14/7/1852 |
| work_keys_str_mv | AT michaelwlewis enhancerrnatranscriptionisessentialforanovelcsf1enhancerintriplenegativebreastcancer AT kamilawisniewska enhancerrnatranscriptionisessentialforanovelcsf1enhancerintriplenegativebreastcancer AT caitlinmking enhancerrnatranscriptionisessentialforanovelcsf1enhancerintriplenegativebreastcancer AT shenli enhancerrnatranscriptionisessentialforanovelcsf1enhancerintriplenegativebreastcancer AT alishacoffey enhancerrnatranscriptionisessentialforanovelcsf1enhancerintriplenegativebreastcancer AT michaelrkelly enhancerrnatranscriptionisessentialforanovelcsf1enhancerintriplenegativebreastcancer AT matthewjregner enhancerrnatranscriptionisessentialforanovelcsf1enhancerintriplenegativebreastcancer AT hectorlfranco enhancerrnatranscriptionisessentialforanovelcsf1enhancerintriplenegativebreastcancer |