Landscape of enhancer disruption and functional screen in melanoma [4C-seq]
Ontology highlight
ABSTRACT: The high mutation rate across the whole melanoma genome provides a major challenge in stratifying true driver events from the background mutations. Many non-coding recurrent events, such as those occurred in enhancer, can shape tumor evolution, emphasizing the importance in systematically deciphering enhancer disruptions in melanoma. Here, we leveraged 297 melanoma whole-genome sequencing (WGS) samples to prioritize highly recurrent regions (HRRs). By performing a genome-scale CRISPR interference (CRISPRi) screen on HRR-associated enhancers in melanoma cells, we identified 66 significant hits which could have tumor-suppressive roles. These functional enhancers show unique mutational patterns independent of classical significantly mutated genes in melanoma. Target gene analysis for the essential enhancers revealed many known and hidden mechanisms underlying melanoma development. We demonstrated that a super enhancer element could modulate melanoma cell proliferation by targeting MEF2A and another distal enhancer was able to sustain PTEN tumor-suppressive potential via long-range interaction. Our study established a catalogue of crucial enhancers and their target genes in melanoma development and progression, which illuminates the identification of novel mechanism of dysregulation for melanoma driver genes and new therapeutic targeting strategy.
Project description:The high mutation rate across the whole melanoma genome provides a major challenge in stratifying true driver events from the background mutations. Many non-coding recurrent events, such as those occurred in enhancer, can shape tumor evolution, emphasizing the importance in systematically deciphering enhancer disruptions in melanoma. Here, we leveraged 297 melanoma whole-genome sequencing (WGS) samples to prioritize highly recurrent regions (HRRs). By performing a genome-scale CRISPR interference (CRISPRi) screen on HRR-associated enhancers in melanoma cells, we identified 66 significant hits which could have tumor-suppressive roles. These functional enhancers show unique mutational patterns independent of classical significantly mutated genes in melanoma. Target gene analysis for the essential enhancers revealed many known and hidden mechanisms underlying melanoma development. We demonstrated that a super enhancer element could modulate melanoma cell proliferation by targeting MEF2A and another distal enhancer was able to sustain PTEN tumor-suppressive potential via long-range interaction. Our study established a catalogue of crucial enhancers and their target genes in melanoma development and progression, which illuminates the identification of novel mechanism of dysregulation for melanoma driver genes and new therapeutic targeting strategy.
Project description:The high mutation rate across the whole melanoma genome provides a major challenge in stratifying true driver events from the background mutations. Many non-coding recurrent events, such as those occurred in enhancer, can shape tumor evolution, emphasizing the importance in systematically deciphering enhancer disruptions in melanoma. Here, we leveraged 297 melanoma whole-genome sequencing (WGS) samples to prioritize highly recurrent regions (HRRs). By performing a genome-scale CRISPR interference (CRISPRi) screen on HRR-associated enhancers in melanoma cells, we identified 66 significant hits which could have tumor-suppressive roles. These functional enhancers show unique mutational patterns independent of classical significantly mutated genes in melanoma. Target gene analysis for the essential enhancers revealed many known and hidden mechanisms underlying melanoma development. We demonstrated that a super enhancer element could modulate melanoma cell proliferation by targeting MEF2A and another distal enhancer was able to sustain PTEN tumor-suppressive potential via long-range interaction. Our study established a catalogue of crucial enhancers and their target genes in melanoma development and progression, which illuminates the identification of novel mechanism of dysregulation for melanoma driver genes and new therapeutic targeting strategy.
Project description:Here, we leveraged 297 melanoma whole-genome sequencing (WGS) samples to prioritize highly recurrent regions (HRRs). By performing a genome-scale CRISPR interference (CRISPRi) screen on HRR-associated enhancers in melanoma cells, we identified 66 significant hits which could have tumor-suppressive roles. These functional enhancers show unique mutational patterns independent of classical significantly mutated genes in melanoma. Target gene analysis for the essential enhancers revealed many known and hidden mechanisms underlying melanoma growth. Utilizing extensive functional validation experiments, we demonstrated that a super enhancer element could modulate melanoma cell proliferation by targeting MEF2A and another distal enhancer was able to sustain PTEN tumor-suppressive potential via long-range interaction.
Project description:Myocyte Enhancer Factor 2 (MEF2) proteins are involved in multiple developmental, physiological, and pathological processes in vertebrates. Protein:protein interactions underlie the plethora of biological processes impacted by MEF2A, necessitating a detailed characterization of the MEF2A interactome. A nanobody based affinity-purification/mass spectrometry strategy was employed to achieve this goal. Specifically, the MEF2A protein complexes were captured from myogenic lysates using a GFP-tagged MEF2A protein immobilized with a GBP-nanobody followed by LC-MS/MS proteomic analysis to identify MEF2A interactors.
Project description:The implication of epigenetic alterations in the pathogenesis of melanoma is increasingly recognized. Here we performed genome-wide DNA methylation analysis of primary cutaneous melanoma and benign melanocytic naevus interrogating 14,495 genes using beadchip technology. This first genome-wide view of promoter methylation in primary cutaneous melanoma revealed an array of recurrent DNA methylation alterations with potential diagnostic applications. Among 106 frequently hypermethylated genes there were many novel methylation targets and tumor suppressor genes. Highly recurrent methylation of the HOXA9, MAPK13, CDH11, PLEKHG6, PPP1R3C and CLDN11genes was established. Promoter methylation of MAPK13, encoding p38?, was present in 67% of primary and 85% of metastatic melanomas. Restoration of MAPK13 expression in melanoma cells exhibiting epigenetic silencing of this gene reduced proliferation, indicative of tumor suppressive functions. This study demonstrates that DNA methylation alterations are widespread in melanoma and suggests that epigenetic silencing of MAPK13 contributes to melanoma progression. Bisulphite converted genomic DNA from 5 fresh-frozen benign naevus and 24 fresh-frozen primary melanoma biopsy samples were hybridised to Illumina's Infinium HumanMethylation27 Beadchips
Project description:Thousands of enhancers are characterized in the human genome, yet few have been shown important in cancer. Inhibiting oncokinases, such as EGFR, ALK, HER2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by upregulation of the HGF receptor, MET. The mechanisms mediating such genomic responses to targeted therapy are unknown. Here, we identify lineage-specific MET enhancers for multiple common tumor types, including a melanoma lineage-specific MET enhancer that displays inducible chromatin looping and MET gene induction upon BRAF inhibition. Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function. Targeted genomic deletion (<7bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation. Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of tumor lineage-enriched transcription factors mediating innate resistance to oncokinase therapy. COLO829 human melanoma cell line harboring the BRAFV600E mutation was treated with BRAF inhibtior PLX4032 (Vemurafenib) and/or a hairpin against MITF
Project description:Thousands of enhancers are characterized in the human genome, yet few have been shown important in cancer. Inhibiting oncokinases, such as EGFR, ALK, HER2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by upregulation of the HGF receptor, MET. The mechanisms mediating such genomic responses to targeted therapy are unknown. Here, we identify lineage-specific MET enhancers for multiple common tumor types, including a melanoma lineage-specific MET enhancer that displays inducible chromatin looping and MET gene induction upon BRAF inhibition. Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function. Targeted genomic deletion (<7bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation. Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of tumor lineage-enriched transcription factors mediating innate resistance to oncokinase therapy. MITF ChIP-seq was performed in primary human melanocytes with overexpression of BRAFV600E or a lentiviral control (RFP), and in COLO829 melanoma cells treated with DMSO, or PLX4032
Project description:The implication of epigenetic alterations in the pathogenesis of melanoma is increasingly recognized. Here we performed genome-wide DNA methylation analysis of primary cutaneous melanoma and benign melanocytic naevus interrogating 14,495 genes using beadchip technology. This first genome-wide view of promoter methylation in primary cutaneous melanoma revealed an array of recurrent DNA methylation alterations with potential diagnostic applications. Among 106 frequently hypermethylated genes there were many novel methylation targets and tumor suppressor genes. Highly recurrent methylation of the HOXA9, MAPK13, CDH11, PLEKHG6, PPP1R3C and CLDN11genes was established. Promoter methylation of MAPK13, encoding p38?, was present in 67% of primary and 85% of metastatic melanomas. Restoration of MAPK13 expression in melanoma cells exhibiting epigenetic silencing of this gene reduced proliferation, indicative of tumor suppressive functions. This study demonstrates that DNA methylation alterations are widespread in melanoma and suggests that epigenetic silencing of MAPK13 contributes to melanoma progression.
Project description:Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the cancer process. Here, we engineered lymphoma-prone mice with chromosomal instability to assess the utility of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Integrating with targeted re-sequencing, our comparative oncogenomic studies efficiently identified FBXW7 and PTEN as commonly deleted or mutated tumor suppressors in human T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). More generally, the murine cancers acquire widespread recurrent clonal amplifications and deletions targeting loci syntenic to alterations present in not only human T-ALL but also diverse tumors of hematopoietic, mesenchymal and epithelial types. These results thus support the view that murine and human tumors experience common biological processes driven by orthologous genetic events as they evolve towards a malignant phenotype. The highly concordant nature of genomic events encourages the use of genome unstable murine cancer models in the discovery of biologically relevant driver events in human cancer. Experiment Overall Design: 123 Melanoma samples were analyzed. Normal Human DNA was used as reference. Most samples were hybridized with dye-swap replica.
Project description:Although technological advances now allow increased tumor profiling, a detailed understanding of the mechanisms leading to the development of different cancers remains elusive. Our approach towards understanding the molecular events that lead to cancer is to characterize changes in transcriptional regulatory networks between normal and tumor tissue. Because enhancer activity is thought to be critical in regulating cell fate decisions, we have focused our studies on distal regulatory elements and transcription factors that bind to these elements. Using DNA methylation data, we identified more than 25,000 enhancers that are differentially activated in breast, prostate, and kidney tumor tissues, as compared to normal tissues. We then developed an analytical approach called TENET (Tracing Enhancer Networks using Epigenetic Traits) that correlates DNA methylation levels at enhancers with gene expression to identify more than 800,000 genome-wide links from enhancers to genes and from genes to enhancers. We found more than 1,200 transcription factors to be involved in these tumor-specific enhancer networks. We further characterized several transcription factors linked to a large number of enhancers in each tumor type, including GATA3 in non-basal breast tumors, HOXC6 and DLX1 in prostate tumors, and ZNF395 in kidney tumors. We showed that HOXC6 and DLX1 are associated with different clusters of prostate tumor-specific enhancers and confer distinct transcriptomic changes upon knockdown in C42B prostate cancer cells. We also discovered de novo motifs enriched in enhancers linked to ZNF395 in kidney tumors. Our studies characterized tumor-specific enhancers and revealed key transcription factors involved in enhancer networks for specific tumor types and subgroups. Our findings, which include a large set of identified enhancers and transcription factors linked to those enhancers in breast, prostate, and kidney cancers, will facilitate understanding of enhancer networks and mechanisms leading to the development of these cancers. Examination of FAIRE and ChIP assays in prostate and breast epithelial cells. RNA-seq assays in C42B, prostate cancer cell line after knocking down of TFs (HOXC6, DLX1).