Study / S9OR7V60R2021-12-07

Analysis of long and short enhancers in melanoma cell states

David Mauduit, Ibrahim Ihsan Taskiran, Liesbeth Minnoye, Maxime de Waegeneer, Valerie Christiaens et al.

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About this study

Understanding how enhancers drive cell-type specificity and efficiently identifying them is essential for the development of innovative therapeutic strategies. In melanoma, the melanocytic (MEL) and the mesenchymal-like (MES) states present themselves with different responses to therapy, making the identification of specific enhancers highly relevant. Using massively parallel reporter assays (MPRAs) in a panel of patient-derived melanoma lines (MM lines), we set to identify and decipher melanoma enhancers by first focusing on regions with state-specific H3K27 acetylation close to differentially expressed genes. An in-depth evaluation of those regions was then pursued by investigating the activity of overlapping ATAC-seq peaks along with a full tiling of the acetylated regions with 190 bp sequences. Activity was observed in more than 60% of the selected regions, and we were able to precisely locate the active enhancers within ATAC-seq peaks. Comparison of sequence content with activity, using the deep learning model DeepMEL2, revealed that AP-1 alone is responsible for the MES enhancer activity. In contrast, SOX10 and MITF both influence MEL enhancer function with SOX10 being required to achieve high levels of activity. Overall, our MPRAs shed light on the relationship between long and short sequences in terms of their sequence content, enhancer activity, and specificity across melanoma cell states.

Full author list & citation

David Mauduit, Ibrahim Ihsan Taskiran, Liesbeth Minnoye, Maxime de Waegeneer, Valerie Christiaens, Gert Hulselmans, Jonas Demeulemeester, Jasper Wouters, Stein Aerts. Analysis of long and short enhancers in melanoma cell states. 2021-12-07. https://doi.org/10.7554/eLife.71735

Experiments 8

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H3K27ac-selected melanoma enhancers, intron CHEQ-seq

An episomal CHEQ-seq MPRA tested the same H3K27ac-selected melanoma regions using the intron reporter configuration. Constructs were assayed across seven patient-derived melanoma cell lines representing melanocytic and mesenchymal-like states.

Episomal Plasmid MPRAHumanhg19

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E1FCZKVBE

ATAC-seq peak enhancers, CHEQ-seq

An episomal CHEQ-seq MPRA tested selected 501 bp ATAC-seq peak sequences from melanoma cell states in the CHEQ reporter. The processed table reports enhancer-level activity across the seven melanoma cell lines represented in GEO.

Episomal Plasmid MPRAHumanhg19

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E1YMXPF2E

SOX10-KD-selected MEL enhancer tiles

A CHEQ-seq MPRA tested 190 bp tiles from candidate melanocytic enhancers selected using prior SOX10 knockdown data, in melanoma line MM087. The packaged statistical output compares reporter cDNA with plasmid input for the MPRA library.

Episomal Plasmid MPRAHumanhg19

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E3TV0E7IS

H3K27ac-selected melanoma enhancers, 5′ CHEQ-seq

An episomal CHEQ-seq MPRA tested 5′-configuration reporter constructs containing H3K27ac-selected regions near state-associated melanoma genes. Constructs were assayed across seven patient-derived melanoma cell lines representing melanocytic and mesenchymal-like states.

Episomal Plasmid MPRAHumanhg19

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E6WE06IIQ

ATAC-seq peak enhancers, STARR-seq

A standard episomal STARR-seq assay tested the selected 501 bp ATAC-seq peak sequences in a self-transcribing reporter configuration. The processed table reports enhancer-level activity across the seven melanoma cell lines represented in GEO.

Standard STARR-seqHumanhg19

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E844ZNYQM

H3K27ac enhancer tiling library A

A 190 bp tiling CHEQ-seq MPRA densely sampled H3K27ac-selected melanoma regions with overlapping tiles shifted by 20 bp. Library A includes wild-type tiles, motif-focused mutant tiles, and shuffled negative controls assayed across multiple melanoma cell-line replicates.

Episomal Plasmid MPRAHumanhg19

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E87IFCJ9T

H3K27ac enhancer tiling library B

A 190 bp tiling CHEQ-seq MPRA densely sampled H3K27ac-selected melanoma regions with tiles shifted 10 bp downstream relative to library A. Library B includes wild-type tiles, motif-focused mutant tiles, and shuffled negative controls assayed across multiple melanoma cell-line replicates.

Episomal Plasmid MPRAHumanhg19

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E8GLNIVH5

Synthetic SOX-MITF motif combinations

A synthetic regulatory grammar CHEQ-seq MPRA tested combinations of SOX and MITF motifs placed at defined spacings in synthetic backgrounds. The constructs were assayed in melanoma lines MM001, MM074, and MM087, with random-background controls included.

Synthetic Regulatory Grammar MPRAHuman

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Raw source data 33 files

Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.