Abstract

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 (MPRA) 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.

Data availability

Sequencing data have been deposited in GEO under accession codes GSE180879.Enhancer activity tables for each library is provided as source data.Scripts used for enhancer - barcode assignment, read processing and activity measurement and analysis are provided in the Scripts directory.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. David Mauduit

    Department of Human Genetics, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2045-227X
  2. Ibrahim Ihsan Taskiran

    Department of Human Genetics, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  3. Liesbeth Minnoye

    Department of Human Genetics, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  4. Maxime de Waegeneer

    Department of Human Genetics, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  5. Valerie Christiaens

    Department of Human Genetics, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  6. Gert Hulselmans

    Department of Human Genetics, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  7. Jonas Demeulemeester

    Department of Human Genetics, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2660-2478
  8. Jasper Wouters

    Department of Human Genetics, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7129-2990
  9. Stein Aerts

    Department of Human Genetics, KU Leuven, Leuven, Belgium
    For correspondence
    stein.aerts@kuleuven.be
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8006-0315

Funding

H2020 European Research Council (724226_cis-CONTROL)

  • Stein Aerts

KU Leuven (C14/18/092)

  • Stein Aerts

Fonds Wetenschappelijk Onderzoek (1S03317N)

  • Liesbeth Minnoye

Fonds Wetenschappelijk Onderzoek (12J6916N)

  • Jonas Demeulemeester

Kom op tegen Kanker

  • Jasper Wouters

H2020 European Research Council (724226_cis-CONTROL)

  • David Mauduit

H2020 European Research Council (724226_cis-CONTROL)

  • Valerie Christiaens

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Jian Xu, University of Texas Southwestern Medical Center, United States

Version history

  1. Received: June 28, 2021
  2. Preprint posted: July 27, 2021 (view preprint)
  3. Accepted: December 6, 2021
  4. Accepted Manuscript published: December 7, 2021 (version 1)
  5. Version of Record published: December 21, 2021 (version 2)

Copyright

© 2021, Mauduit et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,171
    views
  • 301
    downloads
  • 17
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. David Mauduit
  2. Ibrahim Ihsan Taskiran
  3. Liesbeth Minnoye
  4. Maxime de Waegeneer
  5. Valerie Christiaens
  6. Gert Hulselmans
  7. Jonas Demeulemeester
  8. Jasper Wouters
  9. Stein Aerts
(2021)
Analysis of long and short enhancers in melanoma cell states
eLife 10:e71735.
https://doi.org/10.7554/eLife.71735

Share this article

https://doi.org/10.7554/eLife.71735

Further reading

    1. Chromosomes and Gene Expression
    Rupam Choudhury, Anuroop Venkateswaran Venkatasubramani ... Axel Imhof
    Research Article

    Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity-biotinylation method targeting the RNA and proteins constituents. The method that we termed Antibody-Mediated-Proximity-Labelling-coupled to Mass Spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. This analysis identified a number of known RNA binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.

    1. Cancer Biology
    2. Chromosomes and Gene Expression
    Gregory Caleb Howard, Jing Wang ... William P Tansey
    Research Article

    The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the ‘WIN’ site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.