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.
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.
Analysis of long and short enhancers in melanoma cell statesNCBI Gene Expression Omnibus, GSE180879.
BRG1 recruitment by transcription factors MITF and SOX10 defines a specific configuration of regulatory elements in the melanocyte lineage (ChIP-seq)NCBI Gene Expression Omnibus, GSE61965.
TFAP2A ChIP-seq in human primary melanocytesNCBI Gene Expression Omnibus, GSE67555.
Prioritization of enhancer mutations by combining allele-specific chromatin accessibility with motif analysis and deep learningNCBI Gene Expression Omnibus, GSE159965.
Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state.NCBI Gene Expression Omnibus, GSE60666.
cisTopic: cis-regulatory topic modelling on single-cell ATAC-seq dataNCBI Gene Expression Omnibus, GSE114557.
Cross-species analysis of melanoma enhancer logic using deep learningNCBI Gene Expression Omnibus, GSE142238.
Single-cell analysis of gene expression variation and phenotype switching in melanomaNCBI Gene Expression Omnibus, GSE134432.
- Stein Aerts
- Stein Aerts
- Liesbeth Minnoye
- Jonas Demeulemeester
- Jasper Wouters
- David Mauduit
- Valerie Christiaens
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Jian Xu, University of Texas Southwestern Medical Center, United States
© 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.
Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome, and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, we profiled active chromatin features in the testes from wildtype and meiotic arrest mutants and integrate this with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, we detail widespread patterns associated with regulation of the sex chromosomes. Our results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome.
Imaging experiments reveal the complex and dynamic nature of the transcriptional hubs associated with Notch signaling.