Analysis of long and short enhancers in melanoma cell states

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 assay (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. A more in-depth evaluation of those regions was then pursued by investigating the activity of ATAC-seq peaks found therein 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 regions 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, while SOX and MITF both influence MEL enhancer activity with SOX being required to achieve high levels of activity. Overall, our MPRA assays shed light on the relationship between long and short sequences in terms of their sequence content, enhancer activity, and specificity as reporters across melanoma cell states. Eight MPRA libraries are used in this study, including 7 CHEQ-seq libraries and 1 STARR-seq library. For each CHEQ-seq library an enhancer - barcode assignment is done by sequencing of the plasmid library with PacBio or Illumina sequencing. Libraries CHEQ-seq 5' H3K27ac, CHEQ-seq Intron H3K27ac, CHEQ-seq ATAC and STARR-seq ATAC are transfected in seven cell lines. Libraries CHEQ-seq enhancer tiling A and B are transfected in 5 cell lines. Library CHEQ-seq SOX10-KD is transfected in one cell line. Library CHEQ-seq SOX-MITF combinations is transfected in 3 cell lines.