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Enhanced IFNy response in dedifferentiated melanoma cells is due to chromatin remodeling as revealed by ATAC-seq

ABSTRACT: Background Differentiation status influences immunomodulatory gene expression in melanoma. Loss of Microphthalmia-Associated Transcription Factor (MITF) drives dedifferentiation and enhances the transcriptional responses to Interferon-γ (IFNγ), leading to non-additive increase in inflammatory cytokine and Programmed Death-Ligand 1 (PD-L1) expression. Whilst these transcriptional responses are well characterized, it remains unclear how shifts in differentiation status affect the epigenetic response to inflammatory stimulus. Methods We used Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) to assess how differentiation status and IFNγ affect chromatin organization in 624Mel melanoma cells. Dedifferentiation was induced by MITF knockdown using siRNA, followed by IFNγ stimulation prior to ATAC-seq. Data were processed with the nfcore/atacseq pipeline and analyzed with DESeq2, Analysis of Motif Enrichment, Gene Ontology and KEGG Pathway analysis. Results Both MITF loss and IFNγ stimulation produced significant but distinct changes in chromatin accessibility. MITF knockdown favored chromatin opening, whereas IFNγ induced milder increases in accessibility. Integration with previously published RNA-seq data revealed concordant regulation of chromatin accessibility and gene expression following MITF loss and IFNγ treatment. Regions of altered accessibility following MITF knockdown were enriched for migration related genes, while IFNγ primarily increased access near inflammatory genes, an effect amplified under MITFlow conditions. MITF loss also enhanced IFNγ-driven transcription factor motif enrichment. Conclusions These findings exemplify the epigenetic consequences of MITF loss in melanoma and demonstrate that the established link between differentiation status and immunomodulatory gene expression extends to chromatin organization. This extends the implication of modulating differentiation to shape inflammatory responsiveness in melanoma cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE325514 | GEO | 2026/05/29

REPOSITORIES: GEO

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Project description:Background Melanoma cells frequently dedifferentiate in response to inflammation which can increase responses to certain cytokines. Interferon-γ (IFNγ) is an integral part of the anti-tumour immune response and can directly induce both differentiational changes and expression of immunosuppressive proteins in melanoma cells. How the differentiation status of melanoma cells affects IFNγ responses remains unclear. Methods Dedifferentiation of melanoma cells was induced via either siRNA or shRNA mediated MITF knockdown and the cells were subsequently treated with IFNγ. Effects of MITF knockdown and IFNγ treatment on gene expression were evaluated via qPCR and RNA sequencing. A Luminex assay was used to analyse the effects of dedifferentiation and IFNγ treatment on cytokine secretion. Effects on PD-L1 protein expression were analysed via flow cytometry and western blotting. Inhibition of the JAK kinases, NF-κB and STAT3 with small molecule inhibitors, and siRNA mediated knockdown of STAT1 and IRF1 was applied to investigate the molecular mechanism behind IFNγ induced PD-L1 expression in dedifferentiated melanoma cells. The effects of inhibitor treatments and siRNA mediated knockdowns were evaluated via qPCR and western blotting. Bioinformatic analyses of publicly available RNA sequencing data, consisting of 45 patient derived melanoma cell lines, with or without IFNγ treatment, was conducted to assess the generalisability of the in vitro results. Results Dedifferentiation renders 624Mel melanoma cells hypersensitive to IFNγ stimulation in a context-dependent manner, resulting in non-additive upregulation of IFNγ-induced genes, increased PD-L1 protein expression and amplified secretion of CCL2, CXCL10 and IL-10. Furthermore, the intensified PD-L1 protein expression occurs through the JAK-STAT1-IRF1 axis. Lastly, dedifferentiated patient derived melanoma cell lines showed enhanced inflammatory signalling in response to IFNγ compared to differentiated cells, and tended to have higher PD-L1 expression, associated with increased IRF1 expression and activity. Conclusions Together, these findings indicate the existence of a molecular context linking dedifferentiation and IFNγ signalling in melanoma which may lead to immune evasion. Furthermore, these results imply that modulating melanoma differentiation may help shape IFNγ responsiveness.

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Enhanced IFNy response in dedifferentiated melanoma cells is due to chromatin remodeling as revealed by ATAC-seq

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