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Temporal Regulation of Cytokine Mediated Gene Expression Changes Upon Genetic Intervention of Histone Deacetylase 6 in Melanoma Open Access

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Previous gene expression studies in melanoma have revealed that several signaling pathways are modulated by specific histone deacetylases (HDAC). Among them, HDAC6 has been shown to tightly control antigen presentation and other immunomodulatory functions. We aim to investigate the effect of HDAC6 on the expression pattern of genes in melanoma cells through a time point dependent manner with the generation of gene clusters, which can then be mapped to specific signaling pathways. WM164 Non-target (NT) and WM164 HDAC6 Knock down (HDAC6 KD) melanoma cells were used to generate RNA sequencing data. RNA was collected at different time points (0,1, 2, 4, 6, 8, 12, and 24hrs) after being stimulated with interferon gamma (IFNg) or interleukin-6 (IL-6). RNA samples were sequenced, followed by a HISAT2-StringTie pipeline processing to give the Fragments per Kilobase of transcript per Million (FPKM) values at different time points. The FPKM values were then inputted to a cluster generating software Graphia Professional, which determines the similarities between individual expression profiles by building a correlation matrix for both gene-to-gene and sample-to-sample comparisons. This matrix was then filtered to remove all correlations below a certain threshold (for the gene-to-gene comparison in the RNA-seq atlas, Pearson's r < 0.8). Markov clustering (MCL) algorithm was used to construct a network graph with an inflation value of 2.2, generating 537 clusters in total. The clusters obtained were then used for pathway analysis studies using the online software MetaCore - an integrated software suite for functional analysis of Next Generation Sequencing, gene expression, microRNA, and screening data. Upon stimulation of WM164 NT with IL-6, 12 clusters showed patterns of up-regulation, 8 clusters showed patterns of down-regulation and the remaining 109 clusters, showed cyclic patterns of gene expression. Initial analysis of NT showed up and down-regulation of multiple pathways such as HIF-1, TGF-B, and IL-12, IL-4, IL-16 and IL-18 upon stimulation with IL-6 at very specific time windows. In the case of IFNg stimulation, there was a progressive up-regulation of pathways such as Hippo, Notch, IL-23 and WNT while OX40L, IL-8, HIF-1 and VEGF pathways were down-regulated at specific intervals of time. Since most immune-related genes are transiently modulated by external stimuli, and in most cases in very defined time windows, we propose that studying the expression of genes at multiple time points after cytokine stimulation would be more significant to build accurate ontology maps of cellular pathways modulated by specific HDACs.

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