Temporal regulation of cytokine-mediated gene expression changes upon genetic intervention of Histone deacetylase 6
Open AccessPrevious 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 control antigen presentation and other immunomodulatory functions. WM164 Non-target (NT) and WM164 HDAC6 Knock down (KDHDAC6) melanoma cells were used to generate transcriptome sequencing data. RNA was collected at different time points (0,1, 2, 4, 6, 8, 12, and 24hrs) after being stimulated with interferon gamma (IFNγ) or interleukin 6 (IL-6). RNA samples were sequenced, followed by a HISAT2-StringTie pipeline processing to assess transcriptome-wide gene expression 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 in order to generate multiple highly correlated clusters. A total of 537 clusters were generated. The clusters obtained were then used for pathway analyses studies using the online tool MetaCore - an integrated software suite for functional analysis of genomic and proteomic data. Upon stimulation of WM164 NT with IL-6, 12 clusters showed patterns of upregulation, 8 clusters showed patterns of downregulation and the remaining 109 clusters, showed cyclic patterns of gene expression. Initial analysis of NT showed up and downregulation of multiple pathways such as HIF-1, TGF-β, and IL-12, IL-4, IL-16 and IL-18 upon stimulation with IL-6 at very specific time windows. In the case of IFNγ stimulation, there was a progressive upregulation of pathways such as Hippo, Notch, IL-23 and WNT while OX40L, IL-8, HIF-1 and VEGF pathways were downregulated at specific intervals of time. We aimed 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. Since most immune-related genes are transiently modulated by external stimuli, and in most cases in very defined time windows, as a result of this research, we propose that studying the expression of genes at multiple time points after cytokine stimulation is a powerful approach to build accurate ontology maps of cellular pathways modulated by specific HDACs.
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