RNA-seq analysis to examine the effect of the iron chelator deferoxamine on gene expression profile in 3T3-L1 pre-adipocyte cells

Adipocyte differentiation has been shown to require iron, but the underlying mechanism remains elusive. Ferrous iron ion is known to function as a co-factor for alpha-ketoglutarate-dependent dioxygenases, including demethylases for histones, DNA, and RNA. Previously we reported several alpha-ketoglutarate-dependent histone demethylases as critical epigenetic regulators during adipogenesis. These lines of evidence led us to hypothesize that iron orchestrates epigenetic/epitranscriptional regulations during adipogenesis by controlling demethylation activities. In this study, we conducted genome-wide analysis on methylation landscapes of histones, DNA, and RNA in differentiation of 3T3-L1 pre-adipocytes. Using the iron chelator deferoxamine, we demonstrate here how dynamically methylation levels of histones, DNA, and RNA are regulated by iron during adipogenesis. Overall design: 3T3-L1 cells were induced for adipogenesis using the differentiation cocktail (insulin, DEX, and IBMX). In the first 2 days of differentiation, cells were cultured in the absence or presence of the iron chelator deferoxamine. Total RNA was extracted on Days 0, 1, 2, 4, and 8, and for each time point RNA samples were prepared as duplicate (n=2). Column-purified RNA samples were subjected to library preparation using KAPA Stranded mRNA-seq Kit. RNA-seq run was done on an Illumina NextSeq 500 platform.