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Human 5-lipoxygenase (5-LO) is the key enzyme of leukotriene biosynthesis, mostly expressed in leukocytes and thus a crucial component of the innate immune system.
In this study, we show that 5-LO, besides its canonical function as an arachidonic acid metabolizing enzyme, is a regulator of gene expression associated with euchromatin. By Crispr-Cas9-mediated 5-LO knockout (KO) in MonoMac6 (MM6) cells and subsequent RNA-Seq analysis, we identified 5-LO regulated genes which could be clustered to immune/defense response, cell adhesion, transcription and growth/developmental processes. Analysis of differentially expressed genes identified cyclooxygenase-2 (COX2, PTGS2) and kynureninase (KYNU) as strongly regulated 5-LO target genes. 5-LO knockout affected MM6 cell adhesion and tryptophan metabolism via inhibition of the degradation of the immunoregulator kynurenine. By subsequent FAIRE-Seq and 5-LO ChIP-Seq analyses, we found an association of 5-LO with euchromatin, with prominent 5-LO binding to promoter regions in actively transcribed genes. By enrichment analysis of the ChIP-Seq results, we identified potential 5-LO interaction partners. Furthermore, 5-LO ChIP-Seq peaks resemble patterns of H3K27ac histone marks, suggesting that 5-LO recruitment mainly takes place at acetylated histones.>
In summary, we demonstrate a noncanonical function of 5-LO as transcriptional regulator in monocytic cells.
The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.