The Mechanism Of LSD1-mediated Epigenetic Modification And Its Target Genes In MCF-7 Breast Cancer Cells

Backgroud: Histone lysine methylation,which plays an important role in the regulation of gene expression,chromosome conformation and cell differentiation,is a dynamic process that is collaboratively regulated by lysine methyltransferases(KMTs)and lysine demethylases(KDMs).LSD1,the first identified KDMs,catalyzes the demethylation of mono-and di-methylated H3K4 and H3K9 to either repress or activate the expression of genes.Eeste homolog 2(EZH2,an essential component of PRC2)is a H3K27 methyltransferase,which can catalyze the methylation of H3K27 to repress the expression of genes.Objective: In addition to H3K4 and H3K9,we systematically investigated the effects of LSD1 knockdown on other methylation sites in histone.Through analyzing the target genes of LSD1,we planned to explore whether LSD1 knockdown could influence the levels of histone methylations in the promoter region of the target genes and therefore regulate the expression of genes.We further clarified whether LSD1 interacted with other transcription factors and histone methyl-transferases to collaboratively regulate the expression of its target genes.Method: LSD1 was knocked down by siRNA in MCF-7 cells,and we performed LC-MS/MS to explore the sites and level of methylation on histone lysine that were affected by LSD1 knockdown.We used Western blot,RT-PCR and ChromatinImmunoprecipitation(ChIP)to analyze the regulation of the target genes by LSD1,and the levels of histone methylations in the promoter region of the target genes.We identified the interaction domains between LSD1 and EZH2 by immunopresipitation(IP),Immunofluorescence(IF),GST-pull down assay.We performed SILAC based quantitative proteomic analysis in MCF-7 cells with EZH2 or LSD1 knockdown,andanalyzed the proteins that were commonly affected by EZH2 and LSD1 knockdown with the method of STRING and Gene Ontology(GO)clustering,and identified the affected signaling pathway.Chromatin-Immunoprecipitation(ChIP)assay was performed to compare the binding levels of LSD1 to the promoter region of the key genes in the IFN signaling pathway with and without EZH2 knockdown.Results: In addition to H3K4 and H3K9,LSD1 knockdown also could affect the methylation level on other histone lysines,such as H3K27,H3K36 and H3K79 in MCF-7 cells.The methyl-transferases EZH2 could interact with the demethylases LSD1,and together targeted genes in IFN signaling pathway to suppress the expression of interferon stimulated genes(IRF9).Conclusion: In breast cancer MCF-7 cells,LSD1 had a broad effect on histone lysine methylation,and it could regulate histone lysine methylation in collaboration with other KMTs as a double lock system,thus regulated the expression of genes,such as interferon stimulated genes(IRF9).