The Role Of LSD1 In DNA Damage Repair Pathway

Histone methylation is an important epigenetic regulation.Lysine specific demethylase 1?LSD1?is a nuclear flavin adenine dinucleotide-dependent monoamine oxidase.Studies have found that LSD1 is closely related to gene transcription,which specifically removes the monomethylation and dimethylation of histone 3 lysine 4?H3K4?and monomethylation and dimethylation of histone 3 lysine 9?H3K9?.Futher research has shown that LSD1 plays an important role in a wide range of physiological processes,including cell proliferation,lipogenesis,DNA damage repair,sperm formation and embryo development.In recent years,it has been found that LSD1 plays an important role in DNA damage repair.LSD1 is involved in DNA damage response?DDR?,which promotes?H2AX ubiquitination at the damage site in the late stage of S/G2.LSD1 promotes H3K4me2 demethylation at the site of DNA damage.LSD1interacts directly with RNF168 and its recruitment to DNA damage sites is dependent on RNF168.In addition,casein kinase 2?CK2?phosphorylates LSD1 at S131 and S137in vitro and in vivo,whereas wild-type p53-induced phosphatase 1?WIP1?dephosphorylates LSD1 at S131 and S137 in vitro and in vivo.CK2-mediated phosphorylation of LSD1 enhances its binding to RNF168,the interaction between RNF168 and 53BP1 and polyubiquitination at 53BP1 K63.This process is dependent on RNF168 and facilitates the direct recruitment of LSD1 and 53BP1 to DNA damage sites.In this study,we determined that LSD1 can participate in DNA damage responses by laser-induced DNA damage and immunofluorescence staining.We then found that LSD1 is a protein that interacts with E3 ubiquitin ligases RNF20 and RNF40 by immunoprecipitation,whereas previous reports did not involve the presence of three proteins in a common signaling pathway.To identify key regions which LSD1 interacts with RNF20 and RNF40,we constructed SFB-LSD1,PHAP-RNF20,PHAP-RNF40wild-type and a series of deletion mutant plasmids.The transiently transfected 293T cells and co-immunoprecipitation confirmed that the near TOWER domain of LSD1?aa428-527?is a key region for the interaction of LSD1 with RNF20 and RNF40.The RNF20 near Coiled-coil region?a.a.508-703?and the RNF40 near Coiled-coil region?a.a.558-760?are key regions for the interaction of RNF20,RNF40 with LSD1.To further investigate the role of the LSD1/RNF20/RNF40 complex in the DNA damage repair pathway,we successfully constructed the HCT116 RNF20^-/-cell line and the HCT116 RNF40^-/-cell line by CRISPR/Cas9 technology.This makes a base for further exploration of changes in LSD1 function in the absence of RNF20 or RNF40.We knocked down LSD1 and RNF20 by siRNA respectively.Studies have shown that knocking down any of the two proteins does not affect the expression level of the other,but whether it will affect H3K4 monomethylation,dimethylation,trimethylation and H2BK120ub1 requires more reseach to prove.In conclusion,we initially validated the mechanism of interaction between LSD1,RNF20 and RNF40 and laid the foundation for further study on how the three synergistically participate in DNA damage repair and their respective functions.