Many non-histone proteins are lysine methylated and a novel function of this modification is to trigger the proteolysis of methylated proteins. Here, we report that the methylated lysine 142 of DNMT1, a major DNA methyltransferase that preserves epigenetic inheritance of DNA methylation patterns during DNA replication, is demethylated by LSD1. A novel methyl-binding protein, L3MBTL3, binds the K142-methylated DNMT1 and recruits a novel CRL4DCAF5 ubiquitin ligase to degrade DNMT1. Both LSD1 and PHF20L1 act primarily in S phase to prevent DNMT1 degradation by L3MBTL3-CRL4DCAF5. Mouse L3MBTL3/MBT-1 deletion causes accumulation of DNMT1 protein, increased genomic DNA methylation, and late embryonic lethality. DNMT1 contains a consensus methylation motif shared by many non-histone proteins including E2F1, a key transcription factor for S phase. We show that the methylation-dependent E2F1 degradation is also controlled by L3MBTL3-CRL4DCAF5. Our studies elucidate for the first time a novel mechanism by which the stability of many methylated non-histone proteins are regulated.

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119 or K119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (K117, equivalent to K119 in mouse Sox2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Downregulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylationdependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotencycontrolling stem-cell protein Sox2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.The Lysine-Specific Demethylase 1 (LSD1, also called KDM1A) was originally identified as a histone demethylase that removes the methyl group from the mono-and di-methylated lysine 4 in histone H3 (H3K4)(1), which is associated http://www.jbc.org/cgi/doi/10.1074/jbc.RA117.000342 The latest version is at JBC Papers in Press. Published on January 22, 2018 as Manuscript RA117.000342Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc.by guest on May 9, 2018 http://www.jbc.org/ Downloaded from 2 with active chromatin structure for gene activation (2). Mouse deletion of both LSD1 gene alleles causes embryonic lethality, indicating that LSD1 is essential for embryonic development (3). LSD1 is also required for the self-renewal and pluripotency of embryonic stem cells and loss or reduced levels of LSD1 cause transcriptional downregulation of pluripotent stem cell protein Sox2, Oct4, and other essential pluripotent stem cell proteins, promoting cellular differentiation (4-6).Sox2 belongs to a family of SRY-related HMG box (Sox) transcription factors that play key roles in development and differentiation (7,8). Sox2 is a master stem cell protein that is essential for the maintenance of pluripotency and self-renewal of embryonic stem cells and induced pluripotent stem cells (iPSCs) (9). Sox2 is also a key factor for var...

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