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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LSD1/KDM1 is a histone demethylase that preferentially removes methyl groups from the mono- and di-methylated lysine 4 in histone H3 (H3K4), key marks for active chromatin for transcriptional activation. LSD1 is essential for pluripotent embryonic stem cells and embryonic teratocarcinoma/carcinoma cells and its expression is often elevated in various cancers. We developed a new LSD1 inhibitor, CBB3001, which potently inhibited LSD1 activity both in vitro and in vivo. CBB3001 also selectively inhibited the growth of human ovarian teratocarcinoma PA-1 and mouse embryonic carcinoma F9 cells, caused the downregulation of pluripotent stem cell proteins SOX2 and OCT4. However, CBB3001 does not have significant inhibition on the growth of human colorectal carcinoma HCT116 cells or mouse fibroblast NIH3T3 cells that do not express these stem cell proteins. Our studies strongly indicate that CBB3001 is a specific LSD1 inhibitor that selectively inhibits teratocarcinoma and embryonic carcinoma cells that express SOX2 and OCT4.

Edited by Xiao-Fan Wang During DNA replication or repair, the DNA polymerase cofactor, proliferating cell nuclear antigen (PCNA), homotrimerizes and encircles the replicating DNA, thereby acting as a DNA clamp that promotes DNA polymerase processivity. The formation of the PCNA trimer is also essential for targeting the replication-licensing protein, chromatin-licensing, and DNA replication factor 1 (CDT1), for ubiquitin-dependent proteolysis to prevent chromosomal DNA re-replication. CDT1 uses its PCNA-interacting peptide box (PIP box) to interact with PCNA, and the CRL4 E3 ubiquitin ligase subunit CDT2 is recruited through the formation of PCNA-CDT1 complexes. However, it remains unclear how CDT1 and many other PIP box-containing proteins are marked for degradation by the CRL4 CDT2 ubiquitin ligase during DNA replication or damage. Here, using recombinant protein expression coupled with site-directed mutagenesis, we report that CDT2 and PCNA directly interact and this interaction depends on the presence of a highly conserved, C-terminal PIP box-like region in CDT2. Deletion or mutation of this region abolished the CDT2-PCNA interaction between CDT2 and PCNA both in vitro and in vivo. Moreover, PCNA-dependent CDT1 degradation in response to DNA damage and replication during the cell cycle requires an intact PIP box in CDT2. The requirement of the PIP boxes in both CDT2 and its substrate CDT1 suggests that the formation of the PCNA trimeric clamp around DNA during DNA replication and repair may bring together CDT1 and CRL4 CDT2 ubiquitin E3 ligase to target CDT1 for proteolysis in a DNA synthesis-dependent manner. Proliferating cell nuclear antigen (PCNA) 2 is central to DNA replication and DNA repair (1, 2). It normally exists in a mono-This work was supported by National Institutes of Health Grants R15 NS096694 (to H. S.), R15GM116087 and R15GM131255 (to H. Z.