Gene amplification of Sox2 at 3q26.33 is a common event in squamous cell carcinomas (SCCs) of the lung and esophagus, as well as several other cancers. Here, we show that the expression of LSD1/KDM1 histone demethylase is significantly elevated in Sox2-expressing lung SCCs. LSD1-specific inhibitors selectively impair the growth of Sox2-expressing lung SCCs, but not that of Sox2-negative cells. Sox2 expression is associated with sensitivity to LSD1 inhibition in lung, breast, ovarian, and other carcinoma cells. Inactivation of LSD1 reduces Sox2 expression, promotes G1 cell-cycle arrest, and induces genes for differentiation by selectively modulating the methylation states of histone H3 at lysines 4 (H3K4) and 9 (H3K9). Reduction of Sox2 further suppresses Sox2-dependent lineage-survival oncogenic potential, elevates trimethylation of histone H3 at lysine 27 (H3K27) and enhances growth arrest and cellular differentiation. Our studies suggest that LSD1 serves as a selective epigenetic target for therapy in Sox2-expressing cancers.

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 CRL4^DCAF5 ubiquitin ligase to degrade DNMT1. Both LSD1 and PHF20L1 act primarily in S phase to prevent DNMT1 degradation by L3MBTL3-CRL4^DCAF5. 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-CRL4^DCAF5. Our studies elucidate for the first time a novel mechanism by which the stability of many methylated non-histone proteins are regulated.

LSD1 is essential for the maintenance of pluripotency of embryonic stem (ES) or embryonic carcinoma/teratocarcinoma (EC) cells. We have previously developed novel LSD1 inhibitors that selectively inhibit ES/EC cells. However, the critical targets of LSD1 remain unclear. Here, we found that LSD1 interacts with histone deacetylase 1 (HDAC1) to regulate the proliferation of ES/EC cells through acetylation of histone H4 at lysine 16 (H4K16), which we show is a critical substrate of HDAC1. The LSD1 demethylase and HDAC1 deacetylase activities were both inactivated if one of them in the complex was chemically inhibited in ES/EC cells or in reconstituted protein complexes. Loss of HDAC1 phenocopied the selective growth-inhibitory effects and increased the levels of H3K4 methylation and H4K16 acetylation of LSD1 inactivation on ES/EC cells. Reduction of acetylated H4K16 by ablation of the acetyltransferase males absent on the first (MOF) is sufficient to rescue the growth inhibition induced by LSD1 inactivation. While LSD1 or HDAC1 inactivation caused the downregulation of Sox2 and Oct4 and induction of differentiation genes, such as FOXA2 or BMP2, depletion of MOF restored the levels of Sox2, Oct4, and FoxA2 in LSD1-deficient cells. Our studies reveal a novel mechanism by which LSD1 acts through the HDAC1- and MOF-mediated regulation of H4K16 acetylation to maintain the pluripotency of ES/EC cells.

SOX2 is a dose-dependent master stem cell protein that controls the self-renewal and pluripotency or multipotency of embryonic stem (ES) cells and many adult stem cells. We have previously found that SOX2 protein is monomethylated at lysine residues 42 (K42) and 117 (K117) by SET7 methyltransferase to promote SOX2 proteolysis, whereas LSD1 and PHF20L1 act on both methylated K42 and K117 to prevent SOX2 proteolysis. However, the mechanism by which the methylated SOX2 protein is degraded remains unclear. Here, we report that L3MBTL3, a protein with the MBT methylation binding domain, is required for SOX2 proteolysis. Our studies showed that L3MBTL3 preferentially binds to the methylated K42 in SOX2, although mutation of K117 also partially reduces the interaction between SOX2 and L3MBTL3. The direct binding of L3MBTL3 to the methylated SOX2 protein leads to the recruitment of the CRL4-DCAF5 ubiquitin E3 ligase to target SOX2 protein for ubiquitin-dependent proteolysis. While loss of either LSD1 or PHF20L1 destabilizes SOX2 protein and impairs the self-renewal and pluripotency of mouse ES cells, knockdown of L3MBTL3 or DCAF5 is sufficient to restore the protein levels of SOX2 and rescue the defects of mouse ES cells caused by LSD1 or PHF20L1 deficiency. We also found that retinoic acid-induced differentiation of mouse ES cells is accompanied with the enhanced degradation of the methylated SOX2 protein at both K42 and K117. Our studies provide novel insights into the mechanism by which the methylation-dependent degradation of SOX2 protein is controlled by the L3MBTL3-CRL4-DCAF5 ubiquitin ligase complex.

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) 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 (equivalent to Lys-119 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. Down-regulation 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 methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4^Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4^Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4^Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4^Cdt2. The proximity of PCNA-bound Cdt1 to CRL4^Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4^Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.