Lysine Methylation-Dependent Proteolysis by the Malignant Brain Tumor (MBT) Domain Proteins.

Lysine methylation is a major post-translational protein modification that occurs in both histones and non-histone proteins. Emerging studies show that the methylated lysine residues in non-histone proteins provide a proteolytic signal for ubiquitin-dependent proteolysis. The SET7 (SETD7) methyltransferase specifically transfers a methyl group from S-Adenosyl methionine to a specific lysine residue located in a methylation degron motif of a protein substrate to mark the methylated protein for ubiquitin-dependent proteolysis. LSD1 (Kdm1a) serves as a demethylase to dynamically remove the methyl group from the modified protein. The methylated lysine residue is specifically recognized by L3MBTL3, a methyl-lysine reader that contains the malignant brain tumor domain, to target the methylated proteins for proteolysis by the CRL4DCAF5 ubiquitin ligase complex. The methylated lysine residues are also recognized by PHF20L1 to protect the methylated proteins from proteolysis. The lysine methylation-mediated proteolysis regulates embryonic development, maintains pluripotency and self-renewal of embryonic stem cells and other stem cells such as neural stem cells and hematopoietic stem cells, and controls other biological processes. Dysregulation of the lysine methylation-dependent proteolysis is associated with various diseases, including cancers. Characterization of lysine methylation should reveal novel insights into how development and related diseases are regulated.

Proteolysis of methylated SOX2 protein is regulated by L3MBTL3 and CRL4DCAF5 ubiquitin ligase.

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 and 117 by SET7 methyltransferase to promote SOX2 proteolysis, whereas LSD1 and PHF20L1 act on both methylated Lys-42 and Lys-117 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 malignant-brain-tumor (MBT) methylation-binding domain, is required for SOX2 proteolysis. Our studies showed that L3MBTL3 preferentially binds to the methylated Lys-42 in SOX2, although mutation of Lys-117 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 CRL4DCAF5 ubiquitin E3 ligase to target SOX2 protein for ubiquitin-dependent proteolysis. Whereas 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 by the enhanced degradation of the methylated SOX2 protein at both Lys-42 and Lys-117. Our studies provide novel insights into the mechanism by which the methylation-dependent degradation of SOX2 protein is controlled by the L3MBTL3-CRL4DCAF5 ubiquitin ligase complex.

Deletions in 14q24.1q24.3 are associated with congenital heart defects, brachydactyly, and mild intellectual disability.

Interstitial deletions of chromosome band 14q24.1q24.3 are apparently very rare. We report on three unrelated patients with overlapping de novo deletions of sizes 5.4, 2.8, and 2.3 Mb in this region. While some clinical problems such as intestinal malrotation, cryptorchidism, and ectopic kidney were only observed in single patients, all three patients had mild intellectual disability, congenital heart defects (truncus arteriosus, pulmonary atresia, atrial septal defect, and/or ventricular septal defect), brachydactyly, hypertelorism, broad nasal bridge, and thin upper lips. Likely haploinsufficiency of one or several of the 19 genes in the common deleted interval (ACTN1, DCAF5, EXD2, GALNTL1, ERH, SLC39A9, PLEKHD1, CCDC177, KIAA0247, LOC100289511, SRSF5, SLC10A1, SMOC1, SLC8A3, ADAM21P1, COX16, SYNJ2BP, SYNJ2BP-COX16, ADAM21) was responsible for these manifestations, but apart from SMOC1, mutations in which cause autosomal recessive Waardenburg anophthalmia syndrome, and ACTN1, mutations in which are associated with congenital macrothrombocytopenia, no disease associations have so far been reported for the other genes. Functional studies and a systematic search for mutations or chromosome aberrations in this region will elucidate the role of individual genes in the clinical manifestations and will provide insight into the underlying biological mechanisms.

A methylation-phosphorylation switch controls EZH2 stability and hematopoiesis.

The Polycomb Repressive Complex 2 (PRC2) methylates H3K27 to regulate development and cell fate by transcriptional silencing. Alteration of PRC2 is associated with various cancers. Here, we show that mouse Kdm1a deletion causes a dramatic reduction of PRC2 proteins, whereas mouse null mutation of L3mbtl3 or Dcaf5 results in PRC2 accumulation and increased H3K27 trimethylation. The catalytic subunit of PRC2, EZH2, is methylated at lysine 20 (K20), promoting EZH2 proteolysis by L3MBTL3 and the CLR4DCAF5 ubiquitin ligase. KDM1A (LSD1) demethylates the methylated K20 to stabilize EZH2. K20 methylation is inhibited by AKT-mediated phosphorylation of serine 21 in EZH2. Mouse Ezh2K20R/K20R mutants develop hepatosplenomegaly associated with high GFI1B expression, and Ezh2K20R/K20R mutant bone marrows expand hematopoietic stem cells and downstream hematopoietic populations. Our studies reveal that EZH2 is regulated by methylation-dependent proteolysis, which is negatively controlled by AKT-mediated S21 phosphorylation to establish a methylation-phosphorylation switch to regulate the PRC2 activity and hematopoiesis.