Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase

Abstract

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.

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

“…Our analysis also revealed the presence of peptides from DDB1 and DCAF5, components of the CRL4 DCAF5 ubiquitin E3 ligase complexes ( Fig. 1B and Table 1) (29). Our independent immunoprecipitation and Western blotting analyses in mouse ES cells confirmed that the components of the CRL4 DCAF5 , such as CUL4A or CUL4B, DDB1, and DCAF5, are associated with endogenous SOX2 protein ( Fig.…”

“…Mouse null mutation of L3MBTL3 causes late embryonic lethality (36). Our recent studies revealed that L3MBTL3 is critical in regulating the protein stability of DNMT1 when it is methylated by SET7 (29). L3MBTL3 is thus a good candidate for the methylated SOX2 proteolysis, although there are substantial differences between the Lys-42 methylation motif and the surrounding region in SOX2 and that of the methylated Lys-142 in DNMT1 ( Fig.…”

“…1B) (28). The SOX2-associated proteins were identified by an Obitrap XL MS system coupled with an Easy nanoliter LC (29).…”

“…L3MBTL3 is thus a good candidate for the methylated SOX2 proteolysis, although there are substantial differences between the Lys-42 methylation motif and the surrounding region in SOX2 and that of the methylated Lys-142 in DNMT1 ( Fig. 1A) (25,29). To verify the interaction, we tested whether endogenous L3MBTL3 protein interacts with SOX2 by using independent immunocoprecipitation and Western blotting analyses.…”

“…L3MBTL3 contains an MBT domain that contains three MBT tandem repeats (3MBT) at its N-terminal half that can bind to the mono-and dimethylated lysine residues (29,31,41). To further determine whether the 3MBT region of L3MBTL3 can directly and preferentially recognize the methylated Lys-42 or Lys-117 in SOX2, we tested the affinity of the recombinant GST-L3MBTL3-3MBT fusion protein, which contains the glutathione S-transferase (GST) fused to the three tandem-repeated MBT region of L3MBTL3, toward pairs of synthetic SOX2 peptides that contain either the monomethylated Lys-42 or monomethylated Lys-117 and their cognate un-methylated control peptides in vitro (Fig.…”