Abstract
Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.
Highlights
SET domain containing lysine methyltransferase 7 (SET7) monomethylates DNA (cytosine-5) methyltransferase 1 (DNMT1), promoting its proteasomal degradation, yet methylated DNMT1 still remains throughout the cell cycle
To validate if the Malignant brain tumor (MBT) domain of PHF20L1 is a true DNMT1K142me1 reader, glutathione S-transferase (GST) pull-down experiments were performed using increasing amounts of purified full-length recombinant DNMT1 incubated with constant amounts of SET7 lysine methyltransferase, S-adenosylmethionine methyl donor, and the GST-MBT domain fusion derived from PHF20L1
Full-length PHF20L1 was bound to DNMT1K142me1 in a dose-dependent manner, and the interaction was virtually abolished without SET7 methylation in the mock-methylated DNMT1 (Fig. 1C, middle)
Summary
SET7 monomethylates DNMT1, promoting its proteasomal degradation, yet methylated DNMT1 still remains throughout the cell cycle. SiRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. There are several interacting proteins of DNMT1, most notably PCNA and UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) [11, 12] Both PCNA and UHRF1 are colocalized with DNMT1 during DNA replication, and deletion of UHRF1 by genetic knockout resulted in a severe loss (Ͼ80%) of 5mC in the embryonic stem cells. UHRF1 has E3 ubiquitin-protein ligase activity that mediates the ubiquitination of target proteins, such as histone H3 and promyelocytic leukemia protein and alters gene expression [13, 14] Another mechanism that influences epigenetic inheritance is the availability and stability of DNA methyltransferases in cells. We studied its role in DNMT1 stability, loading, and epigenetic inheritance in mammalian cells
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