TET Monoubiquitylation by CRL4^VprBP Is Vital for Its DNA Binding

The TET family of methylcytosine dioxygenases (TET1, TET2, and TET3) catalyzes the three-step oxidation of 5-methylcytosine residues in DNA, which repress gene expression, and regulates a number of biological processes via generation of the epigenetic mark 5-hydroxymethylcytosine (5hmC). Deletion of TET enzymes results in developmental defects, and TET2 is frequently mutated in hematopoietic malignancies, yet little is known about the regulation of TET activity. Nakagawa, Lv, and colleagues found that Vpr-binding protein (VprBP), which acts as a substrate recognition subunit in a complex with DNA-binding protein 1 (DDB1) and cullin 4 (CUL4) (the CRL4^VprBP ubiquitin ligase), bound all three TET proteins directly through their C-terminal cysteine-rich dioxygenase domain. VprBP deficiency in fibroblasts reduced 5hmC levels, whereas deletion of Vprbp in murine zygotes resulted in decreased paternal DNA hydroxymethylation and defective zygotic development, similar to the phenotype in Tet3-deficient zygotes, suggesting that VprBP is vital for TET function. Intriguingly, CRL4^VprBP induced the monoubiquitylation of TET proteins; VprBP overexpression enhanced the monoubiquitylation of TET2, whereas depletion of VprBP, DDB1, or CUL4 proteins abolished TET monoubiquitylation, confirming the function of CRL4^VprBP as the main ubiquitin ligase for TET proteins. CRL4^VprBP -mediated monoubiquitylation occurred at a conserved lysine residue found in all TET proteins (K1299 in TET2). Mutation of the TET monoubiquitylation site did not disrupt the interaction between TET and VprBP, but inhibited TET binding to DNA both in vitro and in vivo and diminished 5hmC production. Examination of TET2 mutations found in human acute myeloid leukemia revealed several mutations targeting the CRL4^VprBP monoubiquitylation site, including K1299 mutations, as well as mutations that disrupted TET2 binding to VprBP. These results demonstrate a role for VprBP in regulating TET DNA binding and activity via monoubiquitylation, and suggest this pathway as a novel mechanism for oncogenic inactivation of TET2.

Nakagawa T, Lv L, Nakagawa M, Yu Y, Yu C, D'Alessio AC, et al. CRL4^VprBP E3 ligase promotes monoubiquitylation and chromatin binding of TET dioxygenases. Mol Cell 2014 Dec 31 [Epub ahead of print].

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