The MBT Repeats of L3MBTL1 Link SET8-mediated p53 Methylation at Lysine 382 to Target Gene Repression*

The p53 tumor suppressor protein is regulated by multiple post-translational modifications, including lysine methylation. We previously found that monomethylation of p53 at lysine 382 (p53K382me1) by the protein lysine methyltransferase (PKMT) SET8/PR-Set7 represses p53 transactivation of target genes. However, the molecular mechanism linking p53K382 monomethylation to repression is not known. Here we show in biochemical and crystallographic studies the preferential recognition of p53K382me1 by the triple malignant brain tumor (MBT) repeats of the chromatin compaction factor L3MBTL1. We demonstrate that SET8-mediated methylation of p53 at Lys-382 promotes the interaction between L3MBTL1 and p53 in cells, and the chromatin occupancy of L3MBTL1 at p53 target promoters. In the absence of DNA damage, L3MBTL1 interacts with p53K382me1 and p53-target genes are repressed, whereas depletion of L3MBTL1 results in a p53-dependent increase in p21 and PUMA transcript levels. Activation of p53 by DNA damage is coupled to a decrease in p53K382me1 levels, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from p53-target promoters. Together, we identify L3MBTL1 as the second known methyl-p53 effector protein, and provide a molecular explanation for the mechanism by which p53K382me1 is transduced to regulate p53 activity

Tubulin Tyrosine Ligase Like 12, a TTLL Family Member with SET- and TTL-Like Domains and Roles in Histone and Tubulin Modifications and Mitosis

Contains fulltext : 110068.pdf (publisher's version ) (Open Access)hTTLL12 is a member of the tubulin tyrosine ligase (TTL) family that is highly conserved in phylogeny. It has both SET-like and TTL-like domains, suggesting that it could have histone methylation and tubulin tyrosine ligase activities. Altered expression of hTTLL12 in human cells leads to specific changes in H4K20 trimethylation, and tubulin detyrosination, hTTLL12 does not catalyse histone methylation or tubulin tyrosination in vitro, as might be expected from the lack of critical amino acids in its SET-like and TTLL-like domains. hTTLL12 misexpression increases mitotic duration and chromosome numbers. These results suggest that hTTLL12 has non-catalytic functions related to tubulin and histone modification, which could be linked to its effects on mitosis and chromosome number stability

CBP and p300 are cytoplasmic E4 polyubiquitin ligases for p53

p300 and CREB-binding protein (CBP) act as multifunctional regulators of p53 via acetylase and polyubiquitin ligase (E4) activities. Prior work in vitro has shown that the N-terminal 595 aa of p300 encode both generic ubiquitin ligase (E3) and p53-directed E4 functions. Analysis of p300 or CBP-deficient cells revealed that both coactivators were required for endogenous p53 polyubiquitination and the normally rapid turnover of p53 in unstressed cells. Unexpectedly, p300/CBP ubiquitin ligase activities were absent in nuclear extracts and exclusively cytoplasmic. Consistent with the cytoplasmic localization of its E3/E4 activity, CBP deficiency specifically stabilized cytoplasmic, but not nuclear p53. The N-terminal 616 aa of CBP, which includes the conserved Zn2+-binding C/H1-TAZ1 domain, was the minimal domain sufficient to destabilize p53 in vivo, and it included within an intrinsic E3 autoubiquitination activity and, in a two-step E4 assay, exhibited robust E4 activity for p53. Cytoplasmic compartmentalization of p300/CBP's ubiquitination function reconciles seemingly opposed functions and explains how a futile cycle is avoided—cytoplasmic p300/CBP E4 activities ubiquitinate and destabilize p53, while physically separate nuclear p300/CBP activities, such as p53 acetylation, activate p53

SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation*

SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ϵ-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ϵ-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ϵ-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes