Yanqi Chang

Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation

Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.

Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294

Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells. Here we present the crystal structure of the catalytic SET domain of GLP in complex with BIX-01294 and S-adenosyl-L-homocysteine. The inhibitor is bound in the substrate peptide groove at the location where the histone H3 residues N-terminal to the target lysine lie in the previously solved structure of the complex with histone peptide. The inhibitor resembles the bound conformation of histone H3 Lys4 to Arg8, and is positioned in place by residues specific for G9a and GLP through specific interactions.

Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling

Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation–linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.Protein lysine methylation signaling is implicated in diverse biological and disease processes. Yet the catalytic activity and substrate specificity are unknown for many human protein lysine methyltransferases (PKMTs). We screened over forty candidate PKMTs and identified SETD6 as a methyltransferase that monomethylates chromatin-associated NF-κB RelA at lysine 310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of GLP, which under basal conditions, promoted a repressed chromatin state at RelA target genes through GLP-mediated H3K9 methylation. NF-κB activation-linked phosphorylation of RelA by PKCζ at serine 311 blocked GLP binding to RelAK310me1 and relieved target gene repression. Our findings establish a new mechanism by which chromatin signaling regulates inflammation programs.

A methylation and phosphorylation switch between an adjacent lysine and serine determines human DNMT1 stability

The protein lysine methyltransferase SET7 regulates DNA methyltransferase-1 (DNMT1) activity in mammalian cells by promoting degradation of DNMT1 and thus allows epigenetic changes via DNA demethylation. Here we reveal an interplay between monomethylation of DNMT1 Lys142 by SET7 and phosphorylation of DNMT1 Ser143 by AKT1 kinase. These two modifications are mutually exclusive, and structural analysis suggests that Ser143 phosphorylation interferes with Lys142 monomethylation. AKT1 kinase colocalizes and directly interacts with DNMT1 and phosphorylates Ser143. Phosphorylated DNMT1 peaks during DNA synthesis, before DNMT1 methylation. Depletion of AKT1 or overexpression of dominant-negative AKT1 increases methylated DNMT1, resulting in a decrease in DNMT1 abundance. In mammalian cells, phosphorylated DNMT1 is more stable than methylated DNMT1. These results reveal cross-talk on DNMT1, through modifications mediated by AKT1 and SET7, that affects cellular DNMT1 levels.

Adding a lysine mimic in the design of potent inhibitors of histone lysine methyltransferases.

Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the histone code. One common feature of these activities is the recognition of lysines in methylated and unmethylated states, whether they are substrates, reaction products, or binding partners. We applied the concept of adding a lysine mimic to an established inhibitor (BIX-01294) of histone H3 lysine 9 methyltransferases G9a and G9a-like protein by including a 5-aminopentyloxy moiety, which is inserted into the target lysine-binding channel and becomes methylated by G9a-like protein, albeit slowly. The compound enhances its potency in vitro and reduces cell toxicity in vivo. We suggest that adding a lysine or methyl-lysine mimic should be considered in the design of small-molecule inhibitors for other methyl-lysine writers, erasers, and readers.

MPP8 mediates the interactions between DNA methyltransferase Dnmt3a and H3K9 methyltransferase GLP/G9a

DNA CpG methylation and histone H3 lysine 9 (H3K9) methylation are two major repressive epigenetic modifications, and these methylations are positively correlated with one another in chromatin. Here we show that G9a or G9a-like protein (GLP) dimethylate the amino-terminal lysine 44 (K44) of mouse Dnmt3a (equivalent to K47 of human DNMT3A) in vitro and in cells overexpressing G9a or GLP. The chromodomain of MPP8 recognizes the dimethylated Dnmt3aK44me2. MPP8 also interacts with self-methylated GLP in a methylation-dependent manner. The MPP8 chromodomain forms a dimer in solution and in crystals, suggesting that a dimeric MPP8 molecule could bridge the methylated Dnmt3a and GLP, resulting in a silencing complex of Dnmt3a-MPP8-GLP/G9a on chromatin templates. Together, these findings provide a molecular explanation, at least in part, for the co-occurrence of DNA methylation and H3K9 methylation in chromatin.

An analog of BIX-01294 selectively inhibits a family of histone H3 lysine 9 Jumonji demethylases.

BIX-01294 and its analogs were originally identified and subsequently designed as potent inhibitors against histone H3 lysine 9 (H3K9) methyltransferases G9a and G9a-like protein. Here, we show that BIX-01294 and its analog E67 can also inhibit H3K9 Jumonji demethylase KIAA1718 with half-maximal inhibitory concentrations in low micromolar range. Crystallographic analysis of KIAA1718 Jumonji domain in complex with E67 indicated that the benzylated six-membered piperidine ring was disordered and exposed to solvent. Removing the moiety (generating compound E67-2) has no effect on the potency against KIAA1718 but, unexpectedly, lost inhibition against G9a-like protein by a factor of 1500. Furthermore, E67 and E67-2 have no effect on the activity against histone H3 lysine 4 (H3K4) demethylase JARID1C. Thus, our study provides a new avenue for designing and improving the potency and selectivity of inhibitors against H3K9 Jumonji demethylases over H3K9 methyltransferases and H3K4 demethylases.

Selective non-nucleoside inhibitors of human DNA methyltransferases active in cancer including in cancer stem cells.

DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in cancer, including in cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of myelodysplastic syndromes. However, only a few non-nucleoside DNMTi have been identified so far, and even fewer have been validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as a potent non-nucleoside DNMTi that is also selective toward other AdoMet-dependent protein methyltransferases. Compound 5 was potent at single-digit micromolar concentrations against a panel of cancer cells and was less toxic in peripheral blood mononuclear cells than two other compounds tested. In mouse medulloblastoma stem cells, 5 inhibited cell growth, whereas related compound 2 showed high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line.

Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling

SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-κB). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-l-methionine, reveals a V-like protein structure and suggests a model for NF-κB binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insight into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation–phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.

Structural insights for MPP8 chromodomain interaction with histone H3 lysine 9: potential effect of phosphorylation on methyl-lysine binding

M-phase phosphoprotein 8 (MPP8) harbors an N-terminal chromodomain and a C-terminal ankyrin repeat domain. MPP8, via its chromodomain, binds histone H3 peptide tri- or di-methylated at lysine 9 (H3K9me3/H3K9me2) in submicromolar affinity. We determined the crystal structure of MPP8 chromodomain in complex with H3K9me3 peptide. MPP8 interacts with at least six histone H3 residues from glutamine 5 to serine 10, enabling its ability to distinguish lysine-9-containing peptide (QTARKS) from that of lysine 27 (KAARKS), both sharing the ARKS sequence. A partial hydrophobic cage with three aromatic residues (Phe59, Trp80 and Tyr83) and one aspartate (Asp87) encloses the methylated lysine 9. MPP8 has been reported to be phosphorylated in vivo, including the cage residue Tyr83 and the succeeding Thr84 and Ser85. Modeling a phosphate group onto the side-chain hydroxyl oxygen of Tyr83 suggests that the negatively charged phosphate group could enhance the binding of positively charged methyl-lysine or create a regulatory signal by allowing or inhibiting binding of other protein(s).