TET proteins iteratively convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in a Fe(II)/α-ketoglutarate-dependent manner. Our previous biochemical studies revealed that TET proteins are more active on 5mC than on 5hmC and 5fC. However, the source of the substrate preference of TET proteins still remains largely elusive. Here, we investigated the substrate binding and catalytic mechanisms of oxidation reactions mediated by TET2 on different substrates through computational approaches. In accordance with previous experimental reports, our computational results suggest that TET2 can bind to different substrates with comparable binding affinities and the hydrogen abstraction step in the catalytic cycle acts as the rate-limiting step. Further structural characterization of the intermediate structures revealed that the 5-substitution groups on 5hmC and 5fC adopt an unfavorable orientation for hydrogen abstraction, which leads to a higher energy barrier for 5hmC and 5fC (compared to 5mC) and thus a lower catalytic efficiency. In summary, our mechanical insights demonstrate that substrate preference is the intrinsic property of TET proteins and our theoretical calculation results can guide further dry-lab or wet-lab studies on the catalytic mechanism of TET proteins as well as other Fe(II)/α-ketoglutarate (KG)-dependent dioxygenases.

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from growth factors, cellular energy levels, stress and amino acids to control cell growth and proliferation through regulating translation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 Å resolution. The mTORC1 comprises a dimer of heterotrimer (mTOR-Raptor-mLST8) mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics. Within the complex, the conserved N-terminal caspase-like domain of Raptor faces toward the catalytic cavity of the kinase domain of mTOR. Raptor shows no caspase activity and therefore may bind to TOS motif for substrate recognition. Structural analysis indicates that FKBP12-Rapamycin may generate steric hindrance for substrate entry to the catalytic cavity of mTORC1. The structure provides a basis to understand the assembly of mTORC1 and a framework to characterize the regulatory mechanism of mTORC1 pathway.

The Hippo-YAP pathway is an evolutionally conserved signaling module that controls tissue growth during development and its dysregulation causes cancer1. Core components of the Hippo pathway include a kinase cascade comprising MST1/2 and LATS1/2 kinases, in which MST1/2 phosphorylates and activates LATS1/22. The major downstream effectors of the Hippo pathway are the transcriptional co-activators YAP and TAZ, which are phosphorylated and inhibited by LATS1/23,4. Unphosphorylated YAP/TAZ localizes in the nucleus and promotes target gene expression through binding to the TEAD family transcription factors5,6. Protein kinase C (PKC) controls a broad range of biological processes and can be classified into three sub-groups based on sequence homology and activation mechanisms: the Ca2+- and diacylglycerol (DAG)-dependent conventional PKC (cPKC α, βI, βII, and γ), the DAG-dependent novel PKC (nPKC δ, θ, ε, and η), and the Ca2+- and DAG-independent atypical PKC (aPKC ι and ζ)7. Recent studies have established that extracellular diffusible signals act through G-protein coupled receptors (GPCRs) to regulate the Hippo-YAP pathway8,9. PKC represents one of the major effectors downstream of GPCRs (especially Gq/11-coupled receptors). This led us to investigate whether PKC regulates the Hippo-YAP pathway.We used the DAG analog TPA to activate PKC in HEK293A cells and observed that TPA induced a rapid and robust YAP dephosphorylation as determined by western blotting using the phospho-specific (serine 127) YAP antibody and by differential electrophoretic mobility shift on phos-tag-containing gels. Similarly, TPA also induced TAZ dephosphorylation as indicated by a faster electrophoretic migration (Figure 1A). TPA-induced YAP dephosphorylation was also observed in HeLa and U251MG cells (Supplementary information, Figure S1A and S1B). Phosphorylated YAP localizes in the cytoplasm, whereas dephosphorylated YAP translocates to the nucleus to promote target gene expression. Consistent with this model, TPA induced YAP nuclear localization in HEK293A cells (Figure 1B). Moreover, GO6983, an inhibitor of both cPKC and nPKC, effectively blocked TPA-induced YAP/TAZ dephosphorylation (Figure 1A). These observations suggest a role of PKC in YAP/TAZ activation.Next, we examined whether activation of PKC by physiological stimuli would activate YAP/TAZ. We found that addition of acetylcholine to U251MG cells, which express the Gq/11-coupled muscarinic acetylcholine receptor M3, resulted in significant YAP dephosphorylation (Figure 1C). Inhibition of either M3 receptor by 4-DAMP or PKC by GO6983 suppressed acetylcholine-induced YAP dephosphorylation (Figure 1C), suggesting that YAP activation by acetylcholine is mediated by the M3 receptor and PKC. Moreover, Gq/11 knockdown in U251MG cells suppressed YAP dephosphorylation by acetylcholine (Supplementary information, Figure S1C). Collectively, these data indicate that PKC acts downstream of Gq/11-coupled receptors to activate YAP.In the efforts to examine the effect of TPA on YAP/TAZ phosphorylation in different cell types, we were surprised that TPA induced a dramatic YAP/TAZ phosphorylation in Swiss3T3 cells (Figure 1D), a response completely opposite to what was observed in HEK293A and several other cell lines (Figure 1A and Supplementary information, Figure S1A and S1B). Notably, TPA-induced YAP/TAZ phosphorylation could be blocked by GO6983 (Figure 1D), indicating that PKC activation was also responsible for the increase of YAP/TAZ phosphorylation in Swiss3T3 cells. Similar effects were observed in MEF cells and the lung cancer A549 cells (Supplementary information, Figure S1D and S1E). Consistent with the increased YAP phosphorylation, TPA treatment promoted a YAP cytoplasmic localization in MEF cells (Figure 1E). Although seemingly paradoxical, the above results are very interesting and show that PKC can either positively or negatively regulate YAP activity in a cell-type-dependent manner.The opposing effects of TPA on YAP phosphorylation observed in different cell types prompted us to speculate that different PKC isoforms may exert opposite effects on YAP regulation. To test this, individual cPKC or nPKC isoform was co-transfected with YAP into HEK293A cells and TPA-induced YAP phosphorylation/dephosphorylation was measured. Overexpression of cPKC (α, β1, β2, or γ) had minor effects on YAP phosphorylation in HEK293A cells (Supplementary information, Figure S1F). We then fused the Src membrane-targeting sequence (myristoylation signal) to the N terminus of each individual PKC to render them constitutively active (also referred to as myri-PKC). Overexpression of myristoylated cPKC (α, β1, β2, or γ) was sufficient to induce YAP dephosphorylation even in the absence of TPA. Stimulation with TPA led to further dephosphorylation of YAP (Figure 1F). These results indicate that cPKCs promote YAP dephosphorylation and activation. In contrast to the cPKC, overexpression of nPKC (δ, θ, or ε) blocked TPA-induced YAP dephosphorylation in HEK293A cells (Supplementary information, Figure S1G). Although PKCη expression decreased YAP phosphorylation in the absence of TPA, it also suppressed TPA-induced YAP dephosphorylation. The above results indicate that nPKCs promote YAP phosphorylation. Consistent with our previous studies, serum induced YAP dephosphorylation (comparing the first lanes of Figure 1F and 1G). We examined the effect of nPKC overexpression on YAP phosphorylation in the presence of serum. Ectopic expression of PKCε strongly increased YAP phosphorylation in the presence of serum and expression of PKCδ, PKCθ, and PKCη had resulted in a mild-to-moderate increase of YAP phosphorylation. As expected, TPA had little effect on YAP phosphorylation in the control or PKCα-transfected cells because YAP was already largely dephosphorylated in the presence of serum (Figure 1G). Remarkably, TPA further increased YAP phosphorylation levels in nPKC-transfected HEK293A cells (Figure 1G). These data demonstrate that nPKC activation by TPA induces YAP phosphorylation. Consistently, expression of constitutively active nPKC, increased YAP phosphorylation in the presence of serum (Figure 1H). Collectively, our results show that cPKC and nPKC have opposite effects on YAP phosphorylation, leading to YAP activation and inhibition, respectively. Moreover, ectopic expression of nPKC in HEK293A cells can reverse the cellular response from YAP activation to YAP inhibition in response to TPA.The above observation suggests that the relative expression levels of cPKC and nPKC may account for the cell type-specific TPA response. mRNA levels of PKC isoforms were quantified in representative cell lines (Supplementary information, Figure S1H). However, no simple correlation could be observed between the levels of cPKC vs nPKC mRNA and the TPA-induced YAP dephosphorylation vs phosphorylation. We speculate that YAP regulation by PKC may also be influenced by additional factors that are differentially expressed in a cell type-dependent manner.Rho GTPases have been established as key mediators in transducing GPCR signals to YAP activation8,9. We used botulinum toxin C3 to inactivate RhoA and found that C3 blocked TPA-induced YAP dephosphorylation in HEK293A cells (Figure 1I). Consistently, expression of Rho GDI, which inhibits Rho GTPases, also blocked TPA-induced YAP dephosphorylation (Supplementary information, Figure S1I). Furthermore, overexpression of an active RhoA Q63L mutant suppressed the effect of PKC inhibition (Supplementary information, Figure S1J). Collectively, these data suggest a pathway that cPKC modulates YAP activity through Rho GTPases.We next examined the role of MST and LATS in PKC-induced YAP activation in HEK293A cells. MST1/2 double knockout slightly decreased YAP phosphorylation. However, TPA-induced YAP dephosphorylation was not affected, indicating that MST1/2 are not required for YAP regulation by PKC (Supplementary information, Figure S1K). Moreover, TPA-induced YAP dephosphorylation was blunted by ectopic LATS2 expression (Supplementary information, Figure S1L), supporting a role of LATS in mediating the PKC signal to YAP regulation. The involvement of MST-LATS kinase cascade in PKC-mediated YAP regulation was also examined in MEF cells. TPA-induced YAP phosphorylation was completely abolished in LATS1/2-knockout, but not MST1/2-knockout, MEF cells (Supplementary information, Figure S1M).To test whether PKC modulates LATS activity, LATS1 was immunoprecipitated from HEK293A cells stimulated with TPA for various periods and then subjected to the in vitro kinase assay using GST-YAP as the substrate. As shown in Figure 1J, TPA rapidly decreased LATS1 kinase activity in HEK293A cells. The time course of LATS inhibition by TPA paralleled that of endogenous YAP dephosphorylation, consistent with a role of LATS in TPA response. LATS1 and 2 are activated by phosphorylation of the activation loop (S909 for LATS1) and the hydrophobic motif (T1079 for LATS1). We found that TPA decreased the phosphorylation level of LATS1 on T1079 (Supplementary information, Figure S1N). Interestingly, TPA treatment significantly enhanced LATS1 kinase activity in Swiss3T3 cells, as evidenced by both increased kinase activity and elevated phosphorylation levels of LATS1 on T1079 and S909 (Figure 1K and 1L). To further test the role of cPKC and nPKC, constitutively active myri-PKCα and myri-PKCδ were transfected into HEK293A cells and LATS1 kinase activity was measured. Overexpression of myri-PKCα significantly suppressed LATS1 kinase activity along with the reduced YAP phosphorylation level (Figure 1M). On the contrary, overexpression of myri-PKCδ markedly increased LATS1 kinase activity. Full activation of myri-PKCδ by TPA treatment further enhanced LATS1 kinase activity and YAP phosphorylation (Figure 1N). Taken together, we conclude that cPKC inhibits LATS kinase activity whereas nPKC activates LATS.We examined the expression of YAP target gene CTGF. TPA increased CTGF expression in HEK293A cells in a YAP/TAZ-dependent manner (Figure 1O). On the contrary, TPA decreased CTGF expression level in Swiss3T3 cells and inhibition of PKC with GO6983 blocked the effect of TPA on CTGF protein levels (Figure 1P). These results are consistent with the effects of TPA on YAP/TAZ phosphorylation in HEK293A and Swiss3T3 cells, and support a role of YAP/TAZ in PKC-induced gene regulation, especially cell type-dependent induction or repression.The Hippo-YAP pathway plays a major role in development and organ size control, and its dysregulation is widely observed in human cancers1,10,11. Given the importance of this pathway, it must be tightly controlled. In this study, we have demonstrated that PKC mediates GPCR signaling, likely downstream of Gq/11 activation, to modulate YAP activity. Notably, Gq/11-activating mutation is found in ~70% of uveal melanoma, the most common adult eye cancer. Consistent with our findings, YAP has been shown to be critical in mutant Gq/11-induced tumorigenesis and PKC inhibitors suppresses uveal melanoma in a mouse model12,13,14. An interesting and surprising finding of this study is that different PKC isoforms have different effects on LATS and YAP phosphorylation. The cPKC activates YAP by inducing its dephosphorylation while the nPKC inhibits YAP by stimulating its phosphorylation. These opposing effects are achieved by cPKC-induced LATS inhibition and nPKC-induced LATS activation. A fascinating and challenging issue in biological research is cell type-specific response. For example, one hormone may induce opposite effects in different tissues or cell types. We know very little about the molecular basis for cell type-specific responses. This study provides one possible underlying mechanism by which different cell types can produce different or even opposite responses in response to the same stimulus. Remarkably, a simple ectopic expression of nPKC can actually convert the TPA-induced YAP activation to inhibition in HEK293A cells, which normally show YAP activation upon TPA treatment. Our study provides an intriguing example of molecular engineering the specificity of cellular response to a given signal and reveals a biochemical basis for cell type-specific responses frequently observed in cell biology.We thank Drs Frank B Furnari, Nikhil Rao, and Alexandra Newton (UCSD, USA) for reagents and insightful discussion. This work was supported by NIH grants (GM51586 and EY022611 to KLG, and GM7752 to AWH and SWP) and the National Natural Science Foundation of China (31030019, YX). FXY was supported in part by China “Thousand Youth Talents” and Shanghai “Oriental Scholar” grants.(Supplementary information is linked to the online version of the paper on the Cell Research website.)

Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that post-translational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a “seesaw” model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2Y105E (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2K305Q (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by post-translational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.

N6-methyladenosine (m6A) has been demonstrated to be ubiquitous in several types of eukaryotic RNAs, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (lncRNA), and small nuclear RNA (snRNA)1. The recent discoveries of RNA m6A methyltransferase complex METTL3/METTL14/WTAP and demethylases FTO and ALKBH5 prove the reversibility of m6A modification2,3,4,5,6. This modification plays important roles in various biological processes, including circadian rhythms7, RNA splicing8, yeast meiosis9, and embryonic stem cell self-renewal10. Two recent studies show that YTH domain family 2 (YTHDF2) and other YTHDF proteins preferentially bind to m6A-containing mRNA in vivo and in vitro and regulate localization and stability of the bound mRNA8,11. YTHDF2 is also known to be involved in development of acute myeloid leukemia12. YTHDC1 (splicing factor YT521-B), another YTH domain-containing protein, is known to play an important role in Emery-Dreifuss muscular dystrophy. While the function of YTHDF2 in the regulation of mRNA stability has been explored, the molecular mechanism for specific recognition of m6A by the YTH domain remains elusive.YTHDF2 consists of a C-terminal YTH domain (designated as YTHYTHDF2), which specifically binds to m6A-containing RNA (m6A-RNA) with a preference for those containing a consensus motif of G(m6A)C11. The YTH domains are highly conserved in YTH domain-containing proteins, including YTHDF1-3, YTHDC1, YTHDC2 (CsA-associated helicase-like protein), Mmi1 (Schizosaccharomyces pombe), and MRB1 (Saccharomyces cerevisiae), suggesting an important function of the YTH domain across species (Figure 1A).To verify the specific recognition of m6A-RNA by YTHYTHDF2, we performed fluorescence polarization (FP) assays and electrophoretic mobility shift assays (EMSA) using purified YTHYTHDF2, unmodified RNA (A-RNA) and m6A-RNA. Interestingly, it appears that YTHYTHDF2 binds to A-RNA in a one-step binding mode (with the goodness-of-fit R2 value of 0.9943), but to m6A-RNA in a two-step binding mode (with R2 of 0.9987, while the R2 value is 0.9843 in one-step binding mode.) (Figure 1B, Supplementary information, Figure S1A and Table S1A). Both FP and EMSA experiments show that YTHYTHDF2 bound to m6A-RNA with much higher binding affinity than to A-RNA (Figure 1B and 1C). Note that 30 pmol of YTHYTHDF2 led to an almost complete shift of m6A-RNA (lane 8), while 100 pmol of YTHYTHDF2 only shifted less than half of the A-RNA (lane 5) (Figure 1C). A similar two-step binding mode was observed in other RNA-binding proteins13. It is likely that binding to RNA facilitates the further recognition of m6A.To reveal the molecular mechanism for specific recognition of m6A-RNA by YTHYTHDF2, we solved the crystal structure of YTHYTHDF2 at 2.1 Å resolution (Figure 1D). The YTH domain forms a dimer in the crystal due to crystal packing (Supplementary information, Figure S1B and Table S1B). The overall structure shows a globular fold with a central core of four-stranded β-sheets surrounded by four α helices and flanking regions on two sides. A Dali search indicates that YTHYTHDF2 is structurally similar to YTHDC1 (PDB: 2YUD) with a root-mean-squared deviation (rmsd) of 1.54 Å for 121 Cα atoms, suggesting a conserved mechanism for m6A-RNA recognition (Supplementary information, Figure S1C).Electrostatic potential surface of YTHYTHDF2 shows a patch that is enriched in basic residues, which may be involved in RNA recognition (Figure 1E). The basic patch is formed by residues R411 and K416 on strand β1, R441 on helix α2, and R527 on the loop connecting helices α3 and α4 (Figure 1A and 1D). Close to this basic patch, a hydrophobic pocket is formed by aromatic residues Y418, W432, W486 and W491 and is supported by the loop connecting strand β1 and helix α1, the loop connecting strands β3 and β4, and the loop connecting helices α1 and α2 (Figure 1A and 1D).Based on above structural analyses, we further characterized several residues that are potentially important for m6A-RNA recognition. Wild-type and mutant YTHYTHDF2 were purified and used for the FP assays (Figure 1F-1G and Supplementary information, Table S1A). Consistent with the structural observations, K416A and R527A mutations of YTHYTHDF2 significantly decreased the binding affinity to both A-RNA (~5 and ~10 folds, respectively) and m6A-RNA (~25 folds), suggesting that these two residues are involved in binding to the backbone of RNA, but may not recognize the methyl-group of m6A. Similar results were obtained for the K416A mutant of YTHYTHDF2 in EMSA (Supplementary information, Figure S1D). In contrast, R411A and R441A mutations of YTHYTHDF2 slightly decreased RNA-binding affinity toward A-RNA (~2 folds) and m6A-RNA (~3 folds). It is worthy to note that we calculated binding affinities of wild-type and two mutants (R411A and R441A) of YTHYTHDF2 to m6A-RNA based on both the two-step and one-step binding modes (Supplementary information, Figure S1A and Table S1). The binding affinities of other mutants to m6A-RNA were calculated based on the one-step binding mode (Supplementary information, Table S1A).In the FP assays, mutating two hydrophobic residues W432 and W486 to alanine markedly decreased the binding affinity of YTHYTHDF2 to m6A-RNA, but barely changed the binding affinity to A-RNA, suggesting that W432 and W486 are important for specific recognition of m6A. To test the involvement of these two residues in m6A recognition in vivo, we measured the ratio of m6A/A levels in the RNA products immunoprecipitated by wild-type and mutant YTHDF2 full-length proteins from HEK293T cells. Indeed, W432A and W486A mutations decreased the m6A-RNA selectivity of YTHDF2. Furthermore, the W432A mutant of YTHYTHDF2 was unable to effectively pull down previously identified YTHDF2 targets, SON and CREBBP, in HeLa cells. Taken together, these data suggest that residues W432 and W486 are essential for specific recognition of m6A by YTHDF2 (Figure 1H and Supplementary information, Figure S1E and S1F).Circular dichroism (CD) measurements show that wild-type and mutant YTHYTHDF2 have similar secondary structure composition, suggesting that the overall structure of YTHYTHDF2 is not disrupted by mutations of these residues (Supplementary information, Figure S1G). Notably, residues W432 and W486 are highly conserved among the YTHDF family members from yeast to human (Figure 1A), further supporting their important role in mediating specific recognition of m6A-RNA. When our manuscript was under revision, two crystal structures of m6A-RNA in complex with YTH domains from YTHDC114 and Zygosaccharomyces rouxii MRB1 (ZrMRB1)15 were reported. Structural comparison shows that residues W432 and W486 of YTHYTHDF2 adopt a similar conformation to that of m6A-binding residues in the two complex structures (Supplementary information, Figure S1H), suggesting a conserved mechanism for m6A-RNA recognition by YTH domains.Taken together, our studies indicate that the basic residues K416 and R527 on the surface of YTHYTHDF2 are involved in binding to the RNA backbone, and residues W432 and W486 within the hydrophobic pocket contribute to the specific recognition of m6A. Our study also provides a platform for further investigations of additional YTH-m6A-RNA complex structures, which would reveal molecular mechanisms for specific recognition of m6A-RNA by YTHDF2 and other YTH family proteins.The coordinate and structure factor for the YTHYTHDF2 structure have been deposited into the Protein Data Bank under the accession code of 4WQN.We thank staff members of beamline BL17U at SSRF (Shanghai Synchrotron Radiation Facility, China) for their assistance in data collection, and Mr Lei Zhang and staff members of Biomedical Core Facility in Fudan University for their help on biochemical analyses. We thank Dr Jinbiao Ma for the help on synthesis of m6A-RNA. This work was supported by grants from the National Basic Research Program of China (2011CB965300), the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (2014ZX09507-002), the National Natural Science Foundation of China (U1432242, 91419301, 31270779 and 31030019), the Basic Research Project of Shanghai Science and Technology Commission (12JC1402700), and the “Shu Guang” project (11SG06) supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation.(Supplementary information is linked to the online version of the paper on the Cell Research website.)

KDM5B is a histone H3K4me2/3 demethylase. The PHD1 domain of KDM5B is critical for demethylation, but the mechanism underlying the action of this domain is unclear. In this paper, we observed that PHD1KDM5B interacts with unmethylated H3K4me0. Our NMR structure of PHD1KDM5B in complex with H3K4me0 revealed that the binding mode is slightly different from that of other reported PHD fingers. The disruption of this interaction by double mutations on the residues in the interface (L325A/D328A) decreases the H3K4me2/3 demethylation activity of KDM5B in cells by approximately 50% and increases the transcriptional repression of tumor suppressor genes by approximately twofold. These findings imply that PHD1KDM5B may help maintain KDM5B at target genes to mediate the demethylation activities of KDM5B.

Histone methylation is a reversible histone post-translational modification that plays an important role in various chromatin-based processes, including chromatin structure remodeling, transcription, and DNA repair 1,2. LSD1 (also known as KDM1A) is the first identified histone lysine demethylase. It converts mono- or di-methylated histone H3 (H3K4me1/me2) to unmodified H3 3. LSD1 is highly conserved in eukaryotes and plays important roles in various biological processes, such as development and tumorgenesis. LSD2 (also known as KDM1B or AOF1) is the only other mammalian paralogue of the LSD1 family. Similar to LSD1, LSD2 is also a histone H3K4me1/me2 demethylase 4,5,6,7. LSD1 has been shown to be enriched at promoter regions; in contrast, LSD2 mainly associates with the gene body regions of actively transcribed genes 5. LSD2 is highly expressed in oocytes, and is required for de novo DNA methylation of some imprinted genes, a function dependent on its H3K4 demethylase activity 4. Thus, LSD2 is an important player in epigenetic regulation and has functions distinct from those of LSD1.Previous studies have shown that LSD2 can demethylate histone H3K4me2 peptide corresponding to residues 1-21, but not the one containing only residues 1-16. The observation suggests that residues 17-21 of H3 might be important for substrate recognition and demethylase activity of LSD2 6. In our recent studies, we identified that NPAC/GLYR1 interacts with LSD2, stabilizes the interaction between LSD2 and H3 peptide, and thus enhances LSD2 activity 8. Interestingly, in the LSD2-NPAC-H3K4M(1-20) structure (H3 residues 1-20, replacing K4 with a methionine to mimic the H3K4me2 substrate of LSD2), we found that residues Q19 and L20 of H3 interact with a loop region in LSD2, further supporting the hypothesis that residues 17-20 of H3 are involved in the substrate recognition of LSD2. These studies also suggest that LSD2 may contain a putative non-canonical substrate-binding site to interact with residues 17-20 of H3. In this study, we further investigate how LSD2 recognizes its histone substrate, and whether LSD2 contains an additional substrate-binding site that is functionally relevant.To investigate whether LSD2 contains an additional substrate recognition site, we first performed an in vitro histone demethylation assay using H3K4me2 peptides as substrate. As shown in Supplementary information, Figure S1, wild-type LSD2 demethylated about 100% H3K4me2 (1-21) into 100% H3K4me1, and demethylated about 100% H3K4me2 (1-26) into 50% H3K4me1 and 50% H3K4me0, suggesting that H3K4me2 (1-26) is a better substrate comparing to H3K4me2 (1-21). The result also indicates that residues 22-26 of H3 are involved in LSD2-mediated demethylation. To study the mechanism of substrate recognition by LSD2, we determined the crystal structure of LSD2-H3K4M(1-26) and LSD2-NPAC-H3K4M(1-26) complexes at 2.0 and 3.1 Å resolution, respectively (Supplementary information, Table S1). H3K4M(1-26) peptide was used as an analogue of H3K4me2 for crystallization. Residues 236-263 of LSD2 were not built in the model due to a lack of electron density, which may result from their flexibility in the crystals. LSD2 adopts similar conformations in both structures with a root-mean-square deviation (RMSD) of 0.553 Å for 666-aligned Cα atoms (Figure 1A and Supplementary information, Figures S2-S4). Structure of LSD2 alone has been described previously 8, and thus will not be discussed here.Structural comparison of LSD2-H3K4M(1-26) and LSD1-CoREST-H3K4M (2V1D.PDB) 9 shows that LSD1 and LSD2 share similar folds for the amine oxidase (AO) and SWIRM domains, which is consistent with their conserved primary sequences and our previous findings (Supplementary information, Figures S5 and S6) 8. The most striking finding from the comparison of these two structures is the different fashion for substrate interaction. In the LSD1-CoREST-H3K4M structure, only residues 1-16 of H3K4M were observed, although the peptide used for crystallization contains residues 1-21, suggesting that residues 17-21 are flexible in the crystal 9. However, in the LSD2-H3K4M(1-26) structure, residues 1-26 of the H3K4M peptide were clearly observed. The N-termini (residues 1-16) of the H3K4M peptides in both complex structures adopt similar folds. In the LSD2-H3K4M(1-26) structure, the H3K4M peptide extends away from the catalytic cavity (the “first binding site”) and interacts with LSD2 on the second binding site (Figure 1B and Supplementary information, Figure S4). This “second binding site” is composed of two loops (hereafter referred to as loop 1 and loop 2 for simplicity) within the linker region of LSD2. Structural comparison indicates that LSD1 lacks the second binding site, supporting that LSD1 may not bind to histone H3 in a similar fashion as in the LSD2-H3K4M(1-26) structure.As shown in Figure 1C, the C-terminus (residues 19-26) of the H3K4M peptide packs against the shallow groove on the second binding site of LSD2. Residues 19-22 of H3K4M mainly interact with residues from loop 2, and residues 23-26 interact with residues from loop 1 in the linker region (Figure 1D). In particular, the side chains of residues Q19, T22, and R26 of H3 form hydrogen bonds with side chains of residues N276, D287, and the carbonyl oxygen of residues S221, Y223 of LSD2. The side chain of residue L20 of H3K4M inserts into a hydrophobic pocket formed by residues Y273, R285, and main chains of residues 276-279 of LSD2. In addition, the side chain of residue A24 of H3K4M inserts into a hydrophobic pocket formed by residues Y223, C227, and D287 of LSD2. Comparing to loop 1, more residues from loop 2 are involved in the interaction with H3K4M. In the LSD2-NPAC-H3K4M(1-26) ternary structure, the H3K4M peptide interacts with the LSD2-NPAC complex in a similar fashion as described above (Supplementary information, Figure S7A and S7B). The existence of NPAC facilitates the formation of more hydrogen bonds and hydrophobic interactions (Supplementary information, Figure S7C). NPAC seems to work together with the second binding site to facilitate LSD2-H3K4me2 interaction, and thus enhance the enzymatic activity of LSD2.As residues 1-21 of H3K4me2 are sufficient for demethylation by LSD2, we speculate that loop 2 plays a more important role in histone tail recognition for the demethylase activity of LSD2. Thus, we focused on loop 2 for subsequent biochemical studies. To test whether the interaction between the second binding site of LSD2 and the C-terminus (residues 19-26) of the H3K4M peptide is important for substrate recognition of LSD2, we first performed isothermal titration calorimetry (ITC) analyses using various H3K4M peptides and purified LSD2 protein. The results show that H3K4M(1-26) binds to LSD2 with a binding affinity (Kd) of 0.74 μM. Using H3K4M peptide (residues 1-16) significantly decreased the binding affinity (9.52 μM) to LSD2 by approximately 13-fold. Mutations of three residues (L20A/T22A/R26A) in H3K4M (residues 1-26) critical for the second binding site interaction also decreased the binding affinity (3.48 μM) to LSD2 by approximately 5-fold (Figure 1E). These results indicate that the C-terminus (residues 19-26) of the H3K4M peptide, and in particular, residues L20, T22, and R26 are important for H3K4M to interact with LSD2, and support that the second binding site is important for substrate recognition of LSD2.We further generated LSD2 mutants to test whether the second binding site of LSD2 is important for its histone demethylase activity. The mutants include M1 (residues 273-278 replaced by GSGSGS), M2 (R285A/D287A), and M3 (the combination of M1 and M2), in which residues 273-278, and R285, D287 are all from loop 2 (Figure 1D and Supplementary information, Figures S8 and S9). Two additional LSD2 mutants, M4 (E563A) and M5 (K661A) were used as control. E563 in the AO domain forms hydrogen bond with residue R2 of H3K4M, and K661 is a conserved residue essential for the catalytic activity of LSD2 5,7,8.We first performed an in vitro histone demethylase assay with various peptide substrates, including H3K4me2 (1-21, wild type), H3K4me2 (1-21, Q19A/L20A), H3K4me2 (1-26, wild type), and H3K4me2 (1-26, Q19A/L20A/T22A). The purified wild-type and mutant LSD2 proteins were used in the assay. The results show that mutations of Q19A/L20A in H3K4me2 (1-21) had a moderate effect on LSD2 activity, whereas mutations of Q19A/L20A/T22A in H3K4me2 (1-26) significantly decreased the demethylation efficiency of LSD2 (Figure 1F and Supplementary information, Figure S10). As a control, LSD2 mutant M4 (E563A) did not exhibit enzymatic activity towards H3K4me2 (1-21) peptide (Supplementary information, Figure S10). In addition, the LSD2 mutant M1 exhibited very weak enzymatic activity for both H3K4me2 (1-21) and H3K4me2 (1-26) (Figure 1F and Supplementary information, Figure S10A), indicating that loop 2 is important for the demethylase activity of LSD2. Similar results were obtained when LSD2 was used at a lower protein concentration (0.15 μM) (Supplementary information, Figure S10B).Next, we performed in vitro histone demethylase assays using calf thymus histones or nucleosomes purified from HeLa cells as substrates, and the reactions were monitored by western blotting analyses. Wild-type LSD2 demethylated H3K4me2 from histones or nucleosomes in a dose-dependent manner (Figure 1G). In contrast, equal amounts of the LSD2 mutant M1 showed significantly decreased enzymatic activity towards H3K4me2 from both histones and nucleosomes. As a negative control, histone H3K9me2 level did not change upon the addition of LSD2 protein, which is consistent with previous studies 4,5,7. Together with the mass-spectrometry-based assay, these demethylase assays indicate that for all three forms of substrates including peptides, histones, and nucleosomes, loop 2 within the second binding site is important for LSD2 demethylase activity.To test whether the loop 2 within the second binding site is important for LSD2 demethylase activity in vivo, we overexpressed GFP-LSD2 in 293T cells and performed immunofluorescence staining to detect the global H3K4me2 levels. A significant decrease of H3K4me2 level was observed in cells expressing wild-type GFP-LSD2. As a control, H3K4me2 level did not change in cells expressing the LSD2 mutant M5. As expected, LSD2 mutants M1, M2 and M3 lost their histone demethylase activity in vivo (Figure 1H), which is consistent with the results from in vitro demethylase assays. Thus, loop 2 is required for histone demethylase activity of LSD2 in vivo.In summary, the crystal structure of LSD2-H3K4M (1-26) reveals that LSD2 interacts with H3K4M through two binding sites. Further biochemical analyses indicate that the second binding site is important for substrate recognition and essential for demethylase activity of LSD2. The finding provides an example that an additional substrate-binding site away from the catalytic site plays an important role in substrate recognition and enzymatic activity for a histone demethylase. Based on these findings, we propose a “two binding sites” model (Figure 1I), where the catalytic AO domain, as the first binding site, recognizes the N-terminus of H3K4me2, and the second binding site facilitates the substrate interaction and is essential for demethylation activity of LSD2. Our study not only elucidates the structural basis of substrate recognition by LSD2 but also provides valuable information for designing specific inhibitors targeting LSD2 for potential therapeutic applications.We thank staff members of beamline BL17U at SSRF (Shanghai Synchrotron Radiation Facility, China) for assistance in data collection, and staff members of Biomedical Core Facility, Fudan University for their help on mass spectrometry analyses. This work was supported by grants from the National Basic Research Program of China (2011CB965300 and 2009CB918600), the National Natural Science Foundation of China (31270779, 31030019, 11079016 and 30870493), and Fok Ying Tung Education Foundation (20090071220012). This work was also supported by a grant from the National Institutes of Health (5R01GM078458) to YGS.(Supplementary information is linked to the online version of the paper on the Cell Research website.)

The target of rapamycin (TOR) is a central regulator controlling cell growth. TOR is highly conserved from yeast to mammals, and is deregulated in human cancers and diabetes. TOR complex 1 (TORC1) integrates signals from growth factors, cellular energy status, stress, and amino acids to control cell growth, mitochondrial metabolism, and lipid biosynthesis. The mechanisms of growth factors and cellular energy status in regulating TORC1 have been well established, whereas the mechanism by which amino acid induces TORC1 remains largely unknown. Recent studies revealed that Rag GTPases play a central role in the regulation of TORC1 activation in response to amino acids. In this review, we will discuss the recent progress in our understanding of Rag GTPase-regulated TORC1 activation in response to amino acids. Particular focus will be given to the function of Rag GTPases in TORC1 activation and how Rag GTPases are regulated by amino acids.

UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and stability regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.

H3K9me2 and H3K27me2 are important epigenetic marks associated with transcription repression, while H3K4me3 is associated with transcription activation. It has been shown that active and repressive histone methylations distribute in a mutually exclusive manner, but the underlying mechanism was poorly understood. Here we identified ceKDM7A, a PHD (plant homeodomain)- and JmjC domain-containing protein, as a histone demethylase specific for H3K9me2 and H3K27me2. We further demonstrated that the PHD domain of ceKDM7A bound H3K4me3 and H3K4me3 co-localized with ceKDM7A at the genome-wide level. Disruption of the PHD domain binding to H3K4me3 reduced the demethylase activity in vivo, and loss of ceKDM7A reduced the expression of its associated target genes. These results indicate that ceKDM7A is recruited to the promoter to demethylate H3K9me2 and H3K27me2 and activate gene expression through the binding of the PHD domain to H3K4me3. Thus, our study identifies a dual-specificity histone demethylase and provides novel insights into the regulation of histone methylation.

Histone lysine methylation can be removed by JmjC domain-containing proteins in a sequence- and methylation-state-specific manner. However, how substrate specificity is determined and how the enzymes are regulated were largely unknown. We recently found that ceKDM7A, a PHD- and JmjC domain-containing protein, is a histone demethylase specific for H3K9me2 and H3K27me2, and the PHD finger binding to H3K4me3 guides the demethylation activity in vivo. To provide structural insight into the molecular mechanisms for the enzymatic activity and the function of the PHD finger, we solved six crystal structures of the enzyme in apo form and in complex with single or two peptides containing various combinations of H3K4me3, H3K9me2, and H3K27me2 modifications. The structures indicate that H3K9me2 and H3K27me2 interact with ceKDM7A in a similar fashion, and that the peptide-binding specificity is determined by a network of specific interactions. The geometrical measurement of the structures also revealed that H3K4me3 associated with the PHD finger and H3K9me2 bound to the JmjC domain are from two separate molecules, suggesting a trans-histone peptide-binding mechanism. Thus, our systemic structural studies reveal not only the substrate recognition by the catalytic domain but also more importantly, the molecular mechanism of dual specificity of ceDKM7A for both H3K9me2 and H3K27me2.

Nosiheptide-resistance methyltransferase (NHR) of Streptomyces actuosus is a class IV methyltransferase of the SpoU family and methylates 23S rRNA at nucleotide adenosine corresponding to A1067 in Escherichia coli. Such methylation is essential for resistance against nosiheptide, a sulfur peptide antibiotic, which is produced by the nosiheptide-producing strain, S. actuosus. Here, we report the crystal structures of NHR and NHR in complex with SAM (S-adenosyl-l-methionine) at 2.0 and 2.1 Å resolution, respectively. NHR forms a functional homodimer, and dimerization is required for methyltransferase activity. The monomeric NHR is comprised of the N-terminal RNA binding domain (NTD) and the C-terminal catalytic domain (CTD). Overall, the structure of NHR suggests that the methyltransferase activity is achieved by “reading” the RNA substrate with NTD and “adding” methyl group using CTD. Comprehensive mutagenesis and methyltransferase activity assays reveal critical regions for SAM binding in CTD and loops (L1 and L3) essential for RNA recognition in NTD. Finally, the catalytic mechanism and structural model that NHR recognizes 23S rRNA is proposed based on the structural and biochemical analyses. Thus, our systematic structural studies reveal the substrate recognition and modification by the nosiheptide-resistance methyltransferase.

•The histone demethylase LSD2 possesses unexpected E3 ubiquitin ligase activity•LSD2 targets OGT for polyubiquitylation in vitro and in vivo•LSD2 inhibits lung cancer cell A549 growth by promoting OGT degradation•LSD2 regulates distinct groups of target genesHistone demethylases play important roles in various biological processes in a manner dependent on their demethylase activities. However, little is known about their demethylase-independent activities. Here, we report that LSD2, a well-known histone H3K4me1/me2 demethylase, possesses an unexpected E3 ubiquitin ligase activity. LSD2 directly ubiquitylates and promotes proteasome-dependent degradation of O-GlcNAc transferase (OGT), and inhibits A549 lung cancer cell growth in a manner dependent on its E3 ligase activity, but not demethylase activity. The depletion of LSD2 stabilizes OGT and promotes colony formation of 293T cells. LSD2 regulates distinct groups of target genes through histone demethylase and E3 ligase activities, respectively. Such regulation suggests a mechanism through which LSD2 suppresses tumorigenesis by promoting the degradation of OGT and other substrates yet to be discovered. Our study reveals an antigrowth function of LSD2 dependent on its E3 ligase activity and establishes a connection between histone demethylase and ubiquitin-dependent pathway.Download high-res image (190KB)Download full-size image

TET proteins iteratively convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in a Fe(II)/α-ketoglutarate-dependent manner. Our previous biochemical studies revealed that TET proteins are more active on 5mC than on 5hmC and 5fC. However, the source of the substrate preference of TET proteins still remains largely elusive. Here, we investigated the substrate binding and catalytic mechanisms of oxidation reactions mediated by TET2 on different substrates through computational approaches. In accordance with previous experimental reports, our computational results suggest that TET2 can bind to different substrates with comparable binding affinities and the hydrogen abstraction step in the catalytic cycle acts as the rate-limiting step. Further structural characterization of the intermediate structures revealed that the 5-substitution groups on 5hmC and 5fC adopt an unfavorable orientation for hydrogen abstraction, which leads to a higher energy barrier for 5hmC and 5fC (compared to 5mC) and thus a lower catalytic efficiency. In summary, our mechanical insights demonstrate that substrate preference is the intrinsic property of TET proteins and our theoretical calculation results can guide further dry-lab or wet-lab studies on the catalytic mechanism of TET proteins as well as other Fe(II)/α-ketoglutarate (KG)-dependent dioxygenases.