RESUMO
Increased protein synthesis plays an etiologic role in diverse cancers. Here, we demonstrate that METTL13 (methyltransferase-like 13) dimethylation of eEF1A (eukaryotic elongation factor 1A) lysine 55 (eEF1AK55me2) is utilized by Ras-driven cancers to increase translational output and promote tumorigenesis in vivo. METTL13-catalyzed eEF1A methylation increases eEF1A's intrinsic GTPase activity in vitro and protein production in cells. METTL13 and eEF1AK55me2 levels are upregulated in cancer and negatively correlate with pancreatic and lung cancer patient survival. METTL13 deletion and eEF1AK55me2 loss dramatically reduce Ras-driven neoplastic growth in mouse models and in patient-derived xenografts (PDXs) from primary pancreatic and lung tumors. Finally, METTL13 depletion renders PDX tumors hypersensitive to drugs that target growth-signaling pathways. Together, our work uncovers a mechanism by which lethal cancers become dependent on the METTL13-eEF1AK55me2 axis to meet their elevated protein synthesis requirement and suggests that METTL13 inhibition may constitute a targetable vulnerability of tumors driven by aberrant Ras signaling.
Assuntos
Metiltransferases/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Adulto , Idoso , Animais , Carcinogênese , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Feminino , Células HEK293 , Xenoenxertos , Humanos , Lisina/metabolismo , Masculino , Metilação , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fator 1 de Elongação de Peptídeos/genética , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteômica , Transdução de SinaisRESUMO
Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Neoplasias de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , CarcinogêneseRESUMO
Trimethylation of histone H3 lysine 9 (H3K9me3) is crucial for the regulation of gene repression and heterochromatin formation, cell-fate determination and organismal development1. H3K9me3 also provides an essential mechanism for silencing transposable elements1-4. However, previous studies have shown that canonical H3K9me3 readers (for example, HP1 (refs. 5-9) and MPP8 (refs. 10-12)) have limited roles in silencing endogenous retroviruses (ERVs), one of the main transposable element classes in the mammalian genome13. Here we report that trinucleotide-repeat-containing 18 (TNRC18), a poorly understood chromatin regulator, recognizes H3K9me3 to mediate the silencing of ERV class I (ERV1) elements such as LTR12 (ref. 14). Biochemical, biophysical and structural studies identified the carboxy-terminal bromo-adjacent homology (BAH) domain of TNRC18 (TNRC18(BAH)) as an H3K9me3-specific reader. Moreover, the amino-terminal segment of TNRC18 is a platform for the direct recruitment of co-repressors such as HDAC-Sin3-NCoR complexes, thus enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis that disrupts the TNRC18(BAH)-mediated H3K9me3 engagement caused neonatal death in mice and, in multiple mammalian cell models, led to derepressed expression of ERVs, which affected the landscape of cis-regulatory elements and, therefore, gene-expression programmes. Collectively, we describe a new H3K9me3-sensing and regulatory pathway that operates to epigenetically silence evolutionarily young ERVs and exert substantial effects on host genome integrity, transcriptomic regulation, immunity and development.
Assuntos
Retrovirus Endógenos , Inativação Gênica , Histonas , Peptídeos e Proteínas de Sinalização Intracelular , Lisina , Retroelementos , Animais , Humanos , Camundongos , Cromatina/genética , Cromatina/metabolismo , Proteínas Correpressoras/metabolismo , Retrovirus Endógenos/genética , Epigênese Genética , Perfilação da Expressão Gênica , Genoma/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Metilação , Domínios Proteicos , Retroelementos/genética , Sequências Repetidas Terminais/genética , Animais Recém-Nascidos , Linhagem CelularRESUMO
Although DNA N 6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA in Tetrahymena thermophila In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2'-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.
Assuntos
Adenina , Metilação de DNA , Tetrahymena thermophila , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Adenina/metabolismo , Adenina/análogos & derivados , Replicação do DNA , DNA de Protozoário/genética , DNA de Protozoário/metabolismoRESUMO
DNA methylation is one of the major epigenetic mechanisms crucial for gene regulation and genome stability. De novo DNA methyltransferase DNMT3C is required for silencing evolutionarily young transposons during mice spermatogenesis. Mutation of DNMT3C led to a sterility phenotype that cannot be rescued by its homologues DNMT3A and DNMT3B. However, the structural basis of DNMT3C-mediated DNA methylation remains unknown. Here, we report the structure and mechanism of DNMT3C-mediated DNA methylation. The DNMT3C methyltransferase domain recognizes CpG-containing DNA in a manner similar to that of DNMT3A and DNMT3B, in line with their high sequence similarity. However, two evolutionary covariation sites, C543 and E590, diversify the substrate interaction among DNMT3C, DNMT3A and DNMT3B, resulting in distinct DNA methylation activity and specificity between DNMT3C, DNMT3A and DNMT3B in vitro. In addition, our combined structural and biochemical analysis reveals that the disease-causing rahu mutation of DNMT3C compromises its oligomerization and DNA-binding activities, providing an explanation for the loss of DNA methylation activity caused by this mutation. This study provides a mechanistic insight into DNMT3C-mediated DNA methylation that complements DNMT3A- and DNMT3B-mediated DNA methylation in mice, unravelling a regulatory mechanism by which evolutionary conservation and diversification fine-tunes the activity of de novo DNA methyltransferases.
RESUMO
The MLL1 gene is a frequent target for recurrent chromosomal translocations, resulting in transformation of hematopoietic precursors into leukemia stem cells. Here, we report on structure-function studies that elucidate molecular events in MLL1 binding of histone H3K4me3/2 marks and recruitment of the cyclophilin CyP33. CyP33 contains a PPIase and a RRM domain and regulates MLL1 function through HDAC recruitment. We find that the PPIase domain of CyP33 regulates the conformation of MLL1 through proline isomerization within the PHD3-Bromo linker, thereby disrupting the PHD3-Bromo interface and facilitating binding of the MLL1-PHD3 domain to the CyP33-RRM domain. H3K4me3/2 and CyP33-RRM target different surfaces of MLL1-PHD3 and can bind simultaneously to form a ternary complex. Furthermore, the MLL1-CyP33 interaction is required for repression of HOXA9 and HOXC8 genes in vivo. Our results highlight the role of PHD3-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me3/2 marks and cyclophilin-mediated repression through HDAC recruitment.
Assuntos
Ciclofilinas/metabolismo , Histona Desacetilases/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Sequência de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Prolina/química , Domínios e Motivos de Interação entre ProteínasRESUMO
Oligomerization of DNMT3B, a mammalian de novo DNA methyltransferase, critically regulates its chromatin targeting and DNA methylation activities. However, how the N-terminal PWWP and ADD domains interplay with the C-terminal methyltransferase (MTase) domain in regulating the dynamic assembly of DNMT3B remains unclear. Here, we report the cryo-EM structure of DNMT3B under various oligomerization states. The ADD domain of DNMT3B interacts with the MTase domain to form an autoinhibitory conformation, resembling the previously observed DNMT3A autoinhibition. Our combined structural and biochemical study further identifies a role for the PWWP domain and its associated ICF mutation in the allosteric regulation of DNMT3B tetramer, and a differential functional impact on DNMT3B by potential ADD-H3K4me0 and PWWP-H3K36me3 bindings. In addition, our comparative structural analysis reveals a coupling between DNMT3B oligomerization and folding of its substrate-binding sites. Together, this study provides mechanistic insights into the allosteric regulation and dynamic assembly of DNMT3B.
Assuntos
DNA Metiltransferase 3B , Humanos , Regulação Alostérica , Cromatina , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Mamíferos/genética , DNA Metiltransferase 3B/química , Microscopia CrioeletrônicaRESUMO
DNA methylation provides an important epigenetic mechanism that critically regulates gene expression, genome imprinting, and retrotransposon silencing. In plants, DNA methylation is prevalent not only in a CG dinucleotide context but also in non-CG contexts, namely CHG and CHH (H = C, T, or A) methylation. It has been established that plant non-CG DNA methylation is highly context dependent, with the +1- and +2-flanking sequences enriched with A/T nucleotides. How DNA sequence, conformation, and dynamics influence non-CG methylation remains elusive. Here, we report structural and biochemical characterizations of the intrinsic substrate preference of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a plant DNA methyltransferase responsible for establishing all cytosine methylation and maintaining CHH methylation. Among nine CHH motifs, the DRM2 methyltransferase (MTase) domain shows marked substrate preference toward CWW (W = A or T) motifs, correlating well with their relative abundance in planta. Furthermore, we report the crystal structure of DRM2 MTase in complex with a DNA duplex containing a flexible TpA base step at the +1/+2-flanking sites of the target nucleotide. Comparative structural analysis of the DRM2-DNA complexes provides a mechanism by which flanking nucleotide composition impacts DRM2-mediated DNA methylation. Furthermore, the flexibility of the TpA step gives rise to two alternative DNA conformations, resulting in different interactions with DRM2 and consequently temperature-dependent shift of the substrate preference of DRM2. Together, this study provides insights into how the interplay between the conformational dynamics of DNA and temperature as an environmental factor contributes to the context-dependent CHH methylation by DRM2.
Assuntos
Arabidopsis , Arabidopsis/metabolismo , DNA/metabolismo , Metilação de DNA , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Metiltransferases/genética , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , Nucleotídeos/metabolismoRESUMO
FAM86A is a class I lysine methyltransferase (KMT) that generates trimethylation on the eukaryotic translation elongation factor 2 (EEF2) at Lys525. Publicly available data from The Cancer Dependency Map project indicate high dependence of hundreds of human cancer cell lines on FAM86A expression. This classifies FAM86A among numerous other KMTs as potential targets for future anticancer therapies. However, selective inhibition of KMTs by small molecules can be challenging due to high conservation within the S-adenosyl methionine (SAM) cofactor binding domain among KMT subfamilies. Therefore, understanding the unique interactions within each KMT-substrate pair can facilitate developing highly specific inhibitors. The FAM86A gene encodes an N-terminal FAM86 domain of unknown function in addition to its C-terminal methyltransferase domain. Here, we used a combination of X-ray crystallography, the AlphaFold algorithms, and experimental biochemistry to identify an essential role of the FAM86 domain in mediating EEF2 methylation by FAM86A. To facilitate our studies, we also generated a selective EEF2K525 methyl antibody. Overall, this is the first report of a biological function for the FAM86 structural domain in any species and an example of a noncatalytic domain participating in protein lysine methylation. The interaction between the FAM86 domain and EEF2 provides a new strategy for developing a specific FAM86A small molecule inhibitor, and our results provide an example in which modeling a protein-protein interaction with AlphaFold expedites experimental biology.
Assuntos
Lisina , Metiltransferases , Modelos Moleculares , Domínios Proteicos , Humanos , Lisina/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , Estrutura Terciária de Proteína , Cristalografia por Raios X , Mutação PuntualRESUMO
DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) at cytosines is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-ångström crystal structure of the DNMT3A-DNMT3L-DNA complex in which two DNMT3A monomers simultaneously attack two cytosine-phosphate-guanine (CpG) dinucleotides, with the target sites separated by 14 base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Haematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its aetiological link to human disease.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA/química , DNA/metabolismo , Sítios de Ligação , Proliferação de Células , Ilhas de CpG/genética , Cristalografia por Raios X , DNA/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The DNA guanine quadruplexes (G4) play important roles in multiple cellular processes, including DNA replication, transcription and maintenance of genome stability. Here, we showed that Yin and Yang 1 (YY1) can bind directly to G4 structures. ChIP-seq results revealed that YY1-binding sites overlap extensively with G4 structure loci in chromatin. We also observed that the dimerization of YY1 and its binding with G4 structures contribute to YY1-mediated long-range DNA looping. Displacement of YY1 from G4 structure sites disrupts substantially the YY1-mediated DNA looping. Moreover, treatment with G4-stabilizing ligands modulates the expression of not only those genes with G4 structures in their promoters, but also those associated with distal G4 structures that are brought to close proximity via YY1-mediated DNA looping. Together, we identified YY1 as a DNA G4-binding protein, and revealed that YY1-mediated long-range DNA looping requires its dimerization and occurs, in part, through its recognition of G4 structure.
Assuntos
DNA/química , DNA/genética , Quadruplex G , Expressão Gênica/genética , Fator de Transcrição YY1/genética , Sítios de Ligação , Sistemas CRISPR-Cas , Cromatina/metabolismo , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Dedos de ZincoRESUMO
Replication of the â¼30 kb-long coronavirus genome is mediated by a complex of non-structural proteins (NSP), in which NSP7 and NSP8 play a critical role in regulating the RNA-dependent RNA polymerase (RdRP) activity of NSP12. The assembly of NSP7, NSP8 and NSP12 proteins is highly dynamic in solution, yet the underlying mechanism remains elusive. We report the crystal structure of the complex between NSP7 and NSP8 of SARS-CoV-2, revealing a 2:2 heterotetrameric form. Formation of the NSP7-NSP8 complex is mediated by two distinct oligomer interfaces, with interface I responsible for heterodimeric NSP7-NSP8 assembly, and interface II mediating the heterotetrameric interaction between the two NSP7-NSP8 dimers. Structure-guided mutagenesis, combined with biochemical and enzymatic assays, further reveals a structural coupling between the two oligomer interfaces, as well as the importance of these interfaces for the RdRP activity of the NSP7-NSP8-NSP12 complex. Finally, we identify an NSP7 mutation that differentially affects the stability of the NSP7-NSP8 and NSP7-NSP8-NSP12 complexes leading to a selective impairment of the RdRP activity. Together, this study provides deep insights into the structure and mechanism for the dynamic assembly of NSP7 and NSP8 in regulating the replication of the SARS-CoV-2 genome, with important implications for antiviral drug development.
Assuntos
COVID-19 , RNA-Polimerase RNA-Dependente de Coronavírus/química , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Cromatografia em Gel , RNA-Polimerase RNA-Dependente de Coronavírus/biossíntese , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Complexos Multiproteicos , Mutagênese , Mutação , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Heterocromatina/metabolismo , Histonas/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Heterocromatina/genética , Histonas/química , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Processamento de Proteína Pós-TraducionalRESUMO
DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Sequência de Bases , Domínio Catalítico , Ilhas de CpG , DNA/química , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Humanos , Ligação Proteica , DNA Metiltransferase 3BRESUMO
In mammals, three major DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, have been identified. Dnmt3a and Dnmt3b are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. Dnmt3-like (Dnmt3l), which is a member of the Dnmt3 family but does not possess DNA methylation activity, was reported to be indispensable for global methylation in germ cells. Once the DNA methylation patterns are established, maintenance-type DNA methyltransferase Dnmt1 faithfully propagates them to the next generation via replication. All Dnmts possess multiple domains. For instance, Dnmt3a and Dnmt3b each contain a Pro-Trp-Trp-Pro (PWWP) domain that recognizes the histone H3K36me2/3 mark, an Atrx-Dnmt3-Dnmt3l (ADD) domain that recognizes unmodified histone H3 tail, and a catalytic domain that methylates CpG sites. Dnmt1 contains an N-terminal independently folded domain (NTD) that interacts with a variety of regulatory factors, a replication foci-targeting sequence (RFTS) domain that recognizes the histone H3K9me3 mark and H3 ubiquitylation, a CXXC domain that recognizes unmodified CpG DNA, two tandem Bromo-Adjacent-homology (BAH1 and BAH2) domains that read the H4K20me3 mark with BAH1, and a catalytic domain that preferentially methylates hemimethylated CpG sites. In this chapter, the structures and functions of these domains are described.
Assuntos
Metilação de DNA , Histonas , Animais , Histonas/metabolismo , DNA Metiltransferase 3A , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilases de Modificação do DNA/genética , DNA/metabolismo , Mamíferos/genéticaRESUMO
Poly- and perfluoroalkyl substances (PFASs) are widely used in industrial products and consumer goods. Due to their extremely recalcitrant nature and potential bioaccumulation and toxicity, exposure to PFASs may result in adverse health outcomes in humans and wildlife. In this study, we developed a chemoproteomic strategy, based on the use of isotope-coded desthiobiotin-perfluorooctanephosphonic acid (PFOPA) probe and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, to profile PFAS-binding proteins. Targeted proteins were labeled with the desthiobiotin-PFOPA probe, digested with trypsin, and the ensuing desthiobiotin-conjugated peptides were enriched with streptavidin beads for LC-MS/MS analysis. We were able to identify 469 putative PFOPA-binding proteins. By conducting competitive binding experiments using low (10 µM) and high (100 µM) concentrations of stable isotope-labeled PFOPA probes, we further identified 128 nonredundant peptides derived from 75 unique proteins that exhibit selective binding toward PFOPA. Additionally, we demonstrated that one of these proteins, fatty acid-binding protein 5 (FABP5), could interact directly with PFASs, including perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), and perfluorobutanesulfonic acid (PFBS). Furthermore, desthiobiotin-labeled lysine residues are located close to the fatty acid-binding pocket of FABP5, and the binding affinity varies with the structures of PFASs. Taken together, we developed a novel chemoproteomic method for interrogating the PFAS-interacting proteome. The identification of these proteins sets the stage for understanding the mechanisms through which exposure to PFASs confers adverse human health effects.
Assuntos
Ácidos Alcanossulfônicos , Fluorocarbonos , Proteoma , Cromatografia Líquida , Poluentes Ambientais , Proteínas de Ligação a Ácido Graxo , Humanos , Espectrometria de Massas em TandemRESUMO
Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation, and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3) binding activity is harbored in the Tudor motifs of PRC2-associated polycomb-like (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of poised developmental genes and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Motivos de Aminoácidos , Animais , Calorimetria , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Núcleo Celular/metabolismo , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Loci Gênicos , Humanos , Metilação , Camundongos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteínas do Grupo Polycomb , Ligação Proteica , Transporte Proteico , Relação Estrutura-Atividade , Termodinâmica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Metilação de DNA/genética , Neoplasias/genética , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/análise , Epigênese Genética/genética , Células HEK293 , Células HeLa , Humanos , Neoplasias/tratamento farmacológicoRESUMO
Glucose is a critical nutrient for cell proliferation. However, the molecular pathways that regulate glucose metabolism are still elusive. We discovered that co-activator-associated arginine methyltransferase 1 (CARM1) suppresses glucose metabolism toward serine biosynthesis. By tracing the 13C-labeled glucose, we found that Carm1 knockout mouse embryonic fibroblasts exhibit significantly increased de novo serine synthesis than WT cells. This is caused, at least in part, by the reduced pyruvate kinase (PK) activity in these cells. The M2 isoform of PK (PKM2) is arginine-methylated by CARM1, and methylation enhances its activity. Mechanistically, CARM1 methylates PKM2 at arginines 445 and 447, which enhances PKM2 tetramer formation. Consequently, Carm1 knockout cells exhibit significant survival advantages over WT cells when extracellular serine is limited, likely due to their enhanced de novo serine synthesis capacity. Altogether, we identified CARM1 as an important regulator of glucose metabolism and serine synthesis.
Assuntos
Proteínas de Transporte/genética , Glucose/genética , Proteínas de Membrana/genética , Proteína-Arginina N-Metiltransferases/genética , Piruvato Quinase/genética , Serina/biossíntese , Hormônios Tireóideos/genética , Animais , Arginina/biossíntese , Proteínas de Transporte/química , Linhagem Celular Tumoral , Proliferação de Células/genética , Cristalografia por Raios X , Fibroblastos/metabolismo , Glucose/metabolismo , Glicólise/genética , Humanos , Proteínas de Membrana/química , Metilação , Camundongos , Camundongos Knockout , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteína-Arginina N-Metiltransferases/química , Piruvato Quinase/química , Serina/genética , Hormônios Tireóideos/química , Proteínas de Ligação a Hormônio da TireoideRESUMO
Lysophosphatidic acid (LPA) is an endogenous cell signaling molecule, and dysregulation of LPA signaling pathways is accompanied by several types of cancer. Herein, we developed a chemical proteomic method for the proteome-wide identification of LPA-binding proteins. The method involves the synthesis of a desthiobiotin-conjugated LPA acyl phosphate probe for the covalent labeling, enrichment, and subsequent LC-MS/MS identification of LPA-binding proteins at the proteome-wide level. By conducting labeling reactions at two different probe concentrations (10 and 100 µM) in conjunction with an SILAC (stable isotope labeling by amino acids in cell culture)-based workflow, we characterized the LPA-binding capabilities of these proteins at the entire proteome scale, which led to the identification of 86 candidate LPA-binding proteins in HEK293T cells. Moreover, we validated that two of these proteins, annexin A5 and phosphoglycerate kinase 1, can bind directly with LPA. Together, we developed a novel LPA probe for the identification and characterizations of LPA-binding proteins from the entire human proteome. The method should be adaptable for the identification of other lipid-binding proteins.