RESUMEN
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2 , Proteínas F-Box , N-Metiltransferasa de Histona-Lisina , Histona Demetilasas con Dominio de Jumonji , Proteínas del Grupo Polycomb , Proteína SMARCB1 , Cromatina/genética , Cromatina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patología , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Células Tumorales Cultivadas/metabolismoRESUMEN
Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.
Asunto(s)
Acetilación , Cromatina , Lisina , Metilación , Procesamiento Proteico-Postraduccional , Sitio de Iniciación de la Transcripción , Animales , Humanos , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Péptidos/química , Péptidos/metabolismo , Histona Desacetilasas/metabolismoRESUMEN
Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.
Asunto(s)
Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Heterocromatina/metabolismo , ADN Ribosómico/metabolismo , Epigénesis Genética/fisiología , Eucromatina/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteómica/métodos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Metabolic signaling to chromatin often underlies how adaptive transcriptional responses are controlled. While intermediary metabolites serve as co-factors for histone-modifying enzymes during metabolic flux, how these modifications contribute to transcriptional responses is poorly understood. Here, we utilize the highly synchronized yeast metabolic cycle (YMC) and find that fatty acid ß-oxidation genes are periodically expressed coincident with the ß-oxidation byproduct histone crotonylation. Specifically, we found that H3K9 crotonylation peaks when H3K9 acetylation declines and energy resources become limited. During this metabolic state, pro-growth gene expression is dampened; however, mutation of the Taf14 YEATS domain, a H3K9 crotonylation reader, results in de-repression of these genes. Conversely, exogenous addition of crotonic acid results in increased histone crotonylation, constitutive repression of pro-growth genes, and disrupted YMC oscillations. Together, our findings expose an unexpected link between metabolic flux and transcription and demonstrate that histone crotonylation and Taf14 participate in the repression of energy-demanding gene expression.
Asunto(s)
Acilcoenzima A/metabolismo , Metabolismo Energético , Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIID/metabolismo , Metabolismo Energético/genética , Ácidos Grasos/metabolismo , Histonas/genética , Homeostasis , Lisina , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factor de Transcripción TFIID/genética , Transcripción GenéticaRESUMEN
Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription1-3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID4-6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression.
Asunto(s)
Regulación de la Expresión Génica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Serotonina/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Diferenciación Celular , Línea Celular , Femenino , Proteínas de Unión al GTP/metabolismo , Glutamina/química , Glutamina/metabolismo , Humanos , Metilación , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Neuronas Serotoninérgicas/citología , Transglutaminasas/metabolismoRESUMEN
Monomethylation of histone H3 at lysine 4 (H3K4me1) and acetylation of histone H3 at lysine 27 (H3K27ac) are correlated with transcriptionally engaged enhancer elements, but the functional impact of these modifications on enhancer activity is not well understood. Here we used CRISPR/Cas9 genome editing to separate catalytic activity-dependent and independent functions of Mll3 (Kmt2c) and Mll4 (Kmt2d, Mll2), the major enhancer H3K4 monomethyltransferases. Loss of H3K4me1 from enhancers in Mll3/4 catalytically deficient cells causes partial reduction of H3K27ac, but has surprisingly minor effects on transcription from either enhancers or promoters. In contrast, loss of Mll3/4 proteins leads to strong depletion of enhancer Pol II occupancy and eRNA synthesis, concomitant with downregulation of target genes. Interestingly, downregulated genes exhibit reduced polymerase levels in gene bodies, but not at promoters, suggestive of pause-release defects. Altogether, our results suggest that enhancer H3K4me1 provides only a minor contribution to the long-range coactivator function of Mll3/4.
Asunto(s)
Células Madre Embrionarias/enzimología , Elementos de Facilitación Genéticos , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Regiones Promotoras Genéticas , ARN/biosíntesis , Transcripción Genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Edición Génica , Regulación del Desarrollo de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Masculino , Metilación , Ratones , Mutación , ARN/genética , Factores de Tiempo , Activación Transcripcional , TransfecciónRESUMEN
The amino acid position within a histone sequence and the chemical nature of post-translational modifications (PTMs) are essential for elucidating the "Histone Code". Previous work has shown that PTMs induce specific biological responses and are good candidates as biomarkers for diagnostics. Here, we evaluate the analytical advantages of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation (PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics of model cancer cells. The study also considered the use of nanoliquid chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and PASEF-MS/MS increased the number of detected peptides due to the added separation dimension. All three methods showed high reproducibility and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS separations showed small RSD across samples, the accurate mobility (1/K0) measurements (<0.6% RSD) increased the confidence of peptide assignments. Trends were observed in the retention time and mobility concerning the number and type of PTMs (e.g., ac, me1-3) and their corresponding unmodified, propionylated peptide that aided in peptide assignment. Mobility separation permitted the annotation of coeluting structural and positional isomers and compared with nLC-MS/MS showed several advantages due to reduced chemical noise.
RESUMEN
Heterochromatic DNA domains have important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. From fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway. Typically, sRNAs, originating from long noncoding RNAs, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and trimethylation (H3K9me2 and H3K9me3, respectively) and the formation of heterochromatin. H3K9me is in turn required for the recruitment of RNAi to chromatin to promote the amplification of sRNA. Yet, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast Schizosaccharomyces pombe homologue of mammalian SUV39H H3K9 methyltransferases, we design active-site mutations that block H3K9me3, but allow H3K9me2 catalysis. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing at pericentromeric DNA repeats. Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, the transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct chromatin states and uncover a mechanism for the formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in sRNA-mediated genome defence.
Asunto(s)
Silenciador del Gen , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/química , Histonas/metabolismo , Interferencia de ARN , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Silenciador del Gen/efectos de los fármacos , Heterocromatina/química , N-Metiltransferasa de Histona-Lisina , Ácidos Hidroxámicos/farmacología , Metilación/efectos de los fármacos , Metiltransferasas/metabolismo , Mutación , Proteínas Represoras/metabolismo , Schizosaccharomyces/efectos de los fármacos , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Post-translational modifications (PTMs) on intact histones play a major role in regulating chromatin dynamics and influence biological processes such as DNA transcription, replication, and repair. The nature and position of each histone PTM is crucial to decipher how this information is translated into biological response. In the present work, the potential of a novel tandem top-"double-down" approachâultraviolet photodissociation followed by mobility and mass-selected electron capture dissociation and mass spectrometry (UVPD-TIMS-q-ECD-ToF MS/MS)âis illustrated for the characterization of HeLa derived intact histone H4 proteoforms. The comparison between q-ECD-ToF MS/MS spectra and traditional Fourier-transform-ion cyclotron resonance-ECD MS/MS spectra of a H4 standard showed a similar sequence coverage (â¼75%) with significant faster data acquisition in the ToF MS/MS platform (â¼3 vs â¼15 min). Multiple mass shifts (e.g., 14 and 42 Da) were observed for the HeLa derived H4 proteoforms for which the top-down UVPD and ECD fragmentation analysis were consistent in detecting the presence of acetylated PTMs at the N-terminus and Lys5, Lys8, Lys12, and Lys16 residues, as well as methylated, dimethylated, and trimethylated PTMs at the Lys20 residue with a high sequence coverage (â¼90%). The presented top-down results are in good agreement with bottom-up TIMS ToF MS/MS experiments and allowed for additional description of PTMs at the N-terminus. The integration of a 213 nm UV laser in the present platform allowed for UVPD events prior to the ion mobility-mass precursor separation for collision-induced dissociation (CID)/ECD-ToF MS. Selected c305+ UVPD fragments, from different H4 proteoforms (e.g., Ac + Me2, 2Ac + Me2 and 3Ac + Me2), exhibited multiple IMS bands for which similar CID/ECD fragmentation patterns per IMS band pointed toward the presence of conformers, adopting the same PTM distribution, with a clear assignment of the PTM localization for each of the c305+ UVPD fragment H4 proteoforms. These results were consistent with the biological "zip" model, where acetylation proceeds in the Lys16 to Lys5 direction. This novel platform further enhances the structural toolbox with alternative fragmentation mechanisms (UVPD, CID, and ECD) in tandem with fast, high-resolution mobility separations and shows great promise for global proteoform analysis.
Asunto(s)
Histonas , Espectrometría de Masas en Tándem , Humanos , Histonas/química , Espectrometría de Masas en Tándem/métodos , Electrones , Procesamiento Proteico-Postraduccional , Análisis de FourierRESUMEN
Dysregulation of polycomb repressive complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing trimethylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19; also known as polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. Chromatin immunoprecipitation followed by sequencing profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains, possibly due to impaired H3K27me3 spreading from cytosine guanine dinucleotide islands, which is reminiscent to the reported effect of an "onco"-histone mutation, H3K27 to methionine (H3K27M). RNA-sequencing-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Correlation studies using patient sample data sets further support a clinical relevance of the PHF19-regulated pathways. Lastly, we show that MM cells are generally sensitive to PRC2 inhibitors. Collectively, this study demonstrates that PHF19 promotes MM tumorigenesis through enhancing H3K27me3 deposition and PRC2's gene-regulatory functions, lending support for PRC2 blockade as a means for MM therapeutics.
Asunto(s)
Carcinogénesis/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Mieloma Múltiple/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Factores de Transcripción/metabolismo , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Humanos , Metilación , Ratones , Mieloma Múltiple/patologíaRESUMEN
We present a system-level analysis of proteome, phosphoproteome, and chromatin state of precursors of muscle cells (myoblasts) differentiating into specialized myotubes. Using stable isotope labeling of amino acids in cell culture and nano-liqud chromatography-mass spectrometry/mass spectrometry, we found that phosphorylation motifs targeted by the kinases protein kinase C, cyclin-dependent kinase, and mitogen-activated protein kinase showed increased phosphorylation during myodifferentiation of LHCN-M2 human skeletal myoblast cell line. Drugs known to inhibit these kinases either promoted (PD0325901 and GW8510) or stalled (CHIR99021 and roscovitine) differentiation, resulting in myotube and myoblast phenotypes, respectively. The proteomes, especially the myogenic and chromatin-related proteins including histone methyltransferases, correlated with their phenotypes, leading us to quantify histone post-translational modifications and identify two gene-silencing marks, H3K9me3 and H4K20me3, with relative abundances changing in correlation with these phenotypes. ChIP-quantitative PCR demonstrated that H3K9me3 is erased from the gene loci of myogenic regulatory factors namely MYOD1, MYOG, and MYF5 in differentiating myotubes. Together, our work integrating histone post-translational modification, phosphoproteomics, and full proteome analysis gives a comprehensive understanding of the close connection between signaling pathways and epigenetics during myodifferentiation in vitro.
Asunto(s)
Benzamidas/farmacología , Diferenciación Celular/efectos de los fármacos , Difenilamina/análogos & derivados , Histonas/metabolismo , Indoles/farmacología , Desarrollo de Músculos/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Quinasas/metabolismo , Línea Celular , Difenilamina/farmacología , Humanos , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/metabolismo , Miogenina/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
Microtubule-based molecular motors mediate transport of intracellular cargo to subdomains in neurons. Previous evidence has suggested that the anesthetic propofol decreases the average run-length potential of the major anterograde transporters kinesin-1 and kinesin-2 without altering their velocity. This effect on kinesin has not been observed with other inhibitors, stimulating considerable interest in the underlying mechanism. Here, we used a photoactive derivative of propofol, meta-azipropofol (AziPm), to search for potential propofol-binding sites in kinesin. Single-molecule motility assays confirmed that AziPm and propofol similarly inhibit kinesin-1 and kinesin-2. We then applied AziPm in semiquantitative radiolabeling and MS microsequencing assays to identify propofol-binding sites within microtubule-kinesin complexes. The radiolabeling experiments suggested preferential AziPm binding to the ATP-bound microtubule-kinesin complex. The photolabeled residues were contained within the kinesin motor domain rather than at the motor domain-ß-tubulin interface. No residues within the P-loop of kinesin were photolabeled, indicating an inhibitory mechanism that does not directly affect ATPase activity and has an effect on run length without changing velocity. Our results also indicated that when the kinesin motor interacts with the microtubule during its processive run, a site forms in kinesin to which propofol can then bind and allosterically disrupt the kinesin-microtubule interaction, resulting in kinesin detachment and run termination. The discovery of the propofol-binding allosteric site in kinesin may improve our understanding of the strict coordination of the motor heads during the processive run. We hypothesize that propofol's potent effect on intracellular transport contributes to various components of its anesthetic action.
Asunto(s)
Sitio Alostérico/efectos de los fármacos , Anestésicos Intravenosos/farmacología , Cinesinas/metabolismo , Microtúbulos/metabolismo , Propofol/farmacología , Secuencia de Aminoácidos , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Cinesinas/química , Microtúbulos/química , Simulación del Acoplamiento MolecularRESUMEN
Most general anesthetics enhance GABA type A (GABAA) receptor activity at clinically relevant concentrations. Sites of action of volatile anesthetics on the GABAA receptor remain unknown, whereas sites of action of many intravenous anesthetics have been identified in GABAA receptors by using photolabeling. Here, we used photoactivatable analogs of isoflurane (AziISO) and sevoflurane (AziSEVO) to locate their sites on α1ß3γ2L and α1ß3 GABAA receptors. As with isoflurane and sevoflurane, AziISO and AziSEVO enhanced the currents elicited by GABA. AziISO and AziSEVO each labeled 10 residues in α1ß3 receptors and 9 and 8 residues, respectively, in α1ß3γ2L receptors. Photolabeled residues were concentrated in transmembrane domains and located in either subunit interfaces or in the interface between the extracellular domain and the transmembrane domain. The majority of these transmembrane residues were protected from photolabeling with the addition of excess parent anesthetic, which indicated specificity. Binding sites were primarily located within α+/ß- and ß+/α- subunit interfaces, but residues in the α+/γ- interface were also identified, which provided a basis for differential receptor subtype sensitivity. Isoflurane and sevoflurane did not always share binding sites, which suggests an unexpected degree of selectivity.-Woll, K. A., Zhou, X., Bhanu, N. V., Garcia, B. A., Covarrubias, M., Miller, K. W., Eckenhoff, R. G. Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors.
Asunto(s)
Anestésicos/metabolismo , Sitios de Unión/fisiología , Receptores de GABA-A/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Línea Celular , Humanos , Isoflurano/farmacología , Proteínas de la Membrana/metabolismo , Oocitos/metabolismo , Sevoflurano/farmacología , Xenopus laevis/metabolismoRESUMEN
In addition to inducing anesthesia, propofol activates a key component of the pain pathway, the transient receptor potential ankyrin 1 ion channel (TRPA1). Recent mutagenesis studies suggested a potential activation site within the transmembrane domain, near the A-967079 cavity. However, mutagenesis cannot distinguish between protein-based and ligand-based mechanisms, nor can this site explain the complex modulation by propofol. Thus more direct approaches are required to reveal potentially druggable binding sites. Here we apply photoaffinity labeling using a propofol derivative, meta-azipropofol, for direct identification of binding sites in mouse TRPA1. We confirm that meta-azipropofol activates TRPA1 like the parent anesthetic, and identify two photolabeled residues (V954 and E969) in the S6 helix. In combination with docking to closed and open state models of TRPA1, photoaffinity labeling suggested that the A-967079 cavity is a positive modulatory site for propofol. Further, the photoaffinity labeling of E969 indicated pore block as a likely mechanism for propofol inhibition at high concentrations. The direct identification of drug-binding sites clarifies the molecular mechanisms of important TRPA1 agonists, and will facilitate drug design efforts to modulate TRPA1.
Asunto(s)
Anestésicos/farmacología , Etiquetas de Fotoafinidad/química , Propofol/farmacología , Canal Catiónico TRPA1/química , Canal Catiónico TRPA1/metabolismo , Animales , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , RatasRESUMEN
Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to achieve the same peptide relative quantification. Taken together, sequential window acquisition of all theoretical mass spectra proved to be an easy-to-use MS acquisition method to perform high quality MS/MS-based quantification of histone-modified peptides.
Asunto(s)
Histonas/aislamiento & purificación , Péptidos/química , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Células Madre/metabolismo , Animales , Células Cultivadas , Cromatografía Liquida/métodos , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Humanos , Ratones , Células Madre/citología , Espectrometría de Masas en Tándem/métodos , Trofoblastos/citología , Trofoblastos/metabolismoRESUMEN
Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification. However, analysis of histones with this method is even more challenging because of the large number and variety of isobaric histone peptides and the high dynamic range of histone peptide abundances. Here, we introduce EpiProfile, a software tool that discriminates isobaric histone peptides using the distinguishing fragment ions in their tandem mass spectra and extracts the chromatographic area under the curve using previous knowledge about peptide retention time. The accuracy of EpiProfile was evaluated by analysis of mixtures containing different ratios of synthetic histone peptides. In addition to label-free quantification of histone peptides, EpiProfile is flexible and can quantify different types of isotopically labeled histone peptides. EpiProfile is unique in generating layouts (i.e. relative retention time) of histone peptides when compared with manual quantification of the data and other programs (such as Skyline), filling the need of an automatic and freely available tool to quantify labeled and non-labeled modified histone peptides. In summary, EpiProfile is a valuable nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry-based quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples.
Asunto(s)
Histonas/química , Péptidos/análisis , Cromatografía Liquida , Células HeLa , Humanos , Péptidos/química , Espectrometría de Masas en TándemRESUMEN
In this study, we trace developmental stages using epigenome changes in human embryonic stem cells (hESCs) treated with drugs modulating either self-renewal or differentiation. Based on microscopy, qPCR and flow cytometry, we classified the treatment outcome as inducing pluripotency (hESC, flurbiprofen and gatifloxacin), mesendoderm (sinomenine), differentiation (cyamarin, digoxin, digitoxin, selegeline and theanine) and lineage-commitment (RA). When we analyzed histone PTMs that imprinted these gene and protein expressions, the above classification was reassorted. Hyperacetylation at H3K4, 9, 14, 18, 56 and 122 as well as H4K5, 8, 12 and 16 emerged as the pluripotency signature of hESCs. Methylations especially of H3 at K9, K20, K27 and K36 characterized differentiation initiation as seen in no-drug control and fluribiprofen. Sinomenine-treated cells clustered close to "differentiation initiators", consistent with flow cytometry where it induced mesendoderm, along with cyamarin and possibly selegnine. Neurectoderm, induced by RA and theanine manifested methylations on H3 shifts to H3.3. By both flow cytometry and histone PTM clustering, it appears that cells treated with gatifloxacin, flurbiprofen, digitoxin and digoxin were not yet lineage-committed or mixed cell types. Taken together, our moderate-throughput histone PTM profiling approach highlighted subtle epigenetic signatures that permitted us to predict divergent lineage progression even in differentiating cells with similar phenotype and gene expression.
Asunto(s)
Linaje de la Célula/genética , Epigénesis Genética , Histonas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Digitoxina/farmacología , Digoxina/farmacología , Fluoroquinolonas/farmacología , Flurbiprofeno/farmacología , Gatifloxacina , Perfilación de la Expresión Génica , Glutamatos/farmacología , Histonas/genética , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Metilación , Selegilina/farmacología , Tretinoina/farmacologíaRESUMEN
BACKGROUND: Pluripotent cells can be differentiated into many different cell types in vitro. Successful differentiation is guided in large part by epigenetic reprogramming and regulation of critical gene expression patterns. Recent genome-wide studies have identified the distribution of different histone-post-translational modifications (PTMs) in various conditions and during cellular differentiation. However, our understanding of the abundance of histone PTMs and their regulatory mechanisms still remain unknown. RESULTS: Here, we present a quantitative and comprehensive study of the abundance levels of histone PTMs during the differentiation of mouse embryonic stem cells (ESCs) using mass spectrometry (MS). We observed dynamic changes of histone PTMs including increased H3K9 methylation levels in agreement with previously reported results. More importantly, we found a global decrease of multiply acetylated histone H4 peptides. Brd4 targets acetylated H4 with a strong affinity to multiply modified H4 acetylation sites. We observed that the protein levels of Brd4 decreased upon differentiation together with global histone H4 acetylation. Inhibition of Brd4:histone H4 interaction by the BET domain inhibitor (+)-JQ1 in ESCs results in enhanced differentiation to the endodermal lineage, by disrupting the protein abundance dynamics. Genome-wide ChIP-seq mapping showed that Brd4 and H4 acetylation are co-occupied in the genome, upstream of core pluripotency genes such as Oct4 and Nanog in ESCs and lineage-specific genes in embryoid bodies (EBs). CONCLUSIONS: Together, our data demonstrate the fundamental role of Brd4 in monitoring cell differentiation through its interaction with acetylated histone marks and disruption of Brd4 may cause aberrant differentiation.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Autorrenovación de las Células/genética , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Proteínas Nucleares/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/química , Tretinoina/farmacologíaRESUMEN
In thalassemia, deficient globin-chain production during erythropoiesis results in anemia. Thalassemia may be further complicated by iron overload (frequently exacerbated by blood transfusion), which induces numerous endocrine diseases, hepatic cirrhosis, cardiac failure and even death. Accumulation of iron in the absence of blood transfusions may result from inappropriate suppression of the iron-regulating peptide hepcidin by an erythropoietic mechanism. To test this hypothesis, we examined erythroblast transcriptome profiles from 15 healthy, nonthalassemic donors. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-beta superfamily, showed increased expression and secretion during erythroblast maturation. Healthy volunteers had mean GDF15 serum concentrations of 450 +/- 50 pg/ml. In comparison, individuals with beta-thalassemia syndromes had elevated GDF15 serum levels (mean 66,000 +/- 9,600 pg/ml; range 4,800-248,000 pg/ml; P < 0.05) that were positively correlated with the levels of soluble transferrin receptor, erythropoietin and ferritin. Serum from thalassemia patients suppressed hepcidin mRNA expression in primary human hepatocytes, and depletion of GDF15 reversed hepcidin suppression. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression.