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1.
Nature ; 622(7981): 173-179, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37731000

RESUMO

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.


Assuntos
Acetilação , Cromatina , Lisina , Metilação , Processamento de Proteína Pós-Traducional , Sítio de Iniciação de Transcrição , Animais , Humanos , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Histona Desacetilases/metabolismo
2.
Nature ; 582(7813): 586-591, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494005

RESUMO

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.


Assuntos
Cromatina/metabolismo , Reparo do DNA , Recombinação Homóloga , Neoplasias/metabolismo , Transdução de Sinais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Lisina Acetiltransferase 5/metabolismo , Metilação/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transdução de Sinais/efeitos dos fármacos
3.
Cell Rep ; 29(8): 2473-2488.e5, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31747614

RESUMO

An abnormal number of chromosomes, or aneuploidy, accounts for most spontaneous abortions, causes developmental defects, and is associated with aging and cancer. The molecular mechanisms by which aneuploidy disrupts cellular function remain largely unknown. Here, we show that aneuploidy disrupts the morphology of the nucleus. Mutations that increase the levels of long-chain bases suppress nuclear abnormalities of aneuploid yeast independent of karyotype identity. Quantitative lipidomics indicates that long-chain bases are integral components of the nuclear membrane in yeast. Cells isolated from patients with Down syndrome also show that abnormal nuclear morphologies and increases in long-chain bases not only suppress these abnormalities but also improve their fitness. We obtained similar results with cells isolated from patients with Patau or Edward syndrome, indicating that increases in long-chain bases improve the fitness of aneuploid cells in yeast and humans. Targeting lipid biosynthesis pathways represents an important strategy to suppress nuclear abnormalities in aneuploidy-associated diseases.


Assuntos
Aneuploidia , Síndrome de Down/metabolismo , Membrana Nuclear/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Cariótipo , Esfingolipídeos/metabolismo , Esfingosina/metabolismo , Síndrome da Trissomia do Cromossomo 13/metabolismo , Síndrome da Trissomía do Cromossomo 18/metabolismo , Leveduras/metabolismo
4.
Genetics ; 213(2): 431-447, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31405990

RESUMO

A subset of cancers rely on telomerase-independent mechanisms to maintain their chromosome ends. The predominant "alternative lengthening of telomeres" pathway appears dependent on homology-directed repair (HDR) to maintain telomeric DNA. However, the molecular changes needed for cells to productively engage in telomeric HDR are poorly understood. To gain new insights into this transition, we monitored the state of telomeres during serial culture of fission yeast (Schizosaccharomyces pombe) lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). In this context, degradation of TERRA associated with the telomere in the form of R-loops drives a severe growth crisis, ultimately leading to a novel type of survivor with linear chromosomes and altered cytological telomere characteristics, including the loss of the shelterin component Rap1 (but not the TRF1/TRF2 ortholog, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective in this context, preventing the growth crisis that is otherwise triggered by degradation of telomeric R-loops in survivors with linear chromosomes. These findings suggest that upregulation of telomere-engaged TERRA, or altered recruitment of shelterin components, can support telomerase-independent telomere maintenance.


Assuntos
Proteínas de Schizosaccharomyces pombe/genética , Homeostase do Telômero/genética , Encurtamento do Telômero/genética , Proteínas de Ligação a Telômeros/genética , Telômero/genética , DNA Fúngico/química , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , RNA Fúngico/química , RNA Fúngico/genética , Reparo de DNA por Recombinação/genética , Schizosaccharomyces/genética , Complexo Shelterina , Telomerase/genética
5.
Mol Biol Cell ; 30(14): 1664-1675, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31091167

RESUMO

The cardiomyocyte cytoskeleton, including the sarcomeric contractile apparatus, forms a cohesive network with cellular adhesions at the plasma membrane and nuclear--cytoskeletal linkages (LINC complexes) at the nuclear envelope. Human cardiomyopathies are genetically linked to the LINC complex and A-type lamins, but a full understanding of disease etiology in these patients is lacking. Here we show that SUN2-null mice display cardiac hypertrophy coincident with enhanced AKT/MAPK signaling, as has been described previously for mice lacking A-type lamins. Surprisingly, in contrast to lamin A/C-null mice, SUN2-null mice fail to show coincident fibrosis or upregulation of pathological hypertrophy markers. Thus, cardiac hypertrophy is uncoupled from profibrotic signaling in this mouse model, which we tie to a requirement for the LINC complex in productive TGFß signaling. In the absence of SUN2, we detect elevated levels of the integral inner nuclear membrane protein MAN1, an established negative regulator of TGFß signaling, at the nuclear envelope. We suggest that A-type lamins and SUN2 play antagonistic roles in the modulation of profibrotic signaling through opposite effects on MAN1 levels at the nuclear lamina, suggesting a new perspective on disease etiology.


Assuntos
Cardiomegalia/metabolismo , Cardiomegalia/patologia , Deleção de Genes , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Adesão Celular , Forma do Núcleo Celular , Proteínas de Ligação a DNA/metabolismo , Fibrose , Integrinas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Membrana Nuclear/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Proteínas de Ligação a Telômeros/deficiência , Fator de Crescimento Transformador beta/metabolismo
6.
Nat Commun ; 10(1): 1695, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979898

RESUMO

Actin cytoskeleton is well-known for providing structural/mechanical support, but whether and how it regulates chromatin and cell fate reprogramming is far less clear. Here, we report that MKL1, the key transcriptional co-activator of many actin cytoskeletal genes, regulates genomic accessibility and cell fate reprogramming. The MKL1-actin pathway weakens during somatic cell reprogramming by pluripotency transcription factors. Cells that reprogram efficiently display low endogenous MKL1 and inhibition of actin polymerization promotes mature pluripotency activation. Sustained MKL1 expression at a level seen in typical fibroblasts yields excessive actin cytoskeleton, decreases nuclear volume and reduces global chromatin accessibility, stalling cells on their trajectory toward mature pluripotency. In addition, the MKL1-actin imposed block of pluripotency can be bypassed, at least partially, when the Sun2-containing linker of the nucleoskeleton and cytoskeleton (LINC) complex is inhibited. Thus, we unveil a previously unappreciated aspect of control on chromatin and cell fate reprogramming exerted by the MKL1-actin pathway.


Assuntos
Reprogramação Celular , Cromatina/química , Transativadores/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Feminino , Fibroblastos/citologia , Transferência Ressonante de Energia de Fluorescência , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Proteínas de Fusão Oncogênica/metabolismo , Células-Tronco Pluripotentes/citologia
7.
Elife ; 72018 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-29697047

RESUMO

Poly(ADP ribose) polymerase inhibitors (PARPi) target cancer cells deficient in homology-directed repair of DNA double-strand breaks (DSBs). In preclinical models, PARPi resistance is tied to altered nucleolytic processing (resection) at the 5' ends of a DSB. For example, loss of either 53BP1 or Rev7/MAD2L2/FANCV derepresses resection to drive PARPi resistance, although the mechanisms are poorly understood. Long-range resection can be catalyzed by two machineries: the exonuclease Exo1, or the combination of a RecQ helicase and Dna2. Here, we develop a single-cell microscopy assay that allows the distinct phases and machineries of resection to be interrogated simultaneously in living S. pombe cells. Using this assay, we find that the 53BP1 orthologue and Rev7 specifically repress long-range resection through the RecQ helicase-dependent pathway, thereby preventing hyper-resection. These results suggest that 'rewiring' of BRCA1-deficient cells to employ an Exo1-independent hyper-resection pathway is a driver of PARPi resistance.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Nucleares/metabolismo , RecQ Helicases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Microscopia , Análise de Célula Única
8.
Nat Commun ; 7: 10997, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26975831

RESUMO

Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Movimento Celular , Núcleo Celular/metabolismo , Células Dendríticas , Neutrófilos , Lâmina Nuclear/metabolismo , Animais , Immunoblotting , Lamina Tipo A/metabolismo , Camundongos , Polimerização
10.
PLoS One ; 8(12): e82758, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24312672

RESUMO

Eukaryotic organisms employ a variety of mechanisms during meiosis to assess and ensure the quality of their gametes. Defects or delays in successful meiotic recombination activate conserved mechanisms to delay the meiotic divisions, but many multicellular eukaryotes also induce cell death programs to eliminate gametes deemed to have failed during meiosis. It is generally thought that yeasts lack such mechanisms. Here, we show that in the fission yeast Schizosaccharomyces pombe, defects in meiotic recombination lead to the activation of a checkpoint that is linked to ascus wall endolysis--the process by which spores are released in response to nutritional cues for subsequent germination. Defects in meiotic recombination are sensed as unrepaired DNA damage through the canonical ATM and ATR DNA damage response kinases, and this information is communicated to the machinery that stimulates ascus wall breakdown. Viability of spores that undergo endolysis spontaneously is significantly higher than that seen upon chemical endolysis, demonstrating that this checkpoint contributes to a selective mechanism for the germination of high quality progeny. These results provide the first evidence for the existence of a checkpoint linking germination to meiosis and suggest that analysis solely based on artificial, enzymatic endolysis bypasses an important quality control mechanism in this organism and potentially other ascomycota, which are models widely used to study meiosis.


Assuntos
Meiose/genética , Schizosaccharomyces/genética , Meiose/fisiologia , RNA de Cadeia Dupla/genética , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Esporos Fúngicos/metabolismo , Esporos Fúngicos/fisiologia
11.
Emerg Med Clin North Am ; 30(4): 1007-19, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23137409

RESUMO

Most postmenopausal vaginal bleeding is benign; however, it merits diagnostic evaluation by transvaginal ultrasound or endometrial biopsy after emergency department evaluation. Patients and physicians may treat menopausal symptoms with hormone replacement therapy or other agents, such as venlafaxine or gabapentin. Hormone replacement therapy, when initiated close to the start of menopause and continued at the lowest possible dose for the shortest possible duration, carries less risk than previously believed. Pelvic organ prolapse affects millions of women and may contribute to poor body image and difficulty with urinary, gastrointestinal, and sexual function. Treatment options include Kegel exercises, pessaries, and surgery.


Assuntos
Neoplasias dos Genitais Femininos , Terapia de Reposição Hormonal/métodos , Pós-Menopausa , Hemorragia Uterina , Prolapso Uterino , Idoso , Feminino , Neoplasias dos Genitais Femininos/complicações , Neoplasias dos Genitais Femininos/diagnóstico , Neoplasias dos Genitais Femininos/terapia , Humanos , Hemorragia Uterina/diagnóstico , Hemorragia Uterina/etiologia , Hemorragia Uterina/terapia , Prolapso Uterino/diagnóstico , Prolapso Uterino/etiologia , Prolapso Uterino/terapia
12.
EMBO J ; 23(9): 1922-33, 2004 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-15103325

RESUMO

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P2 effectors, we identified Saccharomyces cerevisiae mutants that display fab1delta-like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P2 binding of exquisite specificity, GFP-Svp1p localises to the vacuole membrane in a Fab1p-dependent manner, and svp1delta cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P2. These observations identify Svp1p as a PtdIns(3,5)P2 effector required for PtdIns(3,5)P2-dependent membrane recycling from the vacuole. Other Svp1p-related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P2 similarly. Svp1p and related proteins almost certainly fold as beta-propellers, and the PtdIns(3,5)P2-binding site is on the beta-propeller. It is likely that many of the Svp1p-related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P2 effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P2 to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P2 effectors must exist.


Assuntos
Endossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Sequência de Aminoácidos , Proteínas Relacionadas à Autofagia , Sequência de Bases , Clonagem Molecular , Escherichia coli , Componentes do Gene , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plasmídeos/genética , Ligação Proteica , Dobramento de Proteína , Transporte Proteico/fisiologia , Rhinovirus , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de DNA
13.
J Biol Chem ; 277(7): 4704-12, 2002 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-11741943

RESUMO

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.


Assuntos
Membrana Celular/metabolismo , Receptores ErbB/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Cloranfenicol O-Acetiltransferase/metabolismo , Análise Mutacional de DNA , Dimerização , Escherichia coli/metabolismo , Vetores Genéticos , Ácido Glutâmico/química , Humanos , Ligantes , Maltose/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/metabolismo , Receptor ErbB-2/química , Receptor ErbB-3/química , Receptor ErbB-4 , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Valina/química
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