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1.
Mol Cell ; 82(14): 2696-2713.e9, 2022 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-35716669

RESUMO

Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.


Assuntos
Neoplasias do Tronco Encefálico , Glioma , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/metabolismo , Neoplasias do Tronco Encefálico/patologia , Cromatina/genética , Epigênese Genética , Glioma/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Mutação
2.
Nucleic Acids Res ; 51(4): 1662-1673, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36156096

RESUMO

The histone H3 variant, H3.3, is localized at specific regions in the genome, especially promoters and active enhancers, and has been shown to play important roles in development. A lysine to methionine substitution in position 27 (H3.3K27M) is a main cause of Diffuse Intrinsic Pontine Glioma (specifically Diffuse Midline Glioma, K27M-mutant), a lethal type of pediatric cancer. H3.3K27M has a dominant-negative effect by inhibiting the Polycomb Repressor Complex 2 (PRC2) activity. Here, we studied the immediate, genome-wide, consequences of the H3.3K27M mutation independent of PRC2 activity. We developed Doxycycline (Dox)-inducible mouse embryonic stem cells (ESCs) carrying a single extra copy of WT-H3.3, H3.3K27M and H3.3K27L, all fused to HA. We performed RNA-Seq and ChIP-Seq at different times following Dox induction in undifferentiated and differentiated ESCs. We find increased binding of H3.3 around transcription start sites in cells expressing both H3.3K27M and H3.3K27L compared with WT, but not in cells treated with PRC2 inhibitors. Differentiated cells carrying either H3.3K27M or H3.3K27L retain expression of ESC-active genes, in expense of expression of genes related to neuronal differentiation. Taken together, our data suggest that a modifiable H3.3K27 is required for proper histone incorporation and cellular maturation, independent of PRC2 activity.


Assuntos
Células-Tronco Embrionárias , Histonas , Animais , Camundongos , Diferenciação Celular , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Glioma/genética , Histonas/metabolismo , Mutação , Proteínas do Grupo Polycomb/metabolismo , Doxiciclina/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo
3.
J Cell Sci ; 133(24)2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33177075

RESUMO

Eukaryotic cells have evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge of molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore crucial for understanding eukaryotic metabolism. Here, we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. This dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sequência de Aminoácidos , Citosol/metabolismo , Glioxilatos , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Peroxissomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Nat Methods ; 16(2): 205, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30602782

RESUMO

The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.

5.
Nat Methods ; 15(8): 617-622, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29988094

RESUMO

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.


Assuntos
Genoma Fúngico , Biblioteca Genômica , Proteoma/genética , Saccharomyces cerevisiae/genética , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Sequências Rotuladas
6.
PLoS Genet ; 9(6): e1003597, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23825967

RESUMO

The Drosophila heart tube represents a structure that similarly to vertebrates' primary heart tube exhibits a large lumen; the mechanisms promoting heart tube morphology in both Drosophila and vertebrates are poorly understood. We identified Multiplexin (Mp), the Drosophila orthologue of mammalian Collagen-XV/XVIII, and the only structural heart-specific protein described so far in Drosophila, as necessary and sufficient for shaping the heart tube lumen, but not that of the aorta. Mp is expressed specifically at the stage of heart tube closure, in a polarized fashion, uniquely along the cardioblasts luminal membrane, and its absence results in an extremely small heart tube lumen. Importantly, Mp forms a protein complex with Slit, and interacts genetically with both slit and robo in the formation of the heart tube. Overexpression of Mp in cardioblasts promotes a large heart lumen in a Slit-dependent manner. Moreover, Mp alters Slit distribution, and promotes the formation of multiple Slit endocytic vesicles, similarly to the effect of overexpression of Robo in these cells. Our data are consistent with Mp-dependent enhancement of Slit/Robo activity and signaling, presumably by affecting Slit protein stabilization, specifically at the lumen side of the heart tube. This activity results with a Slit-dependent, local reduction of F-actin levels at the heart luminal membrane, necessary for forming the large heart tube lumen. Consequently, lack of Mp results in decreased diastolic capacity, leading to reduced heart contractility, as measured in live fly hearts. In summary, these findings show that the polarized localization of Mp controls the direction, timing, and presumably the extent of Slit/Robo activity and signaling at the luminal membrane of the heart cardioblasts. This regulation is essential for the morphogenetic changes that sculpt the heart tube in Drosophila, and possibly in forming the vertebrates primary heart tube.


Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Colágeno/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Morfogênese/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Aorta/crescimento & desenvolvimento , Aorta/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Coração/anatomia & histologia , Coração/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Transdução de Sinais , Proteínas Roundabout
7.
Methods ; 56(1): 63-8, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21963658

RESUMO

The Drosophila heart has become an exciting model for elucidating the molecular basis for cardiac function in higher organisms. To complement the genetic approaches that have recently identified an array of genes essential for cardiac function, we developed a method to obtain optimal semi-thin cross sections of embryonic, larval, and adult fly hearts in a desired orientation. A procedure for fluorescent labeling of these sections with multiple markers has also been developed, allowing the detection of proteins at high subcellular resolution. Sections obtained by our method reveal changes in cell shape between embryonic heart and aorta cardioblasts and elucidate the morphology of the adult heart. Analysis of the adult heart reveals the precise cardiac tube morphology, differential distribution of the extracellular matrix protein Laminin within the cardiac tube, as well as individual hand-positive, and Held Out Wings (HOW)-positive luminal cells that might represent blood cells. In summary, our method enables visualization of cross sections of the embryonic and adult hearts at high resolution while maintaining the ability to co-label the sections with multiple markers, thereby facilitating the analysis of cardiac tube formation and maintenance at different developmental stages.


Assuntos
Drosophila/citologia , Técnicas de Preparação Histocitológica/métodos , Animais , Anticorpos/química , Drosophila/embriologia , Técnicas de Preparação Histocitológica/tendências , Imuno-Histoquímica , Miocárdio/citologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Coloração e Rotulagem
8.
Nat Commun ; 13(1): 7199, 2022 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-36443319

RESUMO

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a "basal-like" state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed "basal-like" genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression.


Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/genética , Genes Reguladores , Proteínas Serina-Treonina Quinases/genética , Mama , Repressão Psicológica , Correpressor 1 de Receptor Nuclear/genética
9.
Curr Biol ; 30(22): 4399-4412.e7, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-32916113

RESUMO

Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance.


Assuntos
Caseína Quinase I/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Fosforilação/fisiologia , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Saccharomyces cerevisiae , Ubiquitinação/fisiologia
10.
J Mol Med (Berl) ; 97(2): 269-279, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30604168

RESUMO

The yeast protein Taz1 is the orthologue of human Tafazzin, a phospholipid acyltransferase involved in cardiolipin (CL) remodeling via a monolyso CL (MLCL) intermediate. Mutations in Tafazzin lead to Barth syndrome (BTHS), a metabolic and neuromuscular disorder that primarily affects the heart, muscles, and immune system. Similar to observations in fibroblasts and platelets from patients with BTHS or from animal models, abolishing yeast Taz1 results in decreased total CL amounts, increased levels of MLCL, and mitochondrial dysfunction. However, the biochemical mechanisms underlying the mitochondrial dysfunction in BTHS remain unclear. To better understand the pathomechanism of BTHS, we searched for multi-copy suppressors of the taz1Δ growth defect in yeast cells. We identified the branched-chain amino acid transaminases (BCATs) Bat1 and Bat2 as such suppressors. Similarly, overexpression of the mitochondrial isoform BCAT2 in mammalian cells lacking TAZ improves their growth. Elevated levels of Bat1 or Bat2 did not restore the reduced membrane potential, altered stability of respiratory complexes, or the defective accumulation of MLCL species in yeast taz1Δ cells. Importantly, supplying yeast or mammalian cells lacking TAZ1 with certain amino acids restored their growth behavior. Hence, our findings suggest that the metabolism of amino acids has an important and disease-relevant role in cells lacking Taz1 function. KEY MESSAGES: Bat1 and Bat2 are multi-copy suppressors of retarded growth of taz1Δ yeast cells. Overexpression of Bat1/2 in taz1Δ cells does not rescue known mitochondrial defects. Supplementation of amino acids enhances growth of cells lacking Taz1 or Tafazzin. Altered metabolism of amino acids might be involved in the pathomechanism of BTSH.


Assuntos
Aciltransferases/genética , Proteínas Mitocondriais/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transaminases/genética , Síndrome de Barth/genética , Deleção de Genes , Humanos , Mitocôndrias/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de Transcrição/genética , Regulação para Cima
11.
J Cell Biol ; 217(1): 269-282, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29187527

RESUMO

Functional heterogeneity within the lipid droplet (LD) pool of a single cell has been observed, yet the underlying mechanisms remain enigmatic. Here, we report on identification of a specialized LD subpopulation characterized by a unique proteome and a defined geographical location at the nucleus-vacuole junction contact site. In search for factors determining identity of these LDs, we screened ∼6,000 yeast mutants for loss of targeting of the subpopulation marker Pdr16 and identified Ldo45 (LD organization protein of 45 kD) as a crucial targeting determinant. Ldo45 is the product of a splicing event connecting two adjacent genes (YMR147W and YMR148W/OSW5/LDO16). We show that Ldo proteins cooperate with the LD biogenesis component seipin and establish LD identity by defining positioning and surface-protein composition. Our studies suggest a mechanism to establish functional differentiation of organelles, opening the door to better understanding of metabolic decisions in cells.


Assuntos
Gotículas Lipídicas/metabolismo , Proteínas de Membrana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Gotículas Lipídicas/classificação , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma , Saccharomyces cerevisiae/metabolismo
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