RESUMO
Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were 'unmasked' only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.
Assuntos
Glucocorticoides , Sequências Reguladoras de Ácido Nucleico , Glucocorticoides/genética , Glucocorticoides/metabolismo , Fatores de Risco , Humanos , Farmacogenética , Locos de Características QuantitativasRESUMO
Pluripotent stem cells can be derived from embryos or induced from adult cells by reprogramming. They are unique among stem cells in that they can give rise to all cell types of the body. Recent findings indicate that a particularly 'open' chromatin state contributes to maintenance of pluripotency. Two principles are emerging: specific factors maintain a globally open chromatin state that is accessible for transcriptional activation; and other chromatin regulators contribute locally to the silencing of lineage-specific genes until differentiation is triggered. These same principles may apply during reacquisition of an open chromatin state upon reprogramming to pluripotency, and during de-differentiation in cancer.
Assuntos
Reprogramação Celular/genética , Cromatina/genética , Células-Tronco Pluripotentes/fisiologia , Animais , Humanos , Modelos BiológicosRESUMO
Histone variants are emerging as key regulatory molecules in cancer. We report a unique role for the H2A.Z isoform H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 is highly expressed in metastatic melanoma, correlates with decreased patient survival, and is required for cellular proliferation. Our integrated genomic analyses reveal that H2A.Z.2 controls the transcriptional output of E2F target genes in melanoma cells. These genes are highly expressed and display a distinct signature of H2A.Z occupancy. We identify BRD2 as an H2A.Z-interacting protein, levels of which are also elevated in melanoma. We further demonstrate that H2A.Z.2-regulated genes are bound by BRD2 and E2F1 in an H2A.Z.2-dependent manner. Importantly, H2A.Z.2 deficiency sensitizes melanoma cells to chemotherapy and targeted therapies. Collectively, our findings implicate H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma, holding translational potential for novel therapeutic strategies.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição E2F1/genética , Histonas/genética , Melanoma/genética , Proteínas Serina-Treonina Quinases/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Fator de Transcrição E2F1/metabolismo , Células HeLa , Histonas/biossíntese , Humanos , Melanócitos/citologia , Melanoma/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Pontos de Checagem da Fase S do Ciclo Celular/genética , Análise de Sequência de RNA , Fatores de Transcrição , Ativação TranscricionalRESUMO
The transcription factor GLI1 (GLI family zinc finger 1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-seq and ATAC-seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene HHIP (Hedgehog-interacting protein) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies.
Assuntos
Cromatina/genética , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Proteína GLI1 em Dedos de Zinco/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Células HEK293 , Humanos , Domínios Proteicos , Mapas de Interação de Proteínas , Fatores de Transcrição/química , Ativação Transcricional , Proteína GLI1 em Dedos de Zinco/químicaRESUMO
An open chromatin largely devoid of heterochromatin is a hallmark of stem cells. It remains unknown whether an open chromatin is necessary for the differentiation potential of stem cells, and which molecules are needed to maintain open chromatin. Here we show that the chromatin remodelling factor Chd1 is required to maintain the open chromatin of pluripotent mouse embryonic stem cells. Chd1 is a euchromatin protein that associates with the promoters of active genes, and downregulation of Chd1 leads to accumulation of heterochromatin. Chd1-deficient embryonic stem cells are no longer pluripotent, because they are incapable of giving rise to primitive endoderm and have a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. Our results indicate that Chd1 is essential for open chromatin and pluripotency of embryonic stem cells, and for somatic cell reprogramming to the pluripotent state.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Eucromatina/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Biomarcadores , Proliferação de Células , Células Cultivadas , Reprogramação Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endoderma/metabolismo , Eucromatina/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas/genética , Interferência de RNARESUMO
Patient-derived cancer organoids (PDOs) hold considerable promise for personalizing therapy selection and improving patient outcomes. However, it is challenging to generate PDOs in sufficient numbers to test therapies in standard culture platforms. This challenge is particularly acute for pancreatic ductal adenocarcinoma (PDAC) where most patients are diagnosed at an advanced stage with non-resectable tumors and where patient tissue is in the form of needle biopsies. Here the development and characterization of microfluidic devices for testing therapies using a limited amount of tissue or PDOs available from PDAC biopsies is described. It is demonstrated that microfluidic PDOs are phenotypically and genotypically similar to the gold-standard Matrigel organoids with the advantages of 1) spheroid uniformity, 2) minimal cell number requirement, and 3) not relying on Matrigel. The utility of microfluidic PDOs is proven by testing PDO responses to several chemotherapies, including an inhibitor of glycogen synthase kinase (GSKI). In addition, microfluidic organoid cultures are used to test effectiveness of immunotherapy comprised of NK cells in combination with a novel biologic. In summary, our microfluidic device offers considerable benefits for personalizing oncology based on cancer biopsies and may, in the future, be developed into a companion diagnostic for chemotherapy or immunotherapy treatments.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Microfluídica , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/tratamento farmacológico , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/tratamento farmacológico , Imunoterapia , Biópsia , Organoides/patologiaRESUMO
The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.
Assuntos
Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Proteômica , Transdução de Sinais , Análise de Célula Única , Análise de Célula Única/métodos , Humanos , Espectrometria de Massas/métodos , Proteômica/métodos , Organelas/metabolismo , Proteoma/metabolismoRESUMO
The snATAC + snRNA platform allows epigenomic profiling of open chromatin and gene expression with single-cell resolution. The most critical assay step is to isolate high-quality nuclei to proceed with droplet-base single nuclei isolation and barcoding. With the increasing popularity of multiomic profiling in various fields, there is a need for optimized and reliable nuclei isolation methods, mainly for human tissue samples. Herein we compared different nuclei isolation methods for cell suspensions, such as peripheral blood mononuclear cells (PBMC, n = 18) and a solid tumor type, ovarian cancer (OC, n = 18), derived from debulking surgery. Nuclei morphology and sequencing output parameters were used to evaluate the quality of preparation. Our results show that NP-40 detergent-based nuclei isolation yields better sequencing results than collagenase tissue dissociation for OC, significantly impacting cell type identification and analysis. Given the utility of applying such techniques to frozen samples, we also tested frozen preparation and digestion (n = 6). A paired comparison between frozen and fresh samples validated the quality of both specimens. Finally, we demonstrate the reproducibility of scRNA and snATAC + snRNA platform, by comparing the gene expression profiling of PBMC. Our results highlight how the choice of nuclei isolation methods is critical for obtaining quality data in multiomic assays. It also shows that the measurement of expression between scRNA and snRNA is comparable and effective for cell type identification.
Assuntos
Epigenômica , Leucócitos Mononucleares , Humanos , Multiômica , Reprodutibilidade dos Testes , RNA Nuclear Pequeno/genéticaRESUMO
Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis-regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined 'macro-Bound Enhancers', that modulate enhancer activity. We find macroH2A variants localized at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and their repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling of normal mammary stem cells derived from mice, we show that macroH2A deficiency facilitates increased activity of transcription factors associated with stem cell activity.
Assuntos
Proteínas Nucleares , Fatores de Transcrição , Camundongos , Animais , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Reprogramação Celular/genética , Elementos Facilitadores Genéticos , Cromatina/genéticaRESUMO
PURPOSE: Preclinical studies in myeloid neoplasms have demonstrated efficacy of bromodomain and extra-terminal protein inhibitors (BETi). However, BETi demonstrates poor single-agent activity in clinical trials. Several studies suggest that combination with other anticancer inhibitors may enhance the efficacy of BETi. EXPERIMENTAL DESIGN: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and patient-derived xenograft models of disease. We used standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models. RESULTS: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Furthermore, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy. CONCLUSIONS: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data support further clinical investigation of this combination.
Assuntos
Leucemia Mielomonocítica Crônica , MicroRNAs , Humanos , Linhagem Celular Tumoral , Proteínas , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1MT) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1MT in chronic myelomonocytic leukemia (CMML). ASXL1MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions.
Assuntos
Leucemia Mielomonocítica Crônica , Epigênese Genética , Expressão Gênica , Humanos , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/patologia , Mutação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Linhagem Celular Tumoral , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/uso terapêutico , Peptidase 7 Específica de Ubiquitina/metabolismoRESUMO
Circadian rhythm disruption (CD) is associated with impaired glucose homeostasis and type 2 diabetes mellitus (T2DM). While the link between CD and T2DM remains unclear, there is accumulating evidence that disruption of fasting/feeding cycles mediates metabolic dysfunction. Here, we used an approach encompassing analysis of behavioral, physiological, transcriptomic, and epigenomic effects of CD and consequences of restoring fasting/feeding cycles through time-restricted feeding (tRF) in mice. Results show that CD perturbs glucose homeostasis through disruption of pancreatic ß cell function and loss of circadian transcriptional and epigenetic identity. In contrast, restoration of fasting/feeding cycle prevented CD-mediated dysfunction by reestablishing circadian regulation of glucose tolerance, ß cell function, transcriptional profile, and reestablishment of proline and acidic amino acidrich basic leucine zipper (PAR bZIP) transcription factor DBP expression/activity. This study provides mechanistic insights into circadian regulation of ß cell function and corresponding beneficial effects of tRF in prevention of T2DM.
RESUMO
Understanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem (ES) cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cells and identified genes upregulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle, and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved overrepresented motifs in genes upregulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and embryonic germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species.
Assuntos
Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/metabolismo , Sequências Reguladoras de Ácido Nucleico , Animais , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/fisiologia , Linhagem Celular , Proliferação de Células , Células Cultivadas , Biologia Computacional/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Família Multigênica , Células NIH 3T3 , Análise de Sequência com Séries de OligonucleotídeosRESUMO
There is increasing interest in utilizing in vitro cultures as patient avatars to develop personalized treatment for cancer. Typical cultures utilize Matrigel-coated plates and media to promote the proliferation of cancer cells as spheroids or tumor explants. However, standard culture conditions operate in large volumes and require a high concentration of cancer cells to initiate this process. Other limitations include variability in the ability to successfully establish a stable line and inconsistency in the dimensions of these microcancers for in vivo drug response measurements. This paper explored the utility of microfluidics in the cultivation of cancer cell spheroids. Six patient-derived xenograft (PDX) tumors of high-grade serous ovarian cancer were used as the source material to demonstrate that viability and epithelial marker expression in the microfluidic cultures was superior to that of Matrigel or large volume 3D cultures. To further demonstrate the potential for miniaturization and multiplexing, we fabricated multichamber microfluidic devices with integrated microvalves to enable serial seeding of several chambers followed by parallel testing of several drug concentrations. These valve-enabled microfluidic devices permitted the formation of spheroids and testing of seven drug concentrations with as few as 100,000 cancer cells per device. Overall, we demonstrate the feasibility of maintaining difficul-to-culture primary cancer cells and testing drugs in a microfluidic device. This microfluidic platform may be ideal for drug testing and personalized therapy when tumor material is limited, such as following the acquisition of biopsy specimens obtained by fine-needle aspiration.
RESUMO
BACKGROUND: The three-dimensional genome organization is critical for gene regulation and can malfunction in diseases like cancer. As a key regulator of genome organization, CCCTC-binding factor (CTCF) has been characterized as a DNA-binding protein with important functions in maintaining the topological structure of chromatin and inducing DNA looping. Among the prolific binding sites in the genome, several events with altered CTCF occupancy have been reported as associated with effects in physiology or disease. However, hitherto there is no comprehensive survey of genome-wide CTCF binding patterns across different human cancers. RESULTS: To dissect functions of CTCF binding, we systematically analyze over 700 CTCF ChIP-seq profiles across human tissues and cancers and identify cancer-specific CTCF binding patterns in six cancer types. We show that cancer-specific lost and gained CTCF binding events are associated with altered chromatin interactions, partially with DNA methylation changes, and rarely with sequence mutations. While lost bindings primarily occur near gene promoters, most gained CTCF binding events exhibit enhancer activities and are induced by oncogenic transcription factors. We validate these findings in T cell acute lymphoblastic leukemia cell lines and patient samples and show that oncogenic NOTCH1 induces specific CTCF binding and they cooperatively activate expression of target genes, indicating transcriptional condensation phenomena. CONCLUSIONS: Specific CTCF binding events occur in human cancers. Cancer-specific CTCF binding can be induced by other transcription factors to regulate oncogenic gene expression. Our results substantiate CTCF binding alteration as a functional epigenomic signature of cancer.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Metilação de DNA , Humanos , Oncogenes , Receptor Notch1/metabolismoRESUMO
The histone variant macroH2A occupies large repressive domains throughout the genome; however, mechanisms underlying its precise deposition remain poorly understood. Here, we characterize de novo chromatin deposition of macroH2A2 using temporal genomic profiling in murine-derived fibroblasts devoid of all macroH2A isoforms. We find that macroH2A2 is first pervasively deposited genome wide at both steady-state domains and adjacent transcribed regions, the latter of which are subsequently pruned, establishing mature macroH2A2 domains. Pruning of macroH2A2 can be counteracted by chemical inhibition of transcription. Further, locus-specific transcriptional manipulation reveals that gene activation depletes pre-existing macroH2A2, while silencing triggers ectopic macroH2A2 accumulation. We demonstrate that the FACT (facilitates chromatin transcription) complex is required for macroH2A2 pruning within transcribed chromatin. Taken together, we have identified active chromatin as a boundary for macroH2A domains through a transcription-associated 'pruning' mechanism that establishes and maintains the faithful genomic localization of macroH2A variants.
Assuntos
Cromatina/metabolismo , Histonas/fisiologia , Transcrição Gênica , Animais , Cromatina/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Histonas/química , Histonas/metabolismo , Masculino , Camundongos , Modelos MolecularesAssuntos
Dioxigenases , Proteogenômica , Humanos , Proteínas de Ligação a DNA/genética , Leucocitose , Hematopoese/genética , MutaçãoRESUMO
Perkinsus atlanticus is a pathogenic protist that infects the clam Ruditapes decussatus. Although it was recently proposed that the genus Perkinsus belongs to a new phylum, Perkinsozoa, in the infra-kingdom Alveolata, there remain different opinions about whether this genus should form a phylum on its own and consequently divergent views about its taxonomic characterization. In this work, we have identified nine chromosomes by pulsed field gel electrophoresis (PFGE) combined with densitometry analysis. The obtained karyotype of Perkinsus atlanticus, like that of other early branches of the dinoflagellate lineage, displays a more conventional chromosome organization, different from that of most dinoflagellates.
Assuntos
Eletroforese em Gel de Campo Pulsado/veterinária , Eucariotos/genética , Cariotipagem/veterinária , Animais , DNA de Protozoário/análise , Densitometria , Eletroforese em Gel de Campo Pulsado/métodos , Cariotipagem/métodosRESUMO
Chromatin-mediated processes influence the development and progression of breast cancer. Using murine mammary carcinoma-derived tumorspheres as a functional readout for an aggressive breast cancer phenotype, we performed a loss-of-function screen targeting 60 epigenetic regulators. We identified the Polycomb protein Cbx8 as a key regulator of mammary carcinoma both in vitro and in vivo. Accordingly, Cbx8 is overexpressed in human breast cancer and correlates with poor survival. Our genomic analyses revealed that Cbx8 positively regulates Notch signaling by maintaining H3K4me3 levels on Notch-network gene promoters. Ectopic expression of Notch1 partially rescues tumorsphere formation in Cbx8-depleted cells. We find that Cbx8 associates with non-PRC1 complexes containing the H3K4 methyltransferase complex component WDR5, which together regulate Notch gene expression. Thus, our study implicates a key non-canonical role for Cbx8 in promoting breast tumorigenesis.