RESUMO
Cachexia is a major cause of morbidity and mortality in individuals with cancer and is characterized by weight loss due to adipose and muscle tissue wasting. Hallmarks of white adipose tissue (WAT) remodeling, which often precedes weight loss, are impaired lipid storage, inflammation and eventually fibrosis. Tissue wasting occurs in response to tumor-secreted factors. Considering that the continuous endothelium in WAT is the first line of contact with circulating factors, we postulated whether the endothelium itself may orchestrate tissue remodeling. Here, we show using human and mouse cancer models that during precachexia, tumors overactivate Notch1 signaling in distant WAT endothelium. Sustained endothelial Notch1 signaling induces a WAT wasting phenotype in male mice through excessive retinoic acid production. Pharmacological blockade of retinoic acid signaling was sufficient to inhibit WAT wasting in a mouse cancer cachexia model. This demonstrates that cancer manipulates the endothelium at distant sites to mediate WAT wasting by altering angiocrine signals.
Assuntos
Tecido Adiposo Branco , Caquexia , Neoplasias , Receptor Notch1 , Animais , Humanos , Masculino , Camundongos , Tecido Adiposo Branco/patologia , Caquexia/patologia , Neoplasias/complicações , Transdução de Sinais , Tretinoína , Receptor Notch1/metabolismoRESUMO
The Notch pathway transmits signals between neighboring cells to elicit downstream transcriptional programs. Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The pathway signals through the transcription factor CSL (RBPJ in mammals), which forms an activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified L3MBTL3 as a bona fide RBPJ-binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here, we define the RBPJ-interacting domain of L3MBTL3 and report the 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA complex. The structure reveals that L3MBTL3 interacts with RBPJ via an unusual binding motif compared to other RBPJ binding partners, which we comprehensively analyze with a series of structure-based mutants. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.
Assuntos
Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Histona Desmetilases/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/genética , Ligação Proteica , Receptores Notch/genética , Receptores Notch/metabolismoRESUMO
Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic cancers worldwide. EOC cells educate tumor-associated macrophages (TAM) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumor microenvironment (TME). In addition, tumor cells frequently activate Notch1 receptors on endothelial cells (EC) to facilitate metastasis. However, further work is required to establish whether the endothelium also influences the education of recruited monocytes. Here, we report that canonical Notch signaling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature; this was associated with stronger cytotoxic activity of T cells and decreased tumor burden. Mechanistically, CXCL2 was identified as a novel Notch/RBPJ target gene that regulated the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. Together, these findings indicate that EOC cells induce the tumor endothelium to secrete CXCL2 to establish an immunosuppressive microenvironment. SIGNIFICANCE: Endothelial Notch signaling favors immunosuppression by increasing CXCL2 secretion to stimulate CD44 expression in macrophages, facilitating their education by tumor cells.
Assuntos
Neoplasias Ovarianas , Macrófagos Associados a Tumor , Humanos , Feminino , Camundongos , Animais , Células Endoteliais/patologia , Carcinoma Epitelial do Ovário/genética , Neoplasias Ovarianas/patologia , Microambiente Tumoral , Endotélio/metabolismo , Colesterol , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genéticaRESUMO
Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor - the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.
Assuntos
Receptor Notch1 , Hipóxia Celular , Humanos , Hidroxilação , Oxigenases de Função Mista/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Enzymes, such as histone methyltransferases and demethylases, histone acetyltransferases and deacetylases, and DNA methyltransferases are known as epigenetic modifiers that are often implicated in tumorigenesis and disease. One of the best-studied chromatin-based mechanism is X chromosome inactivation (XCI), a process that establishes facultative heterochromatin on only one X chromosome in females and establishes the right dosage of gene expression. The specificity factor for this process is the long non-coding RNA Xinactivespecifictranscript (Xist), which is upregulated from one X chromosome in female cells. Subsequently, Xist is bound by the corepressor SHARP/SPEN, recruiting and/or activating histone deacetylases (HDACs), leading to the loss of active chromatin marks such as H3K27ac. In addition, polycomb complexes PRC1 and PRC2 establish wide-spread accumulation of H3K27me3 and H2AK119ub1 chromatin marks. The lack of active marks and establishment of repressive marks set the stage for DNA methyltransferases (DNMTs) to stably silence the X chromosome. Here, we will review the recent advances in understanding the molecular mechanisms of how heterochromatin formation is established and put this into the context of carcinogenesis and disease.
RESUMO
The Notch signal transduction cascade requires cell-to-cell contact and results in the proteolytic processing of the Notch receptor and subsequent assembly of a transcriptional coactivator complex containing the Notch intracellular domain (NICD) and transcription factor RBPJ. In the absence of a Notch signal, RBPJ remains at Notch target genes and dampens transcriptional output. Like in other signaling pathways, RBPJ is able to switch from activation to repression by associating with corepressor complexes containing several chromatin-modifying enzymes. Here, we focus on the recent advances concerning RBPJ-corepressor functions, especially in regard to chromatin regulation. We put this into the context of one of the best-studied model systems for Notch, blood cell development. Alterations in the RBPJ-corepressor functions can contribute to the development of leukemia, especially in the case of acute myeloid leukemia (AML). The versatile role of transcription factor RBPJ in regulating pivotal target genes like c-MYC and HES1 may contribute to the better understanding of the development of leukemia.
Assuntos
Regulação da Expressão Gênica , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Receptores Notch/metabolismo , Cromatina/genética , Cromatina/metabolismo , Humanos , Transdução de SinaisRESUMO
Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.
Assuntos
Histona Desacetilases/metabolismo , Leucemia/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Humanos , Leucemia/enzimologia , Lisina/metabolismo , Camundongos , Mutação , Peptídeos Cíclicos/farmacologia , Estabilidade Proteica , Receptor Notch1/química , Receptor Notch1/genéticaAssuntos
Processamento Alternativo/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , Isoformas de Proteínas/genética , Proteína 1 Parceira de Translocação de RUNX1/genética , Translocação Genética/genética , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The histone variant H2A.Z is involved in several processes such as transcriptional control, DNA repair, regulation of centromeric heterochromatin and, not surprisingly, is implicated in diseases such as cancer. Here, we review the recent developments on H2A.Z focusing on its role in transcriptional activation and repression. H2A.Z, as a replication-independent histone, has been studied in several model organisms and inducible mammalian model systems. Its loading machinery and several modifying enzymes have been recently identified, and some of the long-standing discrepancies in transcriptional activation and/or repression are about to be resolved. The buffering functions of H2A.Z, as supported by genome-wide localization and analyzed in several dynamic systems, are an excellent example of transcriptional control. Posttranslational modifications such as acetylation and ubiquitination of H2A.Z, as well as its specific binding partners, are in our view central players in the control of gene expression. Understanding the key-mechanisms in either turnover or stabilization of H2A.Z-containing nucleosomes as well as defining the H2A.Z interactome will pave the way for therapeutic applications in the future.
Assuntos
Regulação da Expressão Gênica , Histonas/genética , Acetilação , Adenosina Trifosfatases/metabolismo , Animais , Reparo do DNA , Heterocromatina , Histonas/metabolismo , Humanos , Nucleossomos , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Ativação Transcricional , UbiquitinaçãoRESUMO
Sufficient vascular supply is indispensable for brain development and function, whereas dysfunctional blood vessels are associated with human diseases such as vascular malformations, stroke or neurodegeneration. Pericytes are capillary-associated mesenchymal cells that limit vascular permeability and protect the brain by preserving blood-brain barrier integrity. Loss of pericytes has been linked to neurodegenerative changes in genetically modified mice. Here, we report that postnatal inactivation of the Rbpj gene, encoding the transcription factor RBPJ, leads to alteration of cell identity markers in brain pericytes, increases local TGFß signalling, and triggers profound changes in endothelial behaviour. These changes, which are not mimicked by pericyte ablation, imperil vascular stability and induce the acquisition of pathological landmarks associated with cerebral cavernous malformations. In adult mice, loss of Rbpj results in bigger stroke lesions upon ischemic insult. We propose that brain pericytes can acquire deleterious properties that actively enhance vascular lesion formation and promote pathogenic processes.
Assuntos
Encéfalo/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/deficiência , Pericitos/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Progressão da Doença , Feminino , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Masculino , Camundongos KnockoutRESUMO
The Notch signaling pathway plays a pivotal role in development, physiology and diseases such as cancer. In this chapter, we first give an overview of the different molecular mechanisms that regulate Notch signaling. Each subject is covered in more depth in the subsequent chapters of this book. Next, we will use the inflammatory system as an example to discuss the physiological function of Notch signaling. This is followed by a discussion of recent advances in the different pathophysiological roles of Notch signaling in leukemia as well as a wide range of solid cancers. Finally, we discuss how information about pathogenic mutations in Notch pathway components, combined with structural biological data, are beginning to provide important biological and mechanistic insights about the pathway.
Assuntos
Leucemia , Proteínas de Neoplasias , Receptores Notch , Transdução de Sinais/genética , Animais , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismoRESUMO
A fundamental as yet incompletely understood feature of Notch signal transduction is a transcriptional shift from repression to activation that depends on chromatin regulation mediated by transcription factor RBP-J and associated cofactors. Incorporation of histone variants alter the functional properties of chromatin and are implicated in the regulation of gene expression. Here, we show that depletion of histone variant H2A.Z leads to upregulation of canonical Notch target genes and that the H2A.Z-chaperone TRRAP/p400/Tip60 complex physically associates with RBP-J at Notch-dependent enhancers. When targeted to RBP-J-bound enhancers, the acetyltransferase Tip60 acetylates H2A.Z and upregulates Notch target gene expression. Importantly, the Drosophila homologs of Tip60, p400 and H2A.Z modulate Notch signaling response and growth in vivo. Together, our data reveal that loading and acetylation of H2A.Z are required to assure tight control of canonical Notch activation.
Assuntos
Regulação da Expressão Gênica , Histonas/genética , Receptores Notch/genética , Transdução de Sinais/genética , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Lisina Acetiltransferase 5/genética , Lisina Acetiltransferase 5/metabolismo , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismoRESUMO
OBJECTIVES: Enolase-1-dependent cell surface proteolysis plays an important role in cell invasion. Although enolase-1 (Eno-1), a glycolytic enzyme, has been found on the surface of various cells, the mechanism responsible for its exteriorization remains elusive. Here, we investigated the involvement of post-translational modifications (PTMs) of Eno-1 in its lipopolysaccharide (LPS)-triggered trafficking to the cell surface. RESULTS: We found that stimulation of human lung adenocarcinoma cells with LPS triggered the monomethylation of arginine 50 (R50me) within Eno-1. The Eno-1R50me was confirmed by its interaction with the tudor domain (TD) from TD-containing 3 (TDRD3) protein recognizing methylarginines. Substitution of R50 with lysine (R50K) reduced Eno-1 association with epithelial caveolar domains, thereby diminishing its exteriorization. Similar effects were observed when pharmacological inhibitors of arginine methyltransferases were applied. Protein arginine methyltransferase 5 (PRMT5) was identified to be responsible for Eno-1 methylation. Overexpression of PRMT5 and caveolin-1 enhanced levels of membrane-bound extracellular Eno-1 and, conversely, pharmacological inhibition of PRMT5 attenuated Eno-1 cell-surface localization. Importantly, Eno-1R50me was essential for cancer cell motility since the replacement of Eno-1 R50 by lysine or the suppression of PRMT 5 activity diminished Eno-1-triggered cell invasion. CONCLUSIONS: LPS-triggered Eno-1R50me enhances Eno-1 cell surface levels and thus potentiates the invasive properties of cancer cells. Strategies to target Eno-1R50me may offer novel therapeutic approaches to attenuate tumor metastasis in cancer patients.
Assuntos
Adenocarcinoma/enzimologia , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pulmonares/enzimologia , Proteínas de Neoplasias/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Biomarcadores Tumorais/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Lipopolissacarídeos/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/genética , Fosfopiruvato Hidratase/genética , Transporte Proteico/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Histona Desmetilases/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Neuroglia/metabolismo , Receptores Notch/genética , Animais , Evolução Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Neuroglia/citologia , Ligação Proteica , Domínios Proteicos , Receptores Notch/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Endothelial sprouting and proliferation are tightly coordinated processes mediating the formation of new blood vessels during physiological and pathological angiogenesis. Endothelial tip cells lead sprouts and are thought to suppress tip-like behaviour in adjacent stalk endothelial cells by activating Notch. Here, we show with genetic experiments in postnatal mice that the level of active Notch signalling is more important than the direct Dll4-mediated cell-cell communication between endothelial cells. We identify endothelial expression of VEGF-A and of the chemokine receptor CXCR4 as key processes controlling Notch-dependent vessel growth. Surprisingly, genetic experiments targeting endothelial tip cells in vivo reveal that they retain their function without Dll4 and are also not replaced by adjacent, Dll4-positive cells. Instead, activation of Notch directs tip-derived endothelial cells into developing arteries and thereby establishes that Dll4-Notch signalling couples sprouting angiogenesis and artery formation.
Assuntos
Células Endoteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica , Receptor Notch1/metabolismo , Artéria Retiniana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Comunicação Celular , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Proliferação de Células , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Genótipo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Receptor Notch1/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Artéria Retiniana/citologia , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. The infection process involves bacterial cell surface receptors, which interact with host extracellular matrix components to facilitate colonization and dissemination of bacteria. Here, we investigated the role of host-derived extracellular RNA (eRNA) in the process of pneumococcal alveolar epithelial cell infection. Our study demonstrates that eRNA dose-dependently increased S. pneumoniae invasion of alveolar epithelial cells. Extracellular enolase (Eno), a plasminogen (Plg) receptor, was identified as a novel eRNA-binding protein on S. pneumoniae surface, and six Eno eRNA-binding sites including a C-terminal 15 amino acid motif containing lysine residue 434 were characterized. Although the substitution of lysine 434 for glycine (K434G) markedly diminished the binding of eRNA to Eno, the adherence to and internalization into alveolar epithelial cells of S. pneumoniae strain carrying the C-terminal lysine deletion and the mutation of internal Plg-binding motif were only marginally impaired. Accordingly, using a mass spectrometric approach, we identified seven novel eRNA-binding proteins in pneumococcal cell wall. Given the high number of eRNA-interacting proteins on pneumococci, treatment with RNase1 completely inhibited eRNA-mediated pneumococcal alveolar epithelial cell infection. Our data support further efforts to employ RNAse1 as an antimicrobial agent to combat pneumococcal infectious diseases.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Células Endoteliais/microbiologia , Células Epiteliais/microbiologia , Espaço Extracelular/metabolismo , RNA/farmacologia , Streptococcus pneumoniae/citologia , Células A549 , Motivos de Aminoácidos , Animais , Sítios de Ligação , Bovinos , DNA/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Glicina/metabolismo , Humanos , Pulmão/patologia , Lisina/metabolismo , Mutação/genética , Nucleotídeos/metabolismo , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/patologia , Ribonuclease Pancreático/metabolismo , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2ß. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier.
Assuntos
Cromatina/metabolismo , Proteínas Correpressoras/metabolismo , Regulação da Expressão Gênica , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Transcrição Gênica , Animais , Caseína Quinase II/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Código das Histonas , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Proteínas Nucleares/química , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA , Xenopus laevisRESUMO
Notch signaling is a highly conserved signal transduction pathway that regulates stem cell maintenance and differentiation in several organ systems. Upon activation, the Notch receptor is proteolytically processed, its intracellular domain (NICD) translocates into the nucleus and activates expression of target genes. Output, strength and duration of the signal are tightly regulated by post-translational modifications. Here we review the intracellular post-translational regulation of Notch that fine-tunes the outcome of the Notch response. We also describe how crosstalk with other conserved signaling pathways like the Wnt, Hedgehog, hypoxia and TGFß/BMP pathways can affect Notch signaling output. This regulation can happen by regulation of ligand, receptor or transcription factor expression, regulation of protein stability of intracellular key components, usage of the same cofactors or coregulation of the same key target genes. Since carcinogenesis is often dependent on at least two of these pathways, a better understanding of their molecular crosstalk is pivotal.