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1.
PLoS Genet ; 18(8): e1010335, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35951645

RESUMO

Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Proteínas Correpressoras , DNA , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Displasia Ectodérmica , Humanos , Deformidades Congênitas dos Membros , Mamíferos/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Couro Cabeludo/metabolismo , Dermatoses do Couro Cabeludo/congênito , Crânio/metabolismo
2.
Nucleic Acids Res ; 50(22): 13083-13099, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36477367

RESUMO

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/metabolismo
3.
PLoS Genet ; 17(11): e1009890, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34723970

RESUMO

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.


Assuntos
Coração/fisiologia , Histona Desacetilase 1/metabolismo , Miócitos Cardíacos/citologia , Regeneração/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proliferação de Células
4.
Gut ; 72(5): 821-833, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-35613844

RESUMO

OBJECTIVE: Disruption of the epithelial barrier plays an essential role in developing eosinophilic oesophagitis (EoE), a disease defined by type 2 helper T cell (Th2)-mediated food-associated and aeroallergen-associated chronic inflammation. Although an increased expression of interleukin (IL)-20 subfamily members, IL-19, IL-20 and IL-24, in Th2-mediated diseases has been reported, their function in EoE remains unknown. DESIGN: Combining transcriptomic, proteomic and functional analyses, we studied the importance of the IL-20 subfamily for EoE using patient-derived oesophageal three-dimensional models and an EoE mouse model. RESULTS: Patients with active EoE have increased expression of IL-20 subfamily cytokines in the oesophagus and serum. In patient-derived oesophageal organoids stimulated with IL-20 cytokines, RNA sequencing and mass spectrometry revealed a downregulation of genes and proteins forming the cornified envelope, including filaggrins. On the contrary, abrogation of IL-20 subfamily signalling in Il20R2 -/- animals resulted in attenuated experimental EoE reflected by reduced eosinophil infiltration, lower Th2 cytokine expression and preserved expression of filaggrins in the oesophagus. Mechanistically, these observations were mediated by the mitogen-activated protein kinase (MAPK); extracellular-signal regulated kinases (ERK)1/2) pathway. Its blockade prevented epithelial barrier impairment in patient-derived air-liquid interface cultures stimulated with IL-20 cytokines and attenuated experimental EoE in mice. CONCLUSION: Our findings reveal a previously unknown regulatory role of the IL-20 subfamily for oesophageal barrier function in the context of EoE. We propose that aberrant IL-20 subfamily signalling disturbs the oesophageal epithelial barrier integrity and promotes EoE development. Our study suggests that specific targeting of the IL-20 subfamily signalling pathway may present a novel strategy for the treatment of EoE.


Assuntos
Esofagite Eosinofílica , Animais , Camundongos , Citocinas/metabolismo , Proteínas Filagrinas , Interleucinas/farmacologia , Interleucinas/metabolismo , Proteômica , Humanos
5.
Nucleic Acids Res ; 48(7): 3496-3512, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32107550

RESUMO

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/genética
6.
J Transl Med ; 19(1): 237, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078403

RESUMO

BACKGROUND: Recently, increased interleukin (IL)-24 expression has been demonstrated in the colon biopsies of adult patients with inflammatory bowel disease (IBD). However, the role of IL-24 in the pathomechanism of IBD is still largely unknown. METHODS: Presence of IL-24 was determined in the samples of children with IBD and in the colon of dextran sodium sulfate (DSS) treated mice. Effect of inflammatory factors on IL24 expression was determined in peripheral blood (PBMCs) and lamina propria mononuclear cells (LPMCs). Also, the impact of IL-24 was investigated on HT-29 epithelial cells and CCD-18Co colon fibroblasts. Expression of tissue remodeling related genes was investigated in the colon of wild type (WT) mice locally treated with IL-24 and in the colon of DSS treated WT and Il20rb knock out (KO) mice. RESULTS: Increased amount of IL-24 was demonstrated in the serum and colon samples of children with IBD and DSS treated mice compared to that of controls. IL-1ß, LPS or H2O2 treatment increased the expression of IL24 in PBMCs and LPMCs. IL-24 treatment resulted in increased amount of TGF-ß and PDGF-B in HT-29 cells and enhanced the expression of extracellular matrix (ECM)-related genes and the motility of CCD-18Co cells. Similarly, local IL-24 treatment increased the colonic Tgfb1 and Pdgfb expression of WT mice. Moreover, expression of pro-fibrotic Tgfb1 and Pdgfb were lower in the colon of DSS treated Il20rb KO compared to that of WT mice. The disease activity index of colitis was less severe in DSS treated Il20rb KO compared to WT mice. CONCLUSION: Our study suggest that IL-24 may play a significant role in the mucosal remodeling of patients with IBD by promoting pro-fibrotic processes.


Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Colo , Citocinas , Sulfato de Dextrana , Modelos Animais de Doenças , Humanos , Peróxido de Hidrogênio , Interleucinas , Camundongos , Camundongos Endogâmicos C57BL , Mucosa
7.
Blood ; 133(8): 830-839, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30510140

RESUMO

NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.


Assuntos
Proteína 7 com Repetições F-Box-WD , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B , Mutação de Sentido Incorreto , Proteínas de Neoplasias , Receptor Notch1 , Substituição de Aminoácidos , Linhagem Celular Tumoral , Simulação por Computador , Proteína 7 com Repetições F-Box-WD/química , Proteína 7 com Repetições F-Box-WD/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Domínios Proteicos , Receptor Notch1/química , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais/genética
8.
J Biol Chem ; 294(31): 11741-11750, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31186352

RESUMO

The Notch receptor is a key mediator of developmental programs and cell-fate decisions. Imbalanced Notch signaling leads to developmental disorders and cancer. To fully characterize the Notch signaling pathway and exploit it in novel therapeutic interventions, a comprehensive view on the regulation and requirements of Notch signaling is needed. Notch is regulated at different levels, ranging from ligand binding, stability to endocytosis. Using an array of different techniques, including reporter gene assays, immunocytochemistry, and ChIP-qPCR we show here, to the best of our knowledge for the first time, regulation of Notch signaling at the level of the nuclear pore. We found that the nuclear pore protein Nup214 (nucleoporin 214) and its interaction partner Nup88 negatively regulate Notch signaling in vitro and in vivo in zebrafish. In mammalian cells, loss of Nup88/214 inhibited nuclear export of recombination signal-binding protein for immunoglobulin κJ region (RBP-J), the DNA-binding component of the Notch pathway. This inhibition increased binding of RBP-J to its cognate promoter regions, resulting in increased downstream Notch signaling. Interestingly, we also found that NUP214 fusion proteins, causative for certain cases of T-cell acute lymphatic leukemia, potentially contribute to tumorigenesis via a Notch-dependent mechanism. In summary, the nuclear pore components Nup88/214 suppress Notch signaling in vitro, and in zebrafish, nuclear RBP-J levels are rate-limiting factors for Notch signaling in mammalian cells, and regulation of nucleocytoplasmic transport of RBP-J may contribute to fine-tuning Notch activity in cells.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Tumoral , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Morfolinos/genética , Morfolinos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Notch/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição HES-1/antagonistas & inibidores , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
J Transl Med ; 18(1): 172, 2020 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-32306980

RESUMO

BACKGROUND: Recently, the role of IL-19, IL-20 and IL-24 has been reported in renal disorders. However, still little is known about their biological role. METHODS: Localization of IL-20RB was determined in human biopsies and in the kidneys of mice that underwent unilateral ureteral obstruction (UUO). Renal Il19, Il20 and Il24 expression was determined in ischemia/reperfusion, lipopolysaccharide, streptozotocin, or UUO induced animal models of kidney diseases. The effects of H2O2, LPS, TGF-ß1, PDGF-B and IL-1ß on IL19, IL20 and IL24 expression was determined in peripheral blood mononuclear cells (PBMCs). The extents of extracellular matrix (ECM) and α-SMA, Tgfb1, Pdgfb, and Ctgf expression were determined in the kidneys of Il20rb knockout (KO) and wild type (WT) mice following UUO. The effect of IL-24 was also examined on HK-2 tubular epithelial cells and NRK49F renal fibroblasts. RESULTS: IL-20RB was present in the renal biopsies of patients with lupus nephritis, IgA and diabetic nephropathy. Amount of IL-20RB increased in the kidneys of mice underwent UUO. The expression of Il19, Il20 and Il24 increased in the animal models of various kidney diseases. IL-1ß, H2O2 and LPS induced the IL19, IL20 and IL24 expression of PBMCs. The extent of ECM, α-SMA, fibronectin, Tgfb1, Pdgfb, and Ctgf expression was lower in the kidney of Il20rb KO compared to WT mice following UUO. IL-24 treatment induced the apoptosis and TGF-ß1, PDGF-B, CTGF expression of HK-2 cells. CONCLUSIONS: Our data confirmed the significance of IL-19, IL-20 and IL-24 in the pathomechanism of renal diseases. Furthermore, we were the first to demonstrate the pro-fibrotic effect of IL-24.


Assuntos
Nefropatias , Insuficiência Renal Crônica , Obstrução Ureteral , Animais , Modelos Animais de Doenças , Fibrose , Humanos , Peróxido de Hidrogênio , Rim/patologia , Nefropatias/patologia , Leucócitos Mononucleares , Camundongos , Insuficiência Renal Crônica/patologia , Obstrução Ureteral/patologia
10.
Nucleic Acids Res ; 46(16): 8197-8215, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-29986055

RESUMO

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/metabolismo
11.
PLoS Biol ; 14(7): e1002524, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27458807

RESUMO

Notch signaling is iteratively used throughout development to maintain stem cell potential or in other instances allow differentiation. The central transcription factor in Notch signaling is CBF-1/RBP-J, Su(H), Lag-1 (CSL)-Su(H) in Drosophila-which functions as a molecular switch between transcriptional activation and repression. Su(H) represses transcription by forming a complex with the corepressor Hairless (H). The Su(H)-repressor complex not only competes with the Notch intracellular domain (NICD) but also configures the local chromatin landscape. In this issue, Yuan and colleagues determined the structure of the Su(H)/H complex, showing that a major conformational change within Su(H) explains why the binding of NICD and H is mutually exclusive.


Assuntos
Proteínas de Drosophila/química , Drosophila , Animais , Proteínas de Ligação a DNA , Proteínas de Membrana , Receptores Notch/química , Proteínas Repressoras/química , Transdução de Sinais , Fatores de Transcrição/química
12.
J Biol Chem ; 292(25): 10549-10563, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28487372

RESUMO

The Notch pathway is a cell-to-cell signaling mechanism that is essential for tissue development and maintenance, and aberrant Notch signaling has been implicated in various cancers, congenital defects, and cardiovascular diseases. Notch signaling activates the expression of target genes, which are regulated by the transcription factor CSL (CBF1/RBP-J, Su(H), Lag-1). CSL interacts with both transcriptional corepressor and coactivator proteins, functioning as both a repressor and activator, respectively. Although Notch activation complexes are relatively well understood at the structural level, less is known about how CSL interacts with corepressors. Recently, a new RBP-J (mammalian CSL ortholog)-interacting protein termed RITA has been identified and shown to export RBP-J out of the nucleus, thereby leading to the down-regulation of Notch target gene expression. However, the molecular details of RBP-J/RITA interactions are unclear. Here, using a combination of biochemical/cellular, structural, and biophysical techniques, we demonstrate that endogenous RBP-J and RITA proteins interact in cells, map the binding regions necessary for RBP-J·RITA complex formation, and determine the X-ray structure of the RBP-J·RITA complex bound to DNA. To validate the structure and glean more insights into function, we tested structure-based RBP-J and RITA mutants with biochemical/cellular assays and isothermal titration calorimetry. Whereas our structural and biophysical studies demonstrate that RITA binds RBP-J similarly to the RAM (RBP-J-associated molecule) domain of Notch, our biochemical and cellular assays suggest that RITA interacts with additional regions in RBP-J. Taken together, these results provide molecular insights into the mechanism of RITA-mediated regulation of Notch signaling, contributing to our understanding of how CSL functions as a transcriptional repressor of Notch target genes.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/química , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Células A549 , Animais , Cristalografia por Raios X , DNA , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas de Neoplasias/genética , Ligação Proteica , Domínios Proteicos , Relação Estrutura-Atividade
13.
Nucleic Acids Res ; 44(10): 4703-20, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-26912830

RESUMO

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 laevis
14.
Adv Exp Med Biol ; 1066: 279-295, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30030832

RESUMO

The highly conserved Notch signal transduction pathway orchestrates fundamental cellular processes including, differentiation, proliferation, and apoptosis during embryonic development and in the adult organism. Dysregulated Notch signaling underlies the etiology of a variety of human diseases, such as certain types of cancers, developmental disorders and cardiovascular disease. Ligand binding induces proteolytic cleavage of the Notch receptor and nuclear translocation of the Notch intracellular domain (NICD), which forms a ternary complex with the transcription factor CSL and the coactivator MAML to upregulate transcription of Notch target genes. The DNA-binding protein CSL is the centrepiece of transcriptional regulation in the Notch pathway, acting as a molecular hub for interactions with either corepressors or coactivators to repress or activate, respectively, transcription. Here we review previous structure-function studies of CSL-associated coregulator complexes and discuss the molecular insights gleaned from this research. We discuss the functional consequences of both activating and repressing binding partners using the same interaction platforms on CSL. We also emphasize that although there has been a significant uptick in structural information over the past decade, it is still under debate how the molecular switch from repression to activation mediated by CSL occurs at Notch target genes and whether it will be possible to manipulate these transcription complexes therapeutically in the future.


Assuntos
Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Complexos Multiproteicos/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Transcrição Gênica/fisiologia , Animais , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/química , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Domínios Proteicos , Receptores Notch/química , Receptores Notch/genética , Relação Estrutura-Atividade
15.
Genes Dev ; 24(6): 590-601, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20231316

RESUMO

Timely acquisition of cell fates and the elaborate control of growth in numerous organs depend on Notch signaling. Upon ligand binding, the core transcription factor RBP-J activates transcription of Notch target genes. In the absence of signaling, RBP-J switches off target gene expression, assuring the tight spatiotemporal control of the response by a mechanism incompletely understood. Here we show that the histone demethylase KDM5A is an integral, conserved component of Notch/RBP-J gene silencing. Methylation of histone H3 Lys 4 is dynamically erased and re-established at RBP-J sites upon inhibition and reactivation of Notch signaling. KDM5A interacts physically with RBP-J; this interaction is conserved in Drosophila and is crucial for Notch-induced growth and tumorigenesis responses.


Assuntos
Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Receptores Notch/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Camundongos , Linfócitos T , Ubiquitina-Proteína Ligases
16.
Int J Mol Sci ; 19(12)2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30501048

RESUMO

Liver cholestasis is a chronic liver disease and a major health problem worldwide. Cholestasis is characterised by a decrease in bile flow due to impaired secretion by hepatocytes or by obstruction of bile flow through intra- or extrahepatic bile ducts. Thereby cholestasis can induce ductal proliferation, hepatocyte injury and liver fibrosis. Notch signalling promotes the formation and maturation of bile duct structures. Here we investigated the liver regeneration process in the context of cholestasis induced by disruption of the Notch signalling pathway. Liver-specific deletion of recombination signal binding protein for immunoglobulin kappa j region (Rbpj), which represents a key regulator of Notch signalling, induces severe cholestasis through impaired intra-hepatic bile duct (IHBD) maturation, severe necrosis and increased lethality. Deregulation of the biliary compartment and cholestasis are associated with the change of several signalling pathways including a Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set representing the Hippo pathway, further yes-associated protein (YAP) activation and upregulation of SRY (sex determining region Y)-box 9 (SOX9), which is associated with transdifferentiation of hepatocytes. SOX9 upregulation in cholestatic liver injury in vitro is independent of Notch signalling. We could comprehensively address that in vivo Rbpj depletion is followed by YAP activation, which influences the transdifferentiation of hepatocytes and thereby contributing to liver regeneration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colestase/metabolismo , Regeneração Hepática/fisiologia , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ductos Biliares/metabolismo , Ductos Biliares/fisiologia , Western Blotting , Proteínas de Ciclo Celular , Transdiferenciação Celular/genética , Transdiferenciação Celular/fisiologia , Células Cultivadas , Colestase/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Regeneração Hepática/genética , Masculino , Camundongos , Fosfoproteínas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas de Sinalização YAP
17.
Biochim Biophys Acta ; 1863(2): 303-13, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26592459

RESUMO

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.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Hedgehog/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Animais , Humanos , Hipóxia , Modelos Biológicos
18.
J Cell Mol Med ; 19(5): 948-59, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25754106

RESUMO

Hypersecretion is the major symptom of functional neuroendocrine tumours. The mechanisms that contribute to this excessive secretion of hormones are still elusive. A key event in secretion is the exit of secretory products from the Golgi apparatus. ADP-ribosylation factor (Arf) GTPases are known to control vesicle budding and trafficking, and have a leading function in the regulation of formation of secretory granula at the Golgi. Here, we show that Arf1 is the predominant Arf protein family member expressed in the neuroendocrine pancreatic tumour cell lines BON and QGP-1. In BON cells Arf1 colocalizes with Golgi markers as well as chromogranin A, and shows significant basal activity. The inhibition of Arf1 activity or expression significantly impaired secretion of chromogranin A. Furthermore, we show that the insulin-like growth factor 1 (IGF-1), a major regulator of growth and secretion in BON cells, induces Arf1 activity. We found that activation of Arf1 upon IGF-1 receptor stimulation is mediated by MEK/ERK signalling pathway in BON and QGP-1 cells. Moreover, the activity of Arf1 in BON cells is mediated by autocrinely secreted IGF-1, and concomitantly, autocrine IGF1 secretion is maintained by Arf1 activity. In summary, our data indicate an important regulatory role for Arf1 at the Golgi in hypersecretion in neuroendocrine cancer cells.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Cromogranina A/metabolismo , Complexo de Golgi/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator 1 de Ribosilação do ADP/genética , Autoantígenos/metabolismo , Western Blotting , Tumor Carcinoide/genética , Tumor Carcinoide/metabolismo , Tumor Carcinoide/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Ligação Proteica , Interferência de RNA , Receptor IGF Tipo 1/metabolismo
19.
EMBO J ; 30(1): 43-56, 2011 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-21102556

RESUMO

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.


Assuntos
Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Receptores Notch/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Centrossomo/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Expressão Gênica , Células HeLa , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Neurogênese , Ligação Proteica , Transporte Proteico , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores Notch/genética , Transcrição Gênica , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/análise , Proteínas de Xenopus/genética , Xenopus laevis/genética
20.
Mol Ecol ; 24(2): 453-65, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25496144

RESUMO

The genomic framework that enables corals to adjust to unfavourable conditions is crucial for coral reef survival in a rapidly changing climate. We have explored the striking intraspecific variability in the expression of coral pigments from the green fluorescent protein (GFP) family to elucidate the genomic basis for the plasticity of stress responses among reef corals. We show that multicopy genes can greatly increase the dynamic range over which corals can modulate transcript levels in response to the light environment. Using the red fluorescent protein amilFP597 in the coral Acropora millepora as a model, we demonstrate that its expression increases with light intensity, but both the minimal and maximal gene transcript levels vary markedly among colour morphs. The pigment concentration in the tissue of different morphs is strongly correlated with the number of gene copies with a particular promoter type. These findings indicate that colour polymorphism in reef corals can be caused by the environmentally regulated expression of multicopy genes. High-level expression of amilFP597 is correlated with reduced photodamage of zooxanthellae under acute light stress, supporting a photoprotective function of this pigment. The cluster of light-regulated pigment genes can enable corals to invest either in expensive high-level pigmentation, offering benefits under light stress, or to rely on low tissue pigment concentrations and use the conserved resources for other purposes, which is preferable in less light-exposed environments. The genomic framework described here allows corals to pursue different strategies to succeed in habitats with highly variable light stress levels. In summary, our results suggest that the intraspecific plasticity of reef corals' stress responses is larger than previously thought.


Assuntos
Aclimatação/genética , Antozoários/genética , Luz , Proteínas Luminescentes/genética , Pigmentação/genética , Animais , Antozoários/fisiologia , Cor , Dosagem de Genes , Dados de Sequência Molecular , Família Multigênica , Filogenia , Regiões Promotoras Genéticas , Proteína Vermelha Fluorescente
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