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1.
Nat Commun ; 13(1): 5969, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216795

RESUMO

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.


Assuntos
Adenosina Trifosfatases , Fatores de Transcrição , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteólise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
2.
Cell Rep ; 39(2): 110636, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35417719

RESUMO

Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.


Assuntos
Neoplasias , Oncogenes , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Antígenos de Histocompatibilidade Menor/metabolismo , Neoplasias/genética , Proteínas de Ligação a RNA/genética , Cromossomos Sexuais/metabolismo , Cromossomo X , Cromossomo Y
3.
ChemMedChem ; 16(9): 1420-1424, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33275320

RESUMO

Aberrant WNT pathway activation, leading to nuclear accumulation of ß-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of ß-catenin and subsequent nuclear translocation. Restoring cellular degradation of ß-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to ß-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging ß-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein-protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards ß-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.


Assuntos
Bibliotecas de Moléculas Pequenas/química , beta Catenina/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , beta Catenina/metabolismo
4.
Sci Rep ; 9(1): 11661, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31406271

RESUMO

SMARCA4/BRG1 and SMARCA2/BRM, the two mutually exclusive catalytic subunits of the BAF complex, display a well-established synthetic lethal relationship in SMARCA4-deficient cancers. Using CRISPR-Cas9 screening, we identify SMARCA4 as a novel dependency in SMARCA2-deficient esophageal squamous cell carcinoma (ESCC) models, reciprocal to the known synthetic lethal interaction. Restoration of SMARCA2 expression alleviates the dependency on SMARCA4, while engineered loss of SMARCA2 renders ESCC models vulnerable to concomitant depletion of SMARCA4. Dependency on SMARCA4 is linked to its ATPase activity, but not to bromodomain function. We highlight the relevance of SMARCA4 as a drug target in esophageal cancer using an engineered ESCC cell model harboring a SMARCA4 allele amenable to targeted proteolysis and identify SMARCA4-dependent cell models with low or absent SMARCA2 expression from additional tumor types. These findings expand the concept of SMARCA2/SMARCA4 paralog dependency and suggest that pharmacological inhibition of SMARCA4 represents a novel therapeutic opportunity for SMARCA2-deficient cancers.


Assuntos
DNA Helicases/antagonistas & inibidores , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , DNA Helicases/genética , Epigênese Genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Edição de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Mutação com Perda de Função , Terapia de Alvo Molecular/métodos , Proteínas Nucleares/genética , RNA Guia de Cinetoplastídeos/genética , RNA Interferente Pequeno/metabolismo , Mutações Sintéticas Letais , Fatores de Transcrição/deficiência
5.
Nat Chem Biol ; 15(8): 846, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31267096

RESUMO

In the version of this article originally published, several lines of text in the last paragraph of the right column on page 1 of the PDF were transposed into the bottom paragraph of the left column. The affected text of the left column should read "The ATP-dependent activities of the BAF (SWI/SNF) chromatin remodeling complexes affect the positioning of nucleosomes on DNA and thereby many cellular processes related to chromatin structure, including transcription, DNA repair and decatenation of chromosomes during mitosis12,13." The affected text of the right column should read "SMARCA2/4BD inhibitors are thus precluded from use for the treatment of SMARCA4 mutant cancers but could provide attractive ligands for PROTAC conjugation. Small molecules binding to other bromodomains have been successfully converted into PROTACs by conjugating them with structures capable of binding to the E3 ligases von Hippel-Lindau (VHL) or cereblon5,6,10,11,25,26,27." The errors have been corrected in the PDF version of the paper.

6.
Nat Chem Biol ; 15(7): 672-680, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31178587

RESUMO

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Estrutura Molecular , Proteínas Nucleares/metabolismo
7.
Elife ; 82019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30910006

RESUMO

Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent functional screening data of cancer cell lines we identify Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair (MMR), occurs frequently in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in severe genome integrity defects. ATP-binding deficient variants of WRN fail to rescue the viability phenotype of WRN-depleted MSI-H cancer cells. Reconstitution and depletion studies indicate that WRN dependence is not attributable to acute loss of MMR gene function but might arise during sustained MMR-deficiency. Our study suggests that pharmacological inhibition of WRN helicase function represents an opportunity to develop a novel targeted therapy for MSI-H cancers.


Assuntos
Instabilidade de Microssatélites , Neoplasias/terapia , Helicase da Síndrome de Werner/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular , Reparo de Erro de Pareamento de DNA , Humanos , Modelos Teóricos , Helicase da Síndrome de Werner/genética
8.
Oncotarget ; 9(47): 28625-28637, 2018 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-29983885

RESUMO

Genotype specific vulnerabilities of cancer cells constitute a promising strategy for the development of new therapeutics. Deletions of non-essential genes in tumors can generate unique vulnerabilities which could be exploited therapeutically. The MTAP gene is recurrently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. Recent studies have uncovered an increased dependency of MTAP-deleted cancer cells on the function of a PRMT5 containing complex, including WDR77, PRMT5 and the kinase RIOK1. As RIOK1 kinase activity constitutes a potential therapeutic target, we wanted to test if MTAP deletion confers increased sensitivity to RIOK1 inhibition. Using CRISPR/Cas9-mediated genome engineering we generated analog sensitive alleles of RIOK1 in isogenic cell lines differing only by MTAP status. While we were able to independently confirm an increased dependency of MTAP-deleted cells on PRMT5, we did not detect a differential requirement for RIOK1 kinase activity between MTAP-proficient and deficient cells. Our results reveal that the kinase activity of RIOK1 is required for the survival of cancer cell lines irrespective of their MTAP status and cast doubt on the therapeutic exploitability of RIOK1 in the context of MTAP-deleted cancers.

9.
Cell Death Dis ; 8(2): e2631, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28230862

RESUMO

Coordination of energy metabolism is essential for homeostasis of stem cells, whereas an imbalance in energy homeostasis causes disease and accelerated aging. Here we show that deletion or enzymatic inactivation of lysine-specific demethylase 1 (Lsd1) triggers senescence in trophoblast stem cells (TSCs). Genome-wide transcriptional profiling of TSCs following Lsd1 inhibition shows gene set enrichment of aging and metabolic pathways. Consistently, global metabolomic and phenotypic analyses disclose an unbalanced redox status, decreased glutamine anaplerosis and mitochondrial function. Loss of homeostasis is caused by increased expression of sirtuin 4 (Sirt4), a Lsd1-repressed direct target gene. Accordingly, Sirt4 overexpression in wild-type TSCs recapitulates the senescence phenotype initiated by Lsd1 deletion or inhibition. Inversely, absence of Lsd1 enzymatic activity concomitant with knockdown of Sirt4 reestablishes normal glutamine anaplerosis, redox balance and mitochondrial function. In conclusion, by repression of Sirt4, Lsd1 directs the epigenetic control of TSC immortality via maintenance of metabolic flexibility.


Assuntos
Senescência Celular/fisiologia , Histona Desmetilases/metabolismo , Proteínas Mitocondriais/metabolismo , Sirtuínas/metabolismo , Células-Tronco/metabolismo , Trofoblastos/metabolismo , Envelhecimento/metabolismo , Humanos , Redes e Vias Metabólicas/fisiologia , Mitocôndrias/metabolismo , Oxirredução , Células-Tronco/fisiologia , Trofoblastos/fisiologia
10.
PLoS One ; 8(10): e77652, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24204904

RESUMO

Malignant rhabdoid tumors (MRTs) are aggressive pediatric cancers arising in brain, kidney and soft tissues, which are characterized by loss of the tumor suppressor SNF5/SMARCB1. MRTs are poorly responsive to chemotherapy and thus a high unmet clinical need exists for novel therapies for MRT patients. SNF5 is a core subunit of the SWI/SNF chromatin remodeling complex which affects gene expression by nucleosome remodeling. Here, we report that loss of SNF5 function correlates with increased expression of fibroblast growth factor receptors (FGFRs) in MRT cell lines and primary tumors and that re-expression of SNF5 in MRT cells causes a marked repression of FGFR expression. Conversely, siRNA-mediated impairment of SWI/SNF function leads to elevated levels of FGFR2 in human fibroblasts. In vivo, treatment with NVP-BGJ398, a selective FGFR inhibitor, blocks progression of a murine MRT model. Hence, we identify FGFR signaling as an aberrantly activated oncogenic pathway in MRTs and propose pharmacological inhibition of FGFRs as a potential novel clinical therapy for MRTs.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Tumor Rabdoide/metabolismo , Fatores de Transcrição/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Células HEK293 , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Tumor Rabdoide/genética , Proteína SMARCB1 , Transdução de Sinais/genética , Fatores de Transcrição/genética
11.
J Bone Miner Res ; 28(4): 899-911, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23129509

RESUMO

Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25-dihydroxyvitamin D3 (1,25[OH]2 D3 ). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)2 D3 levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X-linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23-mediated hypophosphatemic disorders using NVP-BGJ398, a novel selective, pan-specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1-null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long-term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP-BGJ398 treatment as a novel approach for the therapy of FGF23-mediated hypophosphatemic diseases.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Raquitismo Hipofosfatêmico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Desenvolvimento Ósseo/efeitos dos fármacos , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/metabolismo , Fêmur/efeitos dos fármacos , Fêmur/patologia , Fator de Crescimento de Fibroblastos 23 , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/patologia , Homeostase/efeitos dos fármacos , Íons , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos Endogâmicos C57BL , Minerais/metabolismo , Compostos de Fenilureia/uso terapêutico , Pirimidinas/uso terapêutico , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Raquitismo Hipofosfatêmico/tratamento farmacológico , Raquitismo Hipofosfatêmico/patologia , Cauda/anatomia & histologia , Vitamina D/análogos & derivados , Vitamina D/biossíntese
12.
Cancer Discov ; 2(12): 1118-33, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23002168

RESUMO

UNLABELLED: Patient stratification biomarkers that enable the translation of cancer genetic knowledge into clinical use are essential for the successful and rapid development of emerging targeted anticancer therapeutics. Here, we describe the identification of patient stratification biomarkers for NVP-BGJ398, a novel and selective fibroblast growth factor receptor (FGFR) inhibitor. By intersecting genome-wide gene expression and genomic alteration data with cell line-sensitivity data across an annotated collection of cancer cell lines called the Cancer Cell Line Encyclopedia, we show that genetic alterations for FGFR family members predict for sensitivity to NVP-BGJ398. For the first time, we report oncogenic FGFR1 amplification in osteosarcoma as a potential patient selection biomarker. Furthermore, we show that cancer cell lines harboring FGF19 copy number gain at the 11q13 amplicon are sensitive to NVP-BGJ398 only when concomitant expression of ß-klotho occurs. Thus, our findings provide the rationale for the clinical development of FGFR inhibitors in selected patients with cancer harboring tumors with the identified predictors of sensitivity. SIGNIFICANCE: The success of a personalized medicine approach using targeted therapies ultimately depends on being able to identify the patients who will benefit the most from any given drug. To this end, we have integrated the molecular profiles for more than 500 cancer cell lines with sensitivity data for the novel anticancer drug NVP-BGJ398 and showed that FGFR genetic alterations are the most significant predictors for sensitivity. This work has ultimately endorsed the incorporation of specific patient selection biomakers in the clinical trials for NVP-BGJ398.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Animais , Linhagem Celular Tumoral , Amplificação de Genes/efeitos dos fármacos , Células HEK293 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Camundongos , Modelos Moleculares , Neoplasias/genética , Neoplasias/patologia , Compostos de Fenilureia/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Bone Miner Res ; 26(10): 2486-97, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21812026

RESUMO

The functional interaction between fibroblast growth factor 23 (FGF-23) and Klotho in the control of vitamin D and phosphate homeostasis is manifested by the largely overlapping phenotypes of Fgf23- and Klotho-deficient mouse models. However, to date, targeted inactivation of FGF receptors (FGFRs) has not provided clear evidence for an analogous function of FGFRs in this process. Here, by means of pharmacologic inhibition of FGFRs, we demonstrate their involvement in renal FGF-23/Klotho signaling and elicit their role in the control of phosphate and vitamin D homeostasis. Specifically, FGFR loss of function counteracts renal FGF-23/Klotho signaling, leading to deregulation of Cyp27b1 and Cyp24a1 and the induction of hypervitaminosis D and hyperphosphatemia. In turn, this initiates a feedback response leading to high serum levels of FGF-23. Further, we show that FGFR inhibition blocks Fgf23 transcription in bone and that this is dominant over vitamin D-induced Fgf23 expression, ultimately impinging on systemic FGF-23 protein levels. Additionally, we identify Fgf23 as a specific target gene of FGF signaling in vitro. Thus, in line with Fgf23- and Klotho-deficient mouse models, our study illustrates the essential function of FGFRs in the regulation of vitamin D and phosphate levels. Further, we reveal FGFR signaling as a novel in vivo control mechanism for Fgf23 expression in bone, suggesting a dual function of FGFRs in the FGF-23/Klotho pathway leading to vitamin D and phosphate homeostasis.


Assuntos
Osso e Ossos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Homeostase/fisiologia , Rim/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais/fisiologia , Vitamina D/fisiologia , Animais , Western Blotting , Linhagem Celular , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo Real
14.
Nucleic Acids Res ; 38(6): 1964-81, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20044351

RESUMO

Alternative splicing can produce multiple protein products with variable domain composition from a single gene. The mouse Tcf7l2 gene is subject to alternative splicing. It encodes TCF4, a member of the T-cell factor (TCF) family of DNA-binding proteins and a nuclear interaction partner of beta-catenin which performs essential functions in Wnt growth factor signalling. Multiple TCF4 isoforms, potentially exhibiting cell-type-specific distribution and differing in gene regulatory properties, could strongly influence tissue-specific Wnt responses. Therefore, we have examined mouse Tcf7l2 splice variants in neonatal tissues, embryonic stem cells and neural progenitors. By polymerase chain reaction amplification, cloning and sequencing, we identify a large number of alternatively spliced transcripts and report a highly flexible combinatorial repertoire of alternative exons. Many, but not all of the variants exhibit a broad tissue distribution. Moreover, two functionally equivalent versions of the C-clamp, thought to represent an auxiliary DNA-binding domain, were identified. Depending upon promoter context and precise domain composition, TCF4 isoforms exhibit strikingly different transactivation potentials at natural Wnt/beta-catenin target promoters. However, differences in C-clamp-mediated DNA binding can only partially explain functional differences among TCF4 variants. Still, the cell-type-specific complement of TCF4 isoforms is likely to be a major determinant for the context-dependent transcriptional output of Wnt/beta-catenin signalling.


Assuntos
Processamento Alternativo , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Ativação Transcricional , Proteínas Wnt/farmacologia , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Variação Genética , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição TCF/química , Distribuição Tecidual , Proteína 2 Semelhante ao Fator 7 de Transcrição
15.
Mol Cell Biol ; 27(23): 8164-77, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17923689

RESUMO

Canonical Wnt signaling and its nuclear effectors, beta-catenin and the family of T-cell factor (TCF) DNA-binding proteins, belong to the small number of regulatory systems which are repeatedly used for context-dependent control of distinct genetic programs. The apparent ability to elicit a large variety of transcriptional responses necessitates that beta-catenin and TCFs distinguish precisely between genes to be activated and genes to remain silent in a specific context. How this is achieved is unclear. Here, we examined patterns of Wnt target gene activation and promoter occupancy by TCFs in different mouse cell culture models. Remarkably, within a given cell type only Wnt-responsive promoters are bound by specific subsets of TCFs, whereas nonresponsive Wnt target promoters remain unoccupied. Wnt-responsive, TCF-bound states correlate with DNA hypomethylation, histone H3 hyperacetylation, and H3K4 trimethylation. Inactive, nonresponsive promoter chromatin shows DNA hypermethylation, is devoid of active histone marks, and additionally can show repressive H3K27 trimethylation. Furthermore, chromatin structural states appear to be independent of Wnt pathway activity. Apparently, cell-type-specific regulation of Wnt target genes comprises multilayered control systems. These involve epigenetic modifications of promoter chromatin and differential promoter occupancy by functionally distinct TCF proteins, which together determine susceptibility to Wnt signaling.


Assuntos
Epigênese Genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição TCF/metabolismo , Proteínas Wnt/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Perfilação da Expressão Gênica , Inibidores de Histona Desacetilases , Histonas/metabolismo , Metilação/efeitos dos fármacos , Camundongos , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Células NIH 3T3 , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição 4
16.
Exp Cell Res ; 313(3): 572-87, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17198701

RESUMO

Canonical Wnt signaling triggers the formation of heterodimeric transcription factor complexes consisting of beta-catenin and T cell factors, and thereby controls the execution of specific genetic programs. During the expansion and neurogenic phases of embryonic neural development canonical Wnt signaling initially controls proliferation of neural progenitor cells, and later neuronal differentiation. Whether Wnt growth factors affect neural progenitor cells postnatally is not known. Therefore, we have analyzed the impact of Wnt signaling on neural progenitors isolated from cerebral cortices of newborn mice. Expression profiling of pathway components revealed that these cells are fully equipped to respond to Wnt signals. However, Wnt pathway activation affected only a subset of neonatal progenitors and elicited a limited increase in proliferation and neuronal differentiation in distinct subsets of cells. Moreover, Wnt pathway activation only transiently stimulated S-phase entry but did not support long-term proliferation of progenitor cultures. The dampened nature of the Wnt response correlates with the predominant expression of inhibitory pathway components and the rapid actuation of negative feedback mechanisms. Interestingly, in differentiating cell cultures activation of canonical Wnt signaling reduced Hes1 and Hes5 expression suggesting that during postnatal neural development, Wnt/beta-catenin signaling enhances neurogenesis from progenitor cells by interfering with Notch pathway activity.


Assuntos
Córtex Cerebral/fisiologia , Células-Tronco Multipotentes/metabolismo , Organogênese , Transdução de Sinais , Proteínas Wnt/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular , Proliferação de Células , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Multipotentes/fisiologia , Proteínas Wnt/metabolismo , beta Catenina
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