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1.
Cell Rep ; 36(11): 109705, 2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34525377

RESUMO

Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a ß-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

2.
Oncogene ; 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545187

RESUMO

Canonical Wnt/ß-catenin signaling is an established regulator of cellular state and its critical contributions to tumor initiation, malignant tumor progression and metastasis formation have been demonstrated in various cancer types. Here, we investigated how the binding of ß-catenin to the transcriptional coactivators B-cell CLL/lymphoma 9 (Bcl9) and Bcl9-Like (Bcl9L) affected mammary gland carcinogenesis in the MMTV-PyMT transgenic mouse model of metastatic breast cancer. Conditional knockout of both Bcl9 and Bcl9L resulted into tumor cell death. In contrast, disrupting the interaction of Bcl9/Bcl9L with ß-catenin, either by deletion of their HD2 domains or by a point mutation in the N-terminal domain of ß-catenin (D164A), diminished primary tumor growth and tumor cell proliferation and reduced tumor cell invasion and lung metastasis. In comparison, the disruption of HD1 domain-mediated binding of Bcl9/Bcl9L to Pygopus had only moderate effects. Interestingly, interfering with the ß-catenin-Bcl9/Bcl9L-Pygo chain of adapters only partially impaired the transcriptional response of mammary tumor cells to Wnt3a and TGFß treatments. Together, the results indicate that Bcl9/Bcl9L modulate but are not critically required for canonical Wnt signaling in its contribution to breast cancer growth and malignant progression, a notion consistent with the "just-right" hypothesis of Wnt-driven tumor progression.

3.
Cell Rep ; 36(5): 109484, 2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34348153

RESUMO

We lack a holistic understanding of the genetic programs orchestrating embryonic colon morphogenesis and governing damage response in the adult. A window into these programs is the transcriptomes of the epithelial and mesenchymal cell populations in the colon. Performing unbiased single-cell transcriptomic analyses of the developing mouse colon at different embryonic stages (embryonic day 14.5 [E14.5], E15.5, and E18.5), we capture cellular and molecular profiles of the stages before, during, and after the appearance of crypt structures, as well as in a model of adult colitis. The data suggest most adult lineages are established by E18.5. We find embryonic-specific gene expression profiles and cell populations that reappear in response to tissue damage. Comparison of the datasets from mice and human colitis suggests the processes are conserved. In this study, we provide a comprehensive single-cell atlas of the developing mouse colon and evidence for the reactivation of embryonic genes in disease.

4.
Proc Natl Acad Sci U S A ; 118(34)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34408016

RESUMO

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, ß-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of ß-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of ß-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of ß-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of ß-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by ß-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-ß-induced EMT. Our results uncouple the signaling from the adhesion function of ß-catenin and underline the importance of Wnt/ß-catenin-dependent transcription in malignant tumor progression of breast cancer.

5.
Nat Commun ; 12(1): 5056, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417458

RESUMO

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a-/- melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.


Assuntos
Epigênese Genética , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fator de Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , Micrometástase de Neoplasia , Ligação Proteica , Carga Tumoral
6.
J Bone Miner Res ; 36(10): 2039-2052, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34155688

RESUMO

In hypertrophic chondrocytes, ß-catenin has two roles. First, it locally suppresses the differentiation of osteoclasts at the chondro-osseous junction by maintaining the pro-osteoclastic factor receptor activator of NF-κB ligand (RANKL) at low levels. Second, it promotes the differentiation of osteoblast-precursors from chondrocytes. Yet, ß-catenin is a dual-function protein, which can either participate in cell-cell adherens junctions or serve as a transcriptional co-activator in canonical Wnt signaling interacting with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors. Hence, whenever studying tissue-specific requirements of ß-catenin using a conventional conditional knockout approach, the functional mechanisms underlying the defects in the conditional mutants remain ambiguous. To decipher mechanistically which of the two molecular functions of ß-catenin is required in hypertrophic chondrocytes, we used different approaches. We analyzed the long bones of newborn mice carrying either the null-alleles of Lef1 or Tcf7, or mice in which Tcf7l2 was conditionally deleted in the hypertrophic chondrocytes, as well as double mutants for Lef1 and Tcf7l2, and Tcf7 and Tcf7l2. Furthermore, we analyzed Ctnnb1 mutant newborns expressing a signaling-defective allele that retains the cell adhesion function in hypertrophic chondrocytes. None of the analyzed Tcf/Lef single or double mutants recapitulated the previously published phenotype upon loss of ß-catenin in hypertrophic chondrocytes. However, using this particular Ctnnb1 allele, maintaining cell adhesion function, we show that it is the co-transcriptional activity of ß-catenin, which is required in hypertrophic chondrocytes to suppress osteoclastogenesis and to promote chondrocyte-derived osteoblast differentiation. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

7.
Nat Commun ; 12(1): 1368, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649334

RESUMO

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/ß-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic ß-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of ß-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with ß-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune ß-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/ß-catenin output requires selective modulation of gene expression by transcriptional co-factors.


Assuntos
Mucosa Intestinal/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , beta Catenina/química , beta Catenina/metabolismo , Algoritmos , Animais , Sequência de Bases , Diferenciação Celular , Proliferação de Células , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Homeostase , Hiperplasia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Organoides/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
8.
PLoS Biol ; 18(12): e3001032, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33306673

RESUMO

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity.


Assuntos
Colo/metabolismo , Fibroblastos/classificação , Fibroblastos/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Colo/fisiologia , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Homeostase , Mucosa Intestinal/metabolismo , Intestinos/fisiologia , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Análise de Célula Única/métodos , Células-Tronco/citologia , Transcriptoma/genética
9.
Elife ; 92020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32808927

RESUMO

BCL9 and PYGO are ß-catenin cofactors that enhance the transcription of Wnt target genes. They have been proposed as therapeutic targets to diminish Wnt signaling output in intestinal malignancies. Here we find that, in colorectal cancer cells and in developing mouse forelimbs, BCL9 proteins sustain the action of ß-catenin in a largely PYGO-independent manner. Our genetic analyses implied that BCL9 necessitates other interaction partners in mediating its transcriptional output. We identified the transcription factor TBX3 as a candidate tissue-specific member of the ß-catenin transcriptional complex. In developing forelimbs, both TBX3 and BCL9 occupy a large number of Wnt-responsive regulatory elements, genome-wide. Moreover, mutations in Bcl9 affect the expression of TBX3 targets in vivo, and modulation of TBX3 abundance impacts on Wnt target genes transcription in a ß-catenin- and TCF/LEF-dependent manner. Finally, TBX3 overexpression exacerbates the metastatic potential of Wnt-dependent human colorectal cancer cells. Our work implicates TBX3 as context-dependent component of the Wnt/ß-catenin-dependent transcriptional complex.


Assuntos
Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt , Animais , Feminino , Células HCT116 , Humanos , Masculino , Camundongos , Especificidade de Órgãos , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Peixe-Zebra
10.
Sci Adv ; 6(16): eaay7928, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32494603

RESUMO

The Hedgehog (Hh) signaling pathway controls embryonic development and adult tissue homeostasis in multicellular organisms. In Drosophila melanogaster, the pathway is primed by secretion of a dually lipid-modified morphogen, Hh, a process dependent on a membrane-integral protein Dispatched. Although Dispatched is a critical component of the pathway, the structural basis of its activity has, so far, not been described. Here, we describe a cryo-electron microscopy structure of the D. melanogaster Dispatched at 3.2-Å resolution. The ectodomains of Dispatched adopt an open conformation suggestive of a receptor-chaperone role. A three-dimensional reconstruction of Dispatched bound to Hh confirms the ability of Dispatched to bind Hh but using a unique mode distinct from those previously observed in structures of Hh complexes. The structure may represent the state of the complex that precedes shedding of Hh from the surface of the morphogen-releasing cell.

11.
Cancer Res ; 80(17): 3631-3648, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32586983

RESUMO

Pygopus 2 (Pygo2) is a coactivator of Wnt/ß-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer, we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller, differentiated, and less metastatic tumors, due, in part, to decreased canonical Wnt/ß-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGFß signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically, the Pygo2-histone interaction potentiated Wnt/ß-catenin signaling, in part, by repressing the expression of Wnt signaling antagonists. Furthermore, Pygo2 and ß-catenin regulated the expression of miR-29 family members, which, in turn, repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively, these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer, as its histone-binding capability promotes ß-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation, EMT, and metastasis.


Assuntos
Desdiferenciação Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Mamárias Experimentais/patologia , Animais , Progressão da Doença , Feminino , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL
12.
G3 (Bethesda) ; 10(5): 1745-1752, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32217630

RESUMO

Several techniques have been developed to study specific gene function in loss-of-function situations. In Drosophila melanoga st er, RNAi and the generation of mutant clones are widely used. However, both techniques have the limitation that there is a significant time lag before gene function is abolished. Given the relatively rapid development of Drosophila, such perdurance is a serious impediment to study gene function. Here we describe the adaptation of the anchor-away technique for use in Drosophila Anchor-away was originally developed in yeast to quickly and efficiently abrogate the function of nuclear proteins by sequestering - anchoring - them away in a different cellular compartment. The required components are present in the cells, and the system is triggered by the addition of rapamycin, resulting in a rapid generation of a loss-of-function situation. We provide here proof of principle for the system by producing loss-of-function situations for two nuclear proteins - Pygopus and Brinker. The system allows to study the requirement of any protein during any time window, and at the same time circumvents difficulties, such as off-target effects or variable phenotypes, which are inherent in other techniques, for example RNAi.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fenótipo , Interferência de RNA
13.
Life Sci Alliance ; 2(3)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31196871

RESUMO

CRISPR-Cas9-based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms-often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.


Assuntos
Cromossomos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Drosophila/genética , Edição de Genes , Expressão Gênica , Marcação de Genes , Genes Reporter , Conformação de Ácido Nucleico , Fenótipo , RNA Guia/genética
14.
Nat Commun ; 10(1): 2192, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097699

RESUMO

The transcription factor Yin Yang 1 (YY1) plays an important role in human disease. It is often overexpressed in cancers and mutations can lead to a congenital haploinsufficiency syndrome characterized by craniofacial dysmorphisms and neurological dysfunctions, consistent with a role in brain development. Here, we show that Yy1 controls murine cerebral cortex development in a stage-dependent manner. By regulating a wide range of metabolic pathways and protein translation, Yy1 maintains proliferation and survival of neural progenitor cells (NPCs) at early stages of brain development. Despite its constitutive expression, however, the dependence on Yy1 declines over the course of corticogenesis. This is associated with decreasing importance of processes controlled by Yy1 during development, as reflected by diminished protein synthesis rates at later developmental stages. Thus, our study unravels a novel role for Yy1 as a stage-dependent regulator of brain development and shows that biosynthetic demands of NPCs dynamically change throughout development.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Neurais/fisiologia , Fator de Transcrição YY1/fisiologia , Animais , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Embrião de Mamíferos , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Técnicas de Inativação de Genes , Redes e Vias Metabólicas/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Animais , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo
15.
Cell Rep ; 27(3): 955-970.e7, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995488

RESUMO

Combinatorial interactions among transcription factors (TFs) play essential roles in generating gene expression specificity and diversity in metazoans. Using yeast 2-hybrid (Y2H) assays on nearly all sequence-specific Drosophila TFs, we identified 1,983 protein-protein interactions (PPIs), more than doubling the number of currently known PPIs among Drosophila TFs. For quality assessment, we validated a subset of our interactions using MITOMI and bimolecular fluorescence complementation assays. We combined our interactome with prior PPI data to generate an integrated Drosophila TF-TF binary interaction network. Our analysis of ChIP-seq data, integrating PPI and gene expression information, uncovered different modes by which interacting TFs are recruited to DNA. We further demonstrate the utility of our Drosophila interactome in shedding light on human TF-TF interactions. This study reveals how TFs interact to bind regulatory elements in vivo and serves as a resource of Drosophila TF-TF binary PPIs for understanding tissue-specific gene regulation.


Assuntos
Drosophila melanogaster/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , DNA/química , DNA/metabolismo , Regulação da Expressão Gênica , Microscopia de Fluorescência , Mapas de Interação de Proteínas/genética , Elementos Reguladores de Transcrição , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
16.
Cell Stem Cell ; 24(4): 637-653.e9, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30951662

RESUMO

Increasing evidence suggests that cancer cells highjack developmental programs for disease initiation and progression. Melanoma arises from melanocytes that originate during development from neural crest stem cells (NCSCs). Here, we identified the transcription factor Yin Yang 1 (Yy1) as an NCSCs regulator. Conditional deletion of Yy1 in NCSCs resulted in stage-dependent hypoplasia of all major neural crest derivatives due to decreased proliferation and increased cell death. Moreover, conditional ablation of one Yy1 allele in a melanoma mouse model prevented tumorigenesis, indicating a particular susceptibility of melanoma cells to reduced Yy1 levels. Combined RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and untargeted metabolomics demonstrated that YY1 governs multiple metabolic pathways and protein synthesis in both NCSCs and melanoma. In addition to directly regulating a metabolic gene set, YY1 can act upstream of MITF/c-MYC as part of a gene regulatory network controlling metabolism. Thus, both NCSC development and melanoma formation depend on an intricate YY1-controlled metabolic program.


Assuntos
Melanoma/metabolismo , Melanoma/patologia , Crista Neural/citologia , Crista Neural/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fator de Transcrição YY1/deficiência
17.
Sci Rep ; 9(1): 1335, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718768

RESUMO

CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational methods, we defined a minimal common pharmacophore in established CDC25 inhibitors and performed virtual screening of a proprietary library. Based on the availability of crystal structures for CDC25A and CDC25B, we implemented a molecular docking strategy and carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, the compounds induced differentiation, accompanied by decreased stemness properties, in intestinal crypt stem cell-derived Apc/K-Ras-mutant mouse organoids, and led to tumor regression and reduction of metastatic potential in zebrafish embryo xenografts used as in vivo model.


Assuntos
Proteína Quinase CDC2/genética , Neoplasias/genética , Conformação Proteica , Fosfatases cdc25/genética , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Divisão Celular/genética , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Xenoenxertos , Humanos , Camundongos , Mitose/genética , Simulação de Acoplamento Molecular , Naftoquinonas/farmacologia , Neoplasias/patologia , Fosfatases cdc25/antagonistas & inibidores , Fosfatases cdc25/química , Fosfatases cdc25/ultraestrutura
18.
Elife ; 82019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30735122

RESUMO

Two decades after signals controlling bone length were discovered, the endogenous ligands determining bone width remain unknown. We show that postnatal establishment of normal bone width in mice, as mediated by bone-forming activity of the periosteum, requires BMP signaling at the innermost layer of the periosteal niche. This developmental signaling center becomes quiescent during adult life. Its reactivation however, is necessary for periosteal growth, enhanced bone strength, and accelerated fracture repair in response to bone-anabolic therapies used in clinical orthopedic settings. Although many BMPs are expressed in bone, periosteal BMP signaling and bone formation require only Bmp2 in the Prx1-Cre lineage. Mechanistically, BMP2 functions downstream of Lrp5/6 pathway to activate a conserved regulatory element upstream of Sp7 via recruitment of Smad1 and Grhl3. Consistent with our findings, human variants of BMP2 and GRHL3 are associated with increased risk of fractures.


Assuntos
Proteína Morfogenética Óssea 2/genética , Osteogênese/genética , Periósteo/crescimento & desenvolvimento , Animais , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Fraturas Ósseas/genética , Fraturas Ósseas/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Periósteo/metabolismo , Transdução de Sinais/genética , Proteína Smad1/genética , Fator de Transcrição Sp7/genética , Fatores de Transcrição/genética
19.
EMBO J ; 38(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30425074

RESUMO

During canonical Wnt signalling, the activity of nuclear ß-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view, we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones lacking all four TCF/LEF genes. By performing unbiased whole transcriptome sequencing analysis, we found that a subset of ß-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that ß-catenin occupied specific genomic regions in the absence of TCF/LEF Finally, we revealed the existence of a transcriptional activity of ß-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as ß-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of ß-catenin that bypasses the TCF/LEF transcription factors.


Assuntos
Perfilação da Expressão Gênica/métodos , Fatores de Transcrição TCF/genética , Transcrição Genética , beta Catenina/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fatores de Transcrição TCF/metabolismo , Sequenciamento Completo do Exoma/métodos , Via de Sinalização Wnt
20.
Sci Rep ; 8(1): 17712, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30531963

RESUMO

The elimination of unfit cells from a tissue is a process known in Drosophila and mammals as cell competition. In a well-studied paradigm "loser" cells that are heterozygous mutant for a haploinsufficient ribosomal protein gene are eliminated from developing tissues via apoptosis when surrounded by fitter wild-type cells, referred to as "winner" cells. However, the mechanisms underlying the induction of this phenomenon are not fully understood. Here we report that a CCAAT-Enhancer-Binding Protein (C/EBP), Xrp1, which is known to help maintaining genomic stability after genotoxic stress, is necessary for the elimination of loser clones in cell competition. In loser cells, Xrp1 is transcriptionally upregulated by an autoregulatory loop and is able to trigger apoptosis - driving cell elimination. We further show that Xrp1 acts in the nucleus to regulate the transcription of several genes that have been previously involved in cell competition. We therefore speculate that Xrp1 might play a fundamental role as a molecular caretaker of the genomic integrity of tissues.


Assuntos
Comunicação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica/genética , Ribossomos/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Regulação para Cima/genética
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