Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 10.975
Filtrar
1.
Curr Protoc ; 1(7): e187, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34242493

RESUMO

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) has become one of the most popular methods to study protein-DNA interactions and can be used, for instance, to identify the binding sites of transcription factors or to determine the distributions of histones with specific post-translational modifications throughout the genome. Although standard ChIP-seq protocols work well in most experimental systems, there are exceptions, and one of these is the popular model organism Caenorhabditis elegans. Even though this system is very amenable to genetic and cytological methods, biochemical approaches are challenging. This is due to both the animals' cuticle, which impairs lysis as well as penetration by cross-linkers, and the rather low protein and chromatin content per body weight. These issues have rendered standard ChIP-seq protocols inefficient in C. elegans and raised a need for their improvement. Here, we describe improved protocols, with the most important advances being the efficient breakage of the C. elegans cuticle by freeze-grinding and the use of a very sensitive sequencing library construction procedure, optimized for the relatively low DNA content per body weight of C. elegans. The protocols should therefore improve the reproducibility, sensitivity, and uniformity across tissues of ChIP-seq in this organism. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth and harvesting of synchronized Caenorhabditis elegans Basic Protocol 2: Chromatin immunoprecipitation (ChIP) Basic Protocol 3: Library construction for Illumina sequencing.


Assuntos
Caenorhabditis elegans , Sequenciamento de Cromatina por Imunoprecipitação , Animais , Caenorhabditis elegans/genética , Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Reprodutibilidade dos Testes
2.
BMC Bioinformatics ; 22(1): 323, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34126932

RESUMO

BACKGROUND: Histone modification constitutes a basic mechanism for the genetic regulation of gene expression. In early 2000s, a powerful technique has emerged that couples chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq). This technique provides a direct survey of the DNA regions associated to these modifications. In order to realize the full potential of this technique, increasingly sophisticated statistical algorithms have been developed or adapted to analyze the massive amount of data it generates. Many of these algorithms were built around natural assumptions such as the Poisson distribution to model the noise in the count data. In this work we start from these natural assumptions and show that it is possible to improve upon them. RESULTS: Our comparisons on seven reference datasets of histone modifications (H3K36me3 & H3K4me3) suggest that natural assumptions are not always realistic under application conditions. We show that the unconstrained multiple changepoint detection model with alternative noise assumptions and supervised learning of the penalty parameter reduces the over-dispersion exhibited by count data. These models, implemented in the R package CROCS ( https://github.com/aLiehrmann/CROCS ), detect the peaks more accurately than algorithms which rely on natural assumptions. CONCLUSION: The segmentation models we propose can benefit researchers in the field of epigenetics by providing new high-quality peak prediction tracks for H3K36me3 and H3K4me3 histone modifications.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Sequenciamento de Nucleotídeos em Larga Escala , Algoritmos , Imunoprecipitação da Cromatina , Análise de Sequência de DNA
3.
Nat Commun ; 12(1): 3707, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140478

RESUMO

While the major drivers of melanoma initiation, including activation of NRAS/BRAF and loss of PTEN or CDKN2A, have been identified, the role of key transcription factors that impose altered transcriptional states in response to deregulated signaling is not well understood. The POU domain transcription factor BRN2 is a key regulator of melanoma invasion, yet its role in melanoma initiation remains unknown. Here, in a BrafV600E PtenF/+ context, we show that BRN2 haplo-insufficiency promotes melanoma initiation and metastasis. However, metastatic colonization is less efficient in the absence of Brn2. Mechanistically, BRN2 directly induces PTEN expression and in consequence represses PI3K signaling. Moreover, MITF, a BRN2 target, represses PTEN transcription. Collectively, our results suggest that on a PTEN heterozygous background somatic deletion of one BRN2 allele and temporal regulation of the other allele elicits melanoma initiation and progression.


Assuntos
Carcinogênese/metabolismo , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor , Proteínas de Homeodomínio/metabolismo , Melanoma/metabolismo , Fatores do Domínio POU/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Estudos de Coortes , Variações do Número de Cópias de DNA , Progressão da Doença , Técnicas de Silenciamento de Genes , Haploinsuficiência , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Melanoma/genética , Melanoma/mortalidade , Melanoma/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise em Microsséries , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Fatores do Domínio POU/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , RNA Interferente Pequeno , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/secundário
4.
Nat Commun ; 12(1): 3686, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140498

RESUMO

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.


Assuntos
Hipóxia Celular , Dano ao DNA/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica/genética , Enzimas Multifuncionais/metabolismo , Estruturas R-Loop/genética , RNA Helicases/metabolismo , Resposta a Proteínas não Dobradas/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , DNA Helicases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Enzimas Multifuncionais/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oxigênio/farmacologia , Estruturas R-Loop/efeitos dos fármacos , RNA Helicases/genética , RNA-Seq , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima , Zinostatina/farmacologia , eIF-2 Quinase/metabolismo
5.
Nat Commun ; 12(1): 3621, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131149

RESUMO

Chromatin structure and accessibility, and combinatorial binding of transcription factors to regulatory elements in genomic DNA control transcription. Genetic variations in genes encoding histones, epigenetics-related enzymes or modifiers affect chromatin structure/dynamics and result in alterations in gene expression contributing to cancer development or progression. Gliomas are brain tumors frequently associated with epigenetics-related gene deregulation. We perform whole-genome mapping of chromatin accessibility, histone modifications, DNA methylation patterns and transcriptome analysis simultaneously in multiple tumor samples to unravel epigenetic dysfunctions driving gliomagenesis. Based on the results of the integrative analysis of the acquired profiles, we create an atlas of active enhancers and promoters in benign and malignant gliomas. We explore these elements and intersect with Hi-C data to uncover molecular mechanisms instructing gene expression in gliomas.


Assuntos
Cromatina , Glioma/genética , Sequências Reguladoras de Ácido Nucleico , Sítios de Ligação , Neoplasias Encefálicas/genética , Imunoprecipitação da Cromatina , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética , Epigenômica , Proteína Forkhead Box M1 , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Código das Histonas , Histonas , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
6.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070531

RESUMO

SMAD4, a key regulator of transforming growth factor-ß (TGF-ß) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-ß/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-ß signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-ß/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-ß/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-ß/SMAD4-signaling suppression in tumorigenesis.


Assuntos
Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Transdução de Sinais/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p15/genética , Dimerização , Genes Supressores de Tumor , Humanos , Fator 1 Nuclear Respiratório/genética , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Deleção de Sequência , Proteína Smad4/genética , Fator de Crescimento Transformador beta/farmacologia
7.
Nat Commun ; 12(1): 3542, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112789

RESUMO

R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA/genética , Estruturas R-Loop , RNA Longo não Codificante/metabolismo , Telômero/metabolismo , Proteína BRCA1/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromatografia Líquida , Ilhas de CpG , DNA Helicases/metabolismo , Exorribonucleases/metabolismo , Humanos , Hibridização in Situ Fluorescente , Espectrometria de Massas , Enzimas Multifuncionais/metabolismo , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Estruturas R-Loop/genética , RNA Helicases/metabolismo , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Telômero/genética
8.
BMC Genomics ; 22(1): 482, 2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34174819

RESUMO

BACKGROUND: Transcription factors (TFs) bind specifically to TF binding sites (TFBSs) at cis-regulatory regions to control transcription. It is critical to locate these TF-DNA interactions to understand transcriptional regulation. Efforts to predict bona fide TFBSs benefit from the availability of experimental data mapping DNA binding regions of TFs (chromatin immunoprecipitation followed by sequencing - ChIP-seq). RESULTS: In this study, we processed ~ 10,000 public ChIP-seq datasets from nine species to provide high-quality TFBS predictions. After quality control, it culminated with the prediction of ~ 56 million TFBSs with experimental and computational support for direct TF-DNA interactions for 644 TFs in > 1000 cell lines and tissues. These TFBSs were used to predict > 197,000 cis-regulatory modules representing clusters of binding events in the corresponding genomes. The high-quality of the TFBSs was reinforced by their evolutionary conservation, enrichment at active cis-regulatory regions, and capacity to predict combinatorial binding of TFs. Further, we confirmed that the cell type and tissue specificity of enhancer activity was correlated with the number of TFs with binding sites predicted in these regions. All the data is provided to the community through the UniBind database that can be accessed through its web-interface ( https://unibind.uio.no/ ), a dedicated RESTful API, and as genomic tracks. Finally, we provide an enrichment tool, available as a web-service and an R package, for users to find TFs with enriched TFBSs in a set of provided genomic regions. CONCLUSIONS: UniBind is the first resource of its kind, providing the largest collection of high-confidence direct TF-DNA interactions in nine species.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , DNA , Sítios de Ligação , Imunoprecipitação da Cromatina , Biologia Computacional , DNA/metabolismo , Ligação Proteica
9.
Life Sci ; 278: 119570, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33964295

RESUMO

AIMS: Increasing evidence has shown that hormone secretion is regulated by endocytosis. Eps15 homology domain-containing protein 3 (EHD3) is an endocytic-trafficking regulatory protein, but whether EHD3 is associated with testosterone secretion is not clear. This work aims to explore the role of EHD3 in testosterone synthesis. MAIN METHODS: Testosterone concentration was determined by ELISA. The effects of EHD3 on endocytosis were assessed by exosomes tracing assay and Immunofluorescence. Targeting relationship between EHD3 and NR5A1 was verified by chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene assay in Leydig cells. For in vivo assessments, conditional NR5A1 knockout mouse model was established with CRISPR/Cas9 gene targeting technology. KEY FINDINGS: EHD3 overexpression significantly increased the concentration of testosterone. EHD3 knockdown markedly decreased testosterone synthesis by reducing endocytosis. The activity of the EHD3 promoter was positively regulated by NR5A1, which occupied the conserved sequence "AGGTCA" in the EHD3 promoter. Furthermore, mice with a Leydig cell-specific conditional NR5A1 knockout displayed the blunted levels of EHD3 and clathrin (a key factor for endocytosis), and serum testosterone concentration compared with NR5A1f/f mice. SIGNIFICANCE: This study suggests a potential molecular mechanism of testosterone synthesis to fully understand male reproductive health.


Assuntos
Proteínas de Transporte/metabolismo , Endocitose , Exossomos/metabolismo , Regulação da Expressão Gênica , Fator Esteroidogênico 1/metabolismo , Testosterona/metabolismo , Animais , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Imunoprecipitação da Cromatina , Feminino , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Fator Esteroidogênico 1/genética , Testosterona/farmacologia
10.
Nat Commun ; 12(1): 2584, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972520

RESUMO

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance pathway utilised in 15% of cancers. ALT cancers are strongly associated with inactivating mutations in ATRX; yet loss of ATRX alone is insufficient to trigger ALT, suggesting that additional cooperating factors are involved. We identify H3.3G34R and IDH1/2 mutations as two such factors in ATRX-mutated glioblastomas. Both mutations are capable of inactivating histone demethylases, and we identify KDM4B as the key demethylase inactivated in ALT. Mouse embryonic stem cells inactivated for ATRX, TP53, TERT and KDM4B (KDM4B knockout or H3.3G34R) show characteristic features of ALT. Conversely, KDM4B over-expression in ALT cancer cells abrogates ALT-associated features. In this work, we demonstrate that inactivation of KDM4B, through H3.3G34R or IDH1/2 mutations, acts in tandem with ATRX mutations to promote ALT in glioblastomas.


Assuntos
Células-Tronco Embrionárias/metabolismo , Glioblastoma/genética , Histonas/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Homeostase do Telômero/genética , Proteína Nuclear Ligada ao X/genética , Adulto , Animais , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Replicação do DNA/genética , Técnicas de Inativação de Genes , Glioblastoma/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Isocitrato Desidrogenase/genética , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Mutação , Transdução de Sinais/genética , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima
11.
Nat Commun ; 12(1): 2482, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931647

RESUMO

While oncogenes promote tumorigenesis, they also induce deleterious cellular stresses, such as apoptosis, that cancer cells must combat by coopting adaptive responses. Whether tumor suppressor gene haploinsufficiency leads to such phenomena and their mechanistic basis is unclear. Here, we demonstrate that elevated levels of the anti-apoptotic factor, CASP8 and FADD-like apoptosis regulator (CFLAR), promotes apoptosis evasion in acute myeloid leukemia (AML) cells haploinsufficient for the cut-like homeobox 1 (CUX1) transcription factor, whose loss is associated with dismal clinical prognosis. Genome-wide CRISPR/Cas9 screening identifies CFLAR as a selective, acquired vulnerability in CUX1-deficient AML, which can be mimicked therapeutically using inhibitor of apoptosis (IAP) antagonists in murine and human AML cells. Mechanistically, CUX1 deficiency directly alleviates CUX1 repression of the CFLAR promoter to drive CFLAR expression and leukemia survival. These data establish how haploinsufficiency of a tumor suppressor is sufficient to induce advantageous anti-apoptosis cell survival pathways and concurrently nominate CFLAR as potential therapeutic target in these poor-prognosis leukemias.


Assuntos
Apoptose/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Haploinsuficiência , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , Dipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Genes Supressores de Tumor , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Indóis/farmacologia , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Análise Serial de Proteínas , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
12.
Nat Commun ; 12(1): 2576, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33958593

RESUMO

Nitric oxide (NO) is a diffusible signaling molecule that modulates animal and plant immune responses. In addition, reactive nitrogen species derived from NO can display antimicrobial activities by reacting with microbial cellular components, leading to nitrosative stress (NS) in pathogens. Here, we identify FgAreB as a regulator of the NS response in Fusarium graminearum, a fungal pathogen of cereal crops. FgAreB serves as a pioneer transcription factor for recruitment of the chromatin-remodeling complex SWI/SNF at the promoters of genes involved in the NS response, thus promoting their transcription. FgAreB plays important roles in fungal infection and growth. Furthermore, we show that a transcription repressor (FgIxr1) competes with the SWI/SNF complex for FgAreB binding, and negatively regulates the NS response. NS, in turn, promotes the degradation of FgIxr1, thus enhancing the recruitment of the SWI/SNF complex by FgAreB.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Fusarium/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Doenças das Plantas/microbiologia , Proteína SMARCB1/metabolismo , Fatores de Transcrição/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Fusarium/genética , Fusarium/patogenicidade , Fatores de Transcrição GATA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Óxido Nítrico/metabolismo , Estresse Nitrosativo , Proteína SMARCB1/genética , Fatores de Transcrição/genética , Triticum/microbiologia , Zea mays/microbiologia
13.
Nat Commun ; 12(1): 2505, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947848

RESUMO

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.


Assuntos
Células Epidérmicas/citologia , Proteína Forkhead Box M1/metabolismo , Queratinócitos/citologia , Análise de Célula Única/métodos , Células-Tronco/citologia , Transcriptoma/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Adesão Celular/genética , Linhagem Celular , Autorrenovação Celular/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Células Epidérmicas/metabolismo , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/metabolismo , Proteína Forkhead Box M1/genética , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Queratinócitos/metabolismo , Camundongos , Análise em Microsséries , Família Multigênica , RNA-Seq , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
14.
Nat Commun ; 12(1): 2509, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947861

RESUMO

Differential transcription of identical DNA sequences leads to distinct tissue lineages and then multiple cell types within a lineage, an epigenetic process central to progenitor and stem cell biology. The associated genome-wide changes, especially in native tissues, remain insufficiently understood, and are hereby addressed in the mouse lung, where the same lineage transcription factor NKX2-1 promotes the diametrically opposed alveolar type 1 (AT1) and AT2 cell fates. Here, we report that the cell-type-specific function of NKX2-1 is attributed to its differential chromatin binding that is acquired or retained during development in coordination with partner transcriptional factors. Loss of YAP/TAZ redirects NKX2-1 from its AT1-specific to AT2-specific binding sites, leading to transcriptionally exaggerated AT2 cells when deleted in progenitors or AT1-to-AT2 conversion when deleted after fate commitment. Nkx2-1 mutant AT1 and AT2 cells gain distinct chromatin accessible sites, including those specific to the opposite fate while adopting a gastrointestinal fate, suggesting an epigenetic plasticity unexpected from transcriptional changes. Our genomic analysis of single or purified cells, coupled with precision genetics, provides an epigenetic basis for alveolar cell fate and potential, and introduces an experimental benchmark for deciphering the in vivo function of lineage transcription factors.


Assuntos
Células Epiteliais Alveolares/metabolismo , Diferenciação Celular/genética , Cromatina/metabolismo , Epigênese Genética , Histonas/metabolismo , Células-Tronco/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Epiteliais Alveolares/citologia , Animais , Linhagem da Célula , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Pulmão/metabolismo , Camundongos , Mutação , Ligação Proteica , RNA-Seq , Análise de Célula Única , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Nat Commun ; 12(1): 2507, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947863

RESUMO

Notch1 is a crucial oncogenic driver in T-cell acute lymphoblastic leukemia (T-ALL), making it an attractive therapeutic target. However, the success of targeted therapy using γ-secretase inhibitors (GSIs), small molecules blocking Notch cleavage and subsequent activation, has been limited due to development of resistance, thus restricting its clinical efficacy. Here, we systematically compare GSI resistant and sensitive cell states by quantitative mass spectrometry-based phosphoproteomics, using complementary models of resistance, including T-ALL patient-derived xenografts (PDX) models. Our datasets reveal common mechanisms of GSI resistance, including a distinct kinase signature that involves protein kinase C delta. We demonstrate that the PKC inhibitor sotrastaurin enhances the anti-leukemic activity of GSI in PDX models and completely abrogates the development of acquired GSI resistance in vitro. Overall, we highlight the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Oligopeptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteína Quinase C/metabolismo , Receptor Notch1/antagonistas & inibidores , Acetofenonas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Antineoplásicos/uso terapêutico , Benzopiranos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Cromatografia Líquida de Alta Pressão , Resistencia a Medicamentos Antineoplásicos/genética , Ontologia Genética , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos NOD , Fosforilação , Análise Serial de Proteínas , Biossíntese de Proteínas/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteínas Quinases/metabolismo , Proteômica , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em Tandem , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Mol Cell Biol ; 41(7): e0004721, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-33875574

RESUMO

In budding tunicates, aging accompanies a decrease in the gene expression of mitochondrial transcription factor A (Tfam), and the in vivo transfection of Tfam mRNA stimulates the mitochondrial respiratory activity of aged animals. The gene expression of both the transcriptional repressor Yin-Yang-1 (YY1) and corepressor Sirtuin6 (Sirt6) increased during aging, and the cotransfection of synthetic mRNA of YY1 and Sirt6 synergistically downregulated Tfam gene expression. Pulldown assays of proteins indicated that YY1-associated factor 2 (YAF2) was associated with both YY1 and SIRT6. Protein cross-linking confirmed that YAF2 bound YY1 and SIRT6 with a molar ratio of 1:1. YY1 was bound to CCAT- or ACAT-containing oligonucleotides in the 5' flanking region of the Tfam gene. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) showed that SIRT6 specifically induced the histone H3 lysine 9 (H3K9) deacetylation of the Tfam upstream region. YY1 and YAF2 accelerated SIRT6-induced H3K9 deacetylation. YY1 and Sirt6 mRNA transfection attenuated mitochondrial respiratory gene expression and blocked MitoTracker fluorescence. In contrast, the SIRT6 inhibitor and Tfam mRNA antagonized the inhibitory effects of YY1 and Sirt6, indicating that Tfam acts on mitochondria downstream of YY1 and Sirt6. We concluded that in the budding tunicate Polyandrocarpa misakiensis, YY1 recruits SIRT6 via YAF2 to the TFAM gene, resulting in aging-related mitochondrial downregulation.


Assuntos
Envelhecimento/fisiologia , Mitocôndrias/metabolismo , Urocordados/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Proteínas Mitocondriais/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Urocordados/genética , Fator de Transcrição YY1/genética
17.
Methods Mol Biol ; 2285: 201-216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928555

RESUMO

Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) is an invaluable method to profile of enrichment of histone modifications and transcription factor binding sites across the genome. However, standard ChIP-seq protocols require large numbers of cells (>107) as starting material, which are often impossible to obtain for rare immune populations. Here we describe a streamlined ChIP protocol optimised for small cell numbers in conjunction with transposon-tagging mediated sequencing library preparation (ChIPmentation) which allows the analysis of samples of as low as 105 cells.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Imunoprecipitação da Cromatina , DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica , Projetos de Pesquisa , Fluxo de Trabalho
18.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807548

RESUMO

About 8% of the human genome is covered with candidate cis-regulatory elements (cCREs). Disruptions of CREs, described as "cis-ruptions" have been identified as being involved in various genetic diseases. Thanks to the development of chromatin conformation study techniques, several long-range cystic fibrosis transmembrane conductance regulator (CFTR) regulatory elements were identified, but the regulatory mechanisms of the CFTR gene have yet to be fully elucidated. The aim of this work is to improve our knowledge of the CFTR gene regulation, and to identity factors that could impact the CFTR gene expression, and potentially account for the variability of the clinical presentation of cystic fibrosis as well as CFTR-related disorders. Here, we apply the robust GWAS3D score to determine which of the CFTR introns could be involved in gene regulation. This approach highlights four particular CFTR introns of interest. Using reporter gene constructs in intestinal cells, we show that two new introns display strong cooperative effects in intestinal cells. Chromatin immunoprecipitation analyses further demonstrate fixation of transcription factors network. These results provide new insights into our understanding of the CFTR gene regulation and allow us to suggest a 3D CFTR locus structure in intestinal cells. A better understand of regulation mechanisms of the CFTR gene could elucidate cases of patients where the phenotype is not yet explained by the genotype. This would thus help in better diagnosis and therefore better management. These cis-acting regions may be a therapeutic challenge that could lead to the development of specific molecules capable of modulating gene expression in the future.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Intestinos/fisiologia , Células CACO-2 , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/métodos , Fibrose Cística/genética , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Humanos , Íntrons/genética , Fatores de Transcrição/genética
19.
BMC Bioinformatics ; 22(1): 193, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33858322

RESUMO

BACKGROUND: ChIP-seq combines chromatin immunoprecipitation assays with sequencing and identifies genome-wide binding sites for DNA binding proteins. While many binding sites have strong ChIP-seq 'peak' observations and are well captured, there are still regions bound by proteins weakly, with a relatively low ChIP-seq signal enrichment. These weak binding sites, especially those at promoters and enhancers, are functionally important because they also regulate nearby gene expression. Yet, it remains a challenge to accurately identify weak binding sites in ChIP-seq data due to the ambiguity in differentiating these weak binding sites from the amplified background DNAs. RESULTS: ChIP-BIT2 ( http://sourceforge.net/projects/chipbitc/ ) is a software package for ChIP-seq peak detection. ChIP-BIT2 employs a mixture model integrating protein and control ChIP-seq data and predicts strong or weak protein binding sites at promoters, enhancers, or other genomic locations. For binding sites at gene promoters, ChIP-BIT2 simultaneously predicts their target genes. ChIP-BIT2 has been validated on benchmark regions and tested using large-scale ENCODE ChIP-seq data, demonstrating its high accuracy and wide applicability. CONCLUSION: ChIP-BIT2 is an efficient ChIP-seq peak caller. It provides a better lens to examine weak binding sites and can refine or extend the existing binding site collection, providing additional regulatory regions for decoding the mechanism of gene expression regulation.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Software , Teorema de Bayes , Sítios de Ligação , Imunoprecipitação da Cromatina , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA
20.
J Vis Exp ; (169)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33843935

RESUMO

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on host and/or pathogen chromatin. Chromatin preparation from tissues creates additional challenges that need to be overcome to obtain reproducible and reliable protocols comparable to those used for cell culture. Tissue disruption and fixation are critical steps to achieve efficient shearing of chromatin. Coexistence of different cell types and clusters may also require different shearing times to reach optimal fragment size and hinders shearing reproducibility. The purpose of this method is to achieve reliable and reproducible host chromatin preparations from frozen tissue (liver) suitable for both ChIP-qPCR and next generation sequencing (NGS) applications. We observed that the combination of liquid nitrogen tissue pulverization followed by homogenization leads to increased reproducibility compared to homogenization only, since it provides a suspension consisting mostly of dissociated single cells that can be efficiently sheared. Moreover, the fixation step should be performed under mild rotation to provide homogeneous crosslinking. The fixed material is then suitable for buffer-based nuclei isolation, to reduce contamination of cytoplasmic protein and pathogen DNAs and RNAs (when applicable), avoiding time-consuming centrifugation gradients. Subsequent sonication will complete nuclear lysis and shear the chromatin, producing a specific size range according to the chosen shearing conditions. The size range should fall between 100 and 300 nt for NGS applications, while it could be higher (300-700 nt) for ChIP-qPCR analysis. Such protocol adaptations can greatly improve chromatin analyses from frozen tissue specimens.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/química , Fígado/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...