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


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.

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
EMBO J ; 39(23): e105606, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33433018


Chromosomes have an intrinsic tendency to segregate into compartments, forming long-distance contacts between loci of similar chromatin states. How genome compartmentalization is regulated remains elusive. Here, comparison of mouse ground-state embryonic stem cells (ESCs) characterized by open and active chromatin, and advanced serum ESCs with a more closed and repressed genome, reveals distinct regulation of their genome organization due to differential dependency on BAZ2A/TIP5, a component of the chromatin remodeling complex NoRC. On ESC chromatin, BAZ2A interacts with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. BAZ2A associates with chromatin sub-domains within the active A compartment, which intersect through long-range contacts. We found that ground-state chromatin selectively requires BAZ2A to limit the invasion of active domains into repressive compartments. BAZ2A depletion increases chromatin accessibility at B compartments. Furthermore, BAZ2A regulates H3K27me3 genome occupancy in a TOP2A-dependent manner. Finally, ground-state ESCs require BAZ2A for growth, differentiation, and correct expression of developmental genes. Our results uncover the propensity of open chromatin domains to invade repressive domains, which is counteracted by chromatin remodeling to establish genome partitioning and preserve cell identity.

Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Genoma , Células-Tronco Pluripotentes/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ciclo Celular , Diferenciação Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA Topoisomerases Tipo II/metabolismo , Epigenômica , Regulação da Expressão Gênica , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo
Nat Cell Biol ; 19(7): 763-773, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28604677


Naive pluripotency is established in preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of naive pluripotency. 2i culture has optimized this state, leading to a gene signature and DNA hypomethylation closely comparable to preimplantation epiblast, the developmental ground state. Here we show that Pramel7 (PRAME-like 7), a protein highly expressed in the inner cell mass (ICM) but expressed at low levels in ESCs, targets for proteasomal degradation UHRF1, a key factor for DNA methylation maintenance. Increasing Pramel7 expression in serum-cultured ESCs promotes a preimplantation epiblast-like gene signature, reduces UHRF1 levels and causes global DNA hypomethylation. Pramel7 is required for blastocyst formation and its forced expression locks ESCs in pluripotency. Pramel7/UHRF1 expression is mutually exclusive in ICMs whereas Pramel7-knockout embryos express high levels of UHRF1. Our data reveal an as-yet-unappreciated dynamic nature of DNA methylation through proteasome pathways and offer insights that might help to improve ESC culture to reproduce in vitro the in vivo ground-state pluripotency.

Antígenos de Neoplasias/metabolismo , Blastocisto/enzimologia , Células-Tronco Embrionárias/enzimologia , Epigênese Genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Antígenos de Neoplasias/genética , Blastocisto/citologia , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas Culina/metabolismo , Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fenótipo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , Interferência de RNA , Fatores de Tempo , Transcriptoma , Transfecção , Ubiquitina-Proteína Ligases
EMBO Rep ; 18(7): 1248-1262, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28588071


Long non-coding RNAs (lncRNAs) have been implicated in the regulation of chromatin conformation and epigenetic patterns. lncRNA expression levels are widely taken as an indicator for functional properties. However, the role of RNA processing in modulating distinct features of the same lncRNA is less understood. The establishment of heterochromatin at rRNA genes depends on the processing of IGS-rRNA into pRNA, a reaction that is impaired in embryonic stem cells (ESCs) and activated only upon differentiation. The production of mature pRNA is essential since it guides the repressor TIP5 to rRNA genes, and IGS-rRNA abolishes this process. Through screening for IGS-rRNA-binding proteins, we here identify the RNA helicase DHX9 as a regulator of pRNA processing. DHX9 binds to rRNA genes only upon ESC differentiation and its activity guides TIP5 to rRNA genes and establishes heterochromatin. Remarkably, ESCs depleted of DHX9 are unable to differentiate and this phenotype is reverted by the addition of pRNA, whereas providing IGS-rRNA and pRNA mutants deficient for TIP5 binding are not sufficient. Our results reveal insights into lncRNA biogenesis during development and support a model in which the state of rRNA gene chromatin is part of the regulatory network that controls exit from pluripotency and initiation of differentiation pathways.

Diferenciação Celular , RNA Helicases DEAD-box/metabolismo , Células-Tronco Embrionárias/fisiologia , Heterocromatina/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Proteínas Cromossômicas não Histona , RNA Helicases DEAD-box/genética , DNA Ribossômico , Epigênese Genética , Genes de RNAr , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Proteínas de Neoplasias/genética , RNA Helicases/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
Oncotarget ; 7(16): 21272-86, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-26848772


Calretinin (CALB2) is a diagnostic marker for epithelioid mesothelioma. It is also a prognostic marker since patients with tumors expressing high calretinin levels have better overall survival. Silencing of calretinin decreases viability of epithelioid mesothelioma cells. Our aim was to elucidate mechanisms regulating calretinin expression in mesothelioma. Analysis of calretinin transcript and protein suggested a control at the mRNA level. Treatment with 5-aza-2'-deoxycytidine and analysis of TCGA data indicated that promoter methylation is not likely to be involved. Therefore, we investigated CALB2 promoter by analyzing ~1kb of genomic sequence surrounding the transcription start site (TSS) + 1 using promoter reporter assay. Deletion analysis of CALB2 proximal promoter showed that sequence spanning the -161/+80bp region sustained transcriptional activity. Site-directed analysis identified important cis-regulatory elements within this -161/+80bp CALB2 promoter. EMSA and ChIP assays confirmed binding of NRF-1 and E2F2 to the CALB2 promoter and siRNA knockdown of NRF-1 led to decreased expression of calretinin. Cell synchronization experiment showed that calretinin expression was cell cycle regulated with a peak of expression at G1/S phase. This study provides the first insight in the regulation of CALB2 expression in mesothelioma cells.

Calbindina 2/genética , Regulação Neoplásica da Expressão Gênica , Mesotelioma/genética , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , Calbindina 2/metabolismo , Células Cultivadas , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Humanos , Mesotelioma/metabolismo , Mesotelioma/patologia , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo
J Cell Biol ; 208(5): 563-79, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25733714


Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy.

Dano ao DNA , Replicação do DNA , Rad51 Recombinase/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Inibidores da Topoisomerase/toxicidade