RESUMO
The resistance of cancer cells to therapy is responsible for the death of most patients with cancer1. Epithelial-to-mesenchymal transition (EMT) has been associated with resistance to therapy in different cancer cells2,3. However, the mechanisms by which EMT mediates resistance to therapy remain poorly understood. Here, using a mouse model of skin squamous cell carcinoma undergoing spontaneous EMT during tumorigenesis, we found that EMT tumour cells are highly resistant to a wide range of anti-cancer therapies both in vivo and in vitro. Using gain and loss of function studies in vitro and in vivo, we found that RHOJ-a small GTPase that is preferentially expressed in EMT cancer cells-controls resistance to therapy. Using genome-wide transcriptomic and proteomic profiling, we found that RHOJ regulates EMT-associated resistance to chemotherapy by enhancing the response to replicative stress and activating the DNA-damage response, enabling tumour cells to rapidly repair DNA lesions induced by chemotherapy. RHOJ interacts with proteins that regulate nuclear actin, and inhibition of actin polymerization sensitizes EMT tumour cells to chemotherapy-induced cell death in a RHOJ-dependent manner. Together, our study uncovers the role and the mechanisms through which RHOJ acts as a key regulator of EMT-associated resistance to chemotherapy.
Assuntos
Carcinoma de Células Escamosas , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Neoplasias Cutâneas , Proteínas rho de Ligação ao GTP , Actinas/efeitos dos fármacos , Actinas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteômica , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Camundongos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Perfilação da Expressão Gênica , GenomaRESUMO
Melanocytes present in hair follicles are responsible for their pigmentation. Melanocyte differentiation and hair pigmentation depend on the stem cell factor (SCF)/c-Kit signaling pathway, but the niche that regulates melanocyte differentiation is not well characterized. In this issue of Genes & Development, Liao and colleagues (pp. 744-756) identify Krox20+-derived cells of the hair shaft as the niche and the essential source of SCF required for melanocyte maturation. This study delineates the niche factors regulating melanocyte differentiation and hair pigmentation and opens up new avenues to further characterize the cross-talk between the hair follicle and melanocytes that controls melanocyte maintenance and differentiation.
Assuntos
Diferenciação Celular , Folículo Piloso/citologia , Melanócitos/citologia , Animais , Melanócitos/metabolismo , Pigmentação/genética , Pigmentação/fisiologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismoRESUMO
RNAi is a conserved mechanism in which small RNAs induce silencing of complementary targets. We have previously identified priRNAs, a class of Dicer-independent small RNAs in fission yeast. The mechanism by which Dicer-independent small RNAs are generated is not well understood in any species. Here we reconstitute the final steps of priRNA and siRNA biogenesis in vitro. We identify the 3'-5' exonuclease Triman and demonstrate that Argonaute, loaded with longer RNA precursors, recruits Triman to generate mature priRNAs and siRNAs. We show that priRNA and siRNA trimming is required for de novo assembly of heterochromatin at centromeric repeats and the mat locus and for maintenance of heterochromatin at developmental genes. Furthermore, in rrp6Δ cells RNAi targets diverse genes in a Triman-dependent way, indicating that the exosome protects the genome from spurious RNAi. Our results suggest that Argonaute association with RNA degradation products generates priRNAs and triggers RNAi in a process of transcriptome surveillance.
Assuntos
Proteínas Argonautas/genética , Endorribonucleases/genética , Exorribonucleases/genética , Heterocromatina/genética , RNA Fúngico/genética , RNA Interferente Pequeno/genética , Proteínas de Schizosaccharomyces pombe/genética , Sequência de Aminoácidos , Proteínas Argonautas/metabolismo , Western Blotting , Centrômero/genética , Centrômero/metabolismo , Eletroforese em Gel de Poliacrilamida , Endorribonucleases/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Heterocromatina/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Fúngico/metabolismo , RNA Interferente Pequeno/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Heterochromatin is a distinctive chromatin structure that is essential for chromosome segregation, genome stability and regulation of gene expression. H3K9 methylation (H3K9me), a hallmark of heterochromatin, is deposited by the Su(var)3-9 family of proteins; however, the mechanism by which H3K9 methyltransferases bind and methylate the nucleosome is poorly understood. In this work we determined the interaction of Clr4, the fission yeast H3K9 methyltransferase, with nucleosomes using nuclear magnetic resonance, biochemical and genetic assays. Our study shows that the Clr4 chromodomain binds the H3K9me3 tail and that both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. We show that interaction of the disordered region with the nucleosome core is independent of H3K9me and contributes to H3K9me in vitro and in vivo. Moreover, we show that those interactions with the nucleosome core are contributing to de novo deposition of H3K9me and to establishment of heterochromatin.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/metabolismo , Heterocromatina/metabolismo , Código das Histonas , Histona-Lisina N-Metiltransferase/metabolismo , Metiltransferases/metabolismo , Nucleossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Deleção de Genes , Heterocromatina/ultraestrutura , Histona-Lisina N-Metiltransferase/química , Metiltransferases/química , Metiltransferases/genética , Modelos Moleculares , Mutagênese Insercional , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , RNA Fúngico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Long noncoding RNAs (lncRNAs) play a conserved role in regulating gene expression, chromatin dynamics, and cell differentiation. They serve as a platform for RNA interference (RNAi)-mediated heterochromatin formation or DNA methylation in many eukaryotic organisms. We found in Schizosaccharomyces pombe that heterochromatin is lost at transcribed regions in the absence of RNA degradation. We show that heterochromatic RNAs are retained on chromatin, form DNA:RNA hybrids, and need to be degraded by the Ccr4-Not complex or RNAi to maintain heterochromatic silencing. The Ccr4-Not complex is localized to chromatin independently of H3K9me and degrades chromatin-associated transcripts, which is required for transcriptional silencing. Overexpression of heterochromatic RNA, but not euchromatic RNA, leads to chromatin localization and loss of silencing of a distant ade6 reporter in wild-type cells. Our results demonstrate that chromatin-bound RNAs disrupt heterochromatin organization and need to be degraded in a process of heterochromatin formation.
Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Heterocromatina/metabolismo , RNA Fúngico/metabolismo , Schizosaccharomyces/metabolismo , Heterocromatina/genética , RNA Fúngico/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
To maintain genome stability, cells pack large portions of their genome into silent chromatin or heterochromatin. Histone H3 lysine 9 methylation, a hallmark of heterochromatin, is recognized by conserved readers called chromodomains. But how chromodomains interact with their actual binding partner, the H3K9 methylated nucleosome, remains elusive. We have determined the structure of a nucleosome trimethylated at lysine 9 of histone H3 (H3K9me3 Nucleosome) in a complex with the chromodomain of Chp1, a protein required for RNA interference-dependent heterochromatin formation in fission yeast. The cryo-electron microscopy structure reveals that the chromodomain of Chp1 binds the histone H3 lysine 9 methylated tail and the core of the nucleosome, primarily histones H3 and H2B. Mutations in chromodomain of Chp1 loops, which interact with the nucleosome core, abolished this interaction in vitro. Moreover, fission yeast cells with Chp1 loop mutations have a defect in Chp1 recruitment and heterochromatin formation. This study reveals the structural basis for heterochromatic silencing and suggests that chromodomains could read histone code in the H3 tail and the nucleosome core, which would provide an additional layer of regulation.