RESUMO
Alternative lengthening of telomeres, or ALT, is a recombination-based process that maintains telomeres to render some cancer cells immortal. The prevailing view is that ALT is inhibited by heterochromatin because heterochromatin prevents recombination. To test this model, we used telomere-specific quantitative proteomics on cells with heterochromatin deficiencies. In contrast to expectations, we found that ALT does not result from a lack of heterochromatin; rather, ALT is a consequence of heterochromatin formation at telomeres, which is seeded by the histone methyltransferase SETDB1. Heterochromatin stimulates transcriptional elongation at telomeres together with the recruitment of recombination factors, while disrupting heterochromatin had the opposite effect. Consistently, loss of SETDB1, disrupts telomeric heterochromatin and abrogates ALT. Thus, inhibiting telomeric heterochromatin formation in ALT cells might offer a new therapeutic approach to cancer treatment.
Assuntos
Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Encurtamento do Telômero , Telômero/metabolismo , Animais , Linhagem Celular Tumoral , Chaperonas de Histonas/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Humanos , Metiltransferases/deficiência , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Proteína Nuclear Ligada ao X/metabolismoRESUMO
Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II-independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells.
Assuntos
Adesão Celular , Membrana Celular/fisiologia , Movimento Celular , Citoesqueleto/fisiologia , Fibroblastos/fisiologia , Mecanotransdução Celular , Pseudópodes/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Forma Celular , Células Cultivadas , Simulação por Computador , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , Modelos Biológicos , Miosina Tipo II/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Pseudópodes/metabolismo , Estresse Mecânico , Fatores de Tempo , Transfecção , Vinculina/metabolismoRESUMO
The biological functions of given genomic regions are ruled by the local chromatin composition. The Proteomics of Isolated Chromatin segments approach (PICh) is a powerful and unbiased method to analyze the composition of chosen chromatin segments, provided they are abundant (repeated) or that the organism studied has a small genome. PICh can be used to identify novel and unexpected regulatory factors, or when combined with quantitative mass spectrometric approaches, to characterize the function of a defined factor at the chosen locus, by quantifying composition changes at the locus upon removal/addition of that factor.