Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathway
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/130700 |
Resumo: | Telomeres are nucleic acids-protein complexes located at the ends of linear eukaryotic chromosomes comprising 5’-TTAGGG-3’ tandem repeats, the multiprotein complex shelterin and the long non-coding RNA TERRA. Telomeres act both as molecular clocks and capping structures supporting chromosome integrity. Due to the end-replication problem, telomeres progressively shorten with each cell division and ultimately trigger cellular senescence. Cancer cells bypass senescence and divide indefinitely by activating mechanisms that buffer telomere shortening. Most cancers reactivate the reverse transcriptase telomerase, yet about 15% elongate their telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. ALT cells rely on break-induced replication (BIR), a homology-directed repair mechanism activated at damaged telomeric DNA. This damage is induced by replication stress (ALT-specific Telomeric Replication Stress; ATRS) and must be kept at a level that induces enough BIR yet without strongly activating DNA damage checkpoints that would cause cell death. This balance is attained by the counteracting activities of molecular triggers and alleviators of ATRS. The Azzalin laboratory has recently identified the transcriptional regulator positive cofactor 4 (PC4), a multifunctional nuclear protein capable of binding to dsDNA, ssDNA, G-quadruplex structures and RNA, as essential for ALT cell survival. PC4 was shown to localize at telomeres of ALT cells by indirect immunofluorescence, being recruited in response to replication stress. This study aims at elucidating the role played by PC4 at ALT telomeres. I confirmed by chromatin immunoprecipitation that PC4 associates with telomeres and this association increases when cells are treated with drugs inducing replication stress. Furthermore, PC4 depletion exacerbates ATRS and ALT features. TERRA levels in PC4-depleted cells showed no consistent alterations, suggesting that the effects observed upon PC4 depletion are unlikely to derive from changes in telomere transcription. Moreover, loss of PC4 did not substantially alter ssDNA and total telomeric DNA levels and ectopic expression of a PC4 mutant unable to bind ssDNA only partially rescued the defects due to PC4 loss; this indicates that the function played by PC4 at ALT telomeres is not majorly relying on its ability to bind ssDNA. This work strengthens the notion that PC4 is an ATRS alleviator capable of restricting telomeric replication stress and ALT features. In light of this, PC4 emerges as an attractive target for the development of novel therapeutic protocols against ALT cancers. |
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Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathwayPC4telomeresALTATRSDomínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e TecnologiasTelomeres are nucleic acids-protein complexes located at the ends of linear eukaryotic chromosomes comprising 5’-TTAGGG-3’ tandem repeats, the multiprotein complex shelterin and the long non-coding RNA TERRA. Telomeres act both as molecular clocks and capping structures supporting chromosome integrity. Due to the end-replication problem, telomeres progressively shorten with each cell division and ultimately trigger cellular senescence. Cancer cells bypass senescence and divide indefinitely by activating mechanisms that buffer telomere shortening. Most cancers reactivate the reverse transcriptase telomerase, yet about 15% elongate their telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. ALT cells rely on break-induced replication (BIR), a homology-directed repair mechanism activated at damaged telomeric DNA. This damage is induced by replication stress (ALT-specific Telomeric Replication Stress; ATRS) and must be kept at a level that induces enough BIR yet without strongly activating DNA damage checkpoints that would cause cell death. This balance is attained by the counteracting activities of molecular triggers and alleviators of ATRS. The Azzalin laboratory has recently identified the transcriptional regulator positive cofactor 4 (PC4), a multifunctional nuclear protein capable of binding to dsDNA, ssDNA, G-quadruplex structures and RNA, as essential for ALT cell survival. PC4 was shown to localize at telomeres of ALT cells by indirect immunofluorescence, being recruited in response to replication stress. This study aims at elucidating the role played by PC4 at ALT telomeres. I confirmed by chromatin immunoprecipitation that PC4 associates with telomeres and this association increases when cells are treated with drugs inducing replication stress. Furthermore, PC4 depletion exacerbates ATRS and ALT features. TERRA levels in PC4-depleted cells showed no consistent alterations, suggesting that the effects observed upon PC4 depletion are unlikely to derive from changes in telomere transcription. Moreover, loss of PC4 did not substantially alter ssDNA and total telomeric DNA levels and ectopic expression of a PC4 mutant unable to bind ssDNA only partially rescued the defects due to PC4 loss; this indicates that the function played by PC4 at ALT telomeres is not majorly relying on its ability to bind ssDNA. This work strengthens the notion that PC4 is an ATRS alleviator capable of restricting telomeric replication stress and ALT features. In light of this, PC4 emerges as an attractive target for the development of novel therapeutic protocols against ALT cancers.Os telómeros são complexos de ácidos nucleicos e proteínas localizados nas extremidades dos cromossomas das células eucarióticas constituídos pela sequência repetitiva 5’-TTAGGG-3’, pelo complexo proteico shelterin e pelo RNA não-codificante TERRA. Os telómeros funcionam como relógios celulares e estruturas protectoras que mantêm a integridade dos cromossomas. Devido à incapacidade de replicar totalmente as extremidades cromossómicas, os telómeros são encurtados a cada divisão celular até ser atingido um estado de senescência replicativa. As células de cancro conseguem dividir-se de forma ilimitada ao activarem mecanismos que contrariam o encurtamento dos telómeros. Na maior parte dos cancros a enzima telomerase é reactivada, porém cerca de 15% alonga os seus telómeros através de um mecanismo alternativo de alongamento dos telómeros (alternative lengthening of telomeres, ALT). O mecanismo de ALT baseia-se num processo de reparação homóloga de DNA (break-induced replication, BIR) onde é necessário que os telómeros se encontrem fisiologicamente danificados para serem alongados. Assim, as células ALT acumulam stress replicativo nos telómeros (ALT-specific telomeric replication stress, ATRS) que tem de ser mantido num nível que assegure o seu alongamento sem que desencadeie mecanismos de morte celular, um equilíbrio mantido por proteínas que despoletam ou mitigam este ATRS. Recentemente, o regulador transcricional PC4 (positive cofactor 4), uma proteína multifuncional com capacidade de ligação a DNA de cadeia simples e dupla (ssDNA e dsDNA, respectivamente), a RNA e a estruturas secundárias de ácidos nucleicos (G-quadruplexes) foi identificado pelo Laboratório Azzalin como sendo essencial para a sobrevivência de células ALT, sendo recrutado em resposta a stress replicativo. Este estudo tem como objetivo elucidar o papel desempenhado por PC4 nos telómeros de células ALT. Através de imunoprecipitação de cromatina, eu confirmei que PC4 se associa a DNA telomérico e que esta associação aumenta quando as células são tratadas com compostos que induzem stress replicativo. Demonstrei ainda que a depleção de PC4 leva ao aumento de ATRS e marcadores característicos de células ALT. Os níveis de TERRA em células desprovidas de PC4 não apresentaram alterações consistentes, sugerindo que os efeitos causados pela depleção de PC4 não derivam de alterações na transcrição telomérica. Além disso, a ausência de PC4 não alterou substancialmente os níveis de DNA telomérico de cadeia simples e a expressão ectópica de um mutante de PC4 com incapacidade de ligação a ssDNA reverteu apenas de forma parcial os efeitos causados pela perda de PC4 endógeno, indicando que a função desempenhada por PC4 nos telómeros de células ALT não está restrita à sua capacidade de ligação a ssDNA. Este estudo reforça a noção de que PC4 é um mitigador de ATRS capaz de restringir o stress replicativo nos telómeros de células ALT, sendo por isso um alvo interessante para novas abordagens terapêuticas contra cancros ALT.Azzalin, ClausSilva, BrunoRUNSalgado, Sara Barros2022-01-042024-11-01T00:00:00Z2022-01-04T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/130700enginfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T05:09:20Zoai:run.unl.pt:10362/130700Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:46:51.745739Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathway |
title |
Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathway |
spellingShingle |
Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathway Salgado, Sara Barros PC4 telomeres ALT ATRS Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
title_short |
Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathway |
title_full |
Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathway |
title_fullStr |
Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathway |
title_full_unstemmed |
Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathway |
title_sort |
Exploring the roles of PC4 in the Alternative Lengthening of Telomeres pathway |
author |
Salgado, Sara Barros |
author_facet |
Salgado, Sara Barros |
author_role |
author |
dc.contributor.none.fl_str_mv |
Azzalin, Claus Silva, Bruno RUN |
dc.contributor.author.fl_str_mv |
Salgado, Sara Barros |
dc.subject.por.fl_str_mv |
PC4 telomeres ALT ATRS Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
topic |
PC4 telomeres ALT ATRS Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
description |
Telomeres are nucleic acids-protein complexes located at the ends of linear eukaryotic chromosomes comprising 5’-TTAGGG-3’ tandem repeats, the multiprotein complex shelterin and the long non-coding RNA TERRA. Telomeres act both as molecular clocks and capping structures supporting chromosome integrity. Due to the end-replication problem, telomeres progressively shorten with each cell division and ultimately trigger cellular senescence. Cancer cells bypass senescence and divide indefinitely by activating mechanisms that buffer telomere shortening. Most cancers reactivate the reverse transcriptase telomerase, yet about 15% elongate their telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. ALT cells rely on break-induced replication (BIR), a homology-directed repair mechanism activated at damaged telomeric DNA. This damage is induced by replication stress (ALT-specific Telomeric Replication Stress; ATRS) and must be kept at a level that induces enough BIR yet without strongly activating DNA damage checkpoints that would cause cell death. This balance is attained by the counteracting activities of molecular triggers and alleviators of ATRS. The Azzalin laboratory has recently identified the transcriptional regulator positive cofactor 4 (PC4), a multifunctional nuclear protein capable of binding to dsDNA, ssDNA, G-quadruplex structures and RNA, as essential for ALT cell survival. PC4 was shown to localize at telomeres of ALT cells by indirect immunofluorescence, being recruited in response to replication stress. This study aims at elucidating the role played by PC4 at ALT telomeres. I confirmed by chromatin immunoprecipitation that PC4 associates with telomeres and this association increases when cells are treated with drugs inducing replication stress. Furthermore, PC4 depletion exacerbates ATRS and ALT features. TERRA levels in PC4-depleted cells showed no consistent alterations, suggesting that the effects observed upon PC4 depletion are unlikely to derive from changes in telomere transcription. Moreover, loss of PC4 did not substantially alter ssDNA and total telomeric DNA levels and ectopic expression of a PC4 mutant unable to bind ssDNA only partially rescued the defects due to PC4 loss; this indicates that the function played by PC4 at ALT telomeres is not majorly relying on its ability to bind ssDNA. This work strengthens the notion that PC4 is an ATRS alleviator capable of restricting telomeric replication stress and ALT features. In light of this, PC4 emerges as an attractive target for the development of novel therapeutic protocols against ALT cancers. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-01-04 2022-01-04T00:00:00Z 2024-11-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
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http://hdl.handle.net/10362/130700 |
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http://hdl.handle.net/10362/130700 |
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eng |
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