Publicação
Molecular mechanism controlling epigenetic maintenance of centromere identity
| Resumo: | The centromere is a specialized chromatin domain that directs kinetochore assembly and thus forms the site of interaction between DNA and the mitotic spindle. As such, it is required for faithful chromosome segregation during cell division. Centromere position and function are specified by an epigenetic, chromatin-based mechanism, thought to be driven by Centromere Protein A (CENP-A). This histone variant specifically replaces canonical H3 in centromeric nucleosomes and is known to template its own duplication. Its assembly into chromatin is tightly coupled to the cell cycle, ensuring the replenishment of the protein pool following its redistribution among daughter cells. Importantly, CENP-A has been shown to segregate in a semi-conservative manner, with no detectable loss for at least 3 cell divisions. Although currently no external factors are known to be responsible for CENP-A maintenance in human centromeres, this extreme stability suggests that such factors exist. The present study consists of a screen aimed at identifying novel regulators of CENP-A dynamics. We conceived and extensively optimized an experimental procedure that integrates the lentiviral-mediated delivery of shRNAs with in vivo labelling of specific CENP-A pools. This allowed us to quantify, by fluorescent microscopy, potential defects in both maintenance and loading of CENP-A, following depletion of a number of candidate factors. Knockdown of CENP-C, CENP-35, hKpd3, Asf1-b, Rsf-1 or H2AZ led to a reduction in the levels of nascent pools of CENP-A, suggesting the involvement of these proteins in the assembly pathway of the histone. All six proteins were shown to contribute to the deposition of newly-synthesized CENP-A specifically in G1 phase, with CENP-C appearing to have an additional role in CENP-A maintenance during subsequent cell cycle stages. Furthermore, our data argues for the existence of a sensing mechanism that allows cells to control the amount of CENP-A that is stably loaded into centromeric chromatin. |
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| Autores principais: | David, Ana Filipa Claro, 1989- |
| Assunto: | Biologia molecular Centrómero Cromatina Teses de mestrado - 2013 |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | The centromere is a specialized chromatin domain that directs kinetochore assembly and thus forms the site of interaction between DNA and the mitotic spindle. As such, it is required for faithful chromosome segregation during cell division. Centromere position and function are specified by an epigenetic, chromatin-based mechanism, thought to be driven by Centromere Protein A (CENP-A). This histone variant specifically replaces canonical H3 in centromeric nucleosomes and is known to template its own duplication. Its assembly into chromatin is tightly coupled to the cell cycle, ensuring the replenishment of the protein pool following its redistribution among daughter cells. Importantly, CENP-A has been shown to segregate in a semi-conservative manner, with no detectable loss for at least 3 cell divisions. Although currently no external factors are known to be responsible for CENP-A maintenance in human centromeres, this extreme stability suggests that such factors exist. The present study consists of a screen aimed at identifying novel regulators of CENP-A dynamics. We conceived and extensively optimized an experimental procedure that integrates the lentiviral-mediated delivery of shRNAs with in vivo labelling of specific CENP-A pools. This allowed us to quantify, by fluorescent microscopy, potential defects in both maintenance and loading of CENP-A, following depletion of a number of candidate factors. Knockdown of CENP-C, CENP-35, hKpd3, Asf1-b, Rsf-1 or H2AZ led to a reduction in the levels of nascent pools of CENP-A, suggesting the involvement of these proteins in the assembly pathway of the histone. All six proteins were shown to contribute to the deposition of newly-synthesized CENP-A specifically in G1 phase, with CENP-C appearing to have an additional role in CENP-A maintenance during subsequent cell cycle stages. Furthermore, our data argues for the existence of a sensing mechanism that allows cells to control the amount of CENP-A that is stably loaded into centromeric chromatin. |
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