Final Report Summary - BIOSYNCEN (Dissection of centromeric chromatin and components: A biosynthetic approach)
The genome of an organism is packaged in the form of chromosomes, which often appear as an X-shape under the microscope. This structure is formed by two sister chromosomes that are joined together at a specialised region called the centromere. Centromeres play an essential role during cell division by serving as an adapter between the chromosomes and to a cellular structure called the mitotic spindle. This connection allows chromosomes to be segregated equally between two daughter cells thereby ensuring the inheritance of genetic information to subsequent cell generations. The basic building block of chromosome is chromatin, which consists of different histone proteins around which the DNA is wrapped to help to package the long thread-like DNA molecule. Centromeric chromatin contains a specialised histone called CENP-A, which is regarded as a key epigenetic mark to determine centromere identity. Defects in centromere function can result in the gain or loss of chromosomes, also called aneuploidy, which is a hallmark of cancer and a major cause of human miscarriage. Although first described over 130 years ago, we still lack a complete understanding of how centromeres are established and maintained in the cell.
During the life time of this project, we have used fruit flies and human tissue culture cells as a model to study how centromere identity is established and maintained.
To unravel the mechanism of this process in molecular detail, we took advantage of the evolutionary difference between Drosophila and human cells by expressing three Drosophila centromere factors in human cells. This unique setup allowed us to dissect the role of these factors in Drosophila CENP-A loading (dCENP-A) without interfering with the host cell machinery. Indeed, we could reveal how these factors depend on each other and were able to reconstitute inheritance of Drosophila centromere identity in a heterologous system.
We also aimed to understand how centromere factors interact with other processes of the cell. This pointed us to factors that are part of the transcription machinery. Normally the role of transcription is to express genes with the help of an enzyme, the RNA polymerase, to produce a so-called messenger RNA. In order to transcribe genes, the chromatin has to be partially disassembled to allow the enzyme that produces the RNA, the RNA polymerase, to travel along the DNA fiber without being blocked by histones. This remodeling of chromatin can result in the complete loss of histones. We recently presented evidence that transcription at the centromere is required for loading of dCENP-A into the chromatin. This observation is consistent with a model where transcription is re-purposed at the centromere to remove nucleosomes instead of transcribing genes to make space for loading of new dCENP-A histones. Thus, transcription-mediated chromatin remodeling ensures that the centromere mark can be replenished and propagated through multiple cell generations.
Finally, the key to understand any complex machinery is to study its components and how they are put together. Although recent years have identified a large number of centromere factors, how these factors connect to the chromatin fibers is little understood. The major obstacle for high resolution mapping of proteins to the underlying centromeric DNA sequence, is the repetitiveness of DNA in these regions which defy their assembly into longer sequences. In this project we succeeded in developing a novel micrososcopy-based methodology for localizing centromere factors over the repetitive centromeric DNA sequences. This technique will be equally useful for mapping proteins on other genomic regions containing repetitive DNA, such as pericentric heterochromatin or telomeres.
Taken together, the knowledge gained during the lifetime of this project is not only highly relevant to understand how centromeres work and failure might lead to diseases, but could also advance gene therapeutic approaches by providing clues how to efficiently build synthetic centromeres on human artificial chromosomes.
During the life time of this project, we have used fruit flies and human tissue culture cells as a model to study how centromere identity is established and maintained.
To unravel the mechanism of this process in molecular detail, we took advantage of the evolutionary difference between Drosophila and human cells by expressing three Drosophila centromere factors in human cells. This unique setup allowed us to dissect the role of these factors in Drosophila CENP-A loading (dCENP-A) without interfering with the host cell machinery. Indeed, we could reveal how these factors depend on each other and were able to reconstitute inheritance of Drosophila centromere identity in a heterologous system.
We also aimed to understand how centromere factors interact with other processes of the cell. This pointed us to factors that are part of the transcription machinery. Normally the role of transcription is to express genes with the help of an enzyme, the RNA polymerase, to produce a so-called messenger RNA. In order to transcribe genes, the chromatin has to be partially disassembled to allow the enzyme that produces the RNA, the RNA polymerase, to travel along the DNA fiber without being blocked by histones. This remodeling of chromatin can result in the complete loss of histones. We recently presented evidence that transcription at the centromere is required for loading of dCENP-A into the chromatin. This observation is consistent with a model where transcription is re-purposed at the centromere to remove nucleosomes instead of transcribing genes to make space for loading of new dCENP-A histones. Thus, transcription-mediated chromatin remodeling ensures that the centromere mark can be replenished and propagated through multiple cell generations.
Finally, the key to understand any complex machinery is to study its components and how they are put together. Although recent years have identified a large number of centromere factors, how these factors connect to the chromatin fibers is little understood. The major obstacle for high resolution mapping of proteins to the underlying centromeric DNA sequence, is the repetitiveness of DNA in these regions which defy their assembly into longer sequences. In this project we succeeded in developing a novel micrososcopy-based methodology for localizing centromere factors over the repetitive centromeric DNA sequences. This technique will be equally useful for mapping proteins on other genomic regions containing repetitive DNA, such as pericentric heterochromatin or telomeres.
Taken together, the knowledge gained during the lifetime of this project is not only highly relevant to understand how centromeres work and failure might lead to diseases, but could also advance gene therapeutic approaches by providing clues how to efficiently build synthetic centromeres on human artificial chromosomes.