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Nuclear envelope attachment and dynamics of Human telomeres - unravelling nuclear organization

Periodic Reporting for period 3 - TeloHOOK (Nuclear envelope attachment and dynamics of Human telomeres - unravelling nuclear organization)

Reporting period: 2020-11-01 to 2022-04-30

The genome of human cells is divided in distinct linear chromosomes whose ends are caped by a specific structure called telomere. Telomeres correspond to the repetition of a short DNA sequence over a certain length, covered by a specific protein complex called Shelterin. One of the main functions of telomeres is to make sure the cell differentiate natural ends of chromosomes from internal chromosome breaks that need to be repaired. Therefore they play a major role in protecting cells against genome instability, hallmark of cancer cells. In human cells, the chromosomes are held in a specific cellular compartment called the nucleus, in which they are distributed in an organized fashion. The proximity of chromosomes to the nuclear envelope that delimits the nuclear compartment is essential for many aspects of chromosome regulation. In this project, we propose to characterize the interaction that occurs between telomeres and the nuclear envelope, and determine which structures at the nuclear envelope telomeres are tethered to. This interaction occurs only during a key phase of the cell cycle, during the reformation of daughter nuclei after division of the mother cell. Consequently it will be assessed what role this interaction plays in re-organizing chromosomes in the newly formed nucleus during this process. This work will determine the function of the telomeres-nuclear envelope connection in genome stability and maintenance of genetic material, with an emphasis on the growth of cancer cell and during premature aging.
During the period covered by this report, we achieved several aims that were planned in the initial proposal.
One of the first goal of this proposal was to determine the molecular mechanism by which telomeres are attached to the nuclear envelope. In other words, which protein at telomeres and at the nuclear envelope trigger and/or maintain this attachment. For this, we conducted a mass spectrometry screen to identify in a unbiased manner the proteins that are connected to telomeres at this specific time of the cell cycle. We discovered that telomeres are more frequently found together with specific proteins from the nuclear envelope in late mitosis. We are now further analyzing these hits to determine their exact function in telomere tethering. In addition, we are using advanced super-resolution microscopy, the technology 3D-SIM, to get a better view on telomere organization at the nuclear envelope. Are telomeres tethered to specific structures such as nuclear pores (that connect the nucleoplasm and the cytoplasm of the cell), the nuclear lamina (a fine meshwork lining the inner side of the nuclear envelope, and giving mechanical support to the nucleus)? We found that telomeres colocalize with the reforming membrane, suggesting they could play an active role in post-mitotic nuclear assembly.
Two technological challenges that were very important for the progress of this work. First, we developed a technology that we called MadID, which probes telomere-nuclear envelope interaction in a semi-quantitative manner. And we achieved to add a fluorescent protein fused to a protein that specifically bind to telomeres, using CRISPR-Cas9 mediated genome editing.
Finally, our work highlighted a functional connection between telomeres, nuclear envelope integrity, and human aging.
The work we carried out so far brought strong evidence that telomeres have an unprecedented role during mitosis at different levels. First, their structure and organization is peculiar during mitosis, and we propose that this is important to promote proper chromosome condensation and segregation. Second, they are involved in nuclear envelope reformation, suggesting that natural chromosome ends can serve as a nucleation point for membrane reformation after mitosis. Finally, telomeres organization in the 3D nuclear volume is not random, but follow a specific pattern that evolves as the cells progress through the cell cycle. Future work will continue in this direction, in order to bring more molecular mechanisms to these different pathways. Organization of chromatin in the interphase nucleus is essential for the maintenance and stability of cell lineages. This organization needs to be reset after each cell division, and our work demonstrate that human telomeres are involved in several steps of this process: to ensure genome stability, chromosome segregation and reformation of the nuclear envelope around daughter cells chromosomes. As such, these data are relevant for normal cell growth, normal and pathological aging, and for the growth of cancer cells.