Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation.

ObjectivesWe examined major issues associated with sharing of individual clinical trial data and developed a consensus document on providing access to individual participant data from clinical trials, using a broad interdisciplinary approach.Design and methodsThis was a consensus-building process among the members of a multistakeholder task force, involving a wide range of experts (researchers, patient representatives, methodologists, information technology experts, and representatives from funders, infrastructures and standards development organisations). An independent facilitator supported the process using the nominal group technique. The consensus was reached in a series of three workshops held over 1 year, supported by exchange of documents and teleconferences within focused subgroups when needed. This work was set within the Horizon 2020-funded project CORBEL (Coordinated Research Infrastructures Building Enduring Life-science Services) and coordinated by the European Clinical Research Infrastructure Network. Thus, the focus was on non-commercial trials and the perspective mainly European.OutcomeWe developed principles and practical recommendations on how to share data from clinical trials.ResultsThe task force reached consensus on 10 principles and 50 recommendations, representing the fundamental requirements of any framework used for the sharing of clinical trials data. The document covers the following main areas: making data sharing a reality (eg, cultural change, academic incentives, funding), consent for data sharing, protection of trial participants (eg, de-identification), data standards, rights, types and management of access (eg, data request and access models), data management and repositories, discoverability, and metadata.ConclusionsThe adoption of the recommendations in this document would help to promote and support data sharing and reuse among researchers, adequately inform trial participants and protect their rights, and provide effective and efficient systems for preparing, storing and accessing data. The recommendations now need to be implemented and tested in practice. Further work needs to be done to integrate these proposals with those from other geographical areas and other academic domains.

Summary Centromeres are essential chromosomal structures that mediate accurate chromosome segregation during cell division. Centromeres are specified epigenetically by the heritable incorporation of the centromeric histone H3 variant, CENP-A. While many of the primary factors that mediate centromeric deposition of CENP-A are known, the chromatin and DNA requirements in this process have remained elusive. Here, we uncover a role for transcription in Drosophila CENP-A deposition. Using an inducible ectopic centromere system that uncouples CENP-A deposition from endogenous centromere function and cell-cycle progression, we demonstrate that CENP-A assembly by its loading factor, CAL1, requires RNAPII-mediated transcription of the underlying DNA. This transcription depends on the novel CAL1 binding partner FACT, but not on CENP-A incorporation. Our work establishes RNAPII passage as a key step in chaperone-mediated CENP-A chromatin establishment and propagation.

No abstract

No abstract