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Dissecting the chromatin dynamics at telomeres during mouse pre-implantation development.

Periodic Reporting for period 1 - TeloChromatin (Dissecting the chromatin dynamics at telomeres during mouse pre-implantation development.)

Reporting period: 2017-07-01 to 2019-06-30

Telomeres are specialized structures required for the proper protection and replication of the end of linear chromosomes, such as those in eukaryotes. In mammals, telomeres are composed of long tracks of double stranded TTAGGG repeated DNA which can extend up to 10kb and 100kb in human and mice respectively. Telomeric DNA shortens after each round of cell division, leading ultimately to cell cycle arrest. However, stem cells can extend telomere length by the action of the telomerase, a specialized enzyme that adds new telomeric DNA. Telomere length must be properly controlled to ensure normal development, aging and to ensure cellular homeostasis. Interestingly, telomerase is not the only way to maintain telomere length. In about 10-15% of cancer, an alternative mechanism of telomere lengthening takes place. The ALT (Alternative Lengthening of Telomeres) pathway is a recombination based mode of telomere maintenance, which has been so far studied predominantly in cancer cells. How ALT is regulated and initiated is still not known. Previous studies identified a telomerase independent telomere lengthening in early mouse embryos, underlying that an ALT or ALT-like system can be used in a physiological context.
The purpose of this research is to use mouse pre-implantation embryos and other embryonic cellular models to characterize the ALT (or ALT-like) pathway naturally occurring during early embryonic development. The research is organized into three aims: (WP1) developing tools to visualize and track telomere in mouse embryos, (WP2) characterize telomere regulation in mouse genetic models conditionally inactivated for Atrx and Daxx, (WP3) perform biochemical purification of proteins associated to telomeres of Atrx and Daxx conditional KO mouse embryonic stem cells lines. The identified proteins will be tested for their physiological relevance in mouse embryos.
The objective of WP1 is to develop a tool to track and analyze telomere dynamics in mouse preimplantation embryo. A mouse model expressing a Trf1-EGFP was proposed. Prior tests on mouse ESCs transiently expressing a Trf1-EGFP are ongoing, and will be further followed by testing the construct in vivo. Other alternatives will also be considered (CRISPR/Cas9 targeted to telomere) before creation of the appropriate mouse line. Immunofluorescence studies using antibodies against Shelterin proteins are ongoing. In light with the proposed research, a review covering the topic of telomere regulation and dynamic in early embryonic development was written and published in Chromosoma.
The objective of WP2 is to analyze and understand ATRX/DAXX localization, and function on telomeres during mouse pre-implantation development. Immunofluorescence studies using antibodies specific to DAXX and ATRX, allowed to visualize the localization of these proteins during development from the 1-Cell until late morula/blastocyst stage. Other immunofluorescence experiments are still ongoing to determine the precise distribution of ATRX and DAXX with other known/putative candidates for telomere regulation. As described in the proposal, Atrx and Daxx conditional mouse lines will be used to determine the role of these proteins in regulating telomere dynamics and lengthening mode in mouse early embryos. An external company is in charge of transport and rederivation of these lines from the laboratory of prof. Antoine Peters (Friedrich Miescher Institute, Basel, Switzerland) to the animal facility of the host institute is planned for September 2018.
To understand how ATRX and DAXX modify and regulate telomeric repeats (WP3), I performed PICh from mouse ESCs conditional KO (cKO) for Daxx. Proteins purified from both Daxx wild-type and KO telomeres were analyzed by mass spectrometry. These results are now under analysis and will be compared with other datasets generated in the lab. PICh followed by Western Blot analyses and immunofluorescence on Daxx KO mouse ESCs will be performed in order to further validate the identified factors changing upon Daxx removal. Same procedure will be used with Atrx cKO mouse ESCs. These factors will be tested in vivo to asses for their role in mouse embryonic development.
This project aim to understand a yet unknown telomere lengthening mechanisms, which occur naturally in mouse pre-implantation embryos. This mode of telomere maintenance is likely to share similarities with the ALT pathway described so far in cancer cells. The characterization of the ALT (or ALT-like) maintenance of telomere in vivo could therefore open new insights about how ALT can be initiated and regulated in normal development and disease.
Fluorescence microscopy picture of a mouse zygote. m/fPN (male/female pronucleus), PB (polar body).