DS0405 -

Functional analysis of nuclear reorganisation – FANR

Submission summary

The 3D organisation of the genome has emerged as an important contributor of gene regulation. Misregulation of this organisation is often observed in diseases like cancers. The nuclear periphery is a major landmark of nuclear architecture, directly mediating transcriptional repression. A global nuclear reorganisation occurs during embryonic stem cell (ESC) differentiation. 1,600 genes gain or loose association with the nuclear envelope upon transcriptional activation or repression. The mechanisms underlying the repositioning of these genes remain unclear. In a previous study, we demonstrated the direct role of chromatin compaction in gene relocation and in position inheritance. Nevertheless, the factors regulating these processes are still unknown.
The first aim of this proposal is to decipher the roles of two factors, the histone variant H3.3 and Pml during nuclear reorganisation. H3.3 loading induces chromatin decondensation in vitro and has been implicated in epigenetic memory, making this variant a very good candidate to induce decondensation in vivo. Furthermore, our preliminary results demonstrate that gene relocation correlates with H3.3 deposition or depletion, independently of transcription, suggesting it could be a major driver of gene relocation. Daxx and Hira, two chaperones of H3.3 normally localise in Pml nuclear bodies (NBs). Pml-NBs are restructured into “rod” like foci during ESCs differentiation releasing Daxx and Hira into the nucleoplasm. This increase in nucleoplasmic chaperones could contribute to the quantitative changes in H3.3 content observed at relocated gene upon differentiation. Interestingly, Pml “rods” accumulate besides patches of incorporating Lamin A, a known nuclear envelope tethering factor.
We propose to investigate the importance of Pml-NBs and H3.3 in gene relocation and subnuclear position inheritance by using a combination of knockout and knockdown strategies in ESCs and differentiated cells. In Pml-/- ESCs, we will observe whether gene relocation occurs upon differentiation and how it correlates with H3.3 deposition and Lamin A incorporation. H3.3 knockdowns will allow us to strengthen preliminary results showing that H3.3 is essential for central positioning maintenance. We will also address whether H3.3 is sufficient to drive gene relocation by fusing TALEs to H3.3 chaperones.
Our second objective asks the question of the biological function of gene repositioning during mESCs differentiation. We aim to prevent the central relocation of endogenous Nrp1 by artificially anchoring it to the nuclear edge. We propose to develop a new method to achieve nuclear periphery tethering by using TALEs fused to a nuclear envelope protein. We will monitor whether Nrp1 is activated normally when its relocation is impaired and establish if gene movements can facilitate transcriptional regulation during development.
Altogether, our project will increase our knowledge of how the nucleus is reorganised at the implantation stage during development. It will help determine the importance of nuclear reorganisation for early embryogenesis. The proposed experiments will tackle important questions to understand the correlation observed between abnormal nuclear organisation and cancer.

Project coordination

Pierre Therizols (Institut National de la Santé et la Recherche Médicale)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSERM Institut National de la Santé et la Recherche Médicale

Help of the ANR 249,600 euros
Beginning and duration of the scientific project: - 36 Months

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