Epigenome establishment and maintenance of the inactive X chromosome in response to DNA double-strand breaks in mammalian cells

Project Information

XICUT

Grant agreement ID: 101110708

DOI

10.3030/101110708

Project closed

EC signature date 3 August 2023

Start date 1 January 2024

End date 31 December 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 211 754,88

Coordinated by

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France

Periodic Reporting for period 1 - XICUT (Epigenome establishment and maintenance of the inactive X chromosome in response to DNA double-strand breaks in mammalian cells)

Reporting period: 2024-01-01 to 2025-12-31

Summary of the context and overall objectives of the project

The DNA damage response takes place on a chromatin substrate that needs to be disorganized to allow access to the repair factors to the DNA lesion, then restored to preserve epigenome integrity. The chromatin structure differs according to the genomic region, with features impacting DNA repair, both at the molecular level and at the level of the repair pathway choice, and chromatin dynamics that accompany the DNA damage response. In mammalian female cells, one of the two X chromosomes is inactivated early on during embryonic development by the formation of facultative heterochromatin, which molecular components such as post-translational histone modifications and associated proteins are well characterized.

My research project aims at identifying changes in the chromatin organization that accompany the repair of DNA double-strand breaks, and the mechanisms at play in maintaining the structure and function of the chromatin associated to the inactive X chromosome during DNA double-strand break repair.

For that, I induce DNA double-strand breaks on the X chromosome of human female cells using the CRISPR/Cas9 technology, that allows sequence-specific targeting of the Cas9 nuclease. After repair is complete, I analyze the mutational signatures on the targeted genomic regions, because these genomic instability profiles are a proxy of the DNA repair pathway at play. I study the dynamics of the chromatin marks associated with the inactive X chromosome using immunofluorescence and chromatin immunoprecipitation approaches, to better understand the interplay between facultative heterochromatin dynamics and DNA double-strand break repair pathway choice.

Moreover, the inactivation of one of the two X chromosomes, which is a crucial process for cell homeostasis, is random and occurs in embryonic stem cells that proliferate rapidly and are therefore prone to replication stress. This replication stress induced DNA double-strand breaks that could play a role in the choice of the X chromosome that will be inactivated, and could then impact stem cell differentiation. As such, I study the impact of DNA double-strand breaks occurring in the X chromosome of stem cells on their differentiation potential and their propensity to inactivate one of the two X chromosomes.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

So far, I have been developing a Cas9-inducible cell system for the time-controlled targeted generation of DNA double-strand breaks on the X chromosome. For that, I use RPE-1 cells, which are an epithelial human immortalized female cell line, transduced first with a doxycycline-inducible Cas9. This cell line will be used as a control for all experiments as it does not express any guide RNA. In a second step, the RPE-1 Cas9 cell line is transduced to generate three different cell lines expressing a unique guide RNAs, that targets a locus on the X chromosome, and designed close to a mapped SNP that allows the discrimination between the active and the inactive X chromosome.
I am characterizing these cell lines to determine the timing of induction of the Cas9 by looking at its protein expression levels, the timing of repair of the generated DNA double-strand break and the cutting efficiency of the Cas9 by digital droplet PCR. This latter technique allows the detection of a single event of PCR amplification, and with allele-specific primers designed around the cut site, I am able to quantify the proportion of broken vs. unbroken DNA in my cell population.
In parallel, I have implemented a Cut&Run approach in the lab to assess H3K27me3 and gH2A.X levels along the genome of RPE-1 cells, treated or not with neocarzinostatin, a drug that induced random DNA double-strand breaks in the genome. Confirming our preliminary results obtained after laser micro-irradiation, I did not detect any change in H3K27me3 levels during or after the repair of DNA double-strand breaks.
I have supervised a master student working on the molecular dynamics modeling of the catalytic pocket of the enzymes responsible for the demethylation of H3K27me3, in complex H3 tail. The results are being further analyzed to determine whether the complex made by these enzymes and the H3 tail has a different stability according to the H3 histone variant used in the modeling (H3.1 or H3.3).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We have recruited a master student who will help with the project and perform experiments related to the determination of the mutational signatures associated with the repair pathway at play for each guide RNA. We will also perform Cut&Run experiments after a single double-strand break induced by Cas9, and assess the potential changes in H3K27me3 around the cut site, during and after repair.
Further down the line, we will assess the possibility that H3K27me3 demethylases could play a role in directing the repair pathway choice through a H3 variant-specific mechanism. For that, we will use either an inhibitor for H3K27me3 demethylases or nanobodies targeting these enzymes to affect the demethylation process during DNA double-strand break repair, and assess how it affects the repair pathway choice and the levels of H3K27me3 around the cut site.