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Complexity and dynamics of nucleic acids modifications in vivo

Periodic Reporting for period 4 - dynamicmodifications (Complexity and dynamics of nucleic acids modifications in vivo)

Reporting period: 2020-02-01 to 2021-11-30

The development of any organism starts with a totipotent zygote that will through series of cell divisions and differentiation steps generate the whole body containing hundreds of distinct cell types. The fact that one set of genetic information (genotype) generates such a vast diversity of cell functions (phenotypes) has fascinated generations of developmental biologists , geneticists and epigeneticists. One of the key questions towards understanding the restriction and diversification of cell potential at the molecular level is what chemical , structural or spatial modifications of our genome (DNA) provide the crucial restrictive as well as inductive information during the differentiation process.
Additionally, although the unidirectional progression of normal development dictates that the above mentioned changes are stable and irreversible , this paradigm is challenged in the context of epigenetic reprogramming whereby somatic cells can be reprogrammed back into a naive , pluripotent, state resembling cellular state observed in early preimplantation embryos. Experimental work using various in vitro reprogramming systems (somatic cell nuclear transfer , reprogramming to pluripotency using Yamanaka factors - iPS cells, reprogramming using cell fusions) clearly shows that next to the re-wiring of transcription factor networks, changes in chemical modifications of DNA and histones are important components in understanding the reprogramming process.

Our work focuses on the understanding the molecular mechanisms and the biological importance of removing various DNA modifications during the reprogramming process. To avoid cellular heterogeneity observed in the in vitro cellular reprogramming systems, we utilise naturally occurring reprogramming processes : 1) zygotic epigenetic reprogramming, where paternal genome undergoes close to complete removal of DNA methylation and 2) developing embrynic germ line, where primordial germ cells undergo a wave of genome-wide DNA demethylation following their entry into the gonadal anlagen. We study the global and local changes in 5mC and 5hmC and search for and analyse additional chemical modifications to DNA that appear during the reprogramming process. Furthermore, to support our search for the mechanism of active, DNA replication independent, DNA demethylation mechanism we utilise mouse pluripotent stem cells cultured in vitro to follow and measure dynamics and turnover of various DNA modfications using an ultra sensitive LC-MS approach. Last, but not least, we are interested in the mechanistic and functional cross-talk between various DNA and RNA modification systems.

Mechanistic understanding of the DNA modifcation changes will enable us to induce and recapitulate these in the context of cellular reprogramming (reprogramming back to pluripotency, transdifferentiation) in vitro. We envisage that the ability to manipulate the epigenetic memory will greatly improve our ability to direct and re-programme cell fate.
Since the beginning of this project we have set up and enginneered cell based models that allows us to track dynamic changes in DNA modifications. Using Crisp/r approach we have additionally generated a panel of loss-of-function genetic mutants, that will enable us to monitor the contribution of various DNA de/modifications enzymes to the observed DNA modification dynamics.

We have further optimised our ultra-sensitive LC-MS approach to detect DNA and RNA modifications. This enabled us to publish new important findings (Amouroux et al,Nat Cell Biol 2016, Hill et al, Nature 2018, Rosic et al, Nat Genetics 2018) and to contribute to a number of other studies in the context of academic collaboration (Ferry et al, Mol Cell 2017, Benesova et al, Mol Carcinog 2017).

Last, but not least, we have optimised state-of-the-art methods to generate rapid depletion of proteins of interest in the mouse oocyte and zygotes : this will enable us to test candidate factors for their contribution to the observed zygotic DNA demethylation.
Overall, the project has been showing a steady progress.
We have considerably improved our LC/MS expertise and took advantage of additional mass spec based technologies to broaden our experimental approaches.