Periodic Reporting for period 5 - dynamicmodifications (Complexity and dynamics of nucleic acids modifications in vivo)
Berichtszeitraum: 2021-12-01 bis 2022-05-31
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 embryonic 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/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.
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 embryonic 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/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.
In the course of the project we have focused on answering the following questions:
1) What is the role of oxidative DNA demethylation (5mC to 5hmC oxidation) and DNA repair in the processes of developmental DNA demethylation?
To this end, we have demonstrated that neither zygotic, nor germline DNA demethylation require 5hmC formation driven by Tet family of enzymes (Amouroux et al, Nat Cell Biol 2016; Hill et al, Nature 2018). We have shown that in both instances, Tet enzymes are required to protect the newly established DNA hypomethylated state (not to install it) from the incoming de novo DNA methylation. We have also shown that Tet enzymes have a clear role in transcriptional regulation that goes beyond their role in DNA demethylation (see Hill et al, Nature 2018).
We have also addressed the role of DNA repair (the Base Excision DNA Repair pathway in particular) during the zygotic DNA demethylation. We have generated a series of genetic loss of function mutants of DNA glycosylases and investigated their role in the global DNA demethylation process. These results are being prepared for publication (Requena, Hatanaka et al, in preparation).
2) What is the stability/turnover of DNA modifications ?
We have developed a metabolic labelling approach that in combination with our ultra-sensitive LC-MS/MS method allows us to determine and quantify turnover of 5mC and 5hmC in proliferating and postmitotic cells. Our results are being prepared for a publication (Requena et al, in preparation). We have also teamed up with a group of A.Groth (University of Copenhagen) and used a similar labelling approach to address the dynamic of DNA modifications in the context of DNA replication (Stewart-Morgan, Requena et al, Nat Cell Biol -accepted). We have also addressed the stability of epigenetic modifications in the context of oocyte as a model for long lived postmitotic cells (Hatanaka et al, in preparation).
3) What is the interplay between DNA and RNA modifications in the context of epigenetic reprogramming ?
We have investigated the dynamic of RNA modifications during germline development and the interplay between global DNA demethylation and the chromatin dynamics and reprogramming. This revealed that global loss of DNA demethylation is functionally compensated by the relocalisation of the polycomb driven histone repressive marks (H3K27me3). This study was published last year (Huang et al, Nature 2021).
1) What is the role of oxidative DNA demethylation (5mC to 5hmC oxidation) and DNA repair in the processes of developmental DNA demethylation?
To this end, we have demonstrated that neither zygotic, nor germline DNA demethylation require 5hmC formation driven by Tet family of enzymes (Amouroux et al, Nat Cell Biol 2016; Hill et al, Nature 2018). We have shown that in both instances, Tet enzymes are required to protect the newly established DNA hypomethylated state (not to install it) from the incoming de novo DNA methylation. We have also shown that Tet enzymes have a clear role in transcriptional regulation that goes beyond their role in DNA demethylation (see Hill et al, Nature 2018).
We have also addressed the role of DNA repair (the Base Excision DNA Repair pathway in particular) during the zygotic DNA demethylation. We have generated a series of genetic loss of function mutants of DNA glycosylases and investigated their role in the global DNA demethylation process. These results are being prepared for publication (Requena, Hatanaka et al, in preparation).
2) What is the stability/turnover of DNA modifications ?
We have developed a metabolic labelling approach that in combination with our ultra-sensitive LC-MS/MS method allows us to determine and quantify turnover of 5mC and 5hmC in proliferating and postmitotic cells. Our results are being prepared for a publication (Requena et al, in preparation). We have also teamed up with a group of A.Groth (University of Copenhagen) and used a similar labelling approach to address the dynamic of DNA modifications in the context of DNA replication (Stewart-Morgan, Requena et al, Nat Cell Biol -accepted). We have also addressed the stability of epigenetic modifications in the context of oocyte as a model for long lived postmitotic cells (Hatanaka et al, in preparation).
3) What is the interplay between DNA and RNA modifications in the context of epigenetic reprogramming ?
We have investigated the dynamic of RNA modifications during germline development and the interplay between global DNA demethylation and the chromatin dynamics and reprogramming. This revealed that global loss of DNA demethylation is functionally compensated by the relocalisation of the polycomb driven histone repressive marks (H3K27me3). This study was published last year (Huang et al, Nature 2021).
Overall, the project has generated new fundamental insights regarding the DNA demethylation process (Amouroux et al, Nat Cell Biol 2016; Hill et al, Nature 2018), the stability and the turnover of DNA modifications (Stewart-Morgan, Requena et al, Nat Cell Biol - accepted; Reguena et al, in preparation) and the functional interplay between DNA demethylation and heterochromatin silencing (Huang et al, Nature 2021).
Next to the new fundamental insights, we have further optimised our ultra-sensitive LC-MS/MS approach of detection of DNA and RNA modifications and the metabolic labelling approaches, that will be of use to the whole scientific community.
Next to the new fundamental insights, we have further optimised our ultra-sensitive LC-MS/MS approach of detection of DNA and RNA modifications and the metabolic labelling approaches, that will be of use to the whole scientific community.