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Single cell profiling of X chromosome reactivation during primordial germ cell specification in vivo

Periodic Reporting for period 1 - XPGCS (Single cell profiling of X chromosome reactivation during primordial germ cell specification in vivo)

Reporting period: 2016-03-01 to 2018-02-28

"Mammalian primordial germ cells (PGCs) are the precursors of eggs and sperm and transmit genetic and epigenetic information to subsequent generations. Following fertilization, these highly specialised gametic cells combine to form a totipotent zygote, which will develop into a new organism. Epigenetic reprogramming in early PGCs is critical towards generating the totipotent state, which includes reactivation of the inactive X chromosome (Xi) in female germ cells. However, the molecular events associated with XCR remain to be fully elucidated. Indeed, XCR is also a key hallmark of female somatic cell reprogramming to a totipotent/pluripotent state, so this investigation has broader implications for regenerative medicine and epigenetic inheritance in development and disease.

In mammals, one of the two Xs is transcriptionally silenced in females for dosage compensation between gender and enriched in ""repressive"" chromatin marks such as H3K27 methylation. In the mouse, XCR occurs at two different time point: in the inner cell mass of the blastocyst and during the PGC differentiation.
During reactivation, the Xi undergoes reprogramming through the loss of Xist RNA coating, followed by the erasure of the repressive H3K27me3 histone mark, and biallelic expression of X-linked genes. Despite this knowledge, little is known about the gene-activation dynamics, or the mechanisms involved both in the ICM and the PGC.
The overall aim of this project was to study the functional association between changes in X-linked gene expression and the dynamic changes in chromatin associated with reprogramming of the Xi.

My specific objectives were to study the key time points of in vivo X-chromosome reprogramming as follows:
- Investigation of X-linked gene activity, at the scale of the entire X chromosome, during PGC differentiation.
- Mapping of H3K27me3 enriched chromatin and its correlation with gene expression along the X chromosome
- The ability of H3K27me3 enriched chromatin to repress X linked gene expression, and more generally to be a switch against X chromosome reactivation."
During my two years as a Marie Sklodowska-Curie Postdoctoral Fellow in Pr Azim Surani laboratory, I have set up different strategies to study the dynamics of X-linked gene expression and chromatin changes during X-chromosome reprogramming. I have also worked on epigenetic programming during pre-implantation development as a side project. The exploitation of the three main results obtained under the EU project are presented below:

1. Exploring the kinetics of XCR chromosome-wide by deep RNA-seq of single polymorphic PGCs:

To determine the reactivation kinetics of X-linked genes during epigenetic reprogramming of PGCs, I have taken advantage of recent technical and methodological advances concerning single-cell RNA sequencing to analyse allele-specific gene expression within individual polymorphic female PGCs between E8.5 and E12.5 days of gestation. All the samples have been collected, amplified, and sequenced and are currently undergoing bioinformatics analysis.
By exploiting polymorphisms and examining single PGCs at different developmental time points, candidate reactivated X-linked genes will be defined as ‘early’ and ‘late’ reactivated genes. Their kinetics of reactivation will be compared to their behaviour during Xi reactivation in the ICM and implemented with important genetic and epigenetic features (genomic location, transcription factor binding sites, local epigenetic landscapes).

These results will be soon included in a manuscript for publication (Borensztein et al, in preparation).

2. Identifying the link between “repressive” H3K27 methylation mark and X-linked gene reactivation

To gain insight into whether the chromatin state of a gene is associated with its kinetics of reactivation during reprogramming of the Xi, I have taken advantage of in one hand the single-cell RNA sequencing that I had previously produced in polymorphic ICM and in the other hand the published Chromatin Immuno-Precipitation for H3K27me3 mark in the ICM. I have compared the enrichment of the repressive histone mark in our different candidate genes (early and late reactivated genes). Thanks to this analysis, I have been able to emphasize that different epigenetic signatures might underlie the distinct transcriptional behaviours of the X-linked genes during Xi reactivation in the ICM and that H3K27me3 mark could act as a “lock” to maintain silencing.

These results have been included in a manuscript, published in Nature Communications in 2017, in which the EU funding is acknowledged (Borensztein et al, 2017).

3. Studying a epigenetic regulator, G9a, during early developmental progression

G9a is an epigenetic modifier that mediates histone H3 lysine 9 dimethylation (H3K9me2). To study early epigenetic programming in the embryo, we have studied the maternally inherited depletion of G9a protein by immunofluorescence and single embryo RNA sequencing. We have shown that G9a is indispensable for correct establishment of a regulatory network in preimplantation embryos, then allowing developmental progression and cell-fate choices. These results highlight the importance of epigenetic programming at the onset of development.

These results have been written and recently published in Elife and the EU funding is acknowledged (Zylicz*, Borensztein* et al, 2018, * equal contribution).
The work taken under this project has resulted into great advances into the comprehension of early epigenetic programming (G9a maternal mutant) and in developmental reprogramming of the X chromosome. Two papers have been published in open Journals (Nature Communications and Elife), and one is under preparation. All published manuscripts have been advertised on different websites (Gurdon Institute, Curie Institute, ResearchGate, Twitter). I have also participated in dissemination of my work for non-specialists in a Radio show (BBC Cambridge).

We are now performing further explorations to better characterize the Xi reprogramming in the context of development, both in the ICM and the PGCs. In particular, mechanisms of XCI/XCR, and associated chromatin changes in female PGCs are crucial towards making advances in our understanding of germline biology and totipotency, as well as infertility and epigenetic inheritance.
The work of Pr Azim Surani's laboratory is highly important for the impact of germ cell and reprogramming research on society and on the socio-economic world.

Moreover, this fellowship had an invaluable impact on my career as a researcher. I have increased my scientific skills, developed new networks and collaborations.
In parallel, I have also been elected by the Queens' College of Cambridge as a Postdoctoral Research Associate (PDRA 2016-2018). This position gave me the opportunity to be part of a multidisciplinary community of the highest standard, present my work to fellows and students and increase the impact of my work.
During the time of my fellowship, I have reached a position of professional maturity that gave me the opportunity to be elected by the CNRS (France) for a tenured researcher position in the Institut Curie. I now wish to develop my current research towards an independent career to study the reprogramming phenomena, using the X chromosome as a model.
X-chromosome dynamics during mouse development