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).
