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Chromatin dynamics during X-inactivation: assessing the role of different chromatin changes in the loss of gene activity and the gain of gene silencing

Final Report Summary - SIMAOROCHAXCI (Chromatin dynamics during X-inactivation: assessing the role of different chromatin changes in the loss of gene activity and the gain of gene silencing)

My IEF was entitled 'Chromatin dynamics during X-inactivation: assessing the role of different chromatin changes in the loss of gene activity and the gain of gene silencing'. The overall aim of this project is to study the functional significance of the dynamic chromatin changes associated with the inactive X chromosome (Xi) in differentiating female (XX) embryonic stem cells (ESCs). Particularly, we were interested in the mechanism of loss of active marks such as H3K4 methylation during the initiation of XCI. We hypothesized the involvement of the H3K4 demethylases of the Jumonji/ARID1 family. This family includes the H3K4 demethylases JARID1A, JARID1B and JARID1C which are all candidates for mediating the rapid loss of H3K4 methylation that occurs soon after initiation of XCI, started with the coating of the X-chr by Xist non-coding RNA. This family also includes the JARID2 protein, with no reported demethylase activity so far, but that has been implicated in the recruitment of Polycomb (PcG) repressive complex 2 -PRC2 to chromatin, a major player in XCI. We created transgenic XX ESCs stably expressing these proteins fused to GFP. These ESC lines allow us to track protein localization and describe their kinetics during XCI. We successfully generated transgenic ESCs for Jarid1b, Jarid1c and Jarid2, but not for Jarid1a. During this analysis, we did not detect a clear role for the H3K4 demethylases in the process of XCI, nor when Jarid1a was knockdown (kd). In contrast to the H3K4 demethylases, we observed that JARID2 was recruited to the Xist-coated X-chr at early stages of XCI. Therefore, as the link of JARID2 and XCI was clear, we decide to concentrate our study in the role of JARID2 in XCI for the rest of the IEF.

We observed that the JARID2 protein becomes enriched on the Xi during early ESC differentiation during the same time window as PRC2 (Fig. 1). This observation suggested that JARID2 might act to modulate the initial PRC2 recruitment to the Xi. In order to explore the role of JARID2 in XCI, we performed kd experiments by creating stable XX ESC lines expressing shRNA directed against Jarid2 (Fig. 2A and B).. Analysing differentiating Jarid2 kd ESCs, we observed a clear impairment of the recruitment of PRC2 components (EED, SUZ12 and EZH2) to the Xi, as well as slight reduction of H3K27me3 enrichment, the mark laid down by PRC2 (Fig. 2C and D). Therefore, loss of JARID2 clearly affects PRC2 recruitment to the Xi.

Next, we explored the mechanism through which JARID2 is recruited to the Xi. First, we assessed whether it is Xist RNA-dependent. For this, we used an inducible system to express Xist from a transgene integrated in an autosome (chr 11) in male ES cells. Induced Xist expression results in efficient recruitment to the Xist-coated chr 11, meaning that JARID2 is recruited by Xist RNA to the coated chr.

Finally, we addressed whether JARID2 recruitment was dependent on PRC2. For this, we used Eed-/- male ESCs carrying an inducible Xist transgene on chr 11 (Fig. 3A). With this system, we observed that JARID2 recruitment to the Xi was not affected in the absence of EED while all the PRC2 core components (e.g. EZH2) were greatly affected (Fig. 3B). This suggests that JARID2 might be the missing link between Xist and PRC2 recruitment.

In conclusion, during the course of my IEF, we have discovered a new protein player, JARID2, that is associated to the Xi at the onset of XCI. Our work points to a role for JARID2 in recruitment of PRC2 to the Xi. We believe our work has made a substantial progress in understanding how PRC2 becomes recruited to the Xi, a long standing question in the field of XCI. A manuscript on our findings is now in preparation.

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