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

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 Obiettivo del progetto (Objective)

'X-inactivation (XCI) is an epigenetic phenomenon resulting in transcriptional silencing to attain gene dosage parity between XX females and XY males. XCI involves coating of the non-coding Xist RNA followed by depletion of euchromatic marks and shortly after accumulation of heterochromatic marks. The chromatin modifying activities that are involved in these steps and their functional interplay remain poorly understood however. We will specifically assess how euchromatic marks, such as H3K4 methylation and H3 and H4 acetylation, are lost shortly after Xist RNA coating and the importance of this loss in gene silencing and heterochromatin formation. First, we will perform functional studies in differentiating XX ES cells, including knockdown of chromatin modifying candidates, such as H3K4 demethylases and histone deacetylases. Second, we will investigate how specific X-linked loci behave during XCI, using a Lac or Tet operator tagging system. This will allow us to (a) visualise a locus in living cells and assess the exact timing of recruitment of proteins of interest (fused to a fluorescent protein), in control and knockdown situations; and (b) target specific chromatin factors (using the Lac or Tet repressor fusion) to a locus in order to assess the impact of chromatin changes on XCI. In summary, the two complementary strategies outlined here should provide us with new insights into the roles that various chromatin changes associated with XCI actually play in this process. This should also give new insights into the process of gene regulation in other systems.'

Introduzione (Teaser)

In humans, two copies of all somatic chromosomes must be present for normal development. However, this is not the case for our genetic chromosomes X and Y, where only one copy seems to be required.

Descrizione progetto (Article)

To correct the imbalance between XX females and XY males, females have evolved the unique mechanism of X chromosome inactivation (XCI), an epigenetic phenomenon that results in transcriptional silencing to attain equal gene dosage. The expression of the X-inactive specific transcript (XIST) RNA molecule initiates the process of XCI by coating the inactive chromosome and inducing chromatin alterations that turn off gene transcription.

Using embryonic stem (ES) cells, the EU-funded Simaorochaxci project aimed to study the functional significance of the dynamic chromatin changes associated with the inactive X chromosome (Xi). Project scientists were particularly interested in the mechanism of loss of active marks during the initiation of XCI.

The project built on the hypothesis that certain enzymes would remove active chromosomal marks such as methylation of histone 3 at the lysine locus 4 (H3K4 methylation) associated with gene transcription. Loss of H3K4 methylation accompanies initiation of the XCI phenomenon.

By operating a fluorescence-tagging system, project scientists were able to visualise and assess the recruitment of proteins of interest to specific chromosomal locations. Results showed that the XIST RNA molecule was responsible for bringing the protein Jarid2 to the Xi. This triggered the recruitment of the polycomb (PcG) protein repressive complex 2 (PRC2), thereby inducing gene silencing.

The Simaorochaxci project findings significantly advanced our knowledge of the X inactivation phenomenon and the associated chromatin modifications. Apart from helping us understand gene inheritance and transcription, these data could be extrapolated to deduce gene regulation in other organisms.