A SAGE approach to identifying novel trans-acting factors involved in the X inactivation process

X chromosome inactivation ensures the dosage compensation of X-linked genes in XX females compared to their XY male counterpart. It is characterised by the specific recruitment of an inhibitory ribonucleo protein complex involving the non-coding Xist RNA to the presumptive inactive X chromosome and associated chromatin modifications, which result in the transcriptional silencing of the X chromosome. As an approach to the identification of some of the potential molecular players in this process we have performed comparative transcriptional profiling of mouse 6.5-dpc (days post-coitum) female and male embryos using a modified SAGE (Serial analysis of gene expression) technique which allows the analysis of small quantities of biological material. At 6.5 dpc, a moment when random X inactivation of embryonic tissues has just been achieved, some two hundred transcripts that were significantly enriched in the female gastrula compared to its male counterpart could be identified. The validation of an association with the X inactivation process of a subset of these transcripts has been studied, ex vivo, in differentiating female and male ES cells and in female ES cells in which the establishment of X inactivation is interrupted through the post-transcriptional inhibition of Xist synthesis.