XIST Promoter Switch Mediates RNA Stability at Initiation of Random and Imprinted X Chromosome Inactivation

Neil Brockdorff
X inactivation Group, MRC Clinical Sciences Centre; Royal Postgraduate Medical School, Hammersmith Hospital

The Xist gene plays a central role in regulating X chromosome inactivation. Accumulation of the large non-protein coding transcript in cis over the length of the X chromosome is thought to provide the primary signal for propagating the inactive state. The Xist gene is active prior to the onset of random X inactivation but transcripts are highly unstable. As X inactivation proceeds, Xist RNA is stabilized specifically on the inactive X chromosome (Xi) allele. The active X chromosome (Xa) allele continues to transcribe unstable RNA for a short period and is then silenced. I will describe new evidence demonstrating that alternate promoter usage results in distinct stable and unstable Xist RNA isoforms. Stable Xist RNA initiates from the previously published promoter (P1), and also from a novel promoter located 1.5 kb further downstream (P2). Unstable RNA on the other hand initiates from a novel promoter located 6.5 kb upstream (P0). A switch from P0 to P1/P2 at the onset of random X inactivation accounts for accumulation of stable Xist RNA in cis from the Xi allele. Analysis of Xist transgenic and knockout ES cell lines indicates that transcription from P0 correlates with the ability of an Xist allele to be sensed by the X chromosome counting mechanism. Imprinted Xist expression patterns in pre-implantation embryos are also determined by alternate promoter usage. The paternally inherited Xist allele expresses the stable isoform initiated at P1/P2 from the 2-4 cell stage onwards. The maternal allele is silent until mid-late blastocyst stage. This pattern presumably underlies preferential inactivation of the paternal X chromosome which occurs in differentiating trophectoderm and primitive endoderm lineages of the blastocyst. Erasure of the imprint prior to random inactivation in the embryo proper is then attributable to a switch to unstable P0 Xist transcript on both paternal and maternal alleles.