Morey C, Arnaud D, Avner P, Clerc P
Génétique Moléculaire Murine, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, France.
Hum Mol Genet. 2001 Jun 15;10(13):1403-11. doi: 10.1093/hmg/10.13.1403.
During the X inactivation process, one X chromosome in each female embryonic cell is chosen at random to become coated by Xist RNA and silenced. Tsix, a transcript anti-sense to Xist, participates in the choice of the inactive X and in Xist regulation through as yet unknown mechanisms. Undifferentiated female ES cells, which have two active Xs, recapitulate random X inactivation when induced to differentiate. A 65 kb deletion targeted to one of the two Xs in a female ES cell line, and including both the end of the Xist gene and the site of initiation of Tsix, resulted in the exclusive inactivation of the deleted X in differentiated ES cells. We have re-examined the phenotype of the 65 kb deletion and targeted Tsix and the terminal exons of Xist back to the deleted locus using a cre/loxP site-specific re-insertion strategy. We show that prior to inactivation the deleted X is associated in undifferentiated ES cells with both increased Xist expression and diffusion of the Xist transcript away from its site of synthesis. Restoration of Tsix repressed the steady-state level of Xist expression and restricted Xist RNA to its transcription site. At the onset of inactivation in differentiated ES cells, restoration of Tsix failed to restore random X-inactivation, even though the levels of Xist RNA accumulation in cis were markedly reduced. These results identify for the first time a dual function for Tsix as both a repressor of the steady-state level of Xist expression and as a regulator of the distribution of Xist RNA within the nucleus. They also establish that random inactivation requires mechanisms additional to the in cis repression of XIST:
在X染色体失活过程中，每个雌性胚胎细胞中的一条X染色体被随机选择，由Xist RNA覆盖并沉默。Tsix是Xist的反义转录本，通过尚未明确的机制参与失活X染色体的选择和Xist的调控。未分化的雌性胚胎干细胞有两条活跃的X染色体，在诱导分化时会重现随机X染色体失活。在一个雌性胚胎干细胞系中，针对两条X染色体之一进行了65 kb的缺失操作，该缺失区域包括Xist基因的末端和Tsix的起始位点，结果导致分化的胚胎干细胞中被缺失的X染色体被特异性失活。我们重新研究了65 kb缺失的表型，并使用cre/loxP位点特异性重新插入策略，将Tsix和Xist的末端外显子靶向到缺失位点。我们发现，在失活之前，未分化的胚胎干细胞中被缺失的X染色体与Xist表达增加以及Xist转录本从其合成位点扩散有关。Tsix的恢复抑制了Xist表达的稳态水平，并将Xist RNA限制在其转录位点。在分化的胚胎干细胞开始失活时，尽管顺式作用下Xist RNA的积累水平明显降低，但Tsix的恢复未能恢复随机X染色体失活。这些结果首次确定了Tsix的双重功能，既是Xist表达稳态水平的抑制因子，又是Xist RNA在细胞核内分布的调节因子。它们还表明，随机失活需要XIST顺式抑制之外的其他机制：
