C. Morey et al., Tsix-mediated repression of Xist accumulation is not sufficient for normalrandom X inactivation, HUM MOL GEN, 10(13), 2001, pp. 1403-1411
During the X inactivation process, one X chromosome in each female embryoni
c 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 inactiv
e X and in Xist regulation through as yet unknown mechanisms. Undifferentia
ted female ES cells, which have two active Xs, recapitulate random X inacti
vation when induced to differentiate. A 65 kb deletion targeted to one of t
he 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 inactiva
tion of the deleted X in differentiated ES cells, We have re-examined the p
henotype 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 diffusio
n of the Xist transcript away from its site of synthesis. Restoration of Ts
ix repressed the steady-state level of Xist expression and restricted Xist
RNA to its transcription site. At the onset of inactivation in differentiat
ed ES cells, restoration of Tsix failed to restore random X-inactivation, e
ven 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 bot
h a repressor of the steady-state level of Xist expression and as a regulat
or of the distribution of Xist RNA within the nucleus. They also establish
that random inactivation requires mechanisms additional to the in cis repre
ssion of Xist.