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© The Rockefeller University Press,
0021-9525/2001//773 $5.00
The Journal of Cell Biology, Volume 153, Number 4,
, 2001 773-784
Original Article |
Synergism of Xist Rna, DNA Methylation, and Histone Hypoacetylation in Maintaining X Chromosome Inactivation
jaenisch{at}wi.mit.edu
Xist RNA expression, methylation of CpG islands, and hypoacetylation of histone H4 are distinguishing features of inactive X chromatin. Here, we show that these silencing mechanisms act synergistically to maintain the inactive state. Xist RNA has been shown to be essential for initiation of X inactivation, but not required for maintenance. We have developed a system in which the reactivation frequency of individual X-linked genes can be assessed quantitatively. Using a conditional mutant Xist allele, we provide direct evidence for that loss of Xist RNA destabilizes the inactive state in somatic cells, leading to an increased reactivation frequency of an X-linked GFP transgene and of the endogenous hypoxanthine phosphoribosyl transferase (Hprt) gene in mouse embryonic fibroblasts. Demethylation of DNA, using 5-azadC or by introducing a mutation in Dnmt1, and inhibition of histone hypoacetylation using trichostatin A further increases reactivation in Xist mutant fibroblasts, indicating a synergistic interaction of X chromosome silencing mechanisms.
Key Words: X chromosome • Xist gene • DNA methylation • histone deacetylase • gene silencing
© 2001 The Rockefeller University Press
Abbreviations used in this paper: GFP, green fluorescent protein; HAT, hypoxanthine/aminopterin/thymidine; Hprt, hypoxanthine phosphoribosyl transferase; IAP, intracisternal A particle; ICF, immunodeficiency centromeric instability facial anomalies; TSA, trichostatin A; Xa, active X; Xi, inactive X.
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