Györgyi Csankovszki ^a, András Nagy ^b, and Rudolf Jaenisch ^a

^a Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142
^b Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Correspondence to: Rudolf Jaenisch, Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142.  Tel:(617) 258-5186   Fax:(617) 258-6505
E-mail:  jaenisch@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. 

1. "The Role of  Chromosomal RNAs in Marking the X for Dosage Compensation".