Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation.
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.
Csankovszki
G
G
Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142, USA.
Nagy
A
A
Jaenisch
R
R
D000107:Acetylation; D000818:Animals; D019175:DNA Methylation; D004261:DNA Replication / Q000502:physiology; D004303:Dosage Compensation, Genetic; D005260:Female; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000502:physiology; D017353:Gene Deletion; D020868:Gene Silencing / Q000502:physiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D012313:RNA / Q000502:physiology; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
D006657:Histones; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D014157:Transcription Factors; C077885:XIST non-coding RNA; D049452:Green Fluorescent Proteins; D012313:RNA; D007041:Hypoxanthine Phosphoribosyltransferase; D006655:Histone Deacetylases