Hemimethylation and hypersensitivity are early events in transcriptional reactivation of human inactive X-linked genes in a hamster x human somatic cell hybrid.
Reactivation of the hypoxanthine phosphoribosyltransferase (HPRT) gene on an inactive human X chromosome in a somatic cell hybrid was analyzed following exposure to 5-aza-2'-deoxycytidine. Hemimethylation and chromatin hypersensitivity in the 5' CpG island appeared by 6 h after exposure and continued to increase for 24 h in an exponentially growing cell culture. These results imply that the conformation of inactive chromatin requires a symmetrically methylated 5' G+C-rich promoter region. In addition, quantitative analysis of the time course patterns suggest that chromatin sensitivity changes may depend on strand-specific demethylation. Symmetrically demethylated DNA was first detected at 24 h and continued to increase until 48 h. HPRT mRNA was first detected at 24 h and increased in a biphasic pattern until 48 h. These results suggest that hemimethylation permits nuclease attack but not transcription factor binding, which requires symmetrically demethylated DNA. We also show that in G1-arrested cells, 5-aza-2'-deoxycytidine has no effect on methylation, chromatin conformation, or transcription. We conclude that reactivation of the HPRT gene present on the inactive X chromosome of a somatic cell hybrid involves the initial events of DNA hemimethylation and chromatin hypersensitivity at the 5' CpG island, followed by symmetrical demethylation and transcriptional reactivation.
Sasaki
T
T
Department of Medicine, University of Washington, Seattle 98195.
Hansen
R S
RS
Gartler
S M
SM
D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism; D006224:Cricetinae; D004247:DNA; D000077209:Decitabine; D015226:Dinucleoside Phosphates / Q000235:genetics; D004303:Dosage Compensation, Genetic; D008040:Genetic Linkage; D006801:Humans; D006822:Hybrid Cells; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014960:X Chromosome
D001374:Azacitidine; D002843:Chromatin; D004247:DNA; D000077209:Decitabine; D015226:Dinucleoside Phosphates; D007041:Hypoxanthine Phosphoribosyltransferase; D012333:RNA, Messenger; C015772:CpG dinucleotide