Abstract
During the process of 5-aza-2'-deoxycytidine (5aCdr)-induced reactivation of the X-linked human hypoxanthine phosphoribosyltransferase (HPRT) gene on the inactive X chromosome, acquisition of a nuclease-sensitive chromatin conformation in the 5' region occurs before the appearance of HPRT mRNA. In vivo footprinting experiments reported here show that the 5aCdr-induced change in HPRT chromatin structure precedes the appearance of three footprints in the immediate 5' flanking region that are characteristic of the active HPRT allele. These and other data suggest the following sequence of events that lead to the reactivation of the HPRT gene after 5aCdr treatment: (a) hemi-demethylation of the promoter, (b) an "opening" of chromatin structure detectable as increased nuclease sensitivity, (c) transcription factor binding to the promoter, (d) assembly of the transcription complex, and (e) synthesis of HPRT RNA. This sequence of events supports the view that inactive X-linked genes are silenced by a repressive chromatin structure that prevents the binding of transcriptional activators to the promoter.
Highlights
Summary of Nuclease Sensitivity, HPRT mRNA, and Transcription Factor Binding—A graphical summary of the events following 5aCdr treatment of the inactive X hybrid is shown in Fig. 6, in which chromatin structure, transcription factor binding at positions Ϫ91, Ϫ198, and Ϫ210, and HPRT mRNA levels are plotted as a function of time after initiating 5aCdr treatment
The appearance of the Ϫ91, Ϫ198, and Ϫ210 footprints are correlated with the appearance of HPRT mRNA rather than with the earlier change in chromatin structure
This change in chromatin structure, does not require binding of a factor(s) to the Ϫ91 region, a region that is near the multiple sites of transcription initiation and in a location similar to regions previously reported to be critical for silencing other genes by DNA methylation [33, 34]
Summary
5aCdr, 5-aza-2Ј-deoxycytidine; HPRT, hypoxanthine phosphoribosyltransferase; PGK-1, phosphoglycerate kinase 1; DMS, dimethyl sulfate; kb, kilobase; bp, base pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; LMPCR, ligation-mediated PCR. There is no evidence for the interaction of methylated DNA-binding proteins [25] with the 5Ј regions of these genes on the inactive X chromosome. The remodeling of chromatin structure during 5aCdr reactivation of the HPRT gene on the inactive X chromosome precedes and does not require the binding of at least three sequence-specific transcription factors to the promoter region
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