Inactive chromatin spreads from a focus of methylation.

Abstract

The detailed mechanisms of inhibition of transcription by DNA methylation are still unknown, but it has become obvious that the formation of chromatin plays an important role in this process. Using an approach enabling us to methylate, in vitro, chosen regions in a plasmid, we now show that specific methylation of nonpromoter sequences results in transcriptional inhibition of a reporter gene construct and that this inhibition is independent of the position of the methylated region within the plasmid. In plasmid minichromosomes containing a short region of methylated DNA, both methylated and unmethylated sequences are protected from MATERIALS AND METHODS Plasmids. Plasmid pVHCk was constructed by cloning a 2-kb KpnI-EcoRI fragment containing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the SV40 promoter/enhancer and the SV40 terminator region from pVHC1 (7) into pBluescript KSII-. A map of the plasmid is shown in Fig. 1. The promoterless plasmid pPLk was generated by deleting the SV40 promoter/enhancer from * Corresponding author.pVHCk by digestion with K1nI-HindIII, generation of blunt ends with S1 nuclease, and religation. Two constructs containing the SV40 promoter/enhancer at both sides of the CAT gene/terminator region were generated by cloning the BamHI-linkered promoter fragment in both orientations into the BamHI site of pVHCk immediately 3' of the terminator region. They were named pPPk (promoters in same orientation) and pPRk (second promoter in reverse orientation), respectively.Generation of patch-methylated constructs. Phagemid single-stranded DNA (ssDNA) was isolated by the method of Blondel and Thillet (4). Restriction fragments were gel purified and annealed to ssDNA at a molar ratio of 3:1 (fragment DNA:ssDNA) in 20 mM Tris-HCl (pH 7.5)-10 mM MgCl2-50 mM NaCl-1 mM dithiothreitol by heating for 2 min at 95°C and allowing to cool down from 70 to 30°C in 1 h. To prevent binding to and methylation of the singlestranded region by methylase SssI (M.SssI), T4 gene 32 protein (Boehringer Mannheim) was added (10 ,ug/,g of ssDNA), and the reaction mixture was incubated at 37°C for 15 min. The double-stranded DNA patch was methylated for 16 h, using M.SssI (New England Biolabs) under conditions recommended by the manufacturer. After proteinase K treatment and phenol extraction, the unmethylated gap was filled in and ligated by standard procedures (35). For every patch-methylated construct, a mock-methylated control was generated in the same way but omitting M.SssI. To confirm the location of the methylated region, constructs were isolated immediately or 2 days after transfection, and the methylated region was released with the appropriate restriction enzymes and subjected to HpaII digestion. The DNA was then separated on a 1.5% agarose gel, transferred to a nylon membrane (Hybond N+; Amersham), and hybridized to the appropriate labeled fragment. Membranes were reprobed after stripping with boiling sodium dodecyl sulfate (SDS) solution (0.5%).Transfections and analysis of CA...

Characterization of Two Age-induced Intracisternal A-particle-related Transcripts in the Mouse Liver

“…Spreading of an inactive chromatin conformation from a methylated domain to an adjacent non-methylated region was previously shown to inhibit the expression of a reporter gene in mammalian cells (43), and position effect variegation involving heterochromatin spreading and resulting in gene silencing has been well characterized in Drosophila (44). By analogy, the age-dependent induction of the IAP-AR transcripts could therefore be associated with the spreading of an active chromatin conformation from an adjacent gene that would be specifically expressed upon aging.…”

Parental Allele-Specific Chromatin Configuration in a Boundary–Imprinting-Control Element Upstream of the Mouse H19 Gene

“…Their functional nonequivalence is mediated by genomic imprinting, an epigenetic mechanism that gives rise to differential expression of the maternal and paternal alleles of certain genes. To date, 28 imprinted genes have been identified in the mouse (5), and many of these genes are expressed in a parental allele-specific manner in humans as well (41). The precise epigenetic features that allow mammalian cells to distinguish the parental alleles of imprinted genes are still poorly understood (41,47).…”

Demethylation of the progesterone receptor CpG island is not required for progesterone receptor gene expression

“…Several studies have suggested that dense DNA methylation promotes the binding of histone H1 or methyl-speci®c binding proteins that have a wide range of a nity and speci®city for methylated DNA (Levine et al, 1993;Lewis et al, 1992;Meehan et al, 1989;. These proteins may either compete with transcriptional activators for binding to a speci®c cis-acting site or help to stabilize a condensed chromatin structure that renders the DNA inaccessible to transcription factors (Kass et al, 1993).…”

“…Therefore, one possibility is that methylated sequences within the PR CpG island are too far downstream to in¯uence PR gene expression. However, numerous studies have shown that the ability of methylation to repress gene expression is highly dependent on the length and methyl CpG density of a particular promoter, but less dependent on the position and distance of the methylated DNA sequences from the transcriptional initiation site (Hsieh, 1994;Kass et al, 1993;Hsieh, 1997;Rideout III et al, 1994). Speci®cally, it was found that methylation of nonpromoter DNA sequences approximately two kilobase pairs from the transcription initiation site decreased gene expression to the same extent as methylation of the promoter itself and was associated with inactive chromatin in both the nonpromoter and promoter sequences (Kass et al, 1993).…”