A Distinct Sequence (ATAAA)n Separates Methylated and Unmethylated Domains at the 5′-End of theGSTP1 CpG Island*

Douglas S Millar,Cheryl L Paul,Peter L Molloy,Susan J Clark

doi:10.1074/jbc.m906538199

Douglas S Millar, Cheryl L Paul + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m906538199

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Abstract

What defines the boundaries between methylated and unmethylated domains in the genome is unclear. In this study we used bisulfite genomic sequencing to map the boundaries of methylation that flank the 5'- and 3'-ends of the CpG island spanning the promoter region of the glutathione S-transferase (GSTP1) gene. We show that GSTP1 is expressed in a wide range of tissues including brain, lung, skeletal muscle, spleen, pancreas, bone marrow, prostate, heart, and blood and that this expression is associated with the CpG island being unmethylated. In these normal tissues a marked boundary was found to separate the methylated and unmethylated regions of the gene at the 5'-flank of the CpG island, and this boundary correlated with an (ATAAA)(19-24) repeated sequence. In contrast, the 3'-end of the CpG island was not marked by a sharp transition in methylation but by a gradual change in methylation density over about 500 base pairs. In normal tissue the sequences on either side of the 5'-boundary appear to lie in separate domains in which CpG methylation is independently controlled. These separate methylation domains are lost in all prostate cancer where GSTP1 expression is silenced and methylation extends throughout the island and spans across both the 5'- and 3'-boundary regions.

Highlights

Almost all organisms with a genome of greater than 109 base pairs methylate their DNA, generally at the 5Ј-position of cytosine in CpG dinucleotides
Like many “housekeeping” genes the GSTP1 gene contains a typical CpG island that extends from ϳ400 base pairs upstream to 800 base pairs downstream of the transcription initiation site (Fig. 1A)
We have previously shown that the core promoter region is unmethylated in normal prostate cells but becomes methylated in prostate cancer

Summary

Introduction

Almost all organisms with a genome of greater than 109 base pairs methylate their DNA, generally at the 5Ј-position of cytosine in CpG dinucleotides. Detailed bisulfite sequencing analysis of the CpG island spanning the core promoter region of the GSTP1 gene showed that methylation is extensive at essentially all CpG sites in prostate cancer DNA [14]. In this paper we have extended the bisulfite sequence analysis of the methylation profile of the GSTP1 CpG island/promoter to include the flanking regions where the normal and prostate cancer methylation profiles diverge. This has allowed identification of a defined “boundary” region of methylation at the 5Ј-flank, which is marked by a repeating ATAAA sequence

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