Site-specific methylated reporter constructs for functional analysis of DNA methylation

Weiguo Han, Miao Shi, Simon D. Spivack

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Methods to experimentally alter and functionally evaluate cytosine methylation in a site-specific manner have proven elusive. We describe a site-specific DNa methylation method, using synthetically methylated primers and high fidelity PcR coupled with ligation of reporter constructs. We applied this method to introduce methylated cytosines into fragments of the respective DAPK and RASSF1A promoters that had been cloned into luciferase reporters. We found that methylation of 3-7 residue cpG clusters that were 5′ adjacent to the transcription start site (Tss) of the DAPK gene produced up to a 54% decrease in promoter activity (P < 0.01). similarly, for RASSF1A promoter reporter constructs, the methylation of either of two clusters of four cpGs each, but not an intervening cluster, produced a 63% decrease in promoter activity (P < 0.01), suggesting that precise mcpG position is crucial, and factors other than simple proximity to the Tss are at play. chromatin immunoprecipitation analysis of these reporter constructs demonstrated that transcription factor Oct-1 and sp1 preferentially bound the unmethylated vs. methylated DAPK or RASSF1A promoter reporter constructs at the functional cpG sites. histone h1, hnRNP1, and MecP2 showed preferential binding to methylated sequence at functional sites in these reporter constructs, as well as highly preferential (>8-80-fold) binding to native methylated vs. unmethylated chromatin. These results suggest that: (1) site-specific, precision DNa methylation of a reporter construct can be used for functional analysis of commonly observed gene promoter methylation patterns; (2) the reporter system contains key elements of the endogenous chromatin machinery.

Original languageEnglish (US)
Pages (from-to)1176-1187
Number of pages12
JournalEpigenetics
Volume8
Issue number11
DOIs
StatePublished - 2013

Fingerprint

DNA Methylation
Methylation
Cytosine
Chromatin
Transcription Initiation Site
Luciferases
Genes
Ligation

Keywords

  • DNA methylation
  • Epigenetics
  • Functional analysis
  • Site-specific DNA methylation
  • Transcription factor

ASJC Scopus subject areas

  • Molecular Biology
  • Cancer Research

Cite this

Site-specific methylated reporter constructs for functional analysis of DNA methylation. / Han, Weiguo; Shi, Miao; Spivack, Simon D.

In: Epigenetics, Vol. 8, No. 11, 2013, p. 1176-1187.

Research output: Contribution to journalArticle

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