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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.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Funding
This work was supported by NIH grants: RC1 CA145422; R21 CA 121068; R03 CA132145; R01 CA106186; 1 R21 CA104812.
Acknowledgments
David Reynolds and the Genomics Core at Albert Einstein College of Medicine for help with DNA sequencing; Edward Nieves, Myrasol Callaway, and Berta Burd Proteomics Core of Albert Einstein College of Medicine for the LC-MS/MS analysis. Also, we offer gratitude to Drs Matthew Scharff, Eric Bouhassira, Vern Schramm, Quan Du, Joseph Locker, and Arthur Skoltchi for helping to clarify previous drafts of the manuscript.
Patent
Patent application for this method was submitted in 2011.
Supplemental Materials
Supplemental materials may be found here: www.landesbioscience.com/journals/epigenetics/article/26195