Protein binding protects sites on stable episomes and in the chromosome from de novo methylation

Abstract

We have utilized the Escherichia coli lac repressor-operator system to test whether protein binding can interfere with de novo DNA methylation in mammalian cells. We find that a DNA binding protein can protect sites on the episome as well as in the genome from the de novo methylation activity of Dnmt3a. Transcriptional machinery moving through the binding sites does not affect the de novo methylation of these sites, and it does not affect the binding protein protection of these sites from de novo methylation. This study and previous studies provide a possible mechanism for the observation that an Sp1 site can serve as a cis-acting signal for demethylation and for preventing de novo methylation of the CpG island upstream of the mouse adenine phosphoribosyltransferase (Aprt) gene. These findings also support the hypothesis that protein binding may play a crucial role in changes of CpG methylation pattern in mammalian cells.

FIG. 1

De novo methyltransferase Dnmt3a methylates lacO sequences. (A) Diagram of pOLucOriP and lacO sites. The SacII/NheI fragment of 4.1 kb includes the RSV LTR, the SV40 intron harboring three copies of the lacO sequence, and the luciferase gene. (B) Methylation of the lacO sequences by Dnmt3a. A Southern blot of HindIII- or HindIII- and HhaI-digested pOLucOriP DNA harvested 7 days after transfection showed a 467-bp band when it was probed with the 467-bp HindIII fragment, indicating methylation at the HhaI sites in the lacO sequence. The two panels show plasmid DNAs harvested from two independent transfections.

FIG. 2

Transcriptional activity does not affect de novo methylation of the lacO sites. Plasmid DNAs harvested from transfections were doubly digested with HindIII and HhaI, fractionated on a 1% agarose gel, transferred to a Southern blot, probed with the 467-bp HindIII fragment. (A) Plasmid DNAs harvested 7 days after transfection. The two lanes in each bracket are independent transfections. The 467-bp band was observed in DNAs from all three plasmids, indicating lacO methylation on all three plasmids. (B) Plasmid DNAs harvested 11 days after transfection. The two lanes in each bracket reflect results of independent transfections. The 467-bp band is stronger than the same band in panel A relative to the 304-bp band. (C) Quantitation of the fraction of the plasmid that became methylated. The radioactivity in the 467-bp band was divided by the total radioactivity in the lane after correction of the fragment sizes.

FIG. 3

Protection of the lacO sites from de novo methylation by LacI. Shown is a Southern blot of plasmid DNA doubly digested with HindIII and HhaI harvested from LacI-expressing cells after cotransfection with the Dnmt3a or the mutant Dnmt3a expression vector. (A) The 467-bp fragment was absent when LacI was present in the cells, and this band was detected in the cells treated with IPTG. In contrast, the 467-bp fragment was absent in the DNA cotransfected into the LacI-expressing cells with the mutant Dnmt3a expression vector, regardless of the presence or absence of IPTG, indicating no methylation at the lacO sites by the mutant Dnmt3a. The two lanes in each bracket reflect the results of two independent transfections. (B) LacI protection is specific for lacO sequences. The same Southern blot shown in panel A was stripped and rehybridized with a probe containing the luciferase coding region downstream from the lacO sequence. No difference in the increased-size fragments was observed with or without the presence of IPTG. The two lanes in each bracket reflect the results of two independent transfections.

FIG. 4

LacI protection of lacO sites from de novo methylation in the chromosome. (A) The Dnmt3a expression vector was transfected into independent LacI-expressing cell clones containing the lacO sequences treated or not treated with IPTG. A single 304-bp band was detected in the DNA doubly digested with HindIII and HhaI harvested from untransfected cells, indicating no methylation at the lacO sites without the presence of Dnmt3a. A single 304-bp band was detected in the HindIII- and HhaI-digested DNA from transfected cell clones not treated with IPTG. This indicates that no methylation occurred at the HhaI sites in the lacO sequences when IPTG was absent and that LacI was allowed to bind to lacO in the cells. The 467-bp band was present in the DNA doubly digested with HindIII and HhaI from these cell clones when IPTG was present. This result demonstrates that Dnmt3a can methylate lacO when LacI binding to lacO is inhibited by IPTG. (B) LacI protection is specific for lacO sequences. No difference in the increased-size fragments was observed when the Southern blot shown in panel A was stripped and rehybridized with a probe containing the luciferase coding region, regardless of the presence or absence of IPTG.