Identification of imprinting regulators at the Meg3 differentially methylated region

Abstract

Genomic imprinting at the Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) locus is regulated by the Meg3 differentially methylated region (DMR), but the mechanism by which this DMR acts is unknown. The goal of this study was to analyze the Meg3 DMR during imprinting establishment and maintenance for the presence of histone modifications and trans-acting DNA binding proteins using chromatin immunoprecipitation. In embryonic stem (ES) cells, where Meg3 is biallelically expressed, the DMR showed variable DNA methylation, with biallelic methylation at one region but paternal allele-specific methylation at another. All histone modifications detected at the Meg3 DMR of ES cells were biallelic. In embryonic day 12.5 (e12.5) embryos, where Meg3 is maternally expressed, the paternal Meg3 DMR was methylated, and activating histone modifications were specific to the maternal DMR. DNA-binding proteins that represent potential regulatory factors were identified in both ES cells and embryos.

Figure 1. Regions of the Meg3 DMR analyzed for epigenetic status

(A) Genomic organization of the Dlk1-Meg3 domain. Black boxes represent exons and gray boxes represent DMRs. (B) Depiction of the Meg3 DMR. The gray box represents the Meg3 DMR, and the black box represents Meg3 exon 1. Regions of the Meg3 DMR analyzed by ChIP are indicated by horizontal lines below the DMR. (C) Meg3 DMR methylation analysis in ES cells and e12.5 embryos. D, C, D x C ES cell, and D x Cg12 e12.5 embryo genomic DNA was digested with HpaII, amplified with region 3 (R3) and region 9 (R9) primers, and cut with the appropriate restriction enzyme to determine DNA methylation allele specificity. The uncut DNA is represented by “−”, while “+” denotes DNA cut with the appropriate restriction enzyme; %M indicates the amount of methylation on the maternal allele relative to the total methylation. (D) Meg3 expression analysis in e12.5 embryos and ES cells, using D, C, D x C, and C x D e12.5 embryo cDNA and D x C ES cell cDNA for analysis. Uncut DNA is represented by “−”, while “+” denotes SfcI cut DNA, %M indicates the amount of methylation on the maternal allele relative to the total level of methylation.

Figure 2. Analysis of histone modifications at Meg3 DMR regions R1 through R9 in ES cells

ChIP analysis of (A) H3ac, H4ac, H3K4me, H3K4me2, H3K4me3, (B) H3K9me2, H3K9me3, H3K27me3, H4K20me3, H3S10phos, (C) H3K79me, H3K79me3, and H4K91ac. Left panels display a representative ChIP assay for each modification at each region of the DMR. Right panels depict the average percent precipitation for each modification at each region of the DMR. Average percent precipitation was calculated from at least two independent ChIP experiments. Error bars represent standard error of the mean (SEM).

Figure 3. Allele-specific analysis of histone modifications at Meg3 DMR regions R3 and R9 in ES cells

(A) The region 3 product was cut with MboI and the region 9 product was cut with HinfI to distinguish the maternal and paternal alleles of the DMR. Paternal and maternal bands are indicated by “P” and “M” respectively. The uncut PCR product is represented by “−”, while “+” denotes the PCR product cut with the appropriate restriction enzyme. Average percent of maternal and paternal allele (%M and %P, respectively) of each histone modification present at region 3 (B) and region 9 (C) was calculated using data collected from at least two independent ChIP experiments. Error bars represent SEM.

Figure 4. Analysis of histone modifications at Meg3 DMR regions R1 through R9 in e12.5 embryos

ChIP analysis of (A) H3ac, H4ac, H3K4me, H3K4me2, H3K4me3, (B) H3K9me2, H3K9me3, H3K27me3, H4K20me3, H3S10phos, (C) H3K79me, H3K79me3, and H4K91ac. Left panels show a representative ChIP assay for each modification at each region of the DMR. Right panels depict the average percent precipitation of each modification at each region. Average percent precipitation was calculated from at least two independent ChIP experiments. Error bars represent SEM.

Figure 5. Allele-specific analysis of histone modifications at Meg3 DMR regions R3 and R9 in e12.5 embryos

(A) The region 3 product was cut with MboI and the region 9 product was cut with HinfI to distinguish the maternal and paternal alleles of the DMR. Paternal and maternal bands are indicated by “P” and “M” respectively. The uncut PCR product is represented by “−”, while “+” denotes the PCR product cut with the appropriate restriction enzyme. Average percent of maternal and paternal allele (%M and %P, respectively) of each histone modification present at region 3 (B) and region 9 (C) was calculated using data collected from at least two independent ChIP experiments. Error bars represent SEM.

Figure 6. Analysis of transcription factors at Meg3 DMR regions R1 through R9 in ES cells and e12.5 embryos

ChIP analysis of macroH2A1, MeCp2, EED, YY1, MBD3, Kaiso, MBD1, and CTCF in (A) D x C ES cells and (B) D x Cg e12.5 embryos. Each panel depicts the average percent precipitation of a given protein at each region of the Meg3 DMR. Average percent precipitation was calculated from at least two independent ChIP experiments. Error bars represent SEM.