Tissue specific differentially methylated regions (TDMR): Changes in DNA methylation during development

Abstract

Tissue specific differentially methylated regions (TDMRs) were identified and localized in the mouse genome using second generation virtual RLGS (vRLGS). Sequenom MassARRAY quantitative methylation analysis was used to confirm and determine the fine structure of tissue specific differences in DNA methylation. TDMRs have a broad distribution of locations to intragenic and intergenic regions including both CpG islands, and non-CpG islands regions. Somewhat surprising, there is a strong bias for TDMR location in non-promoter intragenic regions. Although some TDMRs are within or close to repeat sequences, overall they are less frequently associated with repetitive elements than expected from a random distribution. Many TDMRs are methylated at early developmental stages, but unmethylated later, suggesting active or passive demethylation, or expansions of populations of cells with unmethylated TDMRs. This is notable during postnatal testis differentiation where many testis specific TDMRs become progressively "demethylated". These results suggest that methylation changes during development are dynamic, involve demethylation and methylation, and may occur at late stages of embryonic development or even postnatally.

Figure 1

RLGS analysis of Testis and ES cell DNA. A full RLGS profile of ES cell DNA and portions of RLGS profiles (NotI-PstI-PvuII) are shown. Arrows indicate position of Pvu 43, 2, 6, 74, and 75 (see Table 2). Testis DNAs are from two different mice (C57BL/6J) and ES1 and ES2 correspond to C57BL/6J Stewart Bruce 4 ES cells, passage 13 and 17 respectively.

Figure 2

TDMRs confirmed by Sequenom MassARRAY methylation analysis. The figure shows the analysis of two sets of mouse tissue samples including muscle (M), brain (B), kidney (K), liver (L), colon (C) and testis (T). The colored circles indicate the degree of methylation with yellow representing 0% methylation and blue representing 100%. The position of the Restriction Landmark NotI site is indicated. The four TDMRs (Pst4, Pst6, Pvu6, Pvu35) shown are located at 5′ promoter, 3′ exon, intergenic and intron, respectively.

Figure 3

Methylation fine structure associated with the Pst6 TDMR. The location of Pst6 TDMR within Hspa1l and a homologous region within the Hspa1a gene are indicated in mouse July, 2007 UCSC assembly. The GC percent, positions of CpG islands and repeat sequences are also shown. The lower panel indicates the percentage of methylation in adult testis (T), liver (L) and ES cells. Other somatic tissues (muscle, brain, kidney, and colon) were essentially identical to liver (see Figure 2). The length (bp) and number of CpGs in each region analyzed by Sequenom MassARRAY methylation analysis are also shown.

Figure 4

MSP analysis of methylation status of TDMRs during development. MSP primers that would amplify methylated (M) or Unmethylated (U) genomic regions that contain, or were close to, the TDMR NotI restriction landmark site were designed by using METHPRIMER (http://www.urogene.org/methprimer/). DNAs from BAC clones (bacterial artificial chromosome) containing the TDMR region were used as a positive control for the U primers (not shown). Sss1 methylase-treated DNA was used as a positive control for the M primers (not shown). Results from 2 independently derived DNA samples from 12 wk liver and testis are shown.

Figure 5

Demethylation of repeat and unique sequences during postnatal testis development. The Bar graph shows a summary of the methylation status obtained from Sequenom MassArray methylation analysis of testis samples at different stages of development from new born (NB), 10 day old (10d), 20 day old (20d), and adult mice (12 wk) at each locus. The regions analyzed are the following: Pvu1, 7 CpGs (400bp); Pst2, 13 CpGs (225bp); Pst5, 19 CpGs (425bp); Pvu4, 43 CpGs (700bp); Pvu8, 25 CpGs (500bp); Pst3, 59 CpGs (525bp); Pst6, 26 CpGs (350bp); Pst4, 16 CpGs (250bp). The y- axis shows the percent of methylation obtained as an average level of methylation of the CpG dinucleotides for each locus for two independent determinations. The x-axis shows loci of interest (loci associated with either repeat or unique sequences). The error bars indicate standard deviation of the mean. It is to be noted that for the NB samples Pvu4 and Pvu8 loci there are no error bars as these were derived from only one set of samples.

Figure 6

Tissue and developmental stage specific differences in methylation. (A) A diagram of 10.1 kb Zfp206 zinc finger gene region is shown. The positions of exons (black bars), the NotI restriction landmark site, and the transcription start site (arrow) are indicated. The locations of a CpG island and the regions analyzed by Sequenom massARRAY methylation analysis (promoter, exon2, two TDMRs [Pst 10 and Pvu 8]) are indicated by black bars under the gene diagram. The size of the regions (bp) and number of CpG analyzed for methylation by Sequenom massARRAY are also shown. (B) The bar graph indicates the percent of methylation in ES cells, 15 day embryo and adult tissues in the indicated regions. The methylation values were calculated as the mean of two independent determinations. The error bars indicate the standard deviation of the mean.