Quantitative analysis of human tissue-specific differences in methylation

Abstract

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.

Figure 1

Sequenom MassARRAY Analysis of percent DNA methylation at mouse testis-specific differentially methylated regions. The EpiTYPER program from Sequenom MassARRAY Analysis provides the results of percent methylation as an epigram. The epigram shows the percent DNA methylation level of each CpG site of the target region. The aligned epigram shows methylation signature differences in the target genomic region. Different colors display relative methylation changes in 10% increments. The yellow circle indicates 100% methylation, and the red circle is 0% methylation at each CpG site. The number of CpG sites, target sequence length and sample names are included in each epigram. Seven DNA samples from various mouse tissues (brain, heart, kidney, liver, lung, spleen and testes) were used in this analysis. The epigrams for a.Ddx4, b.Dact1 and c. Znf324 were shown.

Figure 2

Mouse and human DNA methylation levels at the Hspa1l tDMR. a. Percent methylation levels are indicated by epigrams for the mouse Hspa1l tDMR using two primer sets. Top is Hspa1l-1 set and bottom is Hspa1l-2 set within the Hspa1l-1 fragment. The aligned epigram shows a methylation signature difference in the target genomic region as shown in Figure 1. b. Genomic structure of human HSPA1L (top). Mouse tDMR, previously analyzed regions [14] and the region analyzed in this paper are indicated as dotted lines, shaded boxes and a black box, respectively. Two independent primer sets are designed to cover the region. The average percent methylation level and the standard deviation are indicated as bar graphs for HSPA1L-1 (middle) and HSPA1L-2 (bottom). c. 0%, 50% and 100% methylation control analysis. A bar graph shows an average percent methylation of two primer sets (BC048318-A and BC048318-B). Average percent methylation levels with standard deviations were depicted for 0%, 50% and 100% controls in each primer set.

Figure 3

Average percent methylation level and standard deviation are indicated as bar graphs for the a. TRIM38, b. MBNL2, c. SPESP1, d. PHLDB3, e. CASZ1 and f. CDH22. Three individuals ID numbers (102, 108 and 114) and tissue samples (K: kidney, T: testis, C: colon, H: heart, B: brain, L: liver and M: muscle) are indicated in each bar.

Figure 4

Genomic structure of a. TRIM38, b. MBNL2, c. CDH22 and d. NF755. The area indicated by a black rectangle is the region examined as a T-DMR. The left scatter-graph shows promoter DNA methylation and spermatocytes. The right scatter-graph shows promoter DNA methylation and age. Eight human autopsy cases were analyzed and their number of spermatocytes per seminiferous tubule was examined by the pathologist.