Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression

Abstract

Early studies proposed that DNA methylation could have a role in regulating gene expression during development [Riggs, A.D. (1975) Cytogenet. Cell Genet. 14, 9-25]. However, some studies of DNA methylation in known tissue-specific genes during development do not support a major role for DNA methylation. In the results presented here, tissue-specific differentially methylated regions (TDMs) were first identified, and then expression of genes associated with these regions correlated with methylation status. Restriction landmark genomic scanning (RLGS) was used in conjunction with virtual RLGS to identify 150 TDMs [Matsuyama, T., Kimura, M.T., Koike, K., Abe, T., Nakao, T., Asami, T., Ebisuzaki, T., Held, W.A., Yoshida, S. & Nagase, H. (2003) Nucleic Acids Res. 31, 4490-4496]. Analysis of 14 TDMs by methylation-specific PCR and by bisulfite genomic sequencing confirms that the regions identified by RLGS are differentially methylated in a tissue-specific manner. The results indicate that 5% or more of the CpG islands are TDMs, disputing the general notion that all CpG islands are unmethylated. Some of the TDMs are within 5' promoter CpG islands of genes, which exhibit a tissue-specific expression pattern that is consistent with methylation status and a role in tissue differentiation.

Fig. 4.

Bisulfite genomic sequencing of TDM Pst3. (a) Sequence traces obtained from the PCR products from bisulfite-treated DNA by using primers that would amplify a portion of the Pst3 locus. Because the PCR products were not cloned, the trace represents an approximation of the “average” methylation status at each CpG residue. The normal sequence and bisulfite sequence are shown. CpG sites are highlighted. (b) Diagrammatic representation of Pst3 within the 5′ promoter region of Ddx4 (DEAD-box protein 4) on chromosome 13. The CpG island includes the 5′ promoter, exon 1, and a portion of the first intron. The arrow indicates the direction of transcription. The methylation status of the CpGs (black circles, fully methylated; blue circles, partial methylation; white circles, unmethylated) within a 450-bp region of Pst3 is shown along with the location of the restriction landmark NotI site. Note the dense hypermethylation in liver, kidney, colon, muscle, and brain and the hypomethylation in testis. Fig. 6 shows the bisulfite sequence of a portion of the Pst3 region that was determined from cloned PCR products. The results are in general agreement, although the cloned sequences exhibit considerable heterogeneity in methylation. Fig. 10, which is published as supporting information on the PNAS web site, shows more detail as a larger version of this figure.

Fig. 1.

RLGS identification of TDMs. RLGS was performed with male C57BL/6J DNA (12 weeks of age) from testis, brain, colon, kidney, liver, and muscle by using two different RLGS restriction enzyme combinations (NotI–PstI–PvuII and NotI–PvuII–PstI). (a) The NotI-PstI–PvuII profile from testis is shown. (b) (Left) A section (enclosed section in a) from testis (Top), kidney (Middle), and liver (Bottom) is enlarged, and TDM loci are indicated. (Right)A portion of the RLGS profiles from six tissues showing additional TDM loci that were further confirmed (shown in italics) by MSP or bisulfite sequencing.

Fig. 2.

RLGS-inferred tissue methylation profiles for 150 TDMs identified by RLGS and virtual RLGS. Black squares indicate the RLGS spot was absent (methylated), purple squares indicate reduced intensity (partial methylation), and white squares indicate full diploid intensity (unmethylated). ND, the spot intensity could not be determined. Note that 64 of 150 loci were unmethylated in only one tissue and that 43 were unmethylated in testis only. Table 4 provides a complete listing of the TDMs and their locations in the genome.

Fig. 3.

Tissue-specific DNA methylation. (a) MSP analysis of TDM loci. MSP primers that would amplify methylated (M) or unmethylated (U) genomic regions that contain or were close to the NotI restriction landmark site were designed by using methprimer (18). The presence of unmethylated and methylated products in some tissues probably reflects heterogeneity of cell types and methylation within a tissue. The detection of unmethylated Pst6 in colon by MSP that was not apparent by RLGS is probably due to the greater sensitivity of MSP. However, because MSP is not quantitative, it could represent a small fraction of the cells in colon. As a positive control for unmethylated primers, bacterial artificial chromosome genomic clones containing the genomic regions of interest (RP23-180J7, -285K3, -59D1, -430C5, -288H11, -467M14, -381B4, -278A5, and -446I5) were mixed, bisufite treated, and used as a template for MSP (Cont). (b) MSP analysis of the methylation status of Pst6 TDM and an adjacent CpG island. Genomic DNA was treated with sodium bisulfite and then amplified with primers specific for methylated and unmethylated sequences within the Pst6 TDM (CpG1) and a small CpG island just 3′ of Pst6 (CpG2). CpG1 is strongly methylated in all tissues except testis. In contrast, CpG2 is unmethylated in kidney and brain. See text for further explanation.

Fig. 5.

Graphical representation (SYBR Green RT-PCR) of tissue expression of genes associated with TDMs relative to expression in liver. Expression levels were standardized to GAPDH by calculating ΔCt (ΔCt = Gene Ct - GAPDH Ct). The mean ΔΔCt values were determined by subtracting the ΔCt for liver expression from the ΔCt of each of the other tissues. For most genes, liver expression was low or was the lowest of the tissues (e.g., Pst3 and Pst61).