Age-related changes in subtelomeric methylation in the normal Japanese population

Abstract

Background: The telomeres of somatic cells become shorter with individual aging. However, no significant change in subtelomeric methylation of somatic cells with aging has yet been reported.

Methods: Telomere lengths of the peripheral blood cells of 148 normal Japanese were analyzed by Southern blotting using methylation-sensitive and -insensitive isoschizomers.

Results: With aging, long telomeres decrease and short telomeres increase, and the contents of the telomeres with methylated subtelomere increase in long telomeres, thus leading us to postulate that telomeres with less methylated subtelomeres tend to become shortened faster.

Conclusions: A telomere length distribution analysis with methylation-sensitive and -insensitive isoschizomer seems to be a useful tool to assess the subtelomeric methylation status of the somatic cell population. The subtelomeric methylation of peripheral blood cells is also indicated to be an indicator for aging-associated genomic changes.

Figure 2.

Peak-MspI-TRF and HpaII–MspI subtracted (H-M) TRF distribution with aging. Closed diamonds, peak-MspI-TRF; open squares, H-M-TRF. (A, Whole population; B, Men; C, Women) H-M values are regarded as approximately the mean values of the methylated lengths of subtelomeric region. The MspI-TRF gradually decreased with aging, but the H-M values did not. Note that some H-M values are negative. TRF = terminal restriction fragment.

Figure 3.

COBRA of the subtelomeres specific for chromosome 17p in healthy Japanese. Bisulfite-treated genomic DNA of the participants was polymerase chain reaction–amplified with chromosome arm–specific primers and digested with MspI. Ladder of HaeI-digested φX174 DNA is shown in the left of the each gel. A complete digestion of the amplified bisulfite-untreated product with MspI yields the bands, which are indicated by black arrowheads. The age, TRF, and relative subtelomeric methylation rate are shown on the top of each panel. Note that 6 of the 30 participants (2 in their 20s, 1 in the 40s, and 3 in the 60s) represent extra bands indicated by white arrowheads, which contain thymine residue changed from unmethylated cytosine residue by the bisulfite treatment. Almost all participants, except for those with extra bands, seem to show the subtelomeric region on chromosome 17p to be well methylated. COBRA = combined bisulfite restriction analysis; TRF = terminal restriction fragment.

Figure 4.

Changes of the subdivided MspI-TRF distribution with aging. Southern blotting smear of MspI-TRF was divided into three portions (>9.4, 9.4 ≥ ≥ 4.4, and 4.4 >). The percentages of densitometry of each portion are shown as columns. (A, Whole population; B, Men; C, Women) The longest telomeric portion decreases with aging and the shortest increases beyond the age of 60. The middle subdivided part (9.4 > > 4.4 kb) did not reveal a clear aging-related change. Horizontal bars depict the standard errors. TRF = terminal restriction fragment.

Figure 1.

A schematic drawing of a densitometric analysis of MspI- and HpaII-digested genomic Southern blot probed with a telomeric sequence repeat. DNA digested with MspI and HpaII appeared as smear on the left photo. The densitometry of the smears of MspI digest (A) and HpaII digest (B) is described in the right. The densitometry of the smears is fractioned according to the molecular weight standard, HindIII-digested l phage DNA, showing bands of 23.1, 9.4, 6.6, and 4.4 kb. The standard sizes for the fractionation are shown as horizontal broken (23.1 and 6.6 kb) and solid (9.4 and 4.4 kb) lines. The peak density (dotted lines and arrows) of the smears described as MspI-TRF (dark gray arrow) and HpaII-TRF (pale gray arrow) are used in the following study as representative telomere lengths of the individuals. The subtracted length of peak-MspI-TRF from peak-HpaII-TRF (peak-H-M-TRF) of an individual is used as a representative length of unmethylated subtelomeric region of the individual. (1), (2), (3), and (4) depict restricted densitometric areas of the smears as follows: (1) longer than 9.4 kb in MspI digestion, (2) shorter than 4.4 kb in MspI digestion, (3) longer than 9.4 kb in HpaII digestion, and (4) shorter than 4.4 kb in HpaII digestion. Each of the areas is described as a percentage to the whole smear as 100%, and the subtracted figures (3) − (1) and (2) − (4) are used in the following analysis. {(3) − (1)}/(3) and {(2) − (4)}/(2), which are described as H-M/H (>9.4 kb) and M-H/M (4.4 kb>) in text and the following figures, are used as indices to evaluate how much subtelomeric regions of telomeres shorter than 9.4 kb and shorter than 4.4 kb are methylated, respectively. TRF = terminal restriction fragment.

Figure 5.

H-M-TRF length distribution with aging. Subtracted value of the MspI-TRF from the HpaII-TRF densitometry in the three subdivided parts are shown as columns. (A, Whole population; B, Men; C, Women) The individuals in their 60s apparently have methylated telomeres both in long and in short telomeres. Horizontal bars depict the standard errors. TRF = terminal restriction fragment.

Figure 6.

Change of the relative methylation status of subtelomere with aging. The H-M area/HpaII area of >9.4 kb portion and the M-H area/MspI area of the 4.4 kb>, portion are used as indices indicating the subtelomeric methylation of longer and shorter (than 4.4 kb) telomeres, respectively. (A, Whole population; B, Men; C, Women). In participants in their 60s, the longer (>9.4 kb) telomeres contain more methylated subtelomeres and the shorter telomere (<4.4 kb) contains less methylated subtelomeres. This pattern is opposite to the pattern of the MspI columns >9.4 and <4.4 kb in the participants in their 60s in Figure 1. Horizontal bars depict the standard errors.