Replicative Senescence-Associated LINE1 Methylation and LINE1-Alu Expression Levels in Human Endothelial Cells

Abstract

One of the main challenges of current research on aging is to identify the complex epigenetic mechanisms involved in the acquisition of the cellular senescent phenotype. Despite some evidence suggested that epigenetic changes of DNA repetitive elements, including transposable elements (TE) sequences, are associated with replicative senescence of fibroblasts, data on different types of cells are scarce. We previously analysed genome-wide DNA methylation of young and replicative senescent human endothelial cells (HUVECs), highlighting increased levels of demethylated sequences in senescent cells. Here, we aligned the most significantly demethylated single CpG sites to the reference genome and annotated their localization inside TE sequences and found a significant hypomethylation of sequences belonging to the Long-Interspersed Element-1 (LINE-1 or L1) subfamilies L1M, L1P, and L1HS. To verify the hypothesis that L1 demethylation could be associated with increased transcription/activation of L1s and/or Alu elements (non-autonomous retroelements that usually depend on L1 sequences for reverse transcription and retrotransposition), we quantified the RNA expression levels of both L1 (generic L1 elements or site-specific L1PA2 on chromosome 14) and Alu elements in young and senescent HUVECs and human dermal fibroblasts (NHDFs). The RNA expression of Alu and L1 sequences was significantly increased in both senescent HUVECs and NHDFs, whereas the RNA transcript of L1PA2 on chromosome 14 was not significantly modulated in senescent cells. Moreover, we found an increased amount of TE DNA copies in the cytoplasm of senescent HUVECs and NHDFs. Our results support the hypothesis that TE, which are significantly increased in senescent cells, could be retrotranscribed to DNA sequences.

Keywords: Alu sequences; LINE1; cellular senescence; retrotransposable elements.

Figure 1

Characterization of replicative senescence of NHDFs and HUVECs. Growth curve showing cumulative population doublings (CPDs) of NHDFs (A) and HUVECs (B) undergoing replicative senescence (X axis: cell passages). (C) Telomere length was analysed by Real Time-PCR calculated as telomere/single copy gene ratio (T/S). (D) Representative images of SA- β-Gal in young (a), in an intermediate passage (b) and in senescent (c) NHDFs and HUVECs. (E) p21 and p16 mRNA expression evaluated by RT-PCR. β-actin was used as internal control. (F) miR-21, miR-146a and miR-217 relative expression evaluated by RT-PCR. RNU44 was used as internal control. * p < 0.05; ** p < 0.01; *** p < 0.001 for paired t-test.

Figure 2

Methylation score of LINE subfamilies and Alu. (A,B) Violin plots showing the methylation score of the three LINE subfamilies and Alu in (A) young and (B) senescent HUVECs. ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. L1P. # p < 0.05; #### p < 0.0001 vs. L1M for two-tailed unpaired t-test. (C) Boxplots showing the distribution of differences in the methylation scores of the LINE1 family, subfamilies, and Alu between senescent and young HUVECs. p for Wilcoxon signed-rank tests.

Figure 3

Distribution of significantly demethylated CpGs within LINE1 subfamilies L1M and L1P grouped according to gene density classes. Low gene density (class 1), blue; intermediate gene density (classes 2–5); green; high gene density (classes 6–17), orange.

Figure 4

Relative expression of ORF2, Alu retrotransposable elements, and L1PA2 in (A) HUVEC and (B) NHDF cells. * p < 0.05; ** p < 0.01 for two-tailed paired t-test.

Figure 5

DNA quantification of ORF2 and Alu retrotransposable elements in the cytosolic and nuclear fractions of HUVEC and NHDF cells. * p < 0.05 for two-way ANOVA.