Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

Abstract

Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2'-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5'-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR.

Keywords: Epigenetics; High-LET radiation; Histone methylation; Ionizing radiation; Transposable elements.

Figure 1

Effects of exposure to densely IR on LINE-1 DNA methylation. (A) LINE-1 schematic representation. (B) Analysis of LINE-1 ORF2 DNA methylation. Data are presented as ± SEM. (C) Analysis of LINE-1 ORF1 DNA methylation. Cumulative data from five CpG sites presented as changes in the percentage of methylated CpG sites ± SEM. Asterisks (***) denotes significant (P<0.001) difference from control (one-way ANOVA). P – protons; ⁵⁶Fe – heavy iron ions; P+⁵⁶Fe – consequent exposure to protons and heavy iron ions.

Figure 2

Evolutionary age of LINE-1 elements. (A) Phylogenetic tree, promoter type and evolutionary age of the investigated murine LINE-1 elements. (B) Correlation between the extent of DNA methylation and the evolutionary age of LINE-1 elements in the normal mouse lung. Data normalized to the values of the youngest LINE-1 family, L1MdA_I. Myr – million years.

Figure 3

Schematic representation of the 5′-UTRs of nine LINE-1 elements with methylation-sensitive restriction sites. Arrows define the amplification loci.

Figure 4

Effects of exposure to densely IR on DNA methylation in LINE-1 5′-UTR. (A) Exposure to protons, heavy iron ions (⁵⁶Fe) alone or in combination results in DNA hypermethylation within the 5′-UTRs of selective LINE-1 elements. Fold change for each sample is represented by a color change on the heat map. (B) DNA methylation within the 5′-UTR of selective LINE-1 elements as measured by MeDIP. Asterisks (**) denotes significant (P<0.01) difference from control.

Figure 5

Analysis of mRNA levels of genes that evolutionary acquired L1MdA_II element insertions. The differential expression of genes was determined by quantitative RT-PCR. The fold change data were calculated from the ΔΔCt values. Asterisks (*) denotes significant (P<0.05), difference from control (n=5). All qRT-PCR reactions were repeated twice. P – protons; ⁵⁶Fe – heavy iron ions; P+⁵⁶Fe – consequent exposure to protons and heavy iron ions.

Figure 6

Exposure to densely IR results in LINE-1 reactivation via histone modifications. (A-B) Analysis of LINE-1 ORF1 (A) and ORF2 (B) mRNA levels by reverse transcription qRT-PCR. (C) Western immunoblot analysis of the LINE-1 ORF1p levels in the mouse lung. P – protons; ⁵⁶Fe – heavy iron ions; P+⁵⁶Fe – consequent exposure to protons and heavy iron ions (n=4). (D, E) Analysis of the histone H3 Lysine 9 (H3K9) dimethylation (D) and histone H3 Lysine 4 (H3K4) trimethylation (E) at the L1MdA-I 5′-UTR by ChIP analysis. Samples were normalized to L1Lx_I ORF1. Data are presented as ± SEM (one-way ANOVA). (F) Analysis of the Ehmt2/G9a histone methyltransferase mRNA levels using qRT-PCR. Asterisks (*) denotes significant (P<0.05), and (***) denotes significant (P<0.001) difference from control. P – protons; ⁵⁶Fe – heavy iron ions; P+⁵⁶Fe – consequent exposure to protons and heavy iron ions.