DNA replication and repair kinetics of Alu, LINE-1 and satellite III genomic repetitive elements

Abstract

Background: Preservation of genome integrity by complete, error-free DNA duplication prior to cell division and by correct DNA damage repair is paramount for the development and maintenance of an organism. This holds true not only for protein-encoding genes, but also it applies to repetitive DNA elements, which make up more than half of the human genome. Here, we focused on the replication and repair kinetics of interspersed and tandem repetitive DNA elements.

Results: We integrated genomic population level data with a single cell immunofluorescence in situ hybridization approach to simultaneously label replication/repair and repetitive DNA elements. We found that: (1) the euchromatic Alu element was replicated during early S-phase; (2) LINE-1, which is associated with AT-rich genomic regions, was replicated throughout S-phase, with the majority being replicated according to their particular histone marks; (3) satellite III, which constitutes pericentromeric heterochromatin, was replicated exclusively during the mid-to-late S-phase. As for the DNA double-strand break repair process, we observed that Alu elements followed the global genome repair kinetics, while LINE-1 elements repaired at a slower rate. Finally, satellite III repeats were repaired at later time points.

Conclusions: We conclude that the histone modifications in the specific repeat element predominantly determine its replication and repair timing. Thus, Alu elements, which are characterized by euchromatic chromatin features, are repaired and replicated the earliest, followed by LINE-1 elements, including more variegated eu/heterochromatic features and, lastly, satellite tandem repeats, which are homogeneously characterized by heterochromatic features and extend over megabase-long genomic regions. Altogether, this work reemphasizes the need for complementary approaches to achieve an integrated and comprehensive investigation of genomic processes.

Keywords: Alu; ChIP-Seq; DNA repair; DNA repetitive elements; DNA replication; Genome-wide analysis; Immuno-FISH; LINE; Phosphorylated H2AX; Satellites.

Fig. 1

Genomic features of repetitive DNA elements. Spearman’s rho correlation matrix. The number of each repetitive DNA element copies, or the amount of a given genomic feature is counted in each 10 kb genomic interval. The correlation coefficient is calculated for comparison of repetitive DNA elements with GC content (a), histone modifications (from HeLa, ENCODE tier 2) or genic regions (b) and replication timing from Repli-Seq data (HeLa, ENCODE tier 2) [31] (c). Data are from > 290,000 genomic intervals. For each correlation, P < 2.2 × 10⁻¹⁶. In a, Topo(…): from left to right, topoisomerase I consensus sequences at decreasing GC content. Highlighted Alu and L1 repetitive elements are arbitrarily chosen to define chromatin compartments with opposing chromatin features, and are further investigated in FISH experiments. d Correlation matrix of histone modifications and replication timing in HeLa cells, for L1-rich (> 10 counts per genomic interval) L1-poor (> 1 count per genomic element) genomic regions

Fig. 2

Replication timing of repetitive DNA elements analyzed by FISH and S-phase substages classification. a Schematics of the experiment. HeLa cells were pulse-labeled with EdU for 15 min to allow the classification of different substages of the S-phase of the cell cycle (early, mid and late). Cells are then fixed, the probe is hybridized and microscopy is performed. b (left) Representative confocal and deconvolved micrographs of HeLa cells depicting the DAPI, Alu elements and EdU as inverted gray channels, at the three different S-phase substages. Merge is shown in pseudo-colors. Scale bar: 5 µm. (right) Colocalization analysis of FISH and EdU signal at the three different S-phase substages via H_coefficent and Pearson’s correlation coefficient as indicated. Error bars show the standard error of the mean. Data are from three independent experiments. n combined total number of cells analyzed. sd standard deviation. c, d Represent the same as in b for L1 and satellite III, respectively

Fig. 3

Genome-wide DNA repair kinetics of non-B and repetitive DNA elements. a Spearman’s rho correlation matrix between repetitive a and non-B b DNA elements and γH2AX levels before and after (0.5, 3 and 24 h) IR in HeLa cells. Calculation of the correlation coefficient is as in Fig. 1. c (top) Pie-charts showing the distribution of read counts for Alu, LINEs, satellites and LTR repetitive DNA elements, before and after (0.5, 3 and 24 h) IR. (bottom) Bar-plots showing the relative enrichment for the repeat element indicated after (0.5, 3 and 24 h) IR. The number of reads for a given repetitive element and at a given time point was normalized over the corresponding number of reads before IR (for details see Materials and methods section). The respective GC content of the repeat is indicated (whole human genome GC content is 43%)

Fig. 4

DNA repair kinetics of repetitive DNA elements assessed by FISH. a Schematics of the experiment. HeLa cells were sham-irradiated or irradiated with 2 Gy X-rays and incubated for 0.5, 3 and 24 h. γH2AX immunofluorescence and probe hybridization were performed before confocal micrographs acquisition and deconvolution. b (left) Representative confocal micrographs of HeLa cells depicting the DAPI, Alu elements and EdU as inverted gray channels, at the three time points post-IR. Merge is shown in pseudo-colors. Scale bar: 5 µm. (right) Relative change of Alu fraction in γH2AX foci. Data are normalized to the median of the 0.5 h time point. Boxes represent median, 2nd and 3rd quartile. Whiskers indicate three times the interquartile distance. Data are from three independent experiments. n combined total number of cells analyzed. sd standard deviation. c, d Represent the same as in b for LINE1 and satellite III, respectively. In d, the empty boxes represent the relative change of γH2AX intensity in segmented satellite III regions

Fig. 5

Distribution, replication and repair kinetics of human repetitive elements. a Side-by-side comparison of colocalization analysis of repetitive DNA element and DNA replication signals at the three different S-phase substages. b Similarly, side-by-side comparison of global genome DNA repair kinetics and each of the different repetitive DNA elements indicated. c Graphical summary of the replication and repair kinetics of Alu, L1 and satellite II repetitive elements in the context of their respective chromosomal distribution