Identification of the Telomere elongation Mutation in Drosophila

Abstract

Telomeres in Drosophila melanogaster, which have inspired a large part of Sergio Pimpinelli work, are similar to those of other eukaryotes in terms of their function. Yet, their length maintenance relies on the transposition of the specialized retrotransposons Het-A, TART, and TAHRE, rather than on the activity of the enzyme telomerase as it occurs in most other eukaryotic organisms. The length of the telomeres in Drosophila thus depends on the number of copies of these transposable elements. Our previous work has led to the isolation of a dominant mutation, Tel¹, that caused a several-fold elongation of telomeres. In this study, we molecularly identified the Tel¹ mutation by a combination of transposon-induced, site-specific recombination and next-generation sequencing. Recombination located Tel¹ to a 15 kb region in 92A. Comparison of the DNA sequence in this region with the Drosophila Genetic Reference Panel of wild-type genomic sequences delimited Tel¹ to a 3 bp deletion inside intron 8 of Ino80. Furthermore, CRISPR/Cas9-induced deletions surrounding the same region exhibited the Tel¹ telomere phenotype, confirming a strict requirement of this intron 8 gene sequence for a proper regulation of Drosophila telomere length.

Keywords: Drosophila melanogaster; next-generation sequencing; telomere; transposon-induced recombination.

Figure 1

Localization of Tel¹ by site-specific recombination. (A) The upper chromosome map shows the candidate genes between two P element insertion sites, 05151 and 05113 (vertical green lines). This region was identified as containing Tel¹ based on Table 1, round 1. Positions of transposons used for further mapping are indicated by green arrows. The Tel¹ mutation is boxed in red. The lower chromosome map shows expansion of the 92A3 region. (B) Graphs showing the change in relative HeT-A copy number (telomere length) in recombinants of Tel¹/MI02316, Tel¹/MI03112, and Tel¹/d10097 over 12 generations. The st recombinants are shown as red lines; ca recombinants as purple lines. These data delimit Tel¹ to a 15 kb between inserts MI02316 and d10097 (shown as red rectangles in (A)).

Figure 2

Telomere length in transposon insertion stocks. Q-PCR analysis of HeT-A copy number in different transposon insertion stocks used for mapping Tel¹ mutation. Error bars represent standard deviation measured from the triplicate Q-PCR results. MB09416 was not used for subsequent site-specific recombination mapping.

Figure 3

Dosage effects of Tel on HeT-A copy number. (A) The cytogenetic and physical map of genomic region 91C-91D is displayed, highlighting the 15 kb Tel¹ region as a vertical box. The lower part of the panel shows the extent of chromosome deficiencies. The bottom rectangle, Dp(3:3)cam30T, is a duplication for this region. Dotted lines beyond this rectangle show that the duplication extends beyond the represented region. (B) Shows the relative HeT-A copy number in stocks of the aberrations shown in A. The highlighted box represents a duplication that includes mapped region (Dp(3;3)cam30T covers 90C-93C) and another duplication of a neighboring region of the genome (Dp(3;3)cam35 covers 67C5-69A5) as a control. The mean from three replicates is represented here and error bars represent standard deviation. ** p < 0.005; *** p < 0.0001, one-way ANOVA with Sidak correction.

Figure 4

Telomere lengths in DGRP lines. A bar graph shows the log-normal distribution of telomere lengths among the 162 DGRP lines measured. The blue arrow shows the position of the Oregon-R control, and the red arrow shows the position of Tel¹. Three lines have Het-A copy numbers that exceed three standard deviations from the mean. These are RAL-161, -703, and -882. The red curve indicates the expected distribution.

Figure 5

Genetic activity and conservation of the 15 kb Tel1 region. (A) The genes found in this region are aligned with molecular coordinates. Minos insertions used for mapping are shown in cyan triangles. Minos insertion MB09416, which showed telomere elongation, is highlighted in the red square. (B) The University of California Santa Cruz genome browser map highlights sequence conservation in this region among different insect species. (C) A developmental transcriptome analysis for the same region as determined by the modENCODE project is also shown. The red vertical line spanning all three panels indicates the position of the TGT deletion.

Figure 6

Transcript analysis in the genes near Tel¹. The histogram represents the relative transcript levels from Tel¹ mutant (red) and Oregon-R (blue) adult ovaries. The genes analyzed are found near Tel1 (TGT mutation), which include Ino80 and the genes within its introns. The last bar represents the expression of Ino80 gene spanning exons 8–9, around the position of the TGT deletion in intron 8. The expression levels of CG31244 and CG31245 are very low, similar to the modENCODE data.

Figure 7

Telomere elongation in the CRISPR/Cas 9-induced Tel deletion alleles. (A) anti-HOAP immunostaining on 2L and XL chromosome tips from Tel^∆F2/+ and Tel^∆C11/+ hemyzygotes, respectively, showing protruding telomeric DNA only on the Tel mutant chromosomes (arrows). Note that, as expected, Tel mutant elongated telomeres do not impair HOAP localization. (B) Examples of telomere fusions involving either two or four chromosome tips (arrowheads) from different Tel∆ hemizygotes. (C) qPCR analysis on third instar larval DNA from two representative Tel∆ deletion alleles showing a robust increase in Het-A copy number compared to control (OR-R). Note that Tel^∆B5 bears a deletion that uncovers TGT, while Tel^∆C10 does not include TGT. * (p < 0.05).