Corruption of DNA end-joining in mammalian chromosomes by progerin expression

Abstract

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging and a life expectancy of about 14 years. HGPS is commonly caused by a point mutation in the LMNA gene which codes for lamin A, an essential component of the nuclear lamina. The HGPS mutation alters splicing of the LMNA transcript, leading to a truncated, farnesylated form of lamin A termed "progerin." Progerin is also produced in small amounts in healthy individuals by alternative splicing of RNA and has been implicated in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), suggesting alteration of DNA repair. DSB repair normally occurs by either homologous recombination (HR), an accurate, templated form of repair, or by nonhomologous end-joining (NHEJ), a non-templated rejoining of DNA ends that can be error-prone; however a good portion of NHEJ events occurs precisely with no alteration to joined sequences. Previously, we reported that over-expression of progerin correlated with increased NHEJ relative to HR. We now report on progerin's impact on the nature of DNA end-joining. We used a model system involving a DNA end-joining reporter substrate integrated into the genome of cultured thymidine kinase-deficient mouse fibroblasts. Some cells were engineered to express progerin. Two closely spaced DSBs were induced in the integrated substrate through expression of endonuclease I-SceI, and DSB repair events were recovered through selection for thymidine kinase function. DNA sequencing revealed that progerin expression correlated with a significant shift away from precise end-joining between the two I-SceI sites and toward imprecise end-joining. Additional experiments revealed that progerin did not reduce HR fidelity. Our work suggests that progerin suppresses interactions between complementary sequences at DNA termini, thereby shifting DSB repair toward low-fidelity DNA end-joining and perhaps contributing to accelerated and normal aging through compromised genome stability.

Keywords: DNA double-strand break repair; Genomic instability; Hutchinson-Gilford Progeria Syndrome; Progerin.

Figure 1.

Strategy for recovering precise DNA end-joining (PEJ) events using DNA substrate pTKSce2. As represented schematically at the top of the figure, pTKSce2 contains a tk gene (white rectangle) disrupted by insertion of a 47 bp oligonucleotide containing two 18 bp I-SceI recognition sites (S). If endonuclease I-SceI is transiently expressed in a cell line containing an integrated copy of pTKSce2, two DSBs can be induced. A PEJ event involving joining of the two outermost sticky ends produced by I-SceI will produce a functional tk gene retaining a 24 bp insert containing an I-SceI site, and such an event is recoverable by HAT selection. The nucleotide sequences of the original 47 bp insert and the 24 bp insert produced by PEJ are illustrated. The 18 bp I-SceI recognition sequence is presented in uppercase font, and the actual sites of staggered cleavage by I-SceI are indicated by arrows.

Figure 2.

Expression of GFP-progerin and GFP in derivatives of cell line 13. Panel A: Fluorescence due to expression of GFP-progerin in cell lines alpha, beta, and gamma which are clonal derivatives of parent line 13 that differ only in the level of expression of GFP-progerin from stably integrated pEGFP-D50 lamin A. Also shown is fluorescence due to GFP expression in cell line 13-GFP as well as the virtual absence of background fluorescence in parent line 13. The white bar in each in fluorescence microscopy image represents 200 μM Panel B: Western blot using a GFP-specific antibody to confirm expression of full-length GFP-progerin in cell lines alpha, beta, and gamma (lanes α, β, γ respectively). Lane M displays a sample from cell line “pLB4-progerin” previously shown to express GFP-progerin at a level similar to progerin levels detected in cells derived from an HGPS patient [55] and this lane serves as a marker for GFP-progerin. Lane P displays a sample from parent cell line 13. Cell lines alpha, beta, and gamma each display the expected 100 kD band for GFP-progerin while the parent cell line 13 does not. Each lane contains 30 μg of total cellular protein. Panel C: Equal protein loading on the western blot is demonstrated by Ponceau staining of the membrane.

Figure 3.

Substrate pHOME [60] for studying HeR. The “recipient” is a herpes simplex virus type one tk gene disrupted by an oligonucleotide containing an I-SceI recognition site. The “donor” is a fragment of the herpes simplex virus type two tk gene missing 30% of the tk coding region at the 5′ end. The donor and recipient display about 20% overall sequence divergence with scattered mismatches. Both crossovers and non-crossovers are potentially recoverable under HAT selection following HeR between the recipient and donor.

Figure 4.

DSB-induced HeR events recovered from cell lines containing pHOME. Presented at the top of the figure is the nucleotide sequence in the vicinity of the I-SceI recognition site (denoted by an inverted triangle) in the recipient tk gene from pHOME. Shown immediately below the recipient sequence are “marker” nucleotides from the donor tk sequence from pHOME that do not match the recipient sequence; the nucleotide positions at which these mismatches occur are indicated above the recipient sequence. The sequence of the single HeR event that arose from progerin-expressing cell line #5 following DSB induction with I-SceI is represented. The donor marker nucleotides present in the gene conversion tract are shown. Also shown are the two HeR clones, 1-24-2-10 and 1-24-3-24, which were recovered from parent cell line HOME-1-24 and reported in our previous studies [60].

Figure 5.

Alternative single-strand annealing origin of clones identified as products of PEJ. Clones indistinguishable from PEJ can potentially arise from the following process: i) the upstream I-SceI site in the 47 bp insert in pTKSce2 is cleaved by I-SceI; ii) 5’ ends of DNA strands are resected by exonuclease; iii) end-resection continues, exposing the two boxed 9 bp complementary sequences; iv) the boxed 9 bp sequences anneal. DNA flaps are clipped, and sequence gaps are filled to produce a clone that retains the 24 bp sequence (boxed in blue) that is also retained following PEJ. Short vertical arrows indicate sites of staggered cleavage within the I-SceI site.