Organismal propagation in the absence of a functional telomerase pathway in Caenorhabditis elegans

Abstract

To counteract replication-dependent telomere shortening most eukaryotic cells rely on the telomerase pathway, which is crucial for the maintenance of proliferative potential of germ and stem cell populations of multicellular organisms. Likewise, cancer cells usually engage the telomerase pathway for telomere maintenance to gain immortality. However, in ∼10% of human cancers telomeres are maintained through telomerase-independent alternative lengthening of telomeres (ALT) pathways. Here, we describe the generation and characterization of C. elegans survivors in a strain lacking the catalytic subunit of telomerase and the nematode telomere-binding protein CeOB2. These clonal strains, some of which have been propagated for >180 generations, represent the first example of a multicellular organism with canonical telomeres that can survive without a functional telomerase pathway. The animals display the heterogeneous telomere length characteristic for ALT cells, contain single-stranded C-circles, a transcription profile pointing towards an adaptation to chronic stress and are therefore a unique and valuable tool to decipher the ALT mechanism.

Figure 1

C-circles are elevated in ceob2 mutant C. elegans. (A) C-circle assay using increasing concentrations (25, 75 and 200 ng) of genomic DNA from KMST-6 ALT cells and IMR90 fibroblasts. Long G-strand products migrated minimally from the wells. The presence (+) or absence (−) of Φ29 DNA polymerase has been indicated. Gels were hybridized using a (CCCTAA)₄-labelled probe. (B) C-circle assays using increasing concentrations (25, 75 and 200 ng) of genomic DNA from wild-type (N2) and ceob2 mutant worms. The presence (+) or absence (−) of Φ29 DNA polymerase has been indicated. Gels were hybridized using a (GCCTAA)₄-labelled probe. (C) M13 bacteriophage circular ssDNA (16 and 160 ng) was used as control template (M13T) for Φ29 rolling circle amplification activity. The presence (+) or absence (−) of Φ29 DNA polymerase has been indicated. (D) Dot blot of C-circle assay performed with serial dilutions of genomic DNA from wild-type (N2) and ceob2 mutant worms with (+) and without (−) Φ29 DNA polymerase. Blots were hybridized using a (GCCTAA)₄-labelled probe. (E) Quantification of the C-circle amplification signals detected in ceob2 mutants relative to wild-type N2 animals. The average of two independent experiments using five different concentrations of DNA as in (D) is shown.

Figure 2

A trt-1/ceob2 double mutation supports indefinite propagation without telomerase. (A) Schematic of a cross between trt-1 and ceob2 mutant worms. (B) Brood size assay of ceob2 mutant animals, (C) trt-1 mutant animals and (D) trt-1/ceob2 DM worms. Grey, green and blue bars represent wild-type, medium and small brood sizes, respectively. Red bars indicate sterility. The percentage of surviving clones has been plotted against generation number. The left and right panels represent two independent experiments.

Figure 3

Longer telomeres allow for longer survival without telomerase. (A) Brood size of AB2 animals over 80 generations and (B) trt-1/AB2 animals that have been out-crossed once into the AB2 strain and (C) trt-1/AB2 animals that have been out-crossed four times into the AB2 strain. Grey represents wild-type brood sizes, green medium, blue small brood sizes and red indicates sterility. The percentage of the original clones still surviving has been plotted against generation number.

Figure 4

trt-1/ceob2 double mutants have increased C-circles and heterogeneous telomeres. (A) Dot blot of C-circle assay performed with serial dilutions of genomic DNA from wild-type (wt) and trt-1/ceob2 double mutant (DM) animals with (+) and without (−) Φ29 DNA polymerase. Blots were hybridized using a (GCCTAA)₄-labelled probe. (B) Quantification of the C-circle amplification signals detected in trt-1/ceob2 double mutant (DM) animals relative to wild-type (wt) animals. The average of two independent experiments using five different concentrations of DNA as in (A) is shown. (C) Telomere length analysis of wild-type (wt), ceob2 mutant (ceob2) and trt-1/ceob2 double mutant (DM) animals from two independent experiments. Two strains from the first cross (left panel) and six strains from the second cross (right panel) were analysed. DNA size is indicated on the left and amount loaded on the bottom. (D) Telomere length of wild-type (wt), ceob2 mutant (ceob2) and trt-1/ceob2 double mutant (DM) animals over 80 generations. DNA size is indicated on the left and 3 μg of DNA was loaded per lane.

Figure 5

Survivor strains contain fused chromosomes. (A) DAPI staining of mitotic chromosomes in oocytes in the nematode germ line. Each panel represents chromosomes from one oocyte. (B) Quantification of chromosome numbers in wild-type (wt) animals (n=63) and two trt-1/ceob2 DM survivor strains (DM B (n=37) and DM D (n=82)). (C) Telomere length analysis of wild-type (wt), and trt-1/ceob2 double mutant (DM) animals after incubation of genomic DNA with Bal-31 nuclease for the indicated time. DNA size is indicated on the left.

Figure 6

Similar gene-expression profiles between the survivor strain and late generation trt-1 mutants. (A) Relative expression levels of CTel55X.1, a gene expressed from the telomeric region of the X chromosome. (B) Hierarchical clustering of genes regulated two-fold in either late generation trt-1 mutant or a DM survivor strain. The upper panel shows clustering of 1982 upregulated genes and the lower panel shows a cluster of 735 downregulated genes. Genotypes corresponding to the individual Affymetrix arrays are indicated at the bottom. Expression levels were standardized by shifting the mean to zero. (C) Venn diagrams depict the overlap of genes regulated two-fold in either late generation trt-1 mutants or a DM survivor strain as depicted in (B).