MERISTEM DISORGANIZATION1 encodes TEN1, an essential telomere protein that modulates telomerase processivity in Arabidopsis

Abstract

Telomeres protect chromosome ends from being recognized as DNA damage, and they facilitate the complete replication of linear chromosomes. CST [for CTC1(Cdc13)/STN1/TEN1] is a trimeric chromosome end binding complex implicated in both aspects of telomere function. Here, we characterize TEN1 in the flowering plant Arabidopsis thaliana. We report that TEN1 (for telomeric pathways in association with Stn1, which stands for suppressor of cdc thirteen) is encoded by a previously characterized gene, MERISTEM DISORGANIZATION1 (MDO1). A point mutation in MDO1, mdo1-1/ten1-3 (G77E), triggers stem cell differentiation and death as well as a constitutive DNA damage response. We provide biochemical and genetic evidence that ten1-3 is likely to be a null mutation. As with ctc1 and stn1 null mutants, telomere tracts in ten1-3 are shorter and more heterogeneous than the wild type. Mutants also exhibit frequent telomere fusions, increased single-strand telomeric DNA, and telomeric circles. However, unlike stn1 or ctc1 mutants, telomerase enzyme activity is elevated in ten1-3 mutants due to an increase in repeat addition processivity. In addition, TEN1 is detected at a significantly smaller fraction of telomeres than CTC1. These data indicate that TEN1 is critical for telomere stability and also plays an unexpected role in modulating telomerase enzyme activity.

Figure 1.

Arabidopsis TEN1 Is a Member of CST Complex. (A) Schematic of TEN1 gene structure. The T-DNA insertion in ten1-1 is illustrated, along with the position of two antisense constructs and the point mutation responsible for the G77E mutation in ten1-3. aa, amino acids. (B) Alignment of TEN1 proteins from different eukaryotes. At, Arabidopsis thaliana; Os, Oryza sativa (rice); Pt, Populus trichocarpa (poplar); Mm, Mus musculus; Hs, Homo sapiens; Sp, Schizosaccharomyces pombe. The positions of β-strands of the OB-fold are indicated below the alignment. Green, 100% similarity; chartreuse, 80 to 99% similarity; yellow, 60 to 79% similarity; and gray, below 60% similarity. (C) TEN1 interacts with STN1 in vitro. Results of coimmunoprecipitation performed with recombinant proteins. One protein is [³⁵S]Met labeled (asterisk), and the other is T7 tagged and unlabeled. s, supernatant; p, pellet. Results for the positive (KU70/KU80) and negative (KU70/KU70) controls are shown. (D) Yeast two-hybrid assay results for STN1 and TEN1. The two proteins fused to GAL4-AD and GAL4-BD were coexpressed and grown on selection plates for His auxotrophy (left) or assayed to detect β-galactosidase activity of positive transformants (right). “−” Indicates empty vector. (E) Nuclear localization of TEN1 in purified nuclei. TEN1 was detected by anti-TEN1 antibody in hexaploid Arabidopsis suspension cell culture. Telomeres were labeled by FISH using a rhodamine-labeled telomere probe. 4′,6-Diamidino-2-phenylindole (DAPI)–stained nuclei are shown. In the merge, closed white arrows denote subcentromeric stretches of telomeric DNA on chromosome 1. TEN1 colocalization with telomeres is indicated by the open white arrow.

Figure 2.

The ten1-3 Mutation Causes Severe Morphological Defects. (A) First generation (G1) ten1-3 mutants are smaller in stature than the wild type (WT) and harbor smaller leaves, fused stems, and irregular phyllotaxy (middle panel). These phenotypes are rescued by expression of a wild-type copy of TEN1 (right panel). (B) Second generation (G2) ten1-3 mutants display more severe growth phenotype than G1 mutants and are infertile. Arrowhead denotes aborted siliques. (C) Two-week-old seedlings of the genotypes indicated were grown on Murashige and Skoog medium without selection. G2 ten1-3 mutants exhibit shoot apical meristem abnormalities and fail to produce true leaves. G2 mutants are shown in a 2× zoom to show abnormal apical meristem.

Figure 3.

The TEN1_G77E Mutant Protein Is Unstable and Does Not Interact with STN1 in Vitro. (A) Protein gel blot results for the wild type (WT), ten1-3, and the TEN1 complementation line (TEN1-3 + TEN1) are shown. Ponceau S stain loading controls included. Molecular mass size markers in kilodaltons are on the left. The blot was probed with a polyclonal antibody raised against Arabidopsis TEN1. (B) Native PAGE results for recombinant wild-type TEN1 or TEN1_G77E protein expressed in rabbit reticulocyte lysate. Arrow indicates a higher molecular mass polypeptide in the TEN1_G77E protein sample. (C) Quantification of recombinant TEN1 protein binding to STN1. Shown are results of coimmunoprecipitation experiments with recombinant wild-type TEN1 and TEN1_G77E. The interaction for wild-type TEN1-STN1 was set to 100%. An example of raw data is shown in Supplemental Figure 2B online. [See online article for color version of this figure.]

Figure 4.

TEN1 Is Important for Telomere Length Regulation and Genome Maintenance. TRF analysis of ten1 mutants. Blots were hybridized with a radiolabeled G-rich telomeric probe. (A) Results for first (G1) and second (G2) generation ten1-1 are shown relative to the wild type (WT). (B) Telomere length in first (left) and second (right) generations of two antisense knockdown lines of TEN1. For comparison, results are shown with first generation stn1-1 mutants. (C) TRF analysis of ten1-3 mutants. Results for offspring of ten1-3 heterozygous plants are analyzed. (D) Parent-progeny analysis for two different ten1-3 mutants. (E) BAL31 time course of DNA with the wild type and a G2 ten1-3 mutant. (F) Telomere profile of G1 and G2 stn1-1 mutants. Asterisks indicated abnormally sharp TRF bands. Interstitial telomeric DNA repeats are denoted by the bracket or an arrowhead.

Figure 5.

TEN1 Prevents End-to-End Chromosome Fusions and Promotes Proper Telomere Architecture. (A) Telomere fusion PCR products obtained from the wild type (WT) and ten1-3 mutants are shown. Primer pair used to amplify specific subtelomeric regions are indicated. (B) Cytology of mitotic chromosomes in the wild type (i) and ten1-3 mutants (ii to iv) are shown. DAPI–stained chromosome spreads were prepared from pistils. (C) In-gel hybridization analysis of DNA isolated from the wild type and ten1-3 mutants using a C-strand telomeric probe under native conditions. (D) Quantification of the G-overhang signal for ten1-3 mutants. A DNA gel blot of interstitial telomere DNA or ethidium bromide staining of DNA was used as a DNA loading control for quantification of G-overhang signal. Data represent seven individual biological replicates of ten1-3. Error bars indicate sd for biological replicates. (E) TCA was performed with the wild type, ten1-3 +/− offspring, and stn1-1 DNA in the presence (+) or absence (−) of phi (ϕ) 29 polymerase. Arrow indicates extrachromosomal telomere circles.

Figure 6.

TEN1 Is a Negative Regulator of Telomerase Activity. (A) Telomerase activity in flowers measured by Q-TRAP. Data are normalized to the wild type (WT); each data point represents two or three biological replicates, with three technical replicates. Error bars represent the sd between biological replicates. (B) TRAP products from the wild type and ten1-3 mutants at 24, 30, and 36 cycles of PCR resolved by PAGE. Quantification (right) represents the signal for the corresponding bands of ten1-3 divided by the wild type for the 36-cycle PCR reaction.

Figure 7.

TEN1 Decreases Telomerase Repeat Addition Processivity. (A) Left: Schematic of processivity TRAP (Szatmari and Aradi, 2001). (1) Telomerase extends a forward primer substrate. (2) and (3) Binding of reverse primer 1RPgg, which contains a unique sequence tag on the 5′ end and terminates in two 3′ noncomplementary G nucleotides that precisely position the primer at the terminus of the extension product. Two cycles of PCR are used to tag the telomerase product with the unique sequence tag. (4) Thirty-three cycles of PCR using the forward primer and 2RP, a reverse primer complementary to the unique sequence tag. Right: Results for control reactions with oligonucleotides containing three (PT3) or six (PT6) telomere repeats (see Supplemental Table 2 online) subjected to steps 2 to 4 of processivity TRAP. (B) Results of processivity TRAP for floral extracts from wild-type (WT), stn1, and ten1-3 mutants. Left: Telomerase extension products displayed by PAGE. Asterisk denotes nonspecific PCR amplification products. Right: Quantification of processivity TRAP. Signal was quantified for the individual bands indicated. The average signal was calculated for each genotype (except stn1) and that average was compared with the average for the wild type.