Telomeres, subtelomeres and telomere-related genes

From Purdue Genomics Database Facility

Eugene V. Shakirov and Dorothy E. Shippen

Department of Biochemistry and Biophysics, Texas A&M University

eugene-shakirov[at]neo.tamu.edu, dshippen[at]tamu.edu

Selaginella telomeres

The ends of linear eukaryotic chromosomes terminate with a long stretch of short tandem repeats of GT-rich telomeric DNA. These sequences, together with specific DNA-binding proteins, comprise the telomeres. Telomeres are important for maintaining genome integrity, distinguishing natural DNA ends from double-strand breaks, and preventing illegitimate DNA repair. Proper maintenance of telomeric DNA length and structure is essential for normal cell viability (Zellinger and Riha 2007). Sequence analysis of terminal chromosomal scaffolds indicates that Selaginella telomeres, like those of most other plants, are composed of tandem arrays of (TTTAGGG)n repeats.

To gauge the size of Selaginella telomere tracts, we performed terminal restriction fragment analysis (TRF). As shown in Figure 1A, Selaginella telomeres appear as a smear ranging in size from 1.5 to 5.5 kb, similar to the profile of Arabidopsis telomeres (Richards and Ausubel 1988; Shakirov and Shippen 2004). To verify that the sequences detected by TRF analysis correspond to chromosome ends, the DNA was preincubated with Bal31 nuclease prior to digestion with Tru1I. Bal31 is a non-specific exonuclease that preferentially degrades DNA ends versus more internal genomic regions. After 15 minutes of Bal31 digestion, the hybridization products migrated faster on the gel and showed reduced intensity (Figure 1B, lane 2). With continued Bal31 incubation, the telomeric signal disappeared completely (Figure 1B, lanes 3-6). In contrast, several cross-hybridizing bands, corresponding to interstitial telomeric DNA were insensitive to Bal31 digestion for up to 90 minutes, further supporting the terminal location of Bal31-sensitive telomeric signals.

Selaginella subtelomeres

Immediately adjacent to telomeric DNA tracts are the subtelomeres. Subtelomeric DNA composition varies greatly between organisms, but several features are conserved even between members of different kingdoms (Riethman et al. 2005). These include the presence of short arrays of repetitive elements and multiple transposon insertions. Subtelomeric regions are subject to frequent inter-chromosomal recombination (Gardner et al. 2002; Ming et al. 2008). As a result, some subtelomeric DNA sequences may be shared by several different chromosome arms. This is the case for Selaginella telomeres (Figure 2).

Sequence analysis of 16 available terminal DNA scaffolds indicates the presence of multiple LTR elements on chromosome ends. Interestingly, in addition to insertions into subtelomeric regions and throughout the genome, numerous LTR elements are inserted directly into the centromere-proximal portion of the telomere tracts with stretches of telomere repeats trapped between individual sites of transposon insertions (Figure 2). By contrast, Arabidopsis subtelomeres consist of unique sequences, lacking repetitive elements or arrays of telomeric repeats (Heacock et al. 2004; Kuo et al. 2006).

Telomere-related genes in Selaginella

In each organism, a species-specific telomere length set point is established and maintained through the combined action of telomere-binding proteins and telomerase, a ribonucleoprotein reverse transcriptase responsible for replenishing telomere tracts (Collins 2006; Hug and Lingner 2006). Since components of the telomere maintenance machinery have not been analyzed in plant lineages outside Angiosperms, we looked for sequence homologues of known constituents of the telomerase holoenzyme and other telomere-related factors in Selaginella.

We identified Selaginella genes encoding sequence homologues of the catalytic telomerase subunit TERT (telomerase reverse transcriptase) (Fitzgerald et al. 1999) (Protein ID: 449905; 47% amino acid similarity to Arabidopsis TERT) and dyskerin, a telomerase RNA stability factor (Kannan et al. 2008) (Protein ID: 451005; 73% amino acid similarity to Arabidopsis dyskerin). We also identified the KU70/80 heterodimer (Protein IDs: 449901 and 449903; 65% and 63% similarity to Arabidopsis KU70/80, respectively). KU70/80 is best known for its role in the non-homologous end joining double strand DNA repair pathway, but in Arabidopsis, yeast and mammals, KU is also a major player in telomere biology, responsible for telomere length control and chromosome end protection (Peterson et al. 2001; Riha et al. 2002).

We also identified Selaginella sequence homologues for two classes of telomere DNA binding proteins. The single-strand telomere-binding protein Protection Of Telomeres POT1 represents one class of telomere-specific DNA binding factors (Baumann and Cech 2001), and is characterized by the presence of an oligosaccharide/oligonucleotide binding fold (OB-fold). In contrast to Arabidopsis, which harbors two highly divergent and functionally distinct AtPOT1a and AtPOT1b proteins (Shakirov et al. 2005), the Selaginella genome encodes only a single POT1 protein (Protein ID: 449845) that displays 46% similarity to AtPOT1a and 47% similarity to AtPOT1b. These data suggest that POT1 proteins in plants are diverging faster than other components of the telomere.

The double-strand binding proteins Telomere Repeat Binding Factors TRF represent the second class of evolutionarily conserved telomere-binding proteins that have affinity for the double-stranded portion of the telomere and regulate major pathways for chromosome end protection and telomere length regulation (Smogorzewska and de Lange 2004). This group of proteins is characterized by the presence of C-terminally located Myb-domain that is responsible for DNA binding (Bianchi et al. 1997). TRF-like (TRFL) proteins have been characterized in several plant species (Hong et al. 2007; Hwang and Cho 2007; Karamysheva et al. 2004; Yang et al. 2004) and shown to possess an additional Myb-extension domain which is important for telomeric DNA recognition in vitro (Karamysheva et al. 2004). As shown in Table 1, plants display great variation in the number of TRFL genes. Interestingly, similar to the situation in vertebrates, most plants, including green algae, Physcomitrella, two monocot and several dicot species encode only two TRFL proteins. In contrast, other dicot species have amplified TRFL gene family members, with three genes in grapes, five genes in poplar and six genes in Arabidopsis.

Selaginella appears to be an outlier among non-flowering plant species, as its genome harbors three TRFL genes (Table 1; Protein IDs: 449854, 449900, 449895). This finding indicates that members of the Selaginella lineage were likely to be the first plants to experience amplification of TRFL genes. Although the precise role of individual TRFL gene family members is currently unknown, this finding will provide the framework for understanding the functions of these key components of the telomere complex.

Figure 1. Telomere length analysis in Selaginella moellendorffii. (A) Comparative terminal restriction fragment (TRF) analysis of Selaginella (lane 1) and Arabidopsis (lane 2) telomeres. Molecular weight markers are shown on the left. (B) Bal31 digestion of Selaginella telomeric DNA. Lane 1, Tru1I digestion of genomic DNA without prior Bal31 treatment (0 min). Lanes 2-6, Tru1I digestion of genomic DNA with Bal31 treatment for 15, 30, 45, 60 and 90 minutes, respectively. Asterisks indicate cross-hybridizing interstitial telomeric DNA bands, which are not sensitive to Bal31 digestion for up to 90 min.

Figure 2. Organization of Selaginella subtelomere and telomere DNA regions. A diagram of 7.5 kb of terminal region of scaffold_105 is shown as an example. Scaffold_105 harbors 105 sequenced terminal telomere repeats (black arrow), followed by LTR elements (light blue boxes) and additional inter-dispersed stretches of telomere repeats (black boxes). Telomere-distal region of the subtelomere is shown in yellow.

Methods

Telomere length analysis and Bal31 digestion. Selaginella DNA was extracted as described (Cocciolone and Cone 1993). To detect telomeric DNA repeats, genomic DNA was digested with Tru1I (Fermentas) (recognition sequence TTAA) and subjected to Southern blotting with 32P-labeled (TTTAGGG)4 as a probe (Fitzgerald et al. 1999). Radioactive signals were scanned by a STORM PhosphorImager (Molecular Dynamics), and the data were analyzed by IMAGEQUANT software (Molecular Dynamics). For the Bal31 exonuclease assay, 100 μg of Selaginella genomic DNA was incubated with 50 units of Bal31 (New England Biolabs) or with H2O (0 min time point) in 1X Bal31 reaction buffer at 30˚C. Equal amounts of sample were removed at 15 or 30 min intervals for 90 min. Reactions were stopped by the addition of 20 mM EGTA and heating to 65˚C for 15 min. DNA in each sample was precipitated with isopropanol and ammonium acetate, followed by Tru1I digestion. Digested DNA was separated on 0.8% agarose, blotted onto a nitrocellulose membrane and subjected to hybridization as described above.

BLAST searches and gene predictions. BLAST searches of the Selaginella genome were performed using the tblastn option with Arabidopsis telomere-related proteins as a query. Individual cDNA sequences were deduced using a combination of EST alignments and various gene prediction programs. TRFL sequences from Physcomitrella patens, Populus trichocarpa, Oryza sativa, Carica papaya, Medicago truncatula, Vitis ninifera, Oryza sativa, Sorghum bicolor, Ostreococcus taurus and Ostreococcus lucimarinus were obtained from the corresponding genome portals (http://genome.jgi-psf.org/euk_cur1.html; http://www.medicago.org/genome/cvit_blast.php; http://www.appliedgenomics.org/blast/; http://asgpb.mhpcc.hawaii.edu/tools/tools.php; http://signal.salk.edu/cgi-bin/RiceiGE) using similar approaches.

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