Detailed evolutionary studies have been conducted on specific systems with some surprising results. Two issues remain unclear. First, there is remarkable retention of association of orthologes of proteins involved in other cellular functions at telomeres of other organisms. We will present examples from the literature as well as new research in the area. We will try to address the question as to whether telomeres contain proteins that evolve more rapidly to accommodate the very specific needs of each organisms regardless of phylogenetic relationship.

Tulane University

Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University

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Frontiers in Genetics

Genetics of Aging

The Evolving Telomeres

The study of the evolution of the end of chromosomes, or telomeres, has moved from the abstract to molecular observations and mechanistic possibilities. Although successful end-replication and end-protection are the primary driving forces acting at all telomeres (<a href="#B3">de Lange, 2009</a>), the studies presented in this issue reveal apparent similarities, surprising differences, and new functions for telomere binding proteins (TeloBPs). These advances in molecular genetics of both common and more diverse organisms should lead to specific hypotheses for the roles of these proteins both at telomeres and throughout the genome and toward a broader view of how evolution solves different problems that occur in biology. The next step will be the experimental testing of evolutionary hypotheses.As a reflection of the molecular advances, we framed the series “The Evolving Telomeres”. We have covered information from multiple systems that use a variety of mechanisms. These include studies in Neal Lue's lab regarding the analysis of work in yeasts belonging to Saccharomycotina involving the co-evolution of single-stranded and double-stranded sequence TeloBPs as a function of telomeric sequence (<a href="http://dx.doi.org/10.3389/fgene.2015.00162">Steinberg-Neifach and Lue</a>). They find that proteins accommodate the differing sequence through duplication and divergence of functional proteins, combinatorial site recognition, and greater protein flexibility. David Shore's laboratory reviewed the apparent differences and similarities in the Rif1 protein (<a href="http://dx.doi.org/10.3389/fgene.2016.00045">Mattarocci et al.</a>) in yeasts and humans. Rif1 was first defined in budding yeast as a negative regulator of telomere size that counteracted the activation effects of Tel1 (ATM) binding to short telomeres (<a href="#B5">Hector et al., 2007</a>; <a href="#B15">Sabourin et al., 2007</a>). The multi-functional Rif1, on the other hand, is delivered to the terminus in greater amounts in longer telomeres that have a greater abundance of the major yeast TeloBP, Rap1, thereby displacing Tel1 (<a href="#B2">Chang et al., 2007</a>; <a href="#B6">Hirano et al., 2009</a>; <a href="#B10">Martina et al., 2012</a>). These activities form a feedback mechanism that protects the telomere against non-productive repair such as the formation of end-to-end fusions. This dynamic homeostasis acts in a cap-like function, termed the anti-checkpoint (<a href="#B14">Ribeyre and Shore, 2012</a>). Feedback mechanisms seem to be ubiquitous among telomeres.One major issue is the source of the many discontinuities in the evolution in plant, fungal, and mammalian telomeres. Two studies probed some of the unique characteristics of plants. Dorothy Shippen's laboratory (<a href="http://dx.doi.org/10.3389/fgene.2015.00277">Nelson and Shippen</a>) studied the participation of long nuclear RNAs in plant telomere regulation. Among these is the telomerase RNA and an entire group of related RNAs, many of which act on telomerase, even as a negative regulator. These RNAs are absent from metazoans, illustrating how the metaphyta have likely adapted the system of RNA-based regulation to telomeres. This finding may reflect the high predominance of RNA-based defense mechanisms in plants, especially against transposons present in most of the genome (<a href="#B16">Shabalina and Koonin, 2008</a>). Karel Riha's laboratory contributed an experimental study of another example of differing solutions to end-protection (<a href="http://dx.doi.org/10.3389/fgene.2015.00349">Fulcher and Riha</a>). One issue in Arabidopsis and many other plants has been the lack of TRF-like (TRFL) factors that are so common in vertebrate cells. The major telomere binding proteins in vertebrates is TRF1, and often, TRF2. These proteins form the backbone of the shelterin complex, involved in both end-replication and protection (<a href="#B7">Karlseder et al., 2003</a>; <a href="#B20">Wu and de Lange, 2008</a>). The strangest observation is that TRFL are present and located at telomeres, but serve no obvious function. To rule out the possibility of functional redundancy, the authors' produced genetic knockouts of the possible functional TRF-like proteins with no effect on telomeres or growth. This result is in sharp contrast to the effects of TRF1 and TRF2 loss in vertebrates. Their data all but eliminate the chance for the presence that a homolog to the vertebrate telomere repeat factor (TRF1) that is important at Arabidopsis telomeres (<a href="#B17">Shakirov et al., 2008</a>). Rather, a simple algal-related protein performs many of the TRF1 functions in Arabidopsis (<a href="#B12">Mozgova et al., 2008</a>), leading to speculation on the odd rapid evolution of TeloBPs. Plants appear to have adapted telomeres to physiological requirements since the divergence of the original common ancestor that gave rise to metazoans.Some components of telomeres are conserved such as the Mre11/Rad50/NBS complex and the Cdt1/Stn1Ten1 complex that assist in end protection. However, many others rapidly change with differing physiological and selective forces that maintain genome stability and cell survival. Art Lustig presented a hypothesis that evolution could cause rapid changes as a consequence of formation and divergence of paralogs (<a href="http://dx.doi.org/10.3389/fgene.2016.00010">Lustig</a>). The hypothesis argues that rapid evolution is driven by the requirement for genomic stability and, in some cases, by telomere stress response that increases the rate of paralogy and divergence. In fact, this result helps to explain the TeloBP divergence among fungal, invertebrates, vertebrate and plant species that have been investigated.Evolution has provided multiple solutions to the end-replication problem of linear chromosomes besides telomerase and even telomeres. Some bacteriophages replicate the end by circularization or recombination (<a href="#B9">Lopes et al., 2010</a>). Both adenovirus and the bacterium that causes Lyme disease, Borrelia burgdorferi, have chromosome ends capped by covalently bound proteins (<a href="#B1">Chaconas, 2005</a>), and Drosophila and other dipterans have transposons at their chromosome termini (<a href="#B19">Villasante et al., 2008</a>). The role of non-LTR retro-transposition in the evolution of telomerase has been controversial.Indeed, in analyzing the origin of telomerase, (<a href="http://dx.doi.org/10.3389/fgene.2015.00321">de Lange</a>) proposes a theoretical scheme for type II introns, coupled with the formation of primitive t-loops, to evolve into telomerase, independent of non-LTR retro-transpositions (<a href="#B8">Lambowitz and Belfort, 2015</a>). Nevertheless, the review by <a href="http://dx.doi.org/10.3389/fgene.2015.00321">Servant and Deininger</a> focuses on the use in extant organisms of non-LTR retro-transposition in telomerase-positive cells, providing an example of a mechanism that persists and even co-exists with telomerase through evolution. The bottom line of these studies is the diversity of telomeric processes. This variety could be put into a broader context by a more extensive study of diverse organisms.A major future goal, at least for microbes, is to test hypotheses regarding telomere evolution. These experiments use techniques for growth of cells at a constant density. One of these instruments used for these experiments is the turbidostat (<a href="#B4">Gresham and Dunham, 2014</a>; <a href="#B11">Matteau et al., 2015</a>; <a href="#B18">Takahashi et al., 2015</a>) that can differentiate between the altered molecular changes that arise during the evolution of cells. Another exciting aspect of this work is that these experiments represent real-time (albeit manipulated) evolution. The artificial evolutionary approach is having signs of success in yeast and microbes under different conditions, such as oxidative stress (<a href="#B13">Raso et al., 2012</a>) and these successes will undoubtedly continue.KWR was responsible for background and analysis of contributions. AJL was responsible for the structure and comments in the editorial.Funding for theoretical studies was provided by NIH 5R01 GM069943, the Louisiana Cancer Research Consortium and pilot funds from Tulane University (to AJL). Funding was additionally provided by NSF 1516220 and NIA RO1 AG051601 (to KWR).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.These excellent articles have been written by some of the most talented telomere investigators. We are grateful for their ideas and viewpoints. But those viewpoints could not have reached such a stage of refinement without the dedicated reviewers from the same telomere community who put considerable time into this effort. We also want to thank our Specialty Chief Editor of Frontiers in Genetics of Aging, Blanka Rogina, and the intrepid staff at Frontiers in Genetics. We hope this two-year effort will catalyze some new approaches and ideas within the telomere and evolution communities.

Editorial: The Evolving Telomeres

Rap1-interacting factor 1 (Rif1) was originally identified in the budding yeast Saccharomyces cerevisiae as a telomere-binding protein that negatively regulates telomerase-mediated telomere elongation. Although this function is conserved in the distantly related fission yeast Schizosaccharomyces pombe, recent studies, both in yeasts and in metazoans, reveal that Rif1 also functions more globally, both in the temporal control of DNA replication and in DNA repair. Rif1 proteins are large and characterized by N-terminal HEAT repeats, predicted to form an elongated alpha-helical structure. In addition, all Rif1 homologs contain two short motifs, abbreviated RVxF/SILK, that are implicated in recruitment of the PP1 (yeast Glc7) phosphatase. In yeasts the RVxF/SILK domains have been shown to play a role in control of DNA replication initiation, at least in part through targeted de-phosphorylation of proteins in the pre-Replication Complex. In human cells Rif1 is recruited to DNA double-strand breaks through an interaction with 53BP1 where it counteracts DNA resection, thus promoting repair by non-homologous end-joining. This function requires the N-terminal HEAT repeat-containing domain. Interestingly, this domain is also implicated in DNA end protection at un-capped telomeres in yeast. We conclude by discussing the deployment of Rif1 at telomeres in yeasts from both an evolutionary perspective and in light of its recently discovered global functions.

Rap1-interacting factor 1 (Rif1) was originally identified in the budding yeast Saccharomyces cerevisiae as a telomere-binding protein that negatively regulates telomerase-mediated telomere elongation. Although this function is conserved in the distantly related fission yeast Schizosaccharomyces pombe, recent studies, both in yeasts and in metazoans, reveal that Rif1 also functions more globally, both in the temporal control of DNA replication and in DNA repair. Rif1 proteins are large and characterized by N-terminal HEAT repeats, predicted to form an elongated alpha-helical structure. In addition, all Rif1 homologs contain two short motifs, abbreviated RVxF/SILK, that are implicated in recruitment of the PP1 (yeast Glc7) phosphatase. In yeasts the RVxF/SILK domains have been shown to play a role in control of DNA replication initiation, at least in part through targeted de-phosphorylation of proteins in the pre-Replication Complex. In human cells Rif1 is recruited to DNA double-strand breaks through an interaction with 53BP1 where it counteracts DNA resection, thus promoting repair by non-homologous end-joining. This function requires the N-terminal HEAT repeat-containing domain. Interestingly, this domain is also implicated in DNA end protection at un-capped telomeres in yeast. We conclude by discussing the deployment of Rif1 at telomeres in yeasts from both an evolutionary perspective and in light of its recently discovered global functions.

Rif1: A Conserved Regulator of DNA Replication and Repair Hijacked by Telomeres in Yeasts

Through elegant studies in fungal cells and complex organisms, we propose a unifying paradigm for the rapid evolution of telomere binding proteins (TBPs) that associate with either (or both) telomeric DNA and telomeric proteins. TBPs protect and regulate telomere structure and function. Four critical factors are involved. First, TBPs that commonly bind to telomeric DNA include the c-Myb binding proteins, OB-fold single-stranded binding proteins, and G-G base paired Hoogsteen structure (G4) binding proteins. Each contributes independently or, in some cases, cooperatively, to provide a minimum level of telomere function. As a result of these minimal requirements and the great abundance of homologs of these motifs in the proteome, DNA telomere-binding activity may be generated more easily than expected. Second, telomere dysfunction gives rise to genome instability, through the elevation of recombination rates, genome ploidy, and the frequency of gene mutations. The formation of paralogs that diverge from their progenitor proteins ultimately can form a high frequency of altered TBPs with altered functions. Third, TBPs that assemble into complexes (e.g., mammalian shelterin) derive benefits from the novel emergent functions. Fourth, a limiting factor in the evolution of TBP complexes is the formation of mutually compatible interaction surfaces amongst the TBPs. These factors may have different degrees of importance in the evolution of different phyla, illustrated by the apparently simpler telomeres in complex plants. Selective pressures that can utilize the mechanisms of paralog formation and mutagenesis to drive TBP evolution along routes dependent on the requisite physiologic changes.

Through elegant studies in fungal cells and complex organisms, we propose a unifying paradigm for the rapid evolution of telomere binding proteins (TBPs) that associate with either (or both) telomeric DNA and telomeric proteins. TBPs protect and regulate telomere structure and function. Four critical factors are involved. First, TBPs that commonly bind to telomeric DNA include the c-Myb binding proteins, OB-fold single-stranded binding proteins, and G-G base paired Hoogsteen structure (G4) binding proteins. Each contributes independently or, in some cases, cooperatively, to provide a minimum level of telomere function. As a result of these minimal requirements and the great abundance of homologs of these motifs in the proteome, DNA telomere-binding activity may be generated more easily than expected. Second, telomere dysfunction gives rise to genome instability, through the elevation of recombination rates, genome ploidy, and the frequency of gene mutations. The formation of paralogs that diverge from their progenitor proteins ultimately can form a high frequency of altered TBPs with altered functions. Third, TBPs that assemble into complexes (e.g., mammalian shelterin) derive benefits from the novel emergent functions. Fourth, a limiting factor in the evolution of TBP complexes is the formation of mutually compatible interaction surfaces amongst the TBPs. These factors may have different degrees of importance in the evolution of different phyla, illustrated by the apparently simpler telomeres in complex plants. Selective pressures that can utilize the mechanisms of paralog formation and mutagenesis to drive TBP evolution along routes dependent on the requisite physiologic changes.

Hypothesis: Paralog Formation from Progenitor Proteins and Paralog Mutagenesis Spur the Rapid Evolution of Telomere Binding Proteins

Proteins that bind to telomeric DNA form the key structural and functional constituents of telomeres. While telomere binding proteins have been described in the majority of organisms, their identity in plants remains unknown. Several protein families containing a telomere binding motif known as the telobox have been previously described in Arabidopsis thaliana. Nonetheless, functional evidence for their involvement at telomeres has not been obtained, likely due to functional redundancy. Here we performed genetic analysis on the TRF-like family consisting of six proteins (TRB1, TRP1, TRFL1, TRFL2, TRFL4, and TRF9) which have previously shown to bind telomeric DNA in vitro. We used haploid genetics to create multiple knock-out plants deficient for all six proteins of this gene family. These plants did not exhibit changes in telomere length, or phenotypes associated with telomere dysfunction. This data demonstrates that this telobox protein family is not involved in telomere maintenance in Arabidopsis. Phylogenetic analysis in major plant lineages revealed early diversification of telobox proteins families indicating that telomere function may be associated with other telobox proteins.

Proteins that bind to telomeric DNA form the key structural and functional constituents of telomeres. While telomere binding proteins have been described in the majority of organisms, their identity in plants remains unknown. Several protein families containing a telomere binding motif known as the telobox have been previously described in Arabidopsis thaliana. Nonetheless, functional evidence for their involvement at telomeres has not been obtained, likely due to functional redundancy. Here we performed genetic analysis on the TRF-like family consisting of six proteins (TRB1, TRP1, TRFL1, TRFL2, TRFL4, and TRF9) which have previously shown to bind telomeric DNA in vitro. We used haploid genetics to create multiple knock-out plants deficient for all six proteins of this gene family. These plants did not exhibit changes in telomere length, or phenotypes associated with telomere dysfunction. This data demonstrates that this telobox protein family is not involved in telomere maintenance in Arabidopsis. Phylogenetic analysis in major plant lineages revealed early diversification of telobox proteins families indicating that telomere function may be associated with other telobox proteins.

Using Centromere Mediated Genome Elimination to Elucidate the Functional Redundancy of Candidate Telomere Binding Proteins in Arabidopsis thaliana

The telomerase complex is a specialized reverse transcriptase (RT) that inserts tandem DNA arrays at the linear chromosome ends and contributes to the protection of the genetic information in eukaryotic genomes. Telomerases are phylogenetically related to retrotransposons, encoding also the RT activity required for the amplification of their sequences throughout the genome. Intriguingly the telomerase gene is lost from the Drosophila genome and tandem retrotransposons replace telomeric sequences at the chromosome extremities. This observation suggests the versatility of RT activity in counteracting the chromosome shortening associated with genome replication and that retrotransposons can provide this activity in case of a dysfunctional telomerase. In this review paper, we describe the major classes of retroelements present in eukaryotic genomes in order to point out the differences and similarities with the telomerase complex. In a second part, we discuss the insertion of retroelements at the ends of chromosomes as an adaptive response for dysfunctional telomeres.

The telomerase complex is a specialized reverse transcriptase (RT) that inserts tandem DNA arrays at the linear chromosome ends and contributes to the protection of the genetic information in eukaryotic genomes. Telomerases are phylogenetically related to retrotransposons, encoding also the RT activity required for the amplification of their sequences throughout the genome. Intriguingly the telomerase gene is lost from the Drosophila genome and tandem retrotransposons replace telomeric sequences at the chromosome extremities. This observation suggests the versatility of RT activity in counteracting the chromosome shortening associated with genome replication and that retrotransposons can provide this activity in case of a dysfunctional telomerase. In this review paper, we describe the major classes of retroelements present in eukaryotic genomes in order to point out the differences and similarities with the telomerase complex. In a second part, we discuss the insertion of retroelements at the ends of chromosomes as an adaptive response for dysfunctional telomeres.

Insertion of Retrotransposons at Chromosome Ends: Adaptive Response to Chromosome Maintenance

About a decade ago, I proposed that t-loops, the lariat structures adopted by many eukaryotic telomeres, could explain how the transition from circular to linear chromosomes was successfully negotiated by early eukaryotes. Here I reconsider this loopy hypothesis in the context of the idea that eukaryotes evolved through a period of genome invasion by Group II introns.

About a decade ago, I proposed that t-loops, the lariat structures adopted by many eukaryotic telomeres, could explain how the transition from circular to linear chromosomes was successfully negotiated by early eukaryotes. Here I reconsider this loopy hypothesis in the context of the idea that eukaryotes evolved through a period of genome invasion by Group II introns.

A loopy view of telomere evolution

Long non-coding RNAs (lncRNAs) evolve rapidly and are functionally diverse. The emergence of new lncRNAs is driven by genome disturbance events, including whole genome duplication, and transposition. One of the few lncRNAs with a conserved role throughout eukaryotes is the telomerase RNA, TER. TER works in concert with the telomerase reverse transcriptase (TERT) to maintain telomeres. Here we discuss recent findings from Arabidopsis thaliana and its relatives illustrating the remarkable evolutionary flexibility within TER and the potential for non-canonical TERT-lncRNA interactions. We highlight the two TERs in A. thaliana. One is a conventional telomerase template. The other lncRNA negatively regulates telomerase activity in response to DNA damage, a function mediated by co-option of a transposable element. In addition, we discuss evidence for multiple independent TER loci throughout the plant family Brassicaceae, and how these loci not only reflect rapid convergent evolution, but also the flexibility of having a lncRNA at the core of telomerase. Lastly, we discuss the propensity for TERT to bind a suite of non-templating lncRNAs, and how such RNAs may facilitate telomerase regulation and off-telomere functions.

Long non-coding RNAs (lncRNAs) evolve rapidly and are functionally diverse. The emergence of new lncRNAs is driven by genome disturbance events, including whole genome duplication, and transposition. One of the few lncRNAs with a conserved role throughout eukaryotes is the telomerase RNA, TER. TER works in concert with the telomerase reverse transcriptase (TERT) to maintain telomeres. Here we discuss recent findings from Arabidopsis thaliana and its relatives illustrating the remarkable evolutionary flexibility within TER and the potential for non-canonical TERT-lncRNA interactions. We highlight the two TERs in A. thaliana. One is a conventional telomerase template. The other lncRNA negatively regulates telomerase activity in response to DNA damage, a function mediated by co-option of a transposable element. In addition, we discuss evidence for multiple independent TER loci throughout the plant family Brassicaceae, and how these loci not only reflect rapid convergent evolution, but also the flexibility of having a lncRNA at the core of telomerase. Lastly, we discuss the propensity for TERT to bind a suite of non-templating lncRNAs, and how such RNAs may facilitate telomerase regulation and off-telomere functions.

Evolution of TERT-interacting lncRNAs: expanding the regulatory landscape of telomerase

In principle, alterations in the telomere repeat sequence would be expected to disrupt the protective nucleoprotein complexes that confer stability to chromosome ends, and hence relatively rare events in evolution. Indeed, numerous organisms in diverse phyla share a canonical 6 bp telomere repeat unit (5′-TTAGGG-3′/5′-CCCTAA-3′), suggesting common descent from an ancestor that carries this particular repeat. All the more remarkable, then, are the extraordinarily divergent telomere sequences that populate the Saccharomycotina subphylum of budding yeast. These sequences are distinguished from the canonical telomere repeat in being long, occasionally degenerate, and frequently non-G/C-rich. Despite the divergent telomere repeat sequences, studies to date indicate that the same families of single-strand and double-strand telomere binding proteins (i.e., the Cdc13 and Rap1 families) are responsible for telomere protection in Saccharomycotina yeast. The recognition mechanisms of the protein family members therefore offer an informative paradigm for understanding the co-evolution of DNA-binding proteins and the cognate target sequences. Existing data suggest three potential, inter-related solutions to the DNA recognition problem: (i) duplication of the recognition protein and functional modification; (ii) combinatorial recognition of target site; and (iii) flexibility of the recognition surfaces of the DNA-binding proteins to adopt alternative conformations. Evidence in support of these solutions and the relevance of these solutions to other DNA-protein regulatory systems are discussed.

In principle, alterations in the telomere repeat sequence would be expected to disrupt the protective nucleoprotein complexes that confer stability to chromosome ends, and hence relatively rare events in evolution. Indeed, numerous organisms in diverse phyla share a canonical 6 bp telomere repeat unit (5&#x2032;-TTAGGG-3&#x2032;/5&#x2032;-CCCTAA-3&#x2032;), suggesting common descent from an ancestor that carries this particular repeat. All the more remarkable, then, are the extraordinarily divergent telomere sequences that populate the Saccharomycotina subphylum of budding yeast. These sequences are distinguished from the canonical telomere repeat in being long, occasionally degenerate, and frequently non-G/C-rich. Despite the divergent telomere repeat sequences, studies to date indicate that the same families of single-strand and double-strand telomere binding proteins (i.e., the Cdc13 and Rap1 families) are responsible for telomere protection in Saccharomycotina yeast. The recognition mechanisms of the protein family members therefore offer an informative paradigm for understanding the co-evolution of DNA-binding proteins and the cognate target sequences. Existing data suggest three potential, inter-related solutions to the DNA recognition problem: (i) duplication of the recognition protein and functional modification; (ii) combinatorial recognition of target site; and (iii) flexibility of the recognition surfaces of the DNA-binding proteins to adopt alternative conformations. Evidence in support of these solutions and the relevance of these solutions to other DNA-protein regulatory systems are discussed.