Telomere consists of 6 nucleotide sequences (GGATTT) at the terminal region of the eukaryotic chromosomes. During chromosome replication, DNA polymerase cannot replicate the ends of chromosomes at somatic level, and gradual loss of telomeres occurs after each round of replication [1-3]. Somatic cells have a limited capacity for proliferation known as the Hayflick limit, which is due to this loss of chromosome ends, and the number of replication beyond this limitation leads to apoptosis. Telomerase is a ribonucleoprotein enzyme that is responsible for maintaining the chromosome integrity in proliferative cells, by providing specific replication machinery at the chromosome ends [4,5]. It has essential roles in cell immortalization, tumorigenesis and progression of most cancers, as it is shown that telomerase has a high activity in 70-90% of malignant human tissues and many immortalized cell lines [6-9].

Telomerase is a multi-subunit enzyme, composed of RNA and proteins including Telomerase reverse transcriptase enzyme (TERT), Telomerase RNA (TER), ES1P catalytic subunit ES3P, KU heterodimers, Dyskerin and TEP that are required for assembly and maturation of telomerase. In human, TERT is known as hTERT, which is catalytic subunit of telomerase and is characterized by having reverse transcription (RT) activities. Also, TER subunit in human is known as hTER, which acts as a template for reverse transcription [10-14].

Telomerase is tightly regulated in several levels, including transcriptional and post-transactional. On one side, alternative splicing has an essential role in the regulation of hTERT mRNA expression [13,15], and on the other side, studies have shown that telomerase function is regulated by post-translational regulation in serine/ threonine or tyrosine residue of hTERT [16]. In addition, several factors including p53, c-myc, AKT kinase, sp1, WT1, NF1, c-myb, AP1, and C-Ets2 regulate the catalytic subunits of telomerase [17-22].

It is demonstrated that human telomerase components are largely conserved in different species among mammals, and vertebrates, due to very important function of this enzyme [23-26]. Moreover, an association between regulatory factors in human telomerase and other species is stated [27,28]. Considering the numerous studies in this field, our aim in this review was to explore the relationship between human telomerase activity and functional hTERT with some candidate species. We compared function and sequences of hTERT with several species of mammals, vertebrates and microorganisms, in order to be able to suggest the best possible animal model for telomerase studies.

Yeast

Yeast telomerase is a ribonucleoproteins complex, composed of TERT (catalytic subunit), RNA template and protein subunits, however, its structure is not yet well known. In saccharomyces cerevisiae, there are a number of known subunits including Est1p, Est3p, Sm and Est2p (TERT) proteins in [29]. The comparison of TERT in yeast, compared to vertebrates, mammals and humans shows fundamental conserved residues in among species (Figure 1), which are essential for telomerase function. However, there are few regions in yeast telomerase which show specificity to this organism. The only less conserved in yeast can be considered as “nonconserved region” (N- region) of TERT, which seems to be species-specific characteristics. These regions are essential for maintaining telomerase and its performance, so the deletion based mutation in these regions affects the assembly of telomerase complex. On the other hand, deletion of the entire yeast TERT C-terminal does not interfere with the enzyme functions, but significantly reduces enzyme processivity.

Figure 1: Schematic of sizes and relative locations of TERT protein motifs in human and some species.

In yeast, N-region proximal to GQ domains is an essential package for the telomerase processing and exhibiting the binding capability of nucleic acid. However, in human this package has important role in enzymatic processing. Furthermore, the domain of dissociation activities of telomerase is mapped to amino acid residues 69 to 134 in hTERT” and has a key role in elongation of telomere in vivo [27]. In addition, RT motif in yeast is highly conserved (includes 200-300 amino acids). Based on studies done in yeast and humans, a similar TERT and RT structure implies a similar function.

The main characteristic of yeast telomerase is an inability to synthesize long extension product. In yeast and human, the synthesis of 3’ telomeric DNA terminus is with the help of RNA template and short primers. DNA synthesis stops repeatedly during replication of template, therefore the majority of yeast telomerase products are inefficient, which suggests that yeast telomerase is inefficient not only at the 5’ end of template but also in all templates areas [27,29] (Figure 1).

C-terminal region is highly conserved in human and other species. Note the flexible linker of chicken is longer than that of the mammalian species. In addition structure of TERT between human and dog are more conserved compared to other species.

TERT in fishes

Among fishes, TERT is well-studied in the zebrafish and pufferfish.

Zebrafish (Danio rerio): The length of Telomere in zebrafish is similar to that of human. Zebrafish telomerase reverse transcriptase (zfTERT) cDNA is 126 KD, and the range of other cDNAs is between 103 to 133 KD. Amino acid sequence that is coded by zfTERT is 36% identical to human and 32% to mouse. In addition, zfTERT contains 7 RT motifs and T motif which is especially found in TERT but not in other reverse transcriptase. Unlike mammalians, the zebrafish has a very high telomerase activity in somatic tissues. For this reason, the organs of adult zebrafish grow throughout the life. Telomere lengths are different in several different organs and tissues in zebrafish lifelong, alternative lengthening of telomeres (ALT), mechanism may be involved in this case in order to maintain telomeres in the absence of telomerase [26].

In mouse and c.elegans, mutation in either of the telomerase components including TERT and TR does not affect the phenotype in the first generation, but after several generations defects and chromosomal instability appear. However, because telomeres of zebrafish and humans are shorter than mouse, defects that result from mutation in each of telomerase component appears in the first generation. The TERT-/- zebra fishes show their phenotypes after first generation and stop cell proliferation in many tissues, such as gut and testis, which is controlled by p53 [26].

Pufferfish (fugue): The N-terminal region in pufferfish, recognized as fTERT, contains three regions with functional capacity known as motifs GQ, CP and QFP. The motifs CP and QFP, are accompanied by T- motif locating at the center of TERT, which are capable to mediate, and distinguish the binding of telomerase RNA in human, yeast and tetrahymena. The fTERT promoter contains Transcription Factor Binding Sites which are also traceable in the hTERT promoter including E2F-1, c-Myc, Mad 1, estrogen. In addition, Sp1 and USF are notable as well; and transcription factors play roles in the regulation of hTERT. However, the common transcriptional role of TFBSs in the hTERT and fTERT promoters may reflect the sharing mechanisms in mammalian and fish as two diverse species. Surprisingly, it is reported that TERT has a “single canonical E-box” within the promoters which locates at “position _68 to _72, relative to the TSS” in variety of species including mouse (mTERT), chicken (cTERT) and pufferfish (fTERT) Besides, the Sp1 is capable to cooperate with c-Myc and activate the hTERT transcription. Imperatively, the hTERT promoter including its two response elements lead to bind estrogen to its receptor through which the transcription of hTERT would be enhanced. Furthermore, the Sp1 and estrogen binding have role in the fTERT promoter [30].

Comparison of fTERT between human, chicken, mouse, and hamster showed lack of TATA box in fTERT promoter .However, promoters of fTERT and cTERT, by having the CCAAT box may characterize “TATA-less promoters” with an initiating role. Although the fTERT promoter has no CpG islands, but the human-TERT promoters and chicken seem to be GC-rich [31].

TERT in birds

Chicken: Among birds, TERT is well-studied in the chicken. The chicken telomerase reverse transcriptase (chTERT) proteins have 45% similarity to humans. The chTERT property can be distinguished from hTERT and other vertebrates by larger sizes of chTERT proteins due to greater linker region in N-terminal domain (144 amino acid greater than human). The chTERT genes are located at 2q2, near the telomere. Amongst many transcription factors, those binding to 5’ UTR or promoter of chTERT are similar to human (Table 1). In spite of this fact, there are binding site of p53, WT1 and c-Ets2 in hTERT promoter that not present in chTERT promoters. Exclusively there are many binding site for c-myb in 5’ UTR of chTERT. However, there is only one E-box in -264 of chTERT promoters but two E-box in -34 and -242 of hTERT [24].

Table 1: The comparison of factors binding to hTERT promoters in different species.

Moreover there is CCAAT motifs in chTERT but is not found in hTERT promoter. This motif is essential for transcription initiation of TATA poor promoters, which usually is adjacent to transcription start site. Both hTERT and chTERT have many CpG Island in 5’ UTR and coding region, this shows that their function is regulated by methylation mechanism [23,24,28]. It could be concluded that regulation of chTERT and hTERT expression is similar but they have apparently different structures (Table 1).

TERT in mammalians

Telomerase structure and biology has been studied in several mammalian species include pig and dog.

Pig: Telomere sequences in pig is revealed to be TTAGGG which is similar to human, although its length in pigs is 10-30 KD which is longer than humans’. Lymph node, lung, and kidney tissues of male and female have high telomerase activities, but male pigs unlike females display no or very low levels of telomerase activity in the liver. In contrast, the telomerase activity of spleen in female pigs is higher than males. Moreover telomerase activity is low in ovary of female pigs. In contrast the humans, telomerase activity in ovary is associated to proliferation of endometrial tissue during the menstrual. The pig telomerase biology is more similar to mouse and rat rather than other animals and human [32].

Dog: The dog TERT protein composed of 1123 amino acids with a molecular weight of -124 kDa. Dog telomere length is 12-23 kb. Comparing the dog TERT with other mammals, it seems that the dog TERT have highest level of similarities to human TERT. Besides, telomerase activity in canine cell line is similar to human.

TERT in many eukaryotes such as yeast, ciliates, plants and mammals, is a large catalytic protein domain, which composed of several RT motifs, including 1, 2, A, B, C, D, and E which are essential for catalytic activity. TERT specific motif that is called T-motif, is located at the center of dog TERT proteins. The comparison of canine TERT with vertebrates revealed that the sequences of TERT N-terminal in this species are very similar [33,34]. The hTERT are more closely resembles to dog TERT rather than other mammals. Only Minor differences exist between human and dog sequences and we tried to refer to some of them; the core promoter of hTERT located at 300 bp upstream of start codon (ATG), but in dog TERT core promoter is located 314 bp away from start codon. Besides, there are several Sp1 binding sites located within this region and there is one MZF2 (zinc finger 2 protein) binding site in-605 of canine TERT promoter. MZF2 inhibits the expression of TERT promoters, and several MZF2 binding site exist in the hTERT promoter, four of which located at 200 bp upstream of hTERT core promoter.

In canine TERT there is an E-box at -1397; whilst, two E-boxes present in hTERT promoter. WT1 binding site located in the core promoter region of canine hTERT but in -358 bp of hTERT promoter. Telomerase levels are undetectable or low in both canine and human somatic tissues, and telomere gets shorter through each division. At a glance, differences and similarities between telomerase expressions in various tissues of humans are compared with several species in Table 2. Considering these facts that the dog physiology has many similarities with human [25,35,36] and that telomerase is highly expressed in most canine cancers (up to 90%), it seems that regulation of the dog telomerase catalytic subunits is very similar to humans (Table 2).

Table 2: Comparison of telomerase expression in human, mammalians and vertebrates tissues.

Mouse: The biology of telomerase in humans and mice are very different. Human telomeres are much shorter than that of mice. Telomere shortening has a pivotal role in tumor growth in humans but has limited role in mouse. Most mouse somatic cells, unlike human, gain telomerase activity and express hTERT [25,37]. Transcriptional activity of the hTERT promoter is much less than the TERT promoter in mouse that may be due to no conserved CG-box regions in hTERT promoter which are responsible for this suppression. So cis-regulation has been suggested to be important for regulation of the TERT transcription in tissue-specific manner [38]. By considering these facts, regulation of telomerase function in mouse and humans are highly different. However, mouse TERT (mTERT) promoter like hTERT has two E-boxes and also has p53 binding site. Mutations in E-box reinforce both hTERT and mTERT promoter expression; in fact, the hTERT promoter is activated by c-myc and deactivated by p53 while the mTERT promoter does not respond to either of them [39].

Studies on human ovarian epithelial cells, vascular endothelial cells, and breast cancer cells suggested that an Estrogen receptor Responsive Element (ERE) exists in the promoter region of hTERT, but there is not in the mouse TERT promoter [40]. In another study on MEF and HEK cell lines, it was determined that the hTERT promoter was inhibited by p53, p63 and p73. Although p53 inhibit the hTERT expression through inhibition of c-myc, in mouse cells inhibit the TERT expression via Ebox/E2F pathway [41]. There are 12 CpG Island at 615bp upstream of the transcription start site in mTERT promoter; these regions are hypomethylated (30% in adult liver, 20.8% in fertilized egg). This could be a potential explanation of higher telomerase activity in mice compared to human. Thus, methylation plays significant role in regulation of mTERT expression [42,43]. Finally as an overview, the key characteristics of TERT in different species are provided which is reflective of common behavior and diversity as well (Table 3).

Table 3: Exploration of Key facts in TERT territory in different species at a glance.

It worth’s to highlight the facts of similarity in specific physiologic and anatomic pattern for vertebrates regarding the selected biological characteristics (Table 4).

Table 4: Similarities and difference facts between human and some vertebrate animals.

In spite of the similarities and the differentiae facts amongst these species, the main leading conclusion relies on’ development and evolution’. However, our aim was to achieve the applicability of one of the mentioned animals as experimental tool in human in purpose of modeling the telomerase biological pattern and drug innovation.

As a complementary insight, some regions of N-terminus and C-terminal of TERT that have essential roles in telomerase activity are conserved in human and other species, while some other regions in N-terminus of hTERT, that have critical roles in telomere maintenance and the ability of telomerase process, are not conserved.

Although the structure of the yeast TERT gene is very similar to hTERT, its performance is different due to the extra regulatory proteins in human telomerase. On the other side, the regulation of expression in chicken TERT and hTERT are similar but their structure is different. The hTERT and mouse TERT sequences are similar but remarkable differences in regulation of their functions may be due to the extra proteins in human telomerase structures and its’ different transregulation. Notably, the structure of the catalytic subunit, the regulation of expression and telomerase function in dog is more similar to humans compared to other animals.

According to previously conducted studies (Table 3), telomere length and telomerase function is associated to body size. Since dogs have a size larger than other studied animals, and also that human and dog have lots in common regarding anatomy, metabolism, alimentary tract, reproductive-e, respiratory- and cardiovascular system (Table 4), it is probably why the structure and biology of human telomerase is more similar to dog than other animals (Figures S1 and S2). Tumor development mechanism and also similar cancer stem cells are conserved between humans and dogs, as well (Table 3). Due to the biological similarities, we can predict similar response to treatment in cancers. These results suggest that dog can be an excellent model for exploring telomerase and diseases related to its abnormal activity.