hTERT Promoter Regulation by Differentiation Mechanisms vs Telomerase Activity in Somatic, Embryonic, and Cancerous Cells
Undergraduate Advanced Researcher at Clarkson University, 10 Clarkson Ave, Potsdam, NY 13699, United States
Academic Editor: Michael Fossel
Special Issue: Perspectives on Telomeres and Aging
Received: February 02, 2019 | Accepted: April 02, 2019 | Published: April 04, 2019
OBM Geriatrics 2019, Volume 3, Issue 2 doi:10.21926/obm.geriatr.1902045
Recommended citation: Liebich S. hTERT Promoter Regulation by Differentiation Mechanisms vs Telomerase Activity in Somatic, Embryonic, and Cancerous Cells. OBM Geriatrics 2019; 3(2): 045; doi:10.21926/obm.geriatr.1902045.
© 2019 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.
1. Telomeres and Telomerase Complex
We have never reached closer to understanding the phenomenon of ageing than the discovery of telomeres. The telomere was discovered by Hermann Muller in 1938, and described as a protecting structure at the ends of chromosomes by Barbara McClintock in 1940 . Nearly half a century later, Blackburn and Gall published their study on a repeated tandem sequence in the chromosomal termini in Tetrahymena thermophile, characterized by 5’-CCCCAA-3’ repeats . The tandem hexanucleotide repeat, rich in guanosine with the sequence 5’GGGTTA-3’, is conserved among most vertebrates .
The chromosomal termini, or simply telomeres (gr. ending parts), are composed of the tandem repeats with 3’ guanine rich tails hanging over at both ends of the chromosome varying in lengths: in humans the G-overhangs are found between 100 and 280 nt (reviewed in ). The G-tails fold back to form a displacement loop in homologous recombination mechanism generating a Holliday junction, namely the t-loop [5,6]. Telomeres also form a secondary DNA structure known as G-quadruplex (G4) formed by guanine-guanine interactions of the two anti-parallel strands . This highly variable DNA-DNA chromosome end structure is associated with a special protein complex, shelterin. Shelterin is a complex of six proteins contributing to its core activity, and binding either directly to the telomeric DNA (TRF1, TRF2, POT1) sequences or interact through protein-protein domains (TIN2, TPP1) [5,8]. The telomere and shelterin complex protect the end of chromosomes from the double-strand break repair systems, especially from non-homologous end joining [9,10,11,12,13].
Therefore, the telomeres protect chromosomes from extremely disastrous recombination events. Non-homologous recombination end-joining (NHEJ) leads to the formation of dicentric or circular chromosomes resulting in a severe telomere dysfunction . However, the main role of the telomere is to maintain the chromosome integrity during the replication process . In some cells, this integrity is maintained through one more mechanism.
In the so-called 3rd phase phenomenon of the cell division described by Leonard Hayflick, a differentiated cell has certain mitotic capacity limit of 50 to 70 divisions, after which it becomes quiescent and incapable of further division . This is due to the end replication problem: during the DNA replication, an approximately 200 nucleotides-long RNA primer on the lagging strand is unable to be replaced by a DNA polymerase, the Okazaki fragment [17,18,19,20]. The incomplete replication of the lagging strand is followed by about 50 – 200 bp telomeric DNA loss each cell division, which subsequently leads to critically shortened telomeres and the cell’s growth arrest [21,22,23]. It became clear that strong correlation between the telomeres length, replication machinery and cellular ageing existed [24,25,26]. It had been hypothesized that an intrinsic regulatory pathway must act in favor for the cells with long telomeres. C. W. Greider and E. H. Blackburn identified a “novel” terminal transferase-like enzyme in Tetrahymena that had been able to synthesize and replicate the tandem 5’-TTGGGG-3’ sequence on the shortened telomeres . The ribonucleoprotein (RNP) enzymatic complex had been termed telomerase [28,29,30].
Telomerase is a multicomponent enzymatic complex capable of extending chromosomal termini by synthesizing de novo tandem repeats at two 3’ ends of the same chromosome. In humans, telomerase is composed of two main components: human telomerase reverse transcriptase (hTERT) and human telomerase RNA component (hTERC or simply hTR) . The RNA component (hTR) binds the 5’ C-rich strand, and its 11-nucleotide sequence serves as the replication template for the reverse transcriptase [31,32,33].
Even though both components are crucial for telomerase activity, hTERT component alone is capable to extend the telomeres, as evidenced in multiple studies [34,35,36,37]. This phenomenon can be explained by high abundance of hTR molecules in most of the tissues, with long and short telomeres, while the hTERT component is downregulated in all cells but embryonic, adult germ cells, lymphocytes, and peripheral blood T and B cells [38,39,40,41,42,43]. The convoluted cellular control mechanisms of the hTERT expression secure the cell from stepping into carcinogenesis pathway; however, its high mRNA levels in the developing embryo cells indicate that telomerase is an inherent and crucial factor [44,45].
2. Theory of Biological Ageing: Somatic Cells vs Stem Cells
The origin of life and its ending have more in common than most could think of, and at some point they meet halfway. If cellular ageing could be defined by only two parameters: 1) loss of mitotic potential and 2) shortened telomeres – then the embryonic stem cell (ES) stands the opposite and might be considered “immortal.” Indeed, stricte physical symptoms of ageing body are reflected by incapability of stem cells located in the organs’ niches to replace dying somatic cells . Moreover, it had been shown that the self-renewal capacity of the niches’ stem cells is limited and also affected by truncated telomeres, and global cell cycle dysregulation .
Stem cells are defined as cells with the capacity of self-renewal and the ability to differentiate into multiple cell lineages . Embryonic stem cells have been considered as pluripotent cells, with the ability to generate each cell line except for the extraembryonic tissues. This point of view changed when a series of studies had proven it wrong : The embryonic stem cells, when treated with MEK (MAPK/ERK) and Gsk3 (glycogen synthase kinase 3) inhibitory procedure, they maintain their pluripotency, while some fractions sustain their totipotency. However, once a stem cell enters the path of differentiation and subsequent determination, it becomes a “mortal” somatic cell. It has been shown that the telomerase might function as one of the most important molecular switches in this mechanism .
The rearrangement of the “stemness” identity into the somatic is the driving force in all cells of a developing embryo. Because of the complexity of the entire dynamic system of the embryo, tracking all virtual changes within its cells is challenging. The most convenient approach in studying the morphogenetic changes of the developing embryo in its entirety is the view from the genome (VFG). This is the view of the genome network with respect to all nodes, circuits, subcircuits, and components (proteomic and genomic) at particular time . The networks for specific body parts are composed of specific subcircuit components, namely transcription factors and their effectors (genes regulated at precise nodes and domains) [45,51]. With the progress of embryo development, the distinct clusters of transcription factors constitute and determine distinct body parts networks (lineages) until they reach the maximum saturation point of specification and lineage determination, when there is only one lineage possible to choose for the cell (unidirectional and progressive differentiation) . Therefore, the stem cells found in distinct tissues are capable to differentiate only into this tissue – they become the unipotent stem cells, though with high plasticity. Although a common term, stem cells researchers are divided whether a unipotent stem cell exists at all; the data indicates that most of these cells are able to differentiate into more than just one lineage and the term unipotent does not necessarily imply their actual role [52,53,54].
From this, following conclusions can be drawn: 1) Differentiation is a multicomponent and multilevel process forwarded by subsequent and reciprocal interactions between transcription factors and target genes. 2) As it progresses, the stem cells change their biochemical states (on the basis of the genomic networks) by losing their plasticity and being coursed into more and more specified lineages. 3) The infinite number of doublings is changed to finite as they become fully the determined somatic cells. 4) One of the main cellular markers of their mortality are shortened telomeres, silenced expression of telomerase, and the resulting Hayflick limit.
This is a very simplified schema of the extremely convoluted process of the embryo development, genomic and environmental interactions, and the changes occurring in the developing and somatic cells, but if properly focused and addressed, leads to an interesting observation. Could the telomerase downregulation, consequential telomeres shortening, and the differentiated state of the cell be coupled? If so, what kind of mechanisms could be involved and what is the exact relation between these processes?
3. Regulation of Human Telomerase Expression
The hTERT locus is positioned on chromosome 5 (5p15.33), extending over 40 kb with 16 exons and 15 introns, 2 Mb away from the telomeric region . It has been observed that the telomerase negative cells still generate differently spliced variants of the polymerase, with α-spliced (lacking 36 nucleotides from the 5’ end of exon 6) and β-spliced (lacking exons 7 and 8) variants being the most widespread . Recently it has been shown that cellular ageing and oncogenic processes are coupled with unique chromatin looping directed by telomeres; chromatin of the promoter of hTERT becomes more permissive for transcription elements (through epigenetic modifications) producing truncated and mostly inactive spliced variants of hTERT, thus protecting the cells from perilous telomerase activity . Due to the limited scope of this article, more information on the alternatively spliced variants of the hTERT gene can be found in [41,57].
Interestingly, the hTERT promoter lacks both TATA and CAAT boxes, but is marked by large CpG islands, including two canonical CACGTG (E-box) sequences [15,58]. The CpG islands make the hTERT promoter susceptible to DNA methylation at the C residues . Besides, the promoter has multiple binding sites for a great number of trans-regulatory elements regulating its expression state. Overall, the telomerase activity is controlled almost entirely by the transcription regulation of hTERT via epigenetic and trans-regulatory mechanisms. They briefly will be described and summarized below.
Methylation is the most prevalent epigenetic modification at the hTERT promoter, although not unambiguous [60,61]. In retinoic acid-induced HL60 differentiated and human teratoma cells, hTERT promoter methylation pattern has its corresponding effect on the decreased hTERT expression , while in other studies on cancerous cells the methylation patterns have not always correlated with their telomerase expression levels [63,64,65,66].
Similarly, histones acetylation usually is related to an increased gene activity . Even though downregulated hTERT in most somatic cells is associated with hypoacetylated core histones, this is not always true . It is also known that DNA methylation and histone acetylation are coupled in the telomerase regulation machinery. Methyl-CpG biding proteins, such as MBD1 and MeCP2, act as mediators between those two processes as evidenced in a synergistic co-play between DNMT (DNA methyltransferase) and HDAC (histone deacetylase) repressor complexes recruited by PCNA (proliferating cell nuclear antigen) and interacting with p21 cell cycle regulator .
The transcription factor control over telomerase expression is even more intricate with the following observations. The core example of the whole network of regulators is heterodimeric E-box-binding c-Myc/Max complex, which is highly abundant at the hTERT promoter, and regulates its transcriptional activity [15,70,71,72]. Human papillomavirus 16’s E6 protein is another significant activating factor shown to activate telomerase in human keratinocytes and mammary epithelial cells . As the endometrium cells are telomerase positive, due to the proliferating epithelium cells, it is not surprising that steroidal hormones, including estrogen, positively affect the hTERT expression as well [74,75]. It has been demonstrated that the most engaged initiation site in the hTERT promoter is the GC-box for zinc finger far-flung Sp1 transcription factor, which cooperates with c-Myc in triggering telomerase activity .
On the contrary, the vastness of repression mechanisms and elements is exceeding over the telomerase activating regulators. Another heterodimer, Mad1/Max represses the hTERT transcription through histone deacetylation, and the switch between the two complexes, c-Myc/Max and Mad1/Max, depends the complexes’ members’ instantaneous concentration . Most cell cycle negative regulators are involved in silencing the hTERT promoter, and this includes p16, p21, E2F, and p53 (mostly through Sp1 downregulation) [78,79,80,81]. And finally, the differentiation factors are also actively engaged in the hTERT downregulation, just to count TGF-β1, Activin, and BMP7, with well-studied telomerase regulating properties [82,83,84,85]. The Nature equipped cells with an impressive tool box for silencing the telomerase activity making sure that the enzyme would become quiescent by all means necessary.
More than this, there are other nodes of control over the telomerase activity, including posttranslational modification of the enzyme and the shelterin complex with its six core components interacting with other proteins [8,59,80]. The control occurs at each level of the telomerase activity, affected by hundreds of various biological factors. Very often it is impossible to find a correlation or a pattern among all the overflowing data, and when such a correlation is finally grasped, it is crucial to point it out with proper justification found in the enormous literature collection, and then to prove it with proper experimental evidence. This article makes an exacting attempt to define an exact correlation between regulatory mechanisms of telomerase and differentiating processes in the developmental course. How could they be possibly explained before any experimental procedure is applied?
4. Biomolecular Relationship between Cellular Differentiation and Ageing
Knowing that pluripotent embryonic cells are telomerase positive, we could freely draw conclusion that all of them should have similar telomeres length. However, it has been shown that the telomere length in the oocytes and blastocysts differs greatly from the telomeres in the zygote, more than it had been expected . While an average oocyte’s telomere length has been determined as ~ 11.12 kb and a blastocyst’s as 12.22 kb, the average telomere length for the cleavage stage embryo is ~ 8.43 kb . It is speculated that the alternative lengthening of telomeres (ALT) mechanism through chromosomal recombination in early embryo cells could yield in this discrepancy . The ALT pathway has not yet been observed in human embryo cells, but it already has been shown that both mechanisms (telomerase-dependent and –independent) coexist in the same cell, which possibly could explain the differential telomeres length phenomenon in the human embryo cells [89,90]. Moreover, it has been reported that self-renewal capacity of human embryonic stem cells (and iPSCs) is secured by the telomere length maintenance through chromosomal trimming, a similar mechanism observed in uni- and multicellular organisms [4,79,90].
Strong correlation between the differentiation status of the cell and telomerase activity is indisputable. Unfortunately, this correlation is frequently overlooked by researchers. Most notably, the majority of the data in this spectrum have been reported in the studies on cancerous cells (see below). Only recently, by reason of the induced pluripotent stem cells research outbreak, it has been evidenced that the telomerase activity increases in somatic cells induced to become pluripotent [91,92]. Since the first successful iPSCs establishment, the four main pluripotency transcription factors (Oct3/4, klf4, Sox2, c-Myc) have been recognized as the main “stemness” gate keepers . Now, it is known that c-Myc is a dispensable factor in activating telomerase expression in the induced cells . It is still uncertain how exactly Oct4, klf4, Sox2, or Nanog affect the hTERT promoter. Most likely, local chromatin and histones remodeling is the primary explanation based on the chromatin studies, and additionally complex cell cycle and apoptotic pathways, incorporating a broader range of proteins that had never been expected to be involved in the regulation of telomerase activity [91,92,94,95,96]. Recently, researchers have found a klf4 binding site in the hTERT promoter stimulating its activity through the Wnt signaling [70,97].
Multiple studies, especially on promyelocytic leukemic HL-60 cells, indicate that terminal differentiation induced by differentiating factors results in the telomerase-negative phenotype [98,99,100,101]. Other components of the telomerase complex were not up- or downregulated, proving once again that hTERT is the rate-limiting agent . Apart from well-established epigenetic modifications, factors such as a cell cycle stage (G0 putatively limits the telomerase activity), “stemness” status of a cell (non-dividing stem cells do not express hTERT as well presented in ), and poorly studied trans-acting repressors (and activators), including WT1 transcription factor, might affect the transcription of the hTERT promoter and be responsible for telomerase activity [69,94,100]. Even more importantly, the trans-acting proteins may interact with the hTERT promoter prior to the chromatin changes , and their effect is performed mostly through silencing, although the studies are very often contradicting [103,104].
One could speculate whether the downregulation in telomerase expression might be a trigger for terminal differentiation, but the available data indicates the opposite. Hence, it could be stated that the terminal differentiation of the cell is possible if and only if the telomerase activity is downregulated through the yet unknown regulatory mechanisms. Silencing the hTERT promoter might be one of the final cellular statements towards the terminal differentiation. A few reasons explain this assumption. First, telomerase is active in ~ 90% of all tumors meaning that its role in cellular homeostasis is rather compelling [38,98,105,106,107,108]. Second, hTERT has binding sites for some of the cell cycle regulators as well as its chromatin remodelers are activated and silenced by other cell cycle trans-acting particles, showing that the telomerase activity regulation is strongly coupled with the intracellular control mechanisms [69,70,94,96]. Third, it has been shown that the hTERT locus might affect the activity of other genes, including genes of the cell cycle, through special telomere positional effect, and its chromatin regulation could be another prerequisite for intracellular stabilization [56,109].
Shortening of telomeres leads to the cell’s replicative senescence and in the ultimate cases, their fatal impairment [20,110,111,112]. This process contributes to organ impairment and systemic aging [113,114,115,116,117]. Telomere shortening is not the defining cause of cellular ageing and biological death, but one of the most significant ones. As the data points out and as our understanding of the ageing processes gradually deepens, the telomerase complex is not an isolated genome regulatory module, but a far-reaching part of the entire intracellular regulatory system. It must be yet carefully studied to what definite extent the differentiation mechanisms are correlated with the telomerase activity control. Afterwards, the next logical step is to determine the clinical applications from the gained knowledge in a plethora of medical disciplines. However, all the present data indicates a strong correlation between the differentiation processes and the telomerase gene silencing, which could be concluded in rather a philosophical statement: Death starts there where life begins.
Terminal determination of the cells is necessary to preserve certain global epigenetic marks and direct the cells towards their biological fate. The genomic changes during this complex mechanism are indiscernibly known, and even less is known on how they affect the telomerase activity. Within the chain of consecutive processes taking place in a virtual embryonic cell, the genomic loops of cis- and trans-acting regulators transform the pluripotent stem cells into the well-defined, fully differentiated somatic cell. During these changes, the hTERT promoter has been observed to be modified as well indicating the intriguing role of the telomerase in the developing and differentiating cells. Properly defined factors coupling the regulatory pathways of cellular differentiation and telomerase activity could become of a great importance.
This knowledge could be used in a plethora of medical applications. First, identifying the differentiation factors directly involved in the telomerase regulation gives a chance to develop new clinical regulators of the telomerase promoter. Second, the newly recognized telomerase regulatory pathways could bring closer the understanding of some yet-unknown cancer development mechanisms. Third, the stem cells research and regenerative medicine fields might benefit in understanding the regulatory processes behind iPSCs and their improvement. The ongoing research on the telomerase and its components over the last thirty years has shown an incredible scope of the enzyme activities involved in the cellular homeostasis, contributing to more than just cellular ageing.
The author would like to thank Jerry W. Shay for inspiring motivation. The author would also like to thank Jack W. Szostak for his inestimable inspiration for writing this work and kind words, and Michael Fossel for the future yet to come The author would finally like to thank the beloved ones, especially his wonderful mother.
Steve Liebich is the main and only author of the manuscript.
The author has declared that no competing interests exist.
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