Regulation of telomerase is very complex as many factors can effect hTERT expression and telomerase activity. Telomerase may be regulated through such known methods as transcriptional regulation, post-transcriptional regulation, post-translational regulation, localization within the cell, assembly of the subunits, epigenetic regulation and by telomeric proteins and RNAs [15, 16]. Many new forms of regulation may be yet to be uncovered. In this section we review recent studies of telomerase regulation involving methylation of the hTERT promoter and telomeric regions, the possible role of sirtuins and non-coding RNAs including microRNAs (miRNAs) and telomeric-repeat containing RNA (TERRA).
Regulation of hTERT transcription generally takes place at the promoter region which has been found to contain an abundance of CpG sites, common targets for methylation . DNA methylation and chromatin remodelling are common regulators of gene activity that alter the binding of transcription factors to gene promoters. The field of epigenetics has become very important in the study of telomerase regulation, and several recent studies have begun to shed light on some of the factors involved.
A recent study in HPV-induced carcinogenesis has suggested that a gradual increase in methylation at the hTERT promoter may coincide with the progression to a tumorigenic phenotype in cervical cell lines, but the effect was not as significant in clinical samples . This discrepancy may be the result of selection for cells with greater methylation or, as the authors suggest, may be caused by the presence of normal tissue in clinical samples, although it may be related to another complication: researchers have not been able to agree about whether hTERT methylation follows the expected relationship. Promoter region methylation is commonly associated with gene silencing, but studying the epigenetics affecting the hTERT gene has been difficult. Although some studies have generated the expected results, other researchers have indicated that there is no correlation between methylation and hTERT expression . It has been suggested that methylation status and its relation to hTERT expression is dependent on the cell type [20, 21], but there is some debate.
In 2007, Zinn et al. studied multiple cell types to try to determine if there was some underlying mechanism behind the epigenetic regulation of telomerase in all cancer types . The group tested breast, lung and colon cancer samples using methylation-specific PCR and bisulphite sequencing of the promoter and found that all of the samples maintained at least one allele with less methylation around the transcription start site despite surrounding methylation patterns. They suggest that although much of the hTERT promoter may be heavily methylated in some cancer cell types, a region of about 300 bp around the transcription start site remains unmethylated and following the usual pattern of methylation resulting in gene silencing. Although this is an interesting study providing a possible explanation to the complexity of hTERT promoter epigenetics, only three tissue types were studied, and some research suggests that we are still not seeing the whole picture [20, 23]. Of course, telomerase regulation is complex, and hTERT promoter methylation is just one piece of the puzzle.
Telomerase activity has also been shown to be subjected to regulation by methylation of other regions such as the subtelomeric region . This is the chromosomal region just proximal to the telomeric region which has been found to be highly methylated in mouse and human cells as reviewed previously [24–26]. A recent study has indicated that subtelomeric methylation may influence telomerase activity, and specifically whether a tumour maintains its telomeres with telomerase or through the alternative lengthening of telomeres (ALT) pathway . The specific mechanisms of the ALT pathway are not well known, and it is only utilized by 10–15% of human tumours . Ng et al. analysed subtelomeric cytosine methylation, first determining methylation state in normal cells, and then comparing cells using the ALT pathway to cells that were telomerase positive. They found that the average percentage of methylated loci across all normal samples was relatively constant: 81 ± 3%. When analysing ALT cells, the group found that the amount of methylation varied dramatically, not just across cell lines, but even across loci. However, when analysing telomerase positive cells, it was found that all loci were heavily methylated with little variation: 97 ± 1%. This indicates that increased methylation of subtelomeric regions may somehow allow for an increase in telomerase activity, although it is not known if this is somehow affecting hTERT transcription.
Recently, work has been done by several groups which suggest that sirtuins may have a role in telomerase regulation, but findings have been unclear. Sirtuins are a family of enzymes that have been shown to play a role in increasing overall health and longevity in organisms . A study from 2003 by Lin and Elledge suggested that Sir2 (a yeast protein and the first sirtuin discovered, shown to increase lifespan by guarding against genome instability ) may be an activator of hTERT in a small variety of cancer cell lines tested . SIRT1 (NAD-dependent deacetylase sirtuin-1, the human analogue to Sir2), however, has been suggested to be an inhibitor of telomerase activity . Likewise, a recent study has shown that an isoform of P63, ΔNP63α, can induce TERT promoter activity in mice, and it was suggested that this may be due to the down-regulation of SIRT1. Recent studies with resveratrol have also added to the speculation that SIRT1 may be involved in telomerase regulation. Resveratrol is one of the most commonly used molecules thought to activate sirtuins. It is a small molecule that has been shown to improve health and longevity in many of the same ways as targets of SIRT1. It has been suggested that resveratrol promotes health and longevity by acting through sirtuins, although this idea is still debated [34, 35]. It has also been shown that resveratrol activates telomerase in endothelial progenitor cells  and decreases telomerase activity in breast cancer cells . Although it has not been shown that these modifications of telomerase activity are directly related to SIRT1 activity, it is an interesting area requiring more study. For more information on current studies looking into the pharmaceutical use of resveratrol as a telomerase inhibitor, refer to the section below.
Little is known about which non-coding RNAs might regulate telomerase. When studying these factors, the complexity of telomerase regulation is compounded by the complex networks of miRNAs. MicroRNAs, also known as miRNAs or miRs, are non-coding, single stranded RNAs that have recently been shown to play critical roles in many biological processes such as development, differentiation, apoptosis and proliferation. They commonly down-regulate the target messenger RNA of protein-coding genes in a sequence-specific manner . More than 1000 miRNAs are predicted to work in human biology in a complex regulation network, and several studies have shown that many miRNAs are deregulated in cancers [37–40]. However, to this day, miRNA regulation of telomerase remains elusive.
In 2008, Mitomo et al. found an association between the down-regulation of miR-138 and overexpression of telomerase in anaplastic thyroid carcinoma cell lines . The researchers selected miRNAs of interest reported to be differentially expressed in thyroid carcinoma when compared to normal thyroid. Each miRNA was studied individually using stem-loop-mediated reverse transcription real-time PCR. miR-138 was found to be significantly down-regulated in anaplastic thyroid carcinoma cell lines compared to papillary thyroid carcinoma cell lines. Using the miR-Base online database (http://microrna.sanger.ac.uk), the authors looked for potential targets of miR-138 and focused on hTERT. The thyroid carcinoma cell lines were transfected with miR-138 precursor molecules, and effects on hTERT expression were studied using Western blotting and luciferase assay of the promoter activity . It is important to note, however, that it has been shown that many commercially available antibodies used for Western blotting are not specific to telomerase and may result in inaccurate conclusions . Although increasing the amount of miR-138 molecules in the cells did not completely turn off telomerase activity in the cell lines studied, this is most likely just the first of many miRNAs to be associated with telomerase activity. Also, miRNA activities may be cell-type specific, and the disregulation of miR-138 may be important in telomerase activity in thyroid carcinomas, whereas other miRNAs and mechanisms may be more important in regulating telomerase in other cells and tissues.
It has proven to be a difficult task to pinpoint which miRNAs target telomerase. Since Motimo et al. 2008 findings, very little headway has been made. Although no other specific miRNAs have been found to date, Miura et al. have found a region of interest that they believe alters telomerase activity and is most likely to contain a miRNA precursor gene . Several studies have indicated that human chromosome 10p may contain a gene involved in regulating telomerase activity [44–46]. To determine which genes in this region may be involved, Miura et al. studied the region by exon trapping using bacterial artificial chromosome clones and studied the effect these segments had on telomerase activity using human hepatocytes and hepatoma cell lines. One of the genes cloned (RGM249-RNA gene for miRNAs, 249 bp in length), located at chromosome 10p15.3 was found to be overexpressed in cancer than in normal liver cells, similar to telomerase expression and was found to inhibit more than 80% of hTERT mRNA expression .
Both the Mitomo and the Miura studies shed light on a small portion of non-coding RNA molecules that may regulate telomerase activity. Both studies utilize relatively new techniques and technologies which will need to be continually combined and refined as we continue to study miRNA functions. Telomerase activity is such an important step in development and cancer progression that many complex regulatory pathways play a role. miRNAs have become popular topics of study since their discovery, but even more recently, large non-coding RNAs called TERRA have been suggested to play a role in telomerase regulation.
Telomeres have long been thought to be transcriptionally silent regions of the genome. Recently, this notion has been challenged with the discovery of TERRA, or telomeric repeat-containing RNA, also called TelRNA [47, 48]. Interestingly, these molecules have been found to have regulatory effects on telomerase. Mammalian TERRA molecules are large, non-coding RNA containing UUAGGG repeats and range in size from 100 bases to about 9 kb [47, 48]. At least some of the TERRA also contain subtelomeric-derived RNAs well as the telomeric UUAGGG repeats suggesting that transcription of these elements begins at different starting points in the subtelomeric regions and moves towards the chromosome ends [47–49]. Relatively little is known about the functions of TERRA, but they have been shown to be an integral part of telomeric heterochromatin [47, 48], and possibly help maintain telomere architecture , provide epigenetic protection of telomeres from DNA repair mechanisms and regulation of telomerase activity . It is thought that TERRA block telomerase activity at the telomeres in at least two ways: by blocking the RNA component with its sequence complementarity [27, 48] and by recruiting heterochromatinizing activities to the telomeric regions .
Several recent publications have supported the notion that TERRA molecules may function as telomerase inhibitors. Researchers have found that TERRA are capable of duplexing with hTERC [48, 50], and it has been shown that there are lower levels of TERRA molecules in tumours than in corresponding normal tissue [48, 51]. It has also been found that shorter telomeres have lower levels of TERRA molecules [48, 50]. Shorter telomeres may transcribe lower amounts of TERRA because of the decrease in template space. This would cause less impairment of telomerase activity and help explain why telomerase has been shown to be selectively active at critically short telomeres [48, 50].
To further elucidate how transcription of telomeres is regulated and how it might be linked to telomere length, Caslini et al. have shown that histone 3/lysine 4 (H3/L4) histone methyltransferase and the transcription regulator Mixed-Lineage Leukemia (MLL) may contribute to telomere methylation and their transcription . Local combined activity of H3/L4 and MLL are thought to promote transcription elongation and chromatin maintenance, but MLL had not been previously associated with telomeres. Using ChIP, they found that TTAGGG repeats were capable of pulling down both the N- and C-terminal domains of MLL in human haematopoietic cell lines and human ovarian surface epithelium. The same methods also showed that telomeres were acetylated at H3 and H4 and to be methylated at H3/K4. It was also shown that MLL binding positively correlated to H3K4 methylation and transcription of telomeres, and that knocking down MLL resulted in histone modifications at the telomeres .
Interestingly, it has also been shown that there are lower levels of TERRA transcripts in telomerase positive cells than in cancer cells that maintain their telomere lengths through the ALT pathway . Ng et al. compared the levels of TERRA in normal cells, ALT cells and telomerase positive cells by hybridizing RNA from each sample set to probes against the UUAGGG TERRA sequence and the antisense CCCUAA sequence and normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). They found greater amounts of TERRA molecules in the ALT cells than any others. One possibility is that these amounts were higher because ALT cells have longer telomeres and therefore can transcribe more TERRA molecules. Taking this into account, the group corrected for the additional telomeres by measuring telomere lengths in each sample. ALT cells were still found to have more TERRA transcripts. The group had already shown that telomerase positive cells have increased methylation at the subtelomeric region compared to normal and ALT cells. It is possible that these two findings are connected and indicate a mechanism in which TERRA molecule transcription is dampened to allow for more telomerase activity without hindrance from TERRA in telomerase positive cells, whereas normal TERRA transcription is allowed in ALT cells.