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[ImmInst]

By James P Watson with editorial assistance and comments by Vince Giuliano

This is Part 3 of 3 in the series The View from the Telomere end of the Chromsome.  Because of the length of the telomere story, we divided this discussion into three parts.  Part 1 relates to a number of more-practical and less-technical topics: the history of telomere biology, telomere length testing, telomerase Inhibitors for cancer, and supplements that activate telomerase and their possible roles for inhibiting or preventing cancers.  Part 2 deals in finer detail with newer discoveries related to the molecular biology of telomeres, Sections 1 – 9.  This part 3 covers Sections 10-17 and a concluding discussion related to some of the implications for a Grand Unified Theory of aging and biology, as viewed from the ends of a chromosome. 

The 1994 telomere-telomerase story, and the 2014 one

Introductory comment by Vince: This particular blog series is for those of you seriously interested in the science related to telomeres, above and beyond the telomere romance of two decades ago. This involves tough reading and some times even tougher understanding. I clearly recall the days in 1994 when I could relate telomere-telomerase story over a glass of wine to an artist or massage-therapist friend and have the friend get it: “Telomeres are like inert shoelace tips at the end of chromosomes that grow a little shorter every time a cell divides. They get shorter and shorter until they can’t do their job and drive a cell into senescence or death. They are like clocks counting down. Telomerase is an enzyme that pastes telomeres back on again. Doing this can run the aging clocks backwards and offers a hope for extending life.” The 2014 story told in these blog entries is vastly more complicated and makes that 1994 version seem incredibly naïve as well as downright wrong. I can’t possibly explain the story told here to most of my MD friends even over a whole case of wine, let alone to my artist friends. Yet the 2014 story is incredibly rich and illustrative of the multiple checks and balances and feedback loops in human biology. And it illustrates that there is no center to biology. You can start anywhere and get to most anywhere else. So, for those of you with the patience, we continue to tell that story here. We pick up where Part 2 left off with item 10. And the last part related to a GUT of biology is definitely interesting.

10.  Regulatory Elements Prevent Silencing of Subtelomeric Genes

Using Transgene Experiments for Basic Science Discoveries

Since subtelomeric genes are vulnerable to the Telomeric Position Effect (TPE) which results in unwanted gene silencing, several complex epigenetic mechanisms have evolved to counteract this. The next four Sections here specifically review these, but as an introduction, I must explain that most of these discoveries were made using transgene experiments of genes spliced into telomeric and subtelomeric DNA to study how DNA is silenced in this area of the chromosome. As you may know, the word “transgene” refers to the procedure that most of us refer to as “genetic engineering” or recombinant DNA technology. With genetic engineering, a piece of non-native DNA (the transgene) is introduced into the genome. The location that the transgene is spliced into the DNA is random and not controllable. When the transgene is spliced into the telomeric or subtelomeric DNA, the expression of the transgene is very low or inconsistent, due to epigenetic silencing of the transgene. Once researchers discovered this, they used this as a laboratory tool to figure out why this occurred. Thus came the discoveries detailed below.

Epigenetic regulators

Now that we are about to explore even more “epigenetic tricks” up Mother Nature’s sleeve to counter-act the Telomere Position Effect (TPE), you might want to refer back to Sections 4, 5 and 6 in Part 2 of this series for a more detailed explanation of the TPE.  Using transgene experiments, the following methods for counteracting the TPE were discovered.  This list of new epigenetic “tricks” are called Epigenetic Regulators.  They include the following:

1.  MARs – Matrix attachment regions

2.  UCOE – Ubiquitous chromatin opening element

3.  Insulators – Example: the chicken beta-globin 5′ hypersensitive site 4 (cHS4)

4.  STARs – Stabilizing Anti-repressor elements – I am not going to go over this, though it is yet another one.

The next few sections will explain, MARs, UCOEs and Insulators

The 2013 publication Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genesreports: “In eukaryotic cells, transgene expression levels may be limited by an unfavourable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which may protect transgenes from such position effect. We evaluated different epigenetic regulators for their ability to protect transgene expression at telomeres, which are commonly associated to low or inconsistent expression because of their repressive chromatin environment. Although to variable extents, matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE) and the chicken cHS4 insulator acted as barrier elements, protecting a telomericdistal transgene from silencing. MARs also increased the probability of silent gene reactivation in time-course experiments. Additionally, all MARs improved the level of expression in non-silenced cells, unlike other elements. MARs were associated to histone marks usually linked to actively expressed genes, especially acetylation of histone H3 and H4, suggesting that they may prevent the spread of silencing chromatin by imposing acetylation marks on nearby nucleosomes. Alternatively, an UCOE was found to act by preventing deposition of repressive chromatin marks. We conclude that epigenetic DNA elements used to enhance and stabilize transgene expression all have specific epigenetic signature that might be at the basis of their mode of action.”

11.  Insulators - The partitioners of the genome

This is something new that I had not learned about before.  An “insulator” is a barrier element that partitions the genome into discreet chromatin domains.  Specifically, an insulator may have an enhancer-blocking activity or a barrier activity.  Here is an illustration of these two functions of insulators:

Article, image and legend reference: The 2012 publication insights into insulator function during development

“Schematic overview of the domain model of a linear genome, highlighting insulator locations.  (A) An active chromatin domain is flanked by heterochromatic regions.  Insulator positions are indicated at the domain boundaries (where they can mediate border or barrier function of insulators) as well as within the active domain (where they can mediate enhancer-blocking function).  It is not known whether both functions are established by similar or different mechanisms.  (B) One aspect of insulator function is to organise chromatin looping by promoting contacts between insulators or with other genomic structures.  Depending on the linear and three-dimensional arrangement, looping may interfere with enhancer-promoter interactions (thus mediating the enhancer-blocking function of insulators), resulting in an inactive gene (pink gene and promoter), or it may assist in increasing enhancer-promoter contacts, resulting in an active gene (green gene and promoter on right).  Gene activation can also be achieved by direct enhancer-promoter interactions (green gene on left) that can occur independently of the presence of an insulator.  Insulators are also found between tandem promoters positioned in a head-to-head orientation ensuring that both promoters can be regulated individually.”

In the first case in the above diagram, an insulator can interfere with the enhancer-promoter communication when interposed between them.  This prevents the spread of repressive heterochromatin over adjacent euchromatin domains.  Thus, insulators have been shown to confer stability to the transgene expression over time and are seen as promising tools to increase the safety of gene therapy vectors.

Numerous insulators have been identified in different species.  The most well known insulator is cHS4, which is a potent insulator in chickens.  It combines the enhancer-blocking and barrier activities of insulators.  This is another epigenetic feature that prevents subtelomeric gene silencing due to the TPE.  Here is an illustration of the cHS4 insulator:

Illustration and legend reference;Chromatin domain boundaries and beyond.

“A model for chromatin barrier function of insulator and the histone code.  The cHS4 insulator binding proteins USF1/USF2 (USF) recruit the histone acetyltransferases (HATs) PCAF and P300/CBP (H3 and H4 acetylation) and methyltransferase SET7/9 (H3 lysine 4 methylation), which form a histone hyperacetylation- and H3 lysine 4 methylation-enriched chromatin boundary to block the spread of adjacent heterochromatin structure ^{8, 42}.  The insulator perhaps recruit other putative protein factors to form a barrier complex to prevent heterochromatin spreading.  Ac and Me are abbreviations of acetyl and methyl groups, respectively.  Especially, Me (blue) mean methylation of H3 lysine 4, which are active chromatin marks.  Other Me mean repressive chromatin marks such as methylation of H3 lysine 9.”

12.  MARs – Attachement Points for DNA to the Nuclear Cytoskeleton

“Matrix attachment regions” and scaffold attachment regionsare areas of A + T rich DNA of variable length that serve as “anchoring points’ for DNA to attach to the nuclear skeleton.  They are also called scaffold-attachment regions (SARs).  MARs are thought to organize eukaryotic chromatin into distinct regulatory domains by the formation of 50-200 kb structural loops. Here are two illustrations of MARs.

Illustration and legend reference:Chromatin loops are selectively anchored using scaffold/matrix-attachment regions

“A proposed model for the selective use of S/MAR for transcription/replication regulation.  The left panel shows a gene located on the loop with a S/MAR in close proximity.  When functional demands require the specific association of this gene with the transcriptional machinery located on the nuclear matrix, the S/MAR moves the gene to the nuclear matrix, thereby initiating gene expression (center panel).  Following initiation, the gene is pulled in through the transcriptional machinery, thus completing the process (right panel).  There are two types of S/MARs.  Functional S/MARs serve as mediators to bring genes onto the nuclear matrix.  Structural S/MARs serve as anchors, which are less dynamic compared with functional S/MARs.”

Article, illustration and legend reference for following: Scaffold/matrix attachment region (Wikipedia)

“S/MAR-functions: constitutive and facultative.  A chromatin domain with constitutive S/MARs at its termini (I).  When functional demands require the specific translocation of the constituent gene to the matrix, facultative S/MARs responds to topological changes which are initiated by the association of transcription factors (TF) and supported by histone acetylation.  Topological changes are propagated once the gene is pulled through the transcriptional machinery (II).  Transcription is terminated (III) followed by dissociation of the transcription complex (IV)”

Transcription factor genes contain a significantly higher portion of MARs.  As a result, when researchers find a gene with a nearby MARs, it emphasizes its functional importance.  Also, when a nuclear scaffold/matrix module is included in a genetic vector such as an episome, if the episomal MAR is attached to a transcription unit, the episome will be maintained and the expression not lost over time.  Thus, a MAR increases the stability of a transgene injected into a cell.  It is estimated that 64,000 MARs may be present in the human genome (plus an additional 10,000 replication foci).  Little sequence homology exists, however, and as a consequence, only a minor fraction have met the official criteria for a MARs.  These epigenetic features are evolutionarily conserved.  The chicken lysozyme MARs was one of the first seen which  demonstrated increased and sustained transgene expression.  In humans, the MAR 1-68 and X-29 are two of the most well understood MARs(ref).  In mice, the MAR S4 is one of the most well understood matrix attachment regions.  When you add a MARs to an expression vector, three things happen:

a.  The number of stably expressing clones increases

b.  The expression of the transgene is enhanced

c.  The variability in gene expression within a polyclonal cell population is reduced

In summary, MARs appear to increase gene expression, but the exact mechanism is still not well understood.  This is another epigenetic feature that helps keep subtelomeric DNA from being silenced by the TPE.

13.  UCOEs - A Promoter-based method of stabilizing gene expression

Ubiquitous chromatin opening elements (UCOEs) are regulatory elements derived from promoters that contain CpG islands (remember that only about half of genes have CpG islands) They appear to be present mainly in housekeeping genes.  It appears that UCOEs have chromatin remodeling function.  Here is an illustration of a UCOE:

Article, image and lesgend reference:  Genomic cis-acting Sequences Improve Expression and Establishment of a Nonviral Vector

“pEPI-EGFP and derivatives.  (a) Original episomal vector pEPI-EGFP harboring an active transcription unit running into the S/MAR sequence.  (b) The ubiquitous chromatin-opening element (UCOE) from the HNRPA2B1-CBX3 locus was introduced upstream the CMV promoter resulting in pEPI-UCOE.  (c) The chicken β-globin insulator sequence cHS4 was introduced downstream and in opposite direction to the S/MAR resulting in pEPI-HS4opp.  (d) Episomal maintenance and cellular replication was ascertained in Southern blot analysis.  Complete digestion with MboI indicates cellular replication (lane 1), as shown for all three pEPI derivatives.  C: Plasmid DNA as control; 1: DNA digested with MboI and plasmid single cutter; 2: DNA digested with DpnI and plasmid single cutter.  In no case, we detected hybridization to chromosomal DNA.”

14.  HDAC5 and Telomeres – Required for maintaining normal telomeres in certain cancer cells

The publicationA new role for histone deacetylase 5 in the maintenance of long telomeresreports: “Telomeres are major regulators of genome stability and cell proliferation.  A detailed understanding of the mechanisms involved in their maintenance is of foremost importance.  Of those, telomere chromatin remodeling is probably the least studied; thus, we intended to explore the role of a specific histone deacetylase on telomere maintenance.  We uncovered a new role for histone deacetylase 5 (HDAC5) in telomere biology.  We report that HDAC5 is recruited to the long telomeres of osteosarcoma- and fibrosarcoma-derived cell lines, where it ensures proper maintenance of these repetitive regions.  Indeed, depletion of HDAC5 by RNAi resulted in the shortening of longer telomeres and homogenization of telomere length in cells that use either telomerase or an alternative mechanism of telomere maintenance.  Furthermore, we present evidence for the activation of telomere recombination on depletion of HDAC5 in fibrosarcoma telomerase-positive cancer cells.  Of potential importance, we also found that depletion of HDAC5 sensitizes cancer cells with long telomeres to chemotherapeutic drugs.  Cells with shorter telomeres were used to control the specificity of HDAC5 role in the maintenance of long telomeres.  HDAC5 is essential for the length maintenance of long telomeres and its depletion is required for sensitization of cancer cells with long telomeres to chemotherapy.”

15.  How Caloric Restriction and p53 can activate Telomere Maintenance Genes and DNA repair genes

Reference:Biological_systems_that_control_transcription_of_DNA_repair_and_telomere_maintenance_associated_genes

Summary: Caloric restriction mimetics such as resveratrol and 2-Deoxyglucose have been shown to activate both telomere maintenance genes and DNA repair genes.  These are activated via p53, using a DNA binding sequence found on the 5′ flanking regions of genes for telomere maintenance and DNA repair that do not have TATA-box or TATA-like elements.  The most common binding motif is a duplicated GGAA motif or a GC box.  The mechanism by which this occurs is the binding of p53 to these sites.  Caloric restriction appears to activate these genes via p53 phosphorylation, which is probably induced via the inhibition of cAMP phosphodiesterase, which in turn activates AMPK pathways.

16.  Relationship of telomere dysfunction and mitochondrial dysfunction

The “Axis of Aging” – Telomere Dysfunction and Mitochondrial Dysfunction are linked via DDR-activated p53 pathway

The 2012 publication Axis of ageing: telomeres, p53 and mitochondriareports: “Progressive DNA damage and mitochondrial decline are both considered to be prime instigators of natural ageing. Traditionally, these two pathways have been viewed largely in isolation. However, recent studies have revealed a molecular circuit that directly links DNA damage to compromised mitochondrial biogenesis and function via p53. This axis of ageing may account for both organ decline and disease development associated with advanced age and could illuminate a path for the development of relevant therapeutics.”

The “Axis of Aging” consists of the link between telomere shortening and mitochondrial dysfunction is now well established and explains why mitochondrial dysfunction is a universal “signature of gene expression change” with aging, as well as a universal “signature of metabolic change” with aging.  Specifically, telomere dysfunction due to oxidative stress, genotoxic stress, radiation, UV, or cell divisions can result in telomere shortening and/or uncapping of Shelterin proteins, resulting in the triggering of the DNA damage response (DDR).  The DDR triggers p53 activation which can then inhibit PGC-1, resulting in the inhibition of gene transcription for nuclear-encoded mitochondrial proteins.  Thus, this mechanism can induce high mitochondrial ROS generation, Warburg-type metabolism, increased oxidative stress leading to further negative effects such as DNA damage.  DNA damage can then lead to PARP-1 activation, which then can deplete nuclear NAD+, which then will stop SIRT1, SIRT6, and SIRT7 function.  This then leads to a pseudohypoxic state of the nucleus due to HIF-1a stabilization.  This then leads to inadequate TFAM, which then results in inadequate expression of mitochondrially encoded proteins for electron transport.  Thus, mitochondrial dysfunction can be triggered by a telomere-dependent, DDR-mediated activation of p53, which affects nuclear encoded mitochondrial proteins.  In addition, excessive DNA damage due to increased oxidative stress can deplete nuclear NAD, due to the requirements of PARP-1 for NAD as a cofactor.  As a result, there is inadequate NAD for SIRT1, SIRT6, and SIRT7 function.  This nuclear depletion of NAD then creates a pseudohypoxic nuclear state and a decrease in the expression of mitochondrial encoded proteins.

So, starting with telomere damage as the center it is possible to get to dysfunction of the mitochondria via a nuclear effect, and then the Warburg effect in the cytoplasm, which we know can lead to cancers and other pathologies which can lead to further telomere shortening.  There is no “center” in such a process, only points of reference which lead to other points of reference.  And many cofactors, state conditions and possible interventions can empower or retard such a process.

The villain factor here seems to be P53.  In the final section of this blog entry, we argue that P53 is also the hero for conveying health and strength in the face of stresses.

Reference: The 2011 publication Telomere dysfunction induces metabolic and mitochondrial compromise

Further, the catalytic component of telomerase hTERT can migrate out of the nucleus and if mutated create various forms of cell damage and dysfunction including in the mitochondria.  This is another pathway through which a telomere-related problem can propagate into other cell components including mitochondria.

The 2010 publication A mutant telomerase defective in nuclear-cytoplasmic shuttling fails to immortalize cells and is associated with mitochondrial dysfunction reports: “Telomerase is a reverse transcriptase specialized in telomere synthesis.  The enzyme is primarily nuclear where it elongates telomeres, but many reports show that the catalytic component of telomerase (in humans called hTERT) also localizes outside of the nucleus, including in mitochondria.  Shuttling of hTERT between nucleus and cytoplasm and vice versa has been reported, and different proteins shown to regulate such translocation.  Exactly why telomerase moves between subcellular compartments is still unclear.  In this study we report that mutations that disrupt the nuclear export signal (NES) of hTERT render it nuclear but unable to immortalize cells despite retention of catalytic activity in vitro.  Overexpression of the mutant protein in primary fibroblasts is associated with telomere-based cellular senescence, multinucleated cells and the activation of the DNA damage response genes ATM, Chk2 and p53.  Mitochondria function is also impaired in the cells.  We find that cells expressing the mutant hTERT produce high levels of mitochondrial reactive oxygen species and have damage in telomeric and extratelomeric DNA.  Dysfunctional mitochondria are also observed in an ALT (alternative lengthening of telomeres) cell line that is insensitive to growth arrest induced by the mutant hTERT showing that mitochondrial impairment is not a consequence of the growth arrest.  Our data indicate that mutations involving the NES of hTERT are associated with defects in telomere maintenance, mitochondrial function and cellular growth, and suggest targeting this region of hTERT as a potential new strategy for cancer treatment.”

Another important 2011 article relating telomere dysfunction to profound mitochondrial problems via activation of p53 and repression of PGC-1α and PGC-1β is Telomere dysfunction induces metabolic and mitochondrial compromise“Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1β, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1β promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere–p53–PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.”

The DNA-mitochondrial Axis of Aging

Also, this 2012 article characterizes DNA damage and mitochondrial decline as the “Axis of aging:” Axis of ageing: telomeres, p53 and mitochondria. “Progressive DNA damage and mitochondrial decline are both considered to be prime instigators of natural ageing. Traditionally, these two pathways have been viewed largely in isolation. However, recent studies have revealed a molecular circuit that directly links DNA damage to compromised mitochondrial biogenesis and function via p53. This axis of ageing may account for both organ decline and disease development associated with advanced age and could illuminate a path for the development of relevant therapeutics.”

These articles portray a direct link between telomere dysfunction and mitochondrial dysfunction, perhaps explaining why many old people are so cold-sensitive and tired much of the time.  Recapitulating the steps of what happens:

1. Telomere dysfunction =>
2. DNA damage response =>
3. p53 levels go up =>
4. High p53 inhibits the expression of both PGC-1a and PGC-1b
5. Genes for nuclear encoded mitochondrial proteins will NOT be expressed, as a result
6. Exercise cannot undo this and increase mitochondrial function.
7. Only deleting p53 or restoring telomere length will do this. This can be done with an Ornish/Blackburn lifestyle-type modification program

Telomeres, and why continuous, uninterrupted DNA damage response (DDR) signaling via p53 is so bad!

The reason is that you can’t turn such constitutive signaling off! Here is a 2012 article wrapping this up:Telomeres are favoured targets of a persistent DNA damage response in ageing and stress-induced senescence. “Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been implicated in the ageing process.  Telomere shortening has been shown to contribute to a persistent DNA damage response (DDR) during replicative senescence, the irreversible loss of division potential of somatic cells.  Similarly, persistent DDR foci can be found in stress-induced senescence, although their nature is not understood.  Here we show, using immuno-fluorescent in situ hybridization and ChIP, that up to half of the DNA damage foci in stress-induced senescence are located at telomeres irrespective of telomerase activity.  Moreover, live-cell imaging experiments reveal that all persistent foci are associated with telomeres.  Finally, we report an age-dependent increase in frequencies of telomere-associated foci in gut and liver of mice, occurring irrespectively of telomere length.  We conclude that telomeres are important targets for stress in vitro and in vivo and this has important consequences for the ageing process.”

By this point you should be getting the impression that P53 is a big-time villain when it comes to aging-related telomere attrition and damage that shows up in mitochondria and other systems.  So be this.  However, in the final section of this blog entry, we argue that P53 is also the hero for conveying health and strength in the face of stresses.

Again, in summary, including why telomerase-activating supplements cannot stop the axis of aging:

1.  Live single cell studies showed that 50% of the DDR foci are located at telomeres (I suspect that the rest are associated with aneuploidy-related foci (such as the pericentromeric region) the nucleolus (due to rDNA), and possibly repetitive noncoding DNA.

2.  Telomere-localized DDR foci occurs even if telomerase is activated (see article). This is why telomerase activating supplements such as Astralagus-based products do NOT turn off the DDR. (I.e. TA-65 or Product B). This is a very important point!

3.  Telomere-localized DDR foci occurs in mouse liver and gut cells regardless of telomere length.  This convinces me that the DDR signal from telomeres is NOT due primarily to telomere shortening or a lack of telomerase, but is due to a more complex “failure of telomere maintenance” that may be due to lack of SIRT6 activity, either due to a lack of nuclear NAD+ or a lack of SIRT6 gene expression secondary to SIRT6.  Remember that the promoter site for SIRT6 has a binding site for SIRT1 (I.e.  SIRT1 functions as a transcription factor for SIRT6 gene expression.

So there we have it.  a.  Yes, short telomeres can create all kinds of problems via DDR signaling, b.  Problems due to telomere DDR signaling can also happen when the telomeres are long, c. Telomerase mutations can create serious mitochondrial problems, c. Telomerase=promoter extender supplements cannot solve the problems, and d. We need to look at interactions such as involving NAD+ and SIRT1 and SIRT6 if we want to positively affect the “Axis of aging.”

17.  The catalylitic subunit of telomerase, telomerase reverse transcriptase (TERT) exercises a number of functions independent of telomere elongation.

The 2012 publication Telomere-independent functions of telomerase in nuclei, cytoplasm, and mitochondria reports: “Telomerase canonical activity at telomeres prevents telomere shortening, allowing chromosome stability and cellular proliferation.  To perform this task, the catalytic subunit (telomerase reverse transcriptase, TERT) of the enzyme works as a reverse transcriptase together with the telomerase RNA component (TERC), adding telomeric repeats to DNA molecule ends.  Growing evidence indicates that, besides the telomeric-DNA synthesis activity, TERT has additional functions in tumor development and is involved in many different biological processes, among which cellular proliferation, gene expression regulation, and mitochondrial functionality. TERT has been shown to act independently of TERC in the Wnt-β-catenin signaling pathway, regulating the expression of Wnt target genes, which play a role in development and tumorigenesis. Moreover, TERT RNA-dependent RNA polymerase activity has been found, leading to the genesis of double-stranded RNAs that act as precursor of silencing RNAs. In mitochondria, a TERT TERC-independent reverse transcriptase activity has been described that could play a role in the protection of mitochondrial integrity. In this review, we will discuss some of the extra-telomeric functions of telomerase.” (Italics ours)

The TERT subunit of telomerase and NF-κB p65 appear to be capable of up-regulating each other, at least in cancer cell lines.  In fact, this could be an important positive loop involved in oncogenic transformation.

The up-regulation of TERT by NF-kappaB appears to be mediated by c-Myc.

See

• Nuclear factor {kappa}B-mediated transactivation of telomerase prevents intimal smooth muscle cell from replicative senescence during vascular repair. “These results support a model in which vascular injury induces de novo expression of TERT in intimal SMCs via activation of nuclear factor κB and upregulation of c-Myc. The resumed TERT activity is critical for intimal hyperplasia.”
• NF-κB-mediated transactivation of telomerase prevents intimal smooth muscle cell from replicative senescence during vascular repair 
• Nuclear factor-{kappa}B-mediated regulation of telomerase: the Myc link.

The 2013 publication Human telomerase reverse transcriptase regulates MMP expression independently of telomerase activity via NF-κB-dependent transcriptionreports: “Telomerase plays a pivotal role in the pathology of aging and cancer by controlling telomere length and integrity.  However, accumulating evidence indicates that telomerase reverse transcriptase may have fundamental biological functions independent of its enzymatic activity in telomere maintenance.  In this study, the ectopic expression of human telomerase reverse transcriptase (hTERT) and its catalytic mutant hTERT K626A induced cancer cell invasion accompanied by the up-regulation of the metalloproteinases (MMPs) MMP1, -3, -9, and -10.  Both hTERT and hTERT K626A induced MMP9 mRNA expression and promoter activity in an NF-κB-dependent manner.  hTERT and hTERT K626A also regulated the expression of several NF-κB target genes in cancer cell lines.  Furthermore, both hTERT and hTERT K626A interacted with NF-κB p65 and increased NF-κB p65 nuclear accumulation and DNA binding.  A mammalian 1-hybrid assay showed a functional interplay between hTERT and NF-κB p65 that may mediate NF-κB-dependent transcription activation in cells.  Together, these data reveal a telomere-independent role for telomerase as a transcriptional modulator of the NF-κB signaling pathway and a possible contributor to cancer development and progression.”

The 2013 publication Transcriptional activation of hTERT through the NF-kappaB pathway in HTLV-I-transformed cellsreports: “In immortal cells, the existence of a mechanism for the maintenance of telomere length is critical.  In most cases this is achieved by the reactivation of telomerase, a cellular reverse transcriptase that prevents telomere shortening.  Here we report that the telomerase gene (hTERT) promoter is up-regulated during transmission of human T-cell lymphotropic virus type-I (HTLV-I) to primary T cells in vitro and in ex vivo adult T-cell leukemia/lymphoma (ATLL) samples, but not asymptomatic carriers.  Although Tax impaired induction of human telomerase reverse transcriptase (hTERT) mRNA in response to mitogenic stimulation, transduction of Tax into primary lymphocytes was sufficient to activate and maintain telomerase expression and telomere length when cultured in the absence of any exogenous stimulation.  Transient transfection assays revealed that Tax stimulates the hTERT promoter through the nuclear factor kappaB (NF-kappaB) pathway.  Consistently, Tax mutants inactive for NF-kappaB activation could not activate the hTERT or sustain telomere length in transduced primary lymphocytes.  Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays suggested that an increased binding of c-Myc and Sp1 is involved in the NF-kappaB-mediated activation of the hTERT promoter.  This study establishes the role of Tax in regulation of telomerase expression, which may cooperate with other functions of Tax to promote HTLV-I-associated adult T-cell leukemia.”

Cross-talk between sirtuins

As a final point, we can’t leave the six sister sirtuins of SIRT6 out of the telomere discussion.  The sirtuins are far from independent actors in-vivo, exhibiting much cross talk among themselves as well as with numerous other factors.  From Mammalian Sirtuins and Energy Metabolism (2011):“As one of the most important cellular metabolic sensors in cells, it has been speculated that the seven sirtuins coordinate with each other in various cellular compartments to actively monitor diverse environmental signals, modulating cellular metabolic activity, gene transcription, and genome stability, ultimately affecting aging.  Indeed, perturbation of the activity of one sirtuin has been shown to impact the activities of other sirtuin members.  For example, we reported that in mouse macrophages, deletion of SIRT1 leads to increased levels of chromatin-associated SIRT6 near the NF-κB binding sites, resulting in reduction of local acetyl-H3K9 levels, compensating for the hyperactivation of NF-κB induced by SIRT1 deficiency (119).  Interestingly, this compensatory effect between SIRT1 and SIRT6 does not appear to exist in hepatocytes.  Instead, deletion of SIRT1 causes 50% reduction of both SIRT6 mRNA and protein levels in the liver (97).  This is a result of SIRT1 binding to Foxo3a and nuclear respiratory factor 1 (NRF-1) on the promoter of SIRT6 in hepatocytes, directly promoting the expression of SIRT6 under both basal and fasting conditions (97).  Additional studies will be necessary to address whether the varying coordination patterns between these two nuclear sirtuins are related to the different metabolic profiles of macrophages and hepatocytes.  — Intensive crosstalk between nuclear and mitochondrial sirtuins have also been implied in the literature.  A recent study reported that SIRT3 is a transcriptional target of PGC-1α via an estrogen-related receptor binding element (ERRE) on its promoter (120).  As PGC-1α is a direct deacetylation substrate of SIRT1 (42), this observation suggests that SIRT1 could indirectly modulate the expression of SIRT3 through PGC-1α deacetylation.  Additionally, Nasrin et al.  recently showed that SIRT4 knockdown in hepatocytes induces an increase in fatty acid oxidation through SIRT1 (116).  Although molecular mechanisms underlying these connections remain to be defined, these findings suggest the existence of a sirtuin-network that may be pivotal in the maintenance of systemic metabolic homeostasis(ref).”

Towards a GUT, looked at from the end of the chromosome

Vince comment: We focus here on stress-response pathways, key to an emerging GUT in biology as pointed out in previous blog entries, particularly in the November 2013 Prospectus for a Grand Unified theory of Biology, Health and Aging.  We have often argued in this blog that stress-response pathways offer numerous opportunities for generating and enhancing human health and possibly longevity via hormesis.  But there has been an interesting paradox, which is that very many very different stresses can be used for the same end health objective, say preconditioning to prevent complications from a kind of surgery.  I just attended a meeting of the International Dose-Response Society, where the focus was on stress-induced pre-conditioning(ref).  I learned that among the stresses useful for surgical and other forms of preconditioning are exercise, gamma and x-radiation, pulses of laser light, pulsed DC current, heat, cold, restriction of blood circulation, calorie restriction, intermittent fasting, and hypoxia among others.  It seems that a common denominator in hormesis is stress and that all these and many other stressors activate the same pathways.  But the mechanisms through which this happens have been not known and has long been the subject of speculation.  In the case of remote preconditioning, stress on one part of the body can lead to healthy impacts on other remote and seemingly unrelated parts of the body.  In the conference I attended there was much discussion that there must be a “Factor X” which is the common mechanism for stress conditioning – an illusive factor capable of many pleiotropiceffects that marvelously work over a range of organisms and scale ranging from cells to organs to whole animals.  It is possible that in the following Jim Watson has identified what this factor X is – simply expression of P53 which in small doses exercises beneficial effects and in large doses kills cells.

A good theory must be “summarizable” or “expressable” in a simple form.  The following Summary is a descriptive form:

L = 2(GGAA). Where

L = Longevity

GGAA = transcription start site motif for all of the key genes involving DNA repair for both SS and DS breaks, genes involving the Shelterin proteins (Including Rap1), the hTERT gene, and the Genes affected by CR Mimetics.  Here is the “story:”

Once upon a time, evolution decided that yeast and bacteria were not stress resistant enough to live longer than a few weeks.  Evolution wanted to make organisms that had longer lifespans so evolution could blossom (I.e.  provide a longer time for genetic diversity to evolve).  So evolution decided to create a new, stress-responsive transcription start signal that would be activated by stress responses, such as running away from dinosaurs (exercise), surviving drought (starvation), blizzards (cold stress), and hot weather (the Ideal condition for evolution to flourish).  The old TATA box method of activating gene transcription had too much “baggage”.  The baggage was thousands of genes that were activated by “good times” such as lots of food (Insulin/IGF pathway), sex (hormones), and the lack of stress (I.e.  the housekeeping genes and genes responsible for growth and development).  So “Father Evolution” decided to make a new transcription start site for key genes that would be activated by stress.  This way, only the most robust members of the species that were stress resistant would survive.  For economy’s sake, evolution decided that this new transcription start site would be activated by the same transcription factor that would kill bad cells, such as Cancer cells.  So Father Evolution decided to choose p53 as the transcription factor to bind to this new motif.  After all, P53 being the Guardian of the Genome is the king of apoptosis when things in the cell are going too screwy.  So why not use P53 for a different signaling function that would trigger very different actions in response to stress when the cell is functioning fairly normally? The Motif was GGAA duplicated and Papa Evolution put this right by the Start site for the binding of RNA polymerase II.  He decided to put the genes responsive for DNA repair, Telomere stabilization, and epicentromeric stabilization all under the control of moderate, sublethal levels of P53 expression, below the 2nd NOEL point for p53.  That is for levels of P53 in the hormetic range, below the dose level required to tall a cell to blow its brains out.  (For every stress response there are two NOEL [No Observable Effect Level] points on the stress scale.  For stress level between zero and the 1^st NOEL point, the impact on the organism is negative.  Stress levels between the 1st and 2^nd NOEL points, in the hormetic range result in stress responses such that the net benefit to the organism is positive.  Stress levels above the 2^nd NOEL point result in a negative impact on the organism.)

Thus, evolution created the genetic equivalent of what The German philosopher Nietzsche said: “What does Not kill you (I.e.  P53) makes you stronger (I.e.  activated P53).”

Chapter 12 in the book New Research Directions in DNA Repair discusses the roles of the duplicated GGAA motif.  The authors of the book chapter are Uchiumi, Larsen, and Tanuma.  The title of the chapter, already mentioned. is Biological Systems that Control transcription of DNA Repair and Telomere Maintenance-associated Genes and the text of the chapter is available via this link. Here are the key points:

1. Resveratrol and 2DG are both caloric restriction mimetics which Induce promoter activities of 5′ flanking regions of genes encoding telomere maintenance factors, including Shelterin proteins and hTERT.  This occurs in both normal cells and cancer cells.
2. The protein, p53 is turned on by resveratrol and 2DG via AMPK phosphorylation, which then activates genes via the MAPK kinase ERK/2.
3. The P53 gene (TP53) also has a duplicated GGAA motif on the 5′ side of its transcription start site.
4. 76% of transcription start sites in the human genome have no TATA box or TATA like elements.

(“The TATA box (also called Goldberg-Hogness box)^[1] is a DNA sequence (cis-regulatory element) found in the promoter region of genes in archaea and eukaryotes;^[2] approximately 24% of human genes contain a TATA box within the core promoter.^{[3] ==} Considered to be the core promoter sequence, it is the binding site of either general transcription factors or histones (the binding of a transcription factor blocks the binding of a histone and vice versa) and is involved in the process of transcription by RNA polymerase. == The TATA box has the core DNA sequence 5′-TATAAA-3′ or a variant, which is usually followed by three or more adenine bases. It is usually located 25 base pairs upstream of the transcription start site.(ref Wikipedia))”

5. There are 174 different binding motifs at the transcription start sites in human genes.  TATA box or TATA like elements are one of the 174.
6. A large number of genes that maintain telomere health and DNA repair have a duplicated GGAA motif near their 5′ end of the transcription start site.
7. The protein p53 can bind to this duplicated GGAA motif.  This may be the fundamental mechanism of how many different cellular stressors such as radiation, UV light, calorie restriction, etc induce life span or health span increases via resistance to cellular stress. Specifically, this may be the “Hormetic pathway” of gene activation for genes involved with DNA repair and telomere maintenance.

The next equation for the GUT must be the mathematical description of the biphasic dose response curve.  The X-axis will be “dose” and the Y axis will be p53 gene activation via the double GGAA motif binding site at the 5′ end of genes that do the following:

1. Repair DNA – BRCA1, BRCA2, ATR, WRN, ATM
2. Control the cell cycle – RB, CDKN, etc.
3. Stabilize the end of the chromosome – Shelterin proteins, hTERT, subtelomeric DNA
4. Stabilize the center of the chromosome – I.e.  Centromere and Pericentromeric DNA.

The mathematical formula must include 2 NOEL points which can be moved to the left or right based on the following:

• Nrf2-mediated gene expression (I.e.  Antioxidant levels)

• FOXO3a-mediated gene expression (antioxidant and apoptosis factor levels)

• NF-kB mediated gene expression

• Etc

These factors would alter how much stress (I.e oxidative stress, radiation stress, toxin stress) we can handle before we pass NOEL point 2.  There are likely to be many other factors that could push the NOEL points to the Left or right, such as NAD levels, AMP levels, Acetyl-CoA levels, SAM levels, alpha- ketoglutarate levels, and FAD levels.  Thus the simple formula could have one “Placeholder” constant where each of these variables can be Inserted.  Thus, we keep the formula “Clean” and Simple

Recapitulation and Summary, re. the GUT:

Once upon a time, a new switch for turning on genes was created by evolution to ensure fitness of the larger species that needed to live longer for biodiversity in evolution to occur.  This new “start signal (GGAA x 2) was unique from the old switches (TATA) that turned on genes in response to plentiful times, such as growth factors, hormones, and plentiful food.  This new “switch” would respond to stressors that conferred “fitness” to the species, allowing them to survive famine, radiation, ultraviolet light, drought, deserts, winters, and infections.

The genes that this new switch (GGAAGGAA) would need to turn on would keep DNA repaired, keep the telomeres stable, and keep the centromeres stable.  Otherwise the organisms would have a short lifespan like yeast and bacteria.  The “trigger” for this new “switch” would also have to be able to trigger cell death (apoptosis) if the levels of cellular damage became too high (Ex: Too much DNA damage, telomeres too short, or aneuploidy).  For this reason, evolution decided to make p53 the trigger for both the GGAAGGAA motif at low levels of stress, whereas with high levels of stress, the classical DNA binding motif for p53 would be activated.

This Classical DNA binding motif for apoptosis or cell cycle arrest is as follows: (RRRCWWGYYY

Where R = A/G

W = A/T

Y = C/TThis motif can be separated by an optional spacer of additional base pairs to form a full binding site.

Ref: The 2013 publication Characterization of the p53 Cistrome – DNA Binding Cooperativity Dissects p53′s Tumor Suppressor Functions

This Classical p53 binding motif is found at all transcription start sites for genes involving either p53-mediated apoptosis or p53 mediated cell cycle arrest (cellular senescence).  To activate apoptosis, p53 must be acetylated at lysine 120 (K120) by the protein acetylase, Tip60.

References:

• Acetylation of the p53 DNA-Binding Domain Regulates Apoptosis Induction
• Tip60-Dependent Acetylation of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis

Apoptosis can be inhibited by SIRT1 or SIRT2, which deacetylate p53.

Ref: The 2009 publication SIRT Inhibitors Induce Cell Death and p53 Acetylation through Targeting Both SIRT1 and SIRT2

K120 acetylation activates p53 binding to only apoptosis genes, not to cell cycle arrest genes (I.e.  Cellular senescence).  Thus, apoptosis can be selectively inhibited by deacetylation of K120 on p53 without turning off p53 mediated cell cycle arrest pathway.

Thus, p53 at low doses confers stress resistance.  At higher doses, p53 induces apoptosis or cell cycle arrest.  The cellular decision to undergo apoptosis vs cell cycle arrest is made by the cell and not an extrinsic factor.  This decision depends on the acetylation status of K120.  SIRT1 or SIRT2 deacetylate K120 and therefore induce cell survival, but will not necessarily inhibit cell cycle arrest, since this can occur with a deacetylated p53.  These are the two Janusian faces of P53.

Image source

So, the “axis of fitness” and the “axis of aging” mentioned above are both due to P53 expression.  Which one will be choosen depends on a decision in the cell which depends on dose and the state of the cell.

This is the story of life.

On good vs bad

The more I learn, I now believe that ROS can be good or bad – it depends on the dose, where the ROS is being made (mitochondrial source vs plasma membrane NOX), and the levels of anti-oxidant enzymes produced by both Nrf2 and FoxO3a pathways.

The same for cellular senescence – it depends on what cells are undergoing senescence (white blood cells in wound healing, precancerous cells being shut off, or cells undergoing aging), where they are located (i.e.  chronic inflammation), and how much the STAT3-dependent SASP factors are being expressed (i.e.  the SASPs, like IL-6, IL-8, etc.)

The same for apoptosis – it depends on what cells are undergoing apoptosis (cancer cells vs normal embryonic development vs hippocampal neurons, etc.).  It also depends on whether replacement cells are being made from stem cells (Ex: skin, colon, muscle, brain, etc.  ).

The same for p53 – it depends on the dose (low confers resistance to stress, high confers either apoptosis or cellular senescence), and whether p53 is acetylated at K120 or not (i.e.  apoptosis vs cell cycle arrest).

The same for SIRT – it depends on whether the cell is normal or malignant (with normal cells, activating SIRT1 is good, with cancer cells, it could be bad).

The same for almost everything!

 

 

View the full article at Anti-Aging Firewalls