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Telomere length

Larry H. Bernstein, MD, FCAP, Curator

LPBI

New Enzyme Discovered for Sustaining Telomere Length

http://www.genengnews.com/gen-news-highlights/new-enzyme-discovered-for-sustaining-telomere-length/81251981/

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Researchers developed a novel assay to identify telomere length regulators and showed that ATM inhibition shortens telomeres, whereas ATM activation elongates telomeres. [Lee et al., 2015, Cell Reports 13, 1–10]

In the early years of molecular biology research, scientists studying chromosomal structure and composition noticed that the terminal ends of chromosomes, called telomeres, would gradually become shorter with each successive round of cellular replication. This process would continue until the chromosome reached a certain length, ultimately becoming unstable and causing the cell to die. Conversely, the scientists noticed that for certain genetic disorders, such as cancer, an abnormally long telomere length led to genome anomalies that were closely associated with the cancer phenotype.

In 1984, researchers Elizabeth Blackburn, Ph.D., and Carol Greider, Ph.D., who was at the time a graduate student in Dr. Blackburn’s laboratory, discovered the telomerase enzyme, which was responsible for maintaining the appropriate length of telomerase after chromosomal replication. Drs. Blackburn and Greider would go on to be awarded the 2009 Nobel Prize in Physiology and Medicine, along with Jack Szostak, Ph.D. for their work on molecular mechanisms of the telomerase enzyme.

Yet, even during their seminal work, the investigators quickly realized that other molecules besides telomerase must be involved in maintaining the protective caps at the end of chromosomes. Now, researchers at Johns Hopkins report uncovering the role of an additional enzyme crucial to telomere length and say the novel method they could be used to speed discovery of other proteins and processes that are involved in telomere stability.

“We’ve known for a long time that telomerase doesn’t tell the whole story of why chromosomes’ telomeres are a given length, but with the tools we had, it was difficult to figure out which proteins were responsible for getting telomerase to do its work,” explained Dr. Greider, professor, and director of molecular biology and genetics in the Johns Hopkins Institute for Basic Biomedical Sciences.

The findings from this study were published recently in Cell Reports through an article entitled “ATM Kinase Is Required for Telomere Elongation in Mouse and Human Cells.”

Understanding the mechanisms that are needed to lengthen telomeres has broad health implications, since shortened telomeres have been implicated in aging and diseases as diverse as lung and bone marrow disorders, while overly long telomeres are linked to cancer. Cells need a well-tuned process to keep adding the right number of building blocks back onto telomeres over an organism’s lifetime.

Unfortunately, until recently, the methods researchers used to study telomere length were extremely time-consuming, often taking months of work to study cells grown in vitro, searching for detectable differences in telomere length. However, Dr. Greider’s team developed a new tool for measuring telomere length in yeast. The idea was to artificially cut mammalian cells’ telomeres and then detect elongation by telomerase—a test that would take less than a day, and could be performed even if the blocked proteins were needed for cells to divide.

The new test, dubbed addition of de novo initiated telomeres (ADDIT) was used to observe an enzyme long suspected to be involved in telomere maintenance, ATM kinase. “ATM kinase was known to be involved in DNA repair, but there were conflicting reports about whether it had a role in telomere lengthening,” noted Dr. Greider.

The Hopkins researchers blocked the enzyme in lab-grown mouse cells and used ADDIT to find that it was indeed needed to lengthen telomeres. They confirmed their result by using the old, three-month-long telomere test, which lead to the same outcome.

Additionally, the team also found that in normal mouse cells, a drug that blocks an enzyme called PARP1 would activate ATM kinase and spur telomere lengthening. This finding has the potential to impact drug-based telomere elongation for treating short-telomere diseases, such as bone marrow failure.

Dr. Greider and her team were excited by their findings and plan to use ADDIT to find out more about the telomere-lengthening biochemical pathway that ATM kinase participates.

“The potential applications are very exciting,” stated lead author Stella Lee, Ph.D., postdoctoral fellow in Dr. Greider’s laboratory. “Ultimately ADDIT can help us understand how cells strike a balance between aging and the uncontrolled cell growth of cancer, which is very intriguing.”

Nobel Laureate Blackburn Named Salk Institute’s New President

Elizabeth H. Blackburn, Ph.D., a 2009 Nobel laureate who has specialized in telomere and telomerase research, was named the new president of the Salk Institute for Biological Studies today, effective January 1, 2016.

“The Salk is full of absolutely terrific people and brimming with great science. Building on its distinguished history and current success, I am delighted to be playing a role in continuing and growing its major contributions to science and health research,” Dr. Blackburn said in a statement. “I am truly honored to be asked to be the next president of the Salk Institute.”

Dr. Blackburn has been a non-resident fellow at the institute since 2001, where she has been one of a group of investigators that advise the institute’s leadership and play key decision-making roles in appointing and promoting Salk professors. In addition, she has been the Morris Herzstein Professor of Biology and Physiology in the department of biochemistry and biophysics at the University of California San Francisco (UCSF).

In 2009, she was named one of three co-winners of the Nobel Prize in Physiology or Medicine “for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.” Dr. Blackburn discovered the molecular nature of telomeres—the ends of eukaryotic chromosomes that serve as protective caps essential for preserving the genetic information—and co-discovered the ribonucleoprotein enzyme telomerase.

Those discoveries helped launch new research around telomeres and telomerase, both believed to play central roles in aging and diseases that include cancer.

In addition to the Nobel Prize, Dr. Blackburn has received nearly every major award in science, including the Lasker, Gruber, and Gairdner prizes. In 2007, she was named to the TIME 100 yearly list of the world’s most influential people. Dr. Blackburn is also a member of the National Academy of Sciences, the National Academy of Medicine, and the Royal Society of London.

Dr. Blackburn was born in Hobart, Tasmania, Australia, to a family of doctors and scientists. Her parents were both family physicians, while her grandfather and great-grandfather were geologists.

Inspired by her fascination with animals and a biography of Marie Curie, Dr. Blackburn chose to also pursue a career in science. She earned her B.Sc. degree in 1970 and her M.Sc. degree in biochemistry, both from the University of Melbourne. She earned her Ph.D. in molecular biology from the University of Cambridge in 1975, then conducted postdoctoral research in molecular and cellular biology at Yale University from 1975 to 1977.

ATM Kinase Is Required for Telomere Elongation in Mouse and Human Cells

Vertebrate telomeres are repetitive TTAGGG DNA sequences located at the ends of chromosomes, which protect the coding regions of DNA. In mammalian germline cells and ∼85% of cancers, telomere length is maintained by the dimeric ribonucleoprotein telomerase, which catalyzes the addition of TTAGGG repeats to counteract telomere shortening and cellular senescence (Shay and Bacchetti, 1997, Kim et al., 1994, Wenz et al., 2001). The minimal catalytic core of human telomerase consists of the telomerase reverse transcriptase protein (hTERT), telomerase RNA (hTR), and the protein dyskerin (Cohen et al., 2007).

The differentiation of telomeres from broken chromosome ends is conferred by a family of six telomere-specific binding proteins collectively termed “shelterin” (de Lange, 2005). This complex consists of the double-stranded binding proteins TRF1 and TRF2, the single-stranded binding proteins POT1 and TPP1, the bridging protein TIN2 that links these two groups of proteins, and Rap1 (reviewed in Palm and de Lange, 2008). TRF1 protects the telomere and negatively regulates telomerase-mediated telomere lengthening (van Steensel and de Lange, 1997, Smogorzewska et al., 2000, Ancelin et al., 2002, Karlseder et al., 2002). TRF1 also facilitates the progression of the replication machinery; deletion of TRF1 increases replication fork stalling, leading to ATR kinase activation and a “fragile telomere” phenotype (Sfeir et al., 2009, Martínez et al., 2009). The TRF1-mediated repression of the ATR response requires recruitment of the shelterin components TIN2 and the TPP1/POT1 heterodimer (Zimmermann et al., 2014).

TPP1 and POT1 also have roles in mediating telomere-length regulation. A surface on the N-terminal oligonucleotide/oligosaccharide-binding (OB) domain of TPP1 termed the TEL patch activates telomerase by stimulating telomerase processivity and providing a direct binding site for telomerase recruitment to telomeres; mutation of the TEL patch can lead to telomere shortening syndromes characterized by bone marrow failure (Abreu et al., 2010, Nandakumar et al., 2012, Zhong et al., 2012, Kocak et al., 2014, Guo et al., 2014, Dalby et al., 2015). Additionally, mutation analyses at sites independent of the TEL patch have implicated TPP1 as part of a telomere-length-dependent feedback loop that regulates telomere-length homeostasis (Sexton et al., 2014). A mutant form of POT1 that abrogates binding to single-stranded DNA (POT1ΔOB) deregulated telomere-length control (Loayza and De Lange, 2003), indicating that the DNA-binding capability of POT1 is vital as a negative regulator of telomere length. The impact of human POT1 on telomere length is complex, since both depletion and overexpression of POT1 lead to telomere lengthening (Ye et al., 2004, Veldman et al., 2004, Colgin et al., 2003, Armbruster et al., 2004). POT1 function as a positive or negative regulator of telomerase activity at the telomere depends on its position of binding relative to the DNA 3′ end and is also modulated by its binding partner, TPP1 (Zaug et al., 2005, Wang et al., 2007, Lei et al., 2005, Kelleher et al., 2005).

Telomerase action at the telomere is highly regulated; it preferentially elongates the shortest telomeres, and recruitment of the enzyme complex to the telomere occurs in mid-S phase of the cell cycle (Bianchi and Shore, 2007, Britt-Compton et al., 2009,Teixeira et al., 2004, Hemann et al., 2001, Tomlinson et al., 2006, Jády et al., 2006). In both budding and fission yeasts, the preference of telomerase to extend the shortest telomeres requires the activity of Tel1, the yeast homolog of human ATM (Sabourin et al., 2007, Hector et al., 2007, Arnerić and Lingner, 2007). ATM and ATR are kinases within the phosphatidylinositol-3 kinase-related kinase (PIKK) family, which regulates cellular responses to DNA damage, mRNA decay, and nutrient-dependent signaling (Lovejoy and Cortez, 2009). Activation of these DNA damage pathways is dampened at telomeres; in mammalian cells, TRF2 represses activation of ATM while POT1 represses ATR (Karlseder et al., 2004, Celli and de Lange, 2005, Denchi and de Lange, 2007, Guo et al., 2007, Okamoto et al., 2013). Nevertheless, there is a large amount of evidence that their yeast homologs play a positive role in facilitating telomere extension by telomerase (Moser et al., 2009, Moser et al., 2011, Yamazaki et al., 2012, Churikov et al., 2013).

It is not known whether the role of the ATM and ATR pathways in recruiting telomerase is conserved in mammals. Although ATM deficiency or ATR mutations can induce telomere shortening or instability in human and mouse cells (Metcalfe et al., 1996, Smilenov et al., 1997, Wong et al., 2003, Wu et al., 2007, Pennarun et al., 2010), these kinases were reported to be dispensable for elongation of the shortest telomeres in mouse models (Feldser et al., 2006, McNees et al., 2010). Also, immortalized cell lines from human patients with ATM mutations are able to maintain their telomeres with telomerase, albeit at short lengths (Sprung et al., 1997). Nonetheless, there is evidence that TRF1-mediated telomere-length regulation in human cells involves ATM. Inhibition of human ATM resulted in increased TRF1 at the telomere, and phosphorylation of TRF1 on serine 367, an ATM/ATR target site, reduced the interaction of TRF1 with telomeres and abrogated its ability to inhibit telomere lengthening (McKerlie et al., 2012, Wu et al., 2007).

In this study, we report that both ATM and ATR are required for the recruitment of human telomerase to telomeres.

ATM and ATR Are Both Required for the Presence of Human Telomerase at Telomeres

ATM Effect on Telomerase Recruitment Is Partially Mediated by TRF1

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Figure 7

Model for ATM and ATR Involvement in Human Telomerase Recruitment to Telomeres

(A) During S phase, ATM and/or other PIKKs phosphorylates TRF1 at S367, which leads to partial dissociation of TRF1 from telomeres (McKerlie et al., 2012, Wu et al., 2007). Depletion of TRF1, together with its protein partners Tin2, TPP1, and Pot1, causes telomeric replication forks to stall, leading to recruitment of RPA and ATR (Sfeir et al., 2009, Martínez et al., 2009, Zimmermann et al., 2014). ATR phosphorylates an unknown substrate to mediate telomerase recruitment. Replication fork stalling caused by aphidicolin treatment also leads to telomerase recruitment (this study) and telomere elongation (Sfeir et al., 2009).

(B) An independent role of ATM and ATR involves stimulation of telomerase assembly, which is a prerequisite for telomerase localization to telomeres. This model does not preclude involvement of other unidentified substrates of ATM, ATR, and other PIKKs.

……..

We demonstrated that one TRF1-independent function of ATM is its impact upon the ability of hTR and hTERT to assemble into a functional enzyme complex (Figure 6), which is a prerequisite for localization of hTR to telomeres (Tomlinson et al., 2008). ATR also plays a role in assembly of human telomerase; we do not know if the substrates of these two kinases in this process are the same. This role is reflected in a substantial decrease in the amount of hTR recovered after hTERT immunoprecipitation and in the total immunoprecipitated telomerase activity following ATM and ATR knockdown. The specific activity of telomerase remains unchanged, demonstrating that both ATM and ATR have no effect on telomerase catalytic activity, consistent with results in yeast (Chan et al., 2001). No consensus PIKK phosphorylation motifs exist in the RNA-binding domain of hTERT, implying either that ATM or ATR can mediate telomerase assembly by targeting regions not in the RNA-binding domain or that they can regulate telomerase assembly by phosphorylating unknown substrates (Figure 7B).

Our data support a model incorporating multiple roles for ATM and ATR in the presence of human telomerase at telomeres (Figure 7). One pathway involving both ATM and ATR is mediated by phosphorylation of TRF1 and its removal from the telomere, leading to replication fork stalling in telomeric DNA, which acts as a trigger for telomerase recruitment. A second pathway involves the role of ATM and ATR in facilitating telomerase assembly; additional phosphorylation targets of ATM, ATR, and other PIKKs in the telomerase recruitment process may remain to be identified. These data reveal that although it is important for telomeres to repressDNA damage signaling in order to avoid deleterious fusions, telomeres have also evolved the ability to carefully exploit aspects of DNA damage signaling pathways to regulate telomerase presence at the telomere. Increased understanding of regulation of telomerase assembly and access to the telomere may provide valuable insight in the process of developing highly specific cancer therapeutics.