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Titia de Lange (Rockefeller U.) 1: Telomeres and human disease

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My name is Titia de Lange. I operate at the Rockefeller College in New York, as well as today I'' m mosting likely to speak with you about telomeres, their framework, their function, as well as their duty in human illness and especially in the development of cancer cells in a human system, and also a set of conditions that are triggered by telomere dysfunction. So, telomeres are completions'of chromosomes. They ' re aspects that safeguard chromosome ends and they ' re defined by a particular sequence that is highlighted, here, in this metaphase spread of human chromosomes by a probe that can detect that series. So, allow'' s very first check out the series of telomeres in detail. Telomeres are made up of a really long variety of a simple repeat. The sequence is TTAGGG, as well as this sequence is repeated on and on, along the telomeric DNA, which can be long. In human cells, it can be as long as 10 kB; in mouse cells it can be as long at 50 kB, lots of thousands of copies of this straightforward TTAGGG repeat. And after that, at the actual end of the telomere, there is a special framework, there is a 3' ' projection, that you can see below, of the single-stranded G-rich strand of the telomere that sticks out, so the 5' ' strand is shorter than the 3' ' strand.And this is fairly lengthy, it'' s 50-300 nucleotides, and also this framework is really important for telomere function. Telomeres are needed at the chromosome end to resolve one crucial issue that cells have and also I refer to this issue as the end-protection issue. The basis of this issue is the following: cells have an extremely cautious means to recognize as well as reply to breaks in their DNA. So, if there is a break somewhere in the chromosome, the cell will certainly acknowledge this break as well as trigger what is called the DNA damage reaction. The DNA damage reaction is guided by two kinases, the ATM as well as the ATR kinase, which each can be turned on at this DNA end, and afterwards via a collection of phosphorylation events direct the cell to quit the cellular division, as well as they manage DNA repair work. Currently, if you take a look at a telomere, obviously it looks really much like a double-stranded break, however this structure ought to not turn on the DNA damages action, since if the atm machine and ATR kinases became energetic at our all-natural chromosome ends, the cells would certainly never ever divide and also cells would attempt to fix the ends of their chromosomes, which would certainly result in genome instability, as we will see later in the talk.So, the major feature of telomeres, the important feature of telomeres, is to make certain that they are overlooked by the DNA damage action. So, exactly how do telomeres do this? Well, the telomeric DNA itself doesn'' t'do quite. It ' s bad at repressing the DNA damages reaction. It requires to operate by recruiting onto the telomeric DNA a protein complicated that we'' ve called shelterin, due to the fact that it shelters the chromosome ends. Shelterin binds the whole time the telomeric DNA with the action of two healthy proteins in the complex, TRF1 and TRF2. These healthy proteins bind to the double-stranded TTAGGG repeats. They then assist to hire into the telomeric complex a protein called POT1, the red guy that you see right here. As well as POT1 has the capacity to bind to the single-stranded telomeric DNA. And afterwards there are various other proteins in the facility, TPP1, TIN2, and also Rap1, that hold the facility together. Shelterin is really bountiful, there are lots of numerous copies of shelterin at every telomere, as well as every cell reveals these proteins, so in every cell of our body the telomeres are covered by this healthy protein. The facility is very saved in animals, however it'' s not discovered in some various other microorganisms, yeast or plants, or it ' s different.But since it is conserved in creatures, we have had the ability to use computer mouse version systems, computer mouse cells, to study the complicated carefully. Telomeres also consist of other proteins that I never ever placed it in in my slides since it'' s as well tough to attract. They include nucleosomal chromatin, so the telomeric DNA is concluded right into nucleosomes– you can comprehend much more about nucleosomes if you listen to David Allis' ' talk– as well as telomeres have shelterin accessory healthy proteins, these are proteins shelterin employees onto the telomere in order to fulfill its feature. So, shelterin blocks these two major signal transducing pathways that react to double-stranded breaks. It has to block the ATM kinase path as well as the ATR kinase pathway, so how does this happen? So, the atm machine kinase pathway is turned on by DNA ends.The DNA end is bound by a trimeric healthy protein complex, MRN. This complicated stably connects with the DNA end at the DNA break and afterwards it hires onto the DNA finish the atm machine kinase. And also in methods we put on'' t completely comprehend, the ATM kinase after that becomes triggered. When the atm machine kinase is active, it phosphorylates healthy proteins in the vicinity of the break in a very details way. As well as one of the proteins that'' s a target of atm machine is a histone variant, H2AX.This histone variant is located throughout the chromatin, in the nucleosomes, so whenever a break takes place there is some H2AX in the area. The phosphorylated kind of H2AX now works as a platform for the employment of healthy proteins that are mosting likely to comprise the DNA damage foci. The very first one is MDC1, which initiates a ubiquitylation waterfall that brings about the recruitment of various other healthy proteins, including this protein, right here, 53BP1, which is a healthy protein we'' ll use as a marker for DNA damage in this talk.These DNA damages
foci assist to both enhance the atm machine kinase signal as well as to direct the repair work at the DNA break in suitable means, relying on the cell cycle phase in which the break takes place. So, this is the repair component of the atm machine kinase, yet it likewise strikes cell cycle progression. The ATM kinase will certainly phosphorylate an effect kinase called Chk2. Chk2 after that phosphorylates various other proteins, consisting of p53 and also the Cdc25 phosphatase, and also together these phosphorylation events result in the inactivation of the cyclin-dependent kinases that normally will aid cells to proceed with the cell cycle. So this is the basis of the cell cycle apprehension. Certainly, none of this ought to take place at telomeres, despite the fact that the telomeres look significantly like an MRN binding site, shelterin requires to close down this program. How does it do this? Our current design is that shelterin manages to block the ATM kinase signaling pathway at natural chromosome ends by renovating the telomere right into what we call a t-loop. We uncovered, together with Jack Griffith, who operates at the University of North Carolina, Chapel Hill, who is a specialist electron microscopist, that telomeres, the telomeric DNA from humans and also, in this instance, what is revealed right here, from mouse cells, is not straight, however finishes in a lariat structure.And detailed analysis of these structures, these are protein-free DNA frameworks, suggested to us that telomeres make use of that single-stranded overhang that I discussed earlier to make this t-loop structure. The single-stranded overhang strand attacks right into the double-stranded component of the telomere and it ' s shelterin that does this'task, as well as in doing so it primarily hides the chromosome end from any type of activity that might potentially load on the chromosome end. So, because of this, the end is hidden from the DNA damage response. That is, while at a damaged end MRN can bind and turn on the ATM signaling cascade. At the telomeres MRN can not discover a binding website, and also therefore the ATM kinase doesn'' t ended up being activated. So, this is how shelterin offers with the ATM risk at chromosome ends. Just how about the other transducer, the ATR kinase? The ATR kinase is dedicated to discovering DNA sores in the genome once they have actually achieved single-stranded DNA.So, a double-stranded break can be absorbed by a nuclease, leading to a lengthy single-stranded overhang that can be identified by the ATR kinase, and also it does it with the company of a very plentiful single-stranded DNA binding protein, RPA. RPA is throughout the cell, will gather at single-stranded DNA, and also it acts in conjunction with a ring-shaped particle, 9-1-1. Once these proteins are loaded at a broken DNA end that has actually been resected, they hire and also activate the ATR kinase through a number of communicating factors.And currently this energetic ATR kinase, at the website of damages, will do basically the very same thing that ATM does: it phosphorylates H2AX. And also H2AX after that hires the proteins that create the DNA damage foci. And also ATR, like atm machine, phosphorylates a downstream effect kinase, that will impinge on the cell cycle progression through the phosphorylation and also devastation, or inactivation, of the Cdc25 phosphatase. How did telomeres avoid this kinase from mistakenly recognizing natural chromosome finishes as sites of DNA damage? After all, the telomere includes the exact framework that turns on the ATR kinase: it has a single-stranded DNA that extends 3 ', it has a 5 ' double-stranded/single-stranded change. It has what it requires to turn on the ATR kinase. Shelterin has this remarkable technique in that it consists of POT1. POT1 is a single-stranded binding protein and by going to the telomere and also covering the entire single-stranded component of the telomere, it protects against the loading of RPA onto the telomeric DNA.So, there ' s not just one POT1 sitting there, however as a matter of fact the whole single-stranded part of the telomere is covered by POT1, and also due to that RPA merely can ' t bind and also consequently will not trigger the ATR'kinase. So, currently we ' ve seen how shelterin has two major methods through which it avoids the activation of these DNA-damage reaction paths at telomeres. So, one issue is addressed. But in truth there is a second problem with telomeres.And that is the trouble that telomeres shorten with cellular division.
So, in this Southern blot, an old-fashioned method that my lab still proudly utilizes, we check out the telomeres of key human cells that are undertaking numerous cellular division– they ' re expanded in society– and after that what we do is we cut the telomeric DNA loose from the remainder of the chromosome, different it based on size on a gel, and after that we detect the telomeres with a probe that can anneal to the telomeric DNA. And you see that the human chromosome contains telomeres that are extremely heterogeneously sized, but as you look over the population increasings, this smear of telomeric DNA ends up being much shorter. There are 2 bands in this blot that don ' t come to be shorter, they act as a remarkable control and they are chromosome-internal websites, where there is a little of telomeric DNA. Why does this happen? Well, there are two processes that generate this reducing. The first is the well-known end-replication trouble. It ' s a trouble linked with DNA polymerases that try to duplicate a straight chromosome. DNA polymerases constantly synthesize DNA from 5 ' to 3 ', so they use a 3 ' hydroxyl to add a nucleotide as well as an additional one as well as an additional one, so they relocate the 3 ' direction.So, when telomeres reproduce from an origin somewhere in the chromosome, moving the duplication fork, vacating via the telomere, one polymerase has not a problem, that ' s the polymerase that does leading strand synthesis, as well as it just zooms bent on the end. However the various other strand can not be duplicated here. It has actually to be reproduced in the various other instructions, and that is what is called lagging-strand synthesis. And since the polymerase goes from 5 ' to 3 ', it requires to do it in little steps, and it needs a 3 ' hydroxyl to begin each of these little steps. As well as in this delayed strand synthesis, it ' s the RNA primers that are put down by primase, component of the replication equipment, that allow delaying strand synthesis to synthesize DNA in this instructions. There you go … as well as before you know it you have almost replicated the entire hair, the RNA primers are currently gotten rid of, as well as you can fill up in this little gap, here.Aha. However below is the trouble … at the very end of the telomere, there is a void that can ' t be filled out, due to the fact that this space has a 5 ' end as well as there ' s no polymerase that can enter this means. So, this is an essential trouble, initial talked about by Jim Watson, with DNA replication of linear chromosomes, and also it results in steady as well as dynamic sequence loss. Yet in people cells and also in mouse cells, there is an added problem.Because the product of this DNA synthesis system has actually not recreated the structure that we require at telomeres– there ' s no long 3 ' overhang. So, what shelterin does, it recruits onto the telomere nucleases that are going to eat in the 5 ' ends, both 5' ' ends, and also digest away completion to make this long overhang. And there you go, currently we have reconstituted the telomere in its initial structure, yet in doing so the nucleases have removed fairly a great deal of DNA from each chromosome end that was recently replicated.So, together, the end-replication problem as well as this eating in of the telomere to make the correct structure causes the rapid loss of telomeric DNA that you see in this experiment.
Currently, there are consequences. If you lose telomeric DNA, at some point one or two telomeres, a heterogeneously-sized population of telomeres, will shed its ability to repress the ATM as well as ATR kinases, and after that cells will arrest. This is normally described as the Hayflick Restriction. Len Hayflick first observed this when he was culturing primary human cells, that after 50 population increasings or so, the cells would go into what is called senescence.Senescence is an incurable phase for cells; they can ' t get out of it. They quit dividing as well as they show a great deal of phenotypic changes, consisting of a pen that we ' ve discolored blue, below, in this particular experiment.

So, what is taking place here is that as the cells'are separating as well as their telomeres obtain much shorter and shorter, for a long period of time there are no effects, once a couple of telomeres have lost their capacity to quelch the atm machine and also ATR kinases, they will certainly signify and trigger a program that causes long-term cell cycle apprehension, senescence, or, in some cells kinds, it can bring about apoptosis. So, this is a problem, since our telomeres reduce, which has effects. Just how can we live? Just how can we breathe? Exactly how can we generate progeny if this goes on in our germline? Well, it really does not take place in our germline. In our germline, there is an enzyme, telomerase, that solves this trouble and also includes series back onto the telomere. Telomerase is a reverse transcriptase, it was discovered by Carol Greider and also Liz Blackburn, as well as they obtained the Nobel Reward for this exploration, together with Jack Szostak. Telomerase is a reverse transcriptase, so it copies an RNA into DNA.So, this big orange point below, that ' s the RT, the reverse transcriptase, and afterwards you see the RNA there, and also this RNA has in it a really brief series that encodes the telomeric DNA, and after that it has some added elements that aid in biogenesis, that'I ' ve tinted in blue right here. So, shelterin is recruited to the telomere by … sorry, telomerase is hired to the telomere by shelterin as well as after that it can align its RNA layout series with the 3 ' end of the chromosome, as well as will after that utilize the 3 ' end of the chromosome to include even more telomeric repeats, thus. There is one repeat. This is just 6 nucleotides, yet it can translocate on the DNA and add a growing number of as well as more repeats, till it pushes me out of the slide. So,' telomerase can solve the loss of telomeric DNA by making up for it by including DNA back, of the specific right series, onto the chromosome end, and it does so in stem cells and in the germline. So, both in the germline and also some stem cell areas, telomerase is expressed as well as it keeps the telomeres, undamaged. Nevertheless, during normal growth of our cells, of the somatic cells, the cells that compose the tissues in our body, telomerase is silenced.Somatic cells do not reveal the third element of the enzyme, the 3rd marketer is switched over off in manner ins which aren ' t fully understood, and also, consequently, all our cells reveal this telomere shortening program that I described to you. Now, as I claimed, telomere shortening will certainly set up a proliferative barrier. Whatever cell kind it happens'in, eventually those cells will lose telomeric DNA to an extent where they will trigger the ATM as well as ATR kinases, as well as that ' s completion of that cell lineage.So, why? Why do we have a system in our cells, in our cells– not in the germline, not in the stem cells, in our cells– that ultimately will obstruct the cells from proliferating. Well, it ' s usually thought that this has actually developed as a lump suppressor pathway. If you consider it, if throughout the formation of bust cancer, early on, an epithelial cell in a mammary gland experiences a mutation in a gene that is'associated with cell signaling, that produces a circumstance in which the cell starts multiplying inappropriately, that cell duplicate will go through a variety of cellular division, however considering that it doesn ' t consist of telomerase it will stop splitting after a variety of PDs. The DNA-damage feedback will be activated, p53 as well as Rb will start running, and also you get induction of apoptosis or senescence. This is believed to be a really effective growth suppressor path that can quit tumorigenesis in bust cancer in a very beginning, in a hyperplasic phase. Nonetheless, like all tumor suppressor paths, sometimes and also unfortunately, it fails.And, if you consider it, one method which this path can fail in attempting to stop insipient cancer cells from expanding out, is with the loss of the 2 tumor suppressor paths, p53 as well as Rb, which are regularly shed if you search in scientifically obvious cancer cells. And now something really different happens … the cells continue to divide without telomerase and also continue to shed telomeric DNA, as well as you produce a situation in which the cell includes not just one or two, yet lots of inefficient telomeres, and this state is described as telomere situation. And in telomere crisis, what occurs is that the inefficient telomeres begin merging together, since they have ended up being so bountiful that they can find each various other. And it ' s fundamental DNA repair work that performs this work. It ' s non-homologous end signing up with that does this. This is a repair work pathway that normally gets turned on at broken DNA finishes, it ' s a ligation reaction and it simply takes 2 DNA ends and also merges them together, whatever the sequence is. As well as this extremely exact same response is repressed at telomeres, like the atm machine and the ATR kinase pathways, by shelterin. So, normally, at telomeres, there is no non-homologous end signing up with, however when cells have actually experienced recurring telomere shortening causing the state where now, not simply one, yet lots of telomeres contain too little shelterin, since they ' re also brief, and therefore do not block DNA fixing, non-homologous end joining can happen at the chromosome end.So, what happens under those conditions is that the non-homologous end signing up with pathway finds 2 telomeres, because there are currently numerous and also they can find each various other in the center, and ligates them together. And this is a disaster, due to the fact that the chromosome that has actually been created by this response now has 2 centromeres, as well as centromeres operate to permit chromosomes to set apart in mitosis by affixing to the spindle apparatus, yet the dicentric chromosome– that has two centromeres– can misattach throughout mitosis, as shown below, and result in a trouble in cell division. So, if you have a cell with dicentric chromosomes, similar to this one that we shot in our laboratory– movie made by John Maciejowski in my laboratory– you can see the cell having problem with this connection between both chromosome clusters. It ' s been researched in detail what occurs to these events, and also we currently understand that the telomere dilemma circumstance, with their lots of dysfunctional telomeres, and the issues that dicentrics produce, can result in an entire variety of genome changes. First of all, we know that translocations can result from this circumstance– this was revealed by Rhonda Pinot, modeling in the mouse.We know that regional amplification can occur therefore of telomere crisis. We ' ve lately revealed that two brand-new sensations in cancer genomes, chromothripsis as well as kataegis, can develop in telomere situation. Chromothripsis is a substantial, unexpected shattering of a whole component of a chromosome and after that the pieces are put back with each other in the incorrect alignment as well as order. And also kataegis is a tornado of anomalies that occur at the break points of these chromothriptic occasions. These two can be due to telomere dilemma. And finally, we ' ve found that the recurring DNA damage signaling originating from the several useless telomeres in situation … despite the fact that the cells don ' t arrest, they still experience the atm machine and ATR signaling … this can lead to cells slipping up in the cell cycle, where they put on ' t synthesize DNA and after that divide, they synthesize DNA, put on ' t divide, manufacture DNA once again, and after that divide.And this brings about an endoreduplication,'an increasing of the genome, and also this is called tetraploidization. This is a phenomenon that ' s fairly general in cancer; many cancer cells genomes show proof of tetraploidization, as well as tetraploidization has actually been revealed to promote tumorigenesis. So, just as an example of these kinds of alterations, I show here a metaphase karyotype of a cancer cells cell line. This cancer cells cell line shows clear evidence of tetraploidization. This is a SKY evaluation, in which people have actually tinted each chromosome with a different color, as well as aligned them … they don ' t loss out of the cell in metaphase such as this … and also you can see that, for example, chromosome 4 … oh, sorry, chromosome 2, below, in red … it ' s existing in 4 duplicates, as is chromosome 3, as is chromosome 4. This presence of 4 copies of one chromosome indicates that this cancer cells initially went via a tetraploidization event, as well as after that it sheds chromosomes conveniently because tetraploid cells are quite unpredictable. In this metaphase karyotype, you can also see that there are many translocations in these cells, where shades from one chromosome are currently connected to shades of one more chromosome.So, this type of genome instability can be referred to telomere dilemma; that ' s what we have suggested and also we ' re studying this better. It ' s challenging to research, since telomere dilemma, once more, like the lump reductions, happens extremely early in tumorigenesis. Making use of the example of bust cancer, it has been shown that telomere situation takes place in DCIS– ductal carcinoma sitting– the earliest phase of bust cancer cells that is really rather not likely to take place to be a dangerous disease. So, in that state the telomeres have actually

lacked their safety ability, cells have lost the tumor suppressor paths, there ' s no telomerase yet, as well as they show massive genome instability. But these cells are not going to grow out into a tumor. Cells in telomere crisis are deeply unhappy, they have a hard time to separate, as well as they will just offer increase to a real growth if telomerase ends up being activated, and also it ' s in telomere situation that there ' s really strong choice for awakening of this enzyme. Telomerase is required to resynthesize the telomeres, to recover them.This will certainly lead to a secure genome, cells now can divide suitably, as well as they will certainly bring out this greatly reorganized aneuploid genome that can promote tumorigenesis.And certainly, telomerase is attended be triggered in > 85%of cancers cells. The other cancers cells have a different means to maintain telomeres. In a few of the cancers, telomerase is turned on by mutating the promoter of telomerase, of the 3rd element of telomerase, damaging through the silencing program that usually is set up during development. So, what I discussed with you is quite a facility, twin duty of telomeres in cancer. It begins out with configured telomere shortening, in our regular somatic cells. This is an advantage because it will develop a proliferative barrier for the cells, and it will certainly stop tumorigenesis.But, in unusual situations, when cells damage via this, the exact same telomere shortening program can lead to genome instability, which of course can result in oncogenic changes and also promote tumorigenesis. And then, in the end, telomerase requires to be activated for the cells to come out of this and also form a clinically appropriate growth. Now, this is one element of telomere biology in human condition. I desire to end by speaking about a 2nd disease-relevant facet of telomeres. And those are the inherited illness that are caused by telomere dysfunction. It ' s now remove that there is a large collection of conditions that are inherited that are due to brief telomeres.In some situations, there are developmental irregularities that play out in utero. In the disease that first brought this concept to light, dyskeratosis congenita, the short telomeres generate bone marrow failure early in life, extremely significant, yet in grownups they ' re now also situations of aplastic anemia, idiopathic lung fibrosis, as well as liver cirrhosis, that are ascribed to a problem in telomere upkeep. So, these illness are plainly acquainted, effectively defined, in huge family pedigrees, and also they ' re constantly because of telomeres being as well short.So, if you consider telomere size in the human population, telomeres reduce a little bit with'age … it ' s very little, but they do shorten a bit. What a lot of people wear ' t realize is that there ' s massive variation in telomere length, so at any type of offered age the telomeres can be quite lengthy or brief, depending on the individual. Yet even in the face of this variation in telomere size, it ' s very clear that the people having these conditions, tinted here, have very short telomeres, and these exceptionally brief telomeres, which they obtain at birth, naturally will cause early cell cycle apprehension or cell fatality in the tissues, simply similarly I explained this to you for main human cells expanded in culture.So, the illness that I explained are due to mutations in 2 paths. One is the telomerase path … you see that nearly every part of telomerase is altered in among another of these family members. So, the initial element, the reverse transcriptase, the third part, the RNA of telomerase, NOP10, NHP2, dyskerin, heaven people that are required for biogenesis with telomerase, TCAB1, a trafficking healthy protein, and PARN, a protein required for maturation of the telomerase RNA, see anomalies in these various genetics, and also in each situation the anomalies do not fully eliminate telomerase, but it decreases the telomerase degree to ensure that the telomeres are not totally preserved in the germline, and also in the stem cells in the people. The other path that ' s affected in these illness is shelterin. You see anomalies in TIN2, POT1, as well as TPP1, and also they ' re most likely to impact the method telomerase is recruited onto the telomere, once again resulting in shortage in telomere maintenance.And after that, finally,'there are some proteins that are shelterin-associated factors, whose function in the telomere pathway is not yet completely comprehended. So, let me end up by repeating the logic of telomeres. This is how telomeres function: telomerase, this attractive reverse transcriptase, mosts likely to the chromosome finishes as well as preserves the TTAGGG repeats. These repeats wear ' t do much by themselves. They require to hire this protein facility, shelterin, in order to protect the telomere. Shelterin customizes'the framework of the telomere into the T-loop, quelches the DNA-damage action, likewise, via its POT1 element, as well as ensures that telomeres are not seen as what they actually are– the end of the chromosome. So, I ' ve spoke about work done by a huge variety of laboratories. I sanctuary ' t had the opportunity to state everybody, that ' s not the objective of this type of lecture, yet, please, if you ' re interested, check out the reviews as well as discover the appropriate references.Our work is'done at The Rockefeller University, a lovely university on the Upper East Side of Manhattan, where I ' ve been for 26 years,

and currently we ' re sustained by a variety of financing agencies, for which I am extremely thankful.

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