Tag: Telomere

Show me your Telomeres and I can tell your (remaining) Age

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A Quick Guide to my current work

This article is based on a poster I presented on “Forschungsforum HTW Berlin” in October 2021. The poster is available in German language on DOI: 10.13140/RG.2.2.26595.43048.

What’s so cool about Telomeres?

Flow diagram of the cellular importance of Telomers (see caption)
Telomeres are the Ends of Chromosomes and shorten with each cell division. Once they reach a critical short point the cell becomes senescent. Senescent cells are responsible for the aging of organs. On the other side Telomerase can stabilize or elongate Telomers. It is active in the germ line, stem cells and in about 90 % of all cancer cells. This makes Telomer dynamics an important target for aging and cancer research.

Telomeres are relevant to both aging and cancer. Drugs elongating or stabilizing them could delay aging. Drugs that shorten them could be used in cancer therapy.

Project Idea

Drug development that affects Telomere length and dynamics requires systems to screen for these parameters. In such a system the influence of thousands of substances on Telomeres has to be examined in parallel. It has to be cheap, quick and easy.

Idea: A high-throughput system that uses optical signals to estimate the telomere lengths of living yeast cells. For this purpose, proteins that naturally bind to Telomeres will be labeled fluorescently and/or luminescently.

Problem: Yeast use Telomerase to keep their Telomeres constant. In addition to the marking the Telomers, I need to integrate a switch on the Telomerase of the yeast to activate and deactivate it.

Markers for Telomer Length

Schematic representation of a Telomere and the main proteins associated with it. The three proteins I’m using as markers are marked with green lightbulbs: Rap1 and Rif1/2. Their close association on the Telomer should make it possible to use molecular distance measurement methods like Quenching, FRET and BRET (see below).

Methods for Marking

The marking is done in the genomic code of the target proteins (RAP1, RIF1 and RIF2) by fusion with reporter proteins. The genetic code of the reporter (a fluorescent or luminescent protein) is attached to the code for the target protein in the genome by means of transformation and recombination.

Possibilities for labeling the proteins: Fluorescent markers light up in a different (red-shifted) color when they are illuminated, bioluminescent markers light up when a substrate is converted, two identical fluorescent markers reduce their fluorescence when they come close to each other (quenching), a fluorescent marker can transfer energy to another, which then glows in a different (even further red-shifted) color (FRET), a luminescent label can transfer its energy to a fluorescent label, which then glows in a different color (BRET), these methods allow distance measurement at atomic scale level

Methods to turn Telomerase on/off

In order to make the Telomerase switchable the promoter of one of the genes that together form the Telomerase is exchanged. A promoter is a regulatory sequence in front of the actual gene (the blueprint of a protein). It controls how and when the encoded protein is produced.

The natural promoter of the EST2 gene, which is one of the components of yeast Telomerase, is replaced with the promoter of the DDI2 gene. While Telomerase is always active in yeast, the DDI2 gene is only activated under certain chemical stimuli. If the promoter is successfully exchanged the Telomerase is inactive but can be activated by adding small amounts of the chemical cyanamide.

One way to achieve this is to exchange the promoter directly in the yeast genome via recombination.

Another slightly simpler method would be to destroy (knock-out) the gene in the yeast genome and introduce a functional version with the exchanged promoter on an artificial chromosome (plasmid) into the cell.

Status of work after year 1 of 4

Libraries of yeast strains with GFP-tagged proteins are available. These were used to carry out the first preliminary tests to establish the measurement methods.

A DNA construct was designed and produced that makes it possible to replace the promoter of the EST2 gene. The construct was transformed into strains with different GFP tags.

Transformed colonies are currently being examined to determine whether and where the construct is integrated in the genome. It is also checked at the protein level whether the control works. This has just been confirmed by a Western blot which I will explain in one of my next articles.

Next the new strain will be tested in the fluorescence assay.

Transforming Yeast or how to make a GMO

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Actual footage of me changing the genes of a hapless yeast, own work

In the last article I ended with a DNA fragment, that I created with a PCR. Now I need to get this DNA into my yeast cells to teach them to age like normal people. This is called transformation. There are several different transformation techniques but most common are elctroporation and chemical transformation. You can spark emotional debates if you ask what is better but the more common and generally a bit easier is the chemical transformation. That is also the method I chose for my aging yeast.

How transformation works mechanistically is not completely understood but the main idea is that you have to overcome the barriers protecting cells from their surroundings. These barriers are the cell wall (for all except animal cells who don’t have one) and the cell membrane. The cell wall’s main responsibility is mechanical stability. You can imagine it like a steel frame that prevents the membrane, which you can imagine like a water bomb, from swelling too much until it bursts. On a molecular level it is a relatively wide net, so it’s not a physical barrier for the DNA molecules. What’s problematic however is that it is electrically charged. DNA is negatively charged and would be repelled from the mostly also negatively charged molecules in the cell wall. This obstacle is overcome by adding positively charged ions that neutralize the charges of the cell wall and allow the DNA to get close to the cell’s membrane.

Schematic structure of a budding yeast cell with a special focus on membrane and cell wall on the right, Giulia Coradello, 2021

But than it has another problem which is the cell membrane. The membrane is a double layer of lipids, fat molecules that have a polar and an uncharged tail. This structure is something like a 2-dimensional liquid. It is relatively elastic and allows uncharged molecules that are not too big to path quite well. Since DNA is huge and strongly charged it normally won’t pass easily. To pass this barrier you have to torture your yeast a bit (*laughs manically in evil scientist). You submit them to a sudden increase of temperature to 42 °C from room temperature. Doesn’t sound too bad but yeast likes it best between 20 and 30 °C above 40 °C starts killing them slowly. This is thought to built differences in pressure between the hot outside and cool inside of the cell which is equalized by the formation of pores in the membrane. Through these pores the DNA can now enter the cell.

To avoid another line of defense of the cells which is enzymes that eat DNA, you also add some carrier DNA, which will be eaten first and so protects the DNA you want to get into the cells. As carrier DNA you normally use Salmon Sperm that you boil and then add to the mix. So if you hear molecular biologists talk about cooking some sperm it’s probably not as weird as it might sound.

After overcoming the defenses you now have a lot of dead cells, many that have taken up nothing or only the carrier DNA but you hopefully also have some that have the DNA that you wanted to introduce into them. What happens with this DNA depends on its form. If it is a closed circle and includes some special signals in the code it will be just kept in the cell and will be copied to the daughter cells when the cell replicates. This is called a plasmid and is good if you just want to introduce genes more transiently without permanently changing the genome. If the DNA (like in my case) is a linear open molecule produced by PCR and has ends that resemble a sequence in the genome of the yeast, it can go into the nucleus of the cell, where the genome is kept. There it can jump in while the cell replicates and integrate permanently into the genome.

Now you have to filter for those cells where this has worked from the rest. You do this with selection. You seed your cells to a medium where only those cells can grow that have taken up your DNA. This is done by adding a marker to the DNA which is a gene that either makes your cells resistant to something that is poisonous to the others or that let’s them live of a food source that the others can’t use. I used cells that normally can’t make Uracil (which is a part of DNA) themselves so they can’t grow on medium where there is no Uracil. They only can grow on this medium if they have taken up my construct which includes a gene that helps them make their own Uracil.

The problem is that this integration is very rare. You start with a huge amount of cells, transform them seed them all to the selection plate and sometimes get nothing, sometimes one or two colonies but sometimes, if you’re lucky, you also get something like 100.

Selection plates of the different transformed strains and a control that was not transformed, own work

To verify that the construct is really integrated where I hoped it would, I now need another PCR. I chose primers that normally produce a short fragment of DNA from the region where I want to integrate my construct. If it is successfully integrated the fragment gets longer which I can see in a gel electrophoresis. And that is what I’m currently doing. I’ll soon give you an update when I’ve finally found my aging yeast.

Teaching Yeast to Age like normal People Part III

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Yeast cells growing on a selective plate, own work

In the post “Teaching Yeast to Age Part II” I explained what I tried first to teach my yeast to shorten its Telomers at each cell division. I also told you that it did not work yet. While I have not yet completely given up on Tretrad dissection, I decided to first try another way. It would already be nice to have my fluorescently tagged cells with a knocked out Telomerase but it would be a lot cooler to be able to switch the Telomerase on and off at will.

power on and off switch on wall
Photo by Mikhail Nilov on Pexels.com

The way I’m trying to achieve this is called promoter shuffle. As you might know, Proteins (like the subunit of Telomerase I’m interested in) are encoded in the genome by a fixed code where 3 letters (called a codon) in the DNA (bases) are translated into one letter in the protein (amino acid). This code is well known and understood. All codes for proteins start with the same start codon and end with one of three stop codons. However to really produce this protein in the cell there needs to be more. The gene needs to be flanked by regulatory code that influences if and how strongly the encoded protein is produced. Most important for this is the so called promoter region. This is the code directly before the start codon. It is normally between 50 and a few hundred bases long. Their code is a lot less understood than that for the protein. There are strong and weak promoters, promoters that are always active and promoters that are switched off or on on specific stimuli.

Since I want to make the Telomerase switchable I take the genetic code for one of its subunits and exchange some of the code before its start codon against something else. There are a handful of common switchable promoters in yeast that are well described but most of them have some disadvantages. I identified a relatively newly identified one that I hope will be a good choice.

It is the promoter of the gene DDI2 that is normally almost inactive but increases its activity more than 1000-fold if the cells are incubated with the chemical cyanamide. In the concentration it is used here cyanamide has almost no effect on the yeast cells.

Exchanging code in the yeast genome luckily isn’t too complicated. Yeast quite readily incorporates genetic material that you feed it if a stretch at the beginning and end of the inserted code match the code in the genome. This process is called recombination. Recombination is also the process that makes children unique instead of copies of their parents because it randomly mixes the genetic material of your parents that is passed on to your kids.

So I need a stretch of DNA containing the 500 bases before the start codon of DDI2 with 50 bases on both ends from the area before the start codon of the Telomerase subunit. I did a lot of thinking how to build this myself but it would have been a lot of work. Then I thought about the paper where I first read about the DDI2 promoter. In the nature article “Fine-tuning the expression of target genes using a DDI2 promoter gene switch in budding yeast” Professor Yu Fu of the Chinese Academy of Sciences and colleagues characterized the promoter and explained how they integrated it in genomic locations.

So I just wrote an email to Professor Fu asking if I could get some of the material they used. He actually replied within a day and said that he’d be happy to help me. He sent me the code of the promoter without expecting anything in return. That’s a great thing about the science community. Most people are happy to share and are really nice if you show interest in their work.

So all I have to do now is add the code stretches at the ends that determine the target where I want to integrate it. I’m doing this with a PCR which I’ll explain in my next article.

Finding the fountain of youth and curing cancer

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boy standing on outdoor fountain
Photo by Rene Asmussen on Pexels.com

Well if that’s not one of the click baitiest titles you’ve ever seen. But that’s what I hope being part of with my PhD project.

In the last article I explained what Telomeres are and why they are important as one of the possible keys to aging on one and cancer on the other side. If you haven’t read the article yet, you can find it here.

When it comes to Telomeres it’s all about their length and its changes over time. If Telomeres degrade it is a sign of cellular stress (in some cases the opposite is true) or strong proliferation and if they reach a certain critical length they stop the cell from division and send it into senescence. If a cancer cell wants to divide further it has to overcome this brink and find a way to elongate its Telomeres.

This shows you why drugs that influence Telomere length (in either way) have a huge potential. But how do you find these drugs? There are several answers to this question however often the answer is high throughput screening. Which is a nice word for trial and error.

In high throughput screens you develope a system that gives you an easy, reproducible and cheap read-out for the effect you hope for and then you make tens or even hundreds of thousands of experiments in parallel with huge libraries of chemical compounds. If some of them show a promising effect you try to reproduce that effect first in the same and later in other experimental setups. Then you try to find out how your candidate compounds work, if they are toxic and other questions before you can start developing a drug from them.

For Telomere length there is currently already a problem in the first step. There is currently not yet a really easy, cheap, quick way to measure Telomere length in high throughput assays. For an overview on the currently available options you can read this review paper by Lai et al. 2011

Proteins associated with the Telomere, Picture from Vaiserman and Krasnienkov. 2021

My idea seems to be an easy (if probably not very accurate) option. Telomeres are normally quite tightly packed with proteins. I want to use this Telosome as a molecular ruler. My idea is to optically tag one or more of these proteins and look for a correlation between an optical read out from these tags and the length of the Telomeres. If I find this correlation I only have to calibrate it and I’m ready to screen for the pill against aging or cancer.

In the first step I’m using the bakers yeast Saccharomyces Cerevisiae as a model. I’ll explain a little more about the advantages and disadvantages of this system in another article. For now one of the advantages is that my lab has a library of yeast strains with tagged proteins. These tagged proteins are fusion proteins that were made by adding the genetic code code for Green Fluorescent Protein (GFP) to end of the code for (basically all) yeast proteins.

So in the first step I’m just taking yeast stains out of the – 80 °C freezer and revive them. Now I only have to check the fluorescence signal from cells with different Telomere lengths and see if there is a difference.

It could be so easy but there’s a problem with yeast. It doesn’t age or at least it keeps it’s Telomeres very constant in length using Telomerase. So I first have to teach my yeast to age like our somatic cells. I do this by disabling their Telomerase. I thought about three different methods of doing this that I’m currently working on but I’ll explain them in other articles.

On the other hand I can not only make their Telomeres shorter but also longer. This is interestingly done by cultivating the cells in 5 % alcohol.

If I don’t see a correlation in this easy system I have a few more elegant ideas that I’ll explain in other articles.

What are Telomeres?

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To explain what I’m doing I’ll first explain what Telomeres are and why their length is relatively important.

Telomeres Structure and function from Kupiec, 2014

Telomeres are solving a problem that came up when bacteria became eucariots (plants, mushrooms and animals). At this point they stopped storing their genetic information on DNA-rings and instead developed linear chromosomes. This brings some structural advantages for bigger chromosomes but has a huge disadvantage. Each time a cell divides (which most cells do a lot) it has to replicate it’s genome so each daughter can have a copy. This is done by enzymes called DNA-polymerases. The process only works in one direction on the DNA-string and doesn’t start at the beginning.

For a circular genome this is no problem but for a linear genome this means the loss of a few bases (the building blocks of DNA) at each devision. For a while this is not a huge problem if you don’t write important stuff at the beginning (or end) of the chromosome. And that is already the first function of Telomeres. Aside from mechanically stabilizing the chromosome ends, they are a peace of genetic code at the ends of chromosomes that do not contain vital information.

This is already enough for many of our somatic cells (those cells in the body that form most of our body) they only divide a certain number of times until a tissue (like a muscle or your brain) is formed and then just work and do not or only rarely divide.

Only cells that need to divide unlimited or often (germ line or stem cells but also unicellular life) need another trick. This trick is called Telomerase and earned it’s discoverers a Nobel Prize. Telomerase is an enzyme complex that uses an RNA-template to elongate Telomeres and so completely solve the so called End-Replication-Problem.

The fact that most somatic cells are not using this trick and have telomerase switched off has an interesting implication. It makes Telomeres something like a cellular clock of aging. They limit the number of times a cell can divide and rejuvenate a tissue before it goes into a state called senescence. The more cells of an organ are in this state, the less likely the whole organ gets to repair itself and to function correctly.

Another type of cell that needs to replicate a lot is cancer. This is why about 4 out of 5 cancer cells have Telomerase switched on.

The full picture of the connection between Telomeres, aging and cancer are quite complicated and still under discussion but it is quite clear that influencing Telomere length has quite some potential to treat cancer on one side and influence aging on the other.

If you’re interested in a good overview on the current state of knowledge on the connection between aging and Telomeres there’s a quite new review article from Vaiserman et al., 2021.

If you want further information on Telomers and cancer I’d recommend the article of Hiyama et al., 2003 about Telomerase as tumor marker or Graham et al., 2017 about Telomeres and prostrate cancer.

For an overview on Telomeres as a target for drug discovery see the nature article of Bearss et al., 2000.

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