Home > Lab-blog, Uncategorized > Raiders of the lost G-quadruplexes …in the human genome

Raiders of the lost G-quadruplexes …in the human genome

Small-molecule–induced DNA damage identifies alternative DNA structures in human genes

Raphaël Rodriguez, Kyle M Miller, Josep V Forment, Charles R Bradshaw, Mehran Nikan, Sébastien Britton, Tobias Oelschlaegel, Blerta Xhemalce, Shankar Balasubramanian* & Stephen P. Jackson*

Nature Chemical Biology 8, 301–310 (2012) doi: 10.1038/nchembio.780

A synopsis by Diana Silva Brenes

The authors of this week’s paper play detective to find out -with great detail- what exactly happens to a human cell when it’s treated with the versatile, potent GQ-binder, pyridostatin. Using a combination of biomolecular assays, the authors manage to give strong support for the in vivo formation of GQ-DNA in human cells, and show their role in the activity of the new drug.

Pyridostatin is shown to induce damage to cellular DNA, stumping their proliferation. This happens because cellular checkpoints, which revise DNA before continuing the cellular division cycle, detect the damage and signal to the cell that something is wrong. The cell stops in its tracks to try to correct the problem before it continues the cycle. The drug, however, isn’t too toxic and most cells can survive long-term exposition to it without undergoing apoptosis. Interestingly, inhibition of the checkpoints restores cell proliferation.

Many of the results rely on detecting the presence of γH2AX (a protein that indicates double strand breaks in DNA) as a way to follow damage done to DNA. In cells treated with pyridostatin, γH2AX is present during the DNA transcription and replication processes, pointing at damage to DNA occurring during both stages.

Next, the authors wanted to localize where in the DNA is pyridostatin taking effect. Fluorescence labeling of γH2AX and the telomeres (marked by the labeling of a telomere binding protein) didn’t show co-localization. It was, thus, necessary to modify the drug to add direct fluorescence labeling. Addition of an alkyne group to the drug allowed an in cellulo click reaction with an azide containing fluorescent dye. After making sure that the modified pyridostatin did not affect drug activity, staining of pyridostatin was performed and fluorescent spots (foci) were compared with the a fluorescently labeled human helicase reputed to bind and resolve GQ-DNA during replication. Good co-localization was observed, suggesting that pyridostatin was localized mostly at putative GQ-DNA sites. In another experiment they showed that addition of pyridostatin before of after “freezing” the cellular processes in formaldehyde gave almost identical results, suggesting that GQ structures are pre-folded even without addition of pyridostatin.

They then performed ChIP sequencing to try to figure out which genes (aka, DNA segment) were targeted by pyridostatin. They found several specific genes (mostly away from the telomeres) that sustained pyridostatin induced damage to DNA, and all of them had above average putative GQ sequences. However, not all areas enriched in putative GQ sequences were affected, suggesting that there are other important requirements for interaction.

A particularly affected gene was SRC as confirmed by checking for loss of its corresponding mRNA transcription activity. Out of 25 putative GQ sequences estimated for this gene, 23 of them could be observed to form QGs in vitro using CD and NMR spectra.

The effect of pyridostatin on the bioactivity of SRC was also evaluated. SRC is important for wound healing and motility of cells. Cells treated with pyridostatin displayed a reduced ability to heal. As a control, cells treated with another DNA-damaging drug (DOX), didn’t affect healing, proving that the deficiency was not due merely to DNA damage.

It was previously shown that pyridostatin binds to GQs with enough strength to resist polymerases. It is hypothesized that damage to DNA by pyridostatin is due to mechanical forces breaking the DNA during the cell’s attempt to transcribe or replicate DNA. The findings of this paper support the potential drugability of GQs in cells.

The data reported by this paper is really important for the field of GQ binders and raises large hopes for the future of the field. Being able to use GQ to recognize and regulate specific genes is a dream come true in drug design, and the authors present strong data as to the viability of this approach. As a chemist, it’s difficult to get used to the rather indirect type of evidence that supports these findings, making it hard for me to comment on this paper’s methods. However, the controls and the analyses they did appear to be adequate. Overall, I find the results in this paper to be really important to anyone in the GQ field.

Categories: Lab-blog, Uncategorized Tags: , ,
  1. Marilyn
    March 14, 2012 at 20:57

    In this publication S. Balasubramanian, S.P. Jackson and colleagues report a detailed analysis of the G-quadruplex binder pyridostatin. Cells treated with pyridostatin show DNA damage at specific genomic locations, in specific in regions of sequences that are capable of adopting G-quadruplex conformation. By labeling pyridostatin they were able to visualize the localization of the molecule, revealed an overlap with a DNA helicase that have G-quadruplex binding properties. Finally, they establish that SRC is targeted by pyridostatin, which is indicative of it application as a G-quadruplex binder for certain cancer genes.

    As Diana mention one of their main findings is that; “not all areas enriched in putative GQ sequences were affected, suggesting that there are other important requirements for interaction” which bring as again to the issue of how can we design GQ binders that selectively binds to different GQ topologies. I believed that in order to accomplish this, systems like this need to be fully understood at the structural level to understand it mode of interaction. This is, if the activity is due to a direct interaction to the quadruplex and not thru another mechanism.

    I have to start by saying that I agree with Diana, it is difficult to comment in this type of articles due to the indirect approach use to support their findings. Apart from that I believed the authors present an impressive and relevant work. The lack of familiarity with the methodology makes Diana’s synopsis even more important in order to fully understand the article and even doe she didn’t enter into the experimental detailed the overall results of the article were well presented.

  2. Ana Victoria
    March 14, 2012 at 22:30

    The authors of the article provided an analysis of the genomic targets of the G-quadruplex binding molecule pyridostatin. Cells that were treated with pyridostatin were damaged and this showed that transcription and replication depended on the DNA damage, this resulted in cell cycle arrest. The protein γH2AX was used to indicate wether this damage occurred. It was shown that pyridostatin targets gene bodies that contain sequences that can form a G-quadruplex conformation. Cellular labeling of a pyridostatin analog revealed where the molecule was located and this showed an overlap with a DNA helicase, which supports the existence of functional alternative DNA structures in human cells. However, to achieve the fluorescence needed for labeling, an alkyne group was added through a click reaction in the cell. It was also determined that the proto-oncogene SRC is a target for pyridostatin and it was concluded that GQs may have a potential drugability inside cells.

    This is a different article from what were used to reading because it focuses more on the biological part. GQs have the potential to recognize genes and therefore they have a lot of medical potential. Obviously, more studies are needed but as Diana said, this gives hope to this field. Diana’s synopsis was good, she explained some of the biological part, and since we don’t deal with those concepts regularly we need reminders. The picture has the important elements of the article: the pyridostatin, the cell, GQ and the detective investigating what is going on with all of these things.

  3. March 15, 2012 at 01:35

    Chemical-Biology in its maximum expression can be appreciated in this article of Balasubramanian and Jackson. This article covers how efficient a drug like pyridostatin can be in terms of target-recognition and how the cellular processes can be interpreted or studied by using cellular experiments and fluorescence probes. In brief, they perform studies with pyridostatin that inhibits cancer cell growths by affecting cell replication and transcription dependent DNA damage. By click chemistry, they use a pyridostatin analog that has a fluorescent group to analyze genomic targets of this GQ-binding molecule. With this label, a significant overlaps of human DNA helicase that is know to have GQ-binding properties supports the existence of functional alternative DNA structures.

    Sincerely, I think that is an excellent work due all the experiments that they performed to obtain their conclusions, but the way that was presented this article can be better. It looks complicated by a very specialized content, but the main findings are not hard to understand. But, I think that presenting this work in a more organized way so other fields that have the cancer topic in common can analyze the impact of the presented findings. We are not talking about just an anti-cancer drug; we are observing information about the genetic location of the target of this drug. At the same time, we can know the effectiveness of this drug by measuring the number of cell lines for different groups of treated/untreated by the drug.

    By using this methodology, they will not only known the effectiveness and target location of the drug, but also they are very close to understand a lot of cellular processes that remain as mysteries until now. For me, the most impressive part was figure 3 images when they found unknown target sites. This work opens the doors for studies with other gene drugs to see their target-interactions and how predictions of these cellular processes can be achieved.

    About Diana’s synopsis, I have to say that help me a lot to understand these article and appreciate the high-impact of these findings. I have to admit that I’m not very familiar with some techniques, but I got the article’s main message about the development of GQ-binding drugs and their precise interactions in genes. About the pictures…. fits perfect with the article’s main findings.

  4. Rocio Roque
    March 15, 2012 at 01:38

    In this weeks article, the authors provide a deep analysis of the G-Quadruplex binding molecule pyridostatin. First up, cells treated with pyridostatin showed DNA damage at specific genomic locations. By detecting the presence of γH2AX, it was proved this damage had been done. And by labeling pyridostatin, they discovered its localization and also an overlap with a DNA helicase. ChIP sequencing was performed to figure out the genes that were targeted by pyridostatin, SRC gene being one of them. This pointing towards the potential drugability of GQs in cells.

    I think the article was rather interesting, a mixture of interdisciplinary subjects, biology and chemistry. Also, Diana synopsis was good and it was very well organized. In a sense a reminder of biological concepts we might have forgotten or are not very familiar with. Finally, as Diana pointed out (although I’m still learning about all of this…) I also believe these are very important results in the GQ field.

  5. AJS
    March 15, 2012 at 01:44

    In this paper, the authors report their detailed investigation of the effect on cells of treatment with a quadruplex-binding drug, pyridostatin. They confirm, as in previous studies, that the drug slows growth of cancer cell lines, arresting the cell cycle; this can later be resuscitated, if the mechanism which checks for double-stand breakages (DSBs) is modified. A number of different markers associated with DNA damage response were also found, particularly the phosphorylation of a histone protein (the phosphorylated version termed γH2AX) which indicates), adding considerable weight to the idea that pyridostatin’s effects stem primarily from DNA damage. Since the number of γH2AX loci doesn’t increase much until a very large concentration of the drug is added, they take these interactions to be site-specific, and perform a vast range of tests to look at where the damage, and the interactions leading to it, can be found in the cell. These tests include fluorescent labelling of γH2AX and of pyridostatin itself, but perhaps the crux of the paper (or, at least, the most exciting development) is the ChIP-sequening analysis, which creates a map of γH2AX domains induced by pyridostatin across the genome. Surprisingly they observe that these are mainly away from telomeres, and that the density of (the much-trumpeted) putative G-quadruplex-forming sequences (PQS) is not an accurate predictor of the DNA damage.
    This is clearly an important paper, and a first step towards understanding rather than the final word on the subject. Like many of my colleagues, I am not qualified to judge the extent to which the headaches it gave me are a result of my failings as a reader, or the authors failing to make it an easier read. I suspect most of the fault lies with me (I can only keep so many new acronyms in my head in any one paragraph), but not the whole; while this is a chemical biology journal, a glossary would really have helped.

    In synthetic ligands reported so far, selectivity between known quadruplexes is poor, and we can only imagine poorer between the myriad PQSs. Given this, seeing such apparent selectivity between potential binding sites from a ligand which is known to be unselective is exciting; seeing that the basal level of damage correlates more strongly with PQSs is fascinating. I would be very interested to know how solid we may consider their semi-quantitative approach to mapping interaction (whether the correlation between γH2AX loci and damage is so straightforward, for example), and wonder whether there is perhaps a degree of over-extrapolation. It’s rather beyond my experience, but seeing their statement that the Huisgen reaction “could be used to trace and assess the distribution of ANY small molecule in cells” [my capitalisation] has my hyperbole senses tingling.

    Diana’s synopsis certainly helped me get through the paper, and what little of my memory hasn’t been overwritten by new acronyms seems to indicate that it was fairly thorough. Perhaps where papers like this are being covered in the future, a glossary would be an invaluable addition to the synopsis. I like her picture: it illustrates the content well. I was going to add a bad joke about the paper being well-chosen for Diana, given her initials, but I’ll leave that to someone else.

  6. Mariana
    March 15, 2012 at 02:18

    I have to give a lot of credit to the blogger (Diana), excellent work! As a matter of fact I was able to digest the article after reading your synopsis, because not even the abstract was that clear, at least for me. From my point of view the summary is as follow they took a small molecule GQ binder, Pyridostatin and tracked it inside a cell using a fluorescent tag. There are a lot of chemical biology terminology and abbreviations that make the understating of the article kind of difficult but at the same time I can imagine that performing such studies was not an easy task. They presented strong evidence for the existence of alternative DNA structures (GQs) and identify those areas as potential for druggability (I like this word). We are dealing again with a very common gap/topic among GQ binders the selectivity, but I guess not all the problems can be solve at once. Their contribution was to confirm the potential targeting of invivo GQs, encouraging many researchers in the area to still develop specific drugs for specific GQ sequences. The picture depicted very well the articles message. I think that comparing the work done in this article with the binders that Maxier will be presenting, in terms of structure, will be a good exercise. It seems like a GQ binder week… same long-term goal and purpose…different contributions.

  7. Jean
    March 15, 2012 at 02:37

    This is an extremely important article for the GQ community. The formation of GQ structures in humans was hinted by a large amount of in vitro studies using DNA and RNA sequences derived from the human genome. Everybody knew that GQ structures formed in human cells but no one had been able to prove it experimentally until now.
    In this article by Jackson and co-workers the existence of folded DNA sequences was proven by analyzing what happens when human cells. are exposed to pyridostatin. Right off the bat, growing cells in the prescence of the drug incuces cell cycle arrest. This is shown to be due to DNA damage. This damage is then shown to be caused by stabilization of GQ strucutures during both replication and transcription. The localization of the drug was also assessed via fluorescence by tagging the cell with Alexa Fluor. The localization of the drug was not limited to the telomeres however, an important finding because GQ binders are usually seen as possible telomerase inhibitors. Finally, the drug was shown to down-regulate SRC gene expression a very good example of the possibility of using drugs to treat diseases at the genetical level by targeting secondary DNA structures.
    Dianas synopsis was excellent, which is an achievement considering that the work present in this article is entirely out of her field. I liked the picture since it shows how the whole article is pretty much detective work. Good job overall, I’m looking forward to the GM.

  8. PGMS
    March 15, 2012 at 05:24

    The authors showed that pyridostatin, the drug that interacts with G-quadruplexes promotes growth arrest in human cancer cells by inducing replication-and transcription-dependent DNA damage. G-quadruplex nucleic acids are an attractive paradigm for chemical genetics as they provide structural variations in the genome that are suitable for selective recognition by small molecules Drugs that induce dysfunctional telomeres or perturb ribosome biogenesis in human cells act by stabilizing clusters of G-quadruplex motifs at the ends of chromosomes or in regions containing clusters of ribosomal RNA genes. Furthermore, biologically relevant G-quadruplex motifs have been identified in several organisms, suggesting that functional alternative DNA structures may also occur in human cells. However, a comprehensive genome-wide analysis of these motifs that are accessible to small molecules has not been described.

    Pyridostatin, a highly selective G-quadruplex–binding small molecule was designed to target polymorphic G-quadruplex structures. It was found that the drug decreased the proliferation of simian virus (SV40)-transformed MRC-5 human fibroblasts (MRC-5–SV40 cells) and various cancer cell lines besides rapid accumulation in the G2 phase of the cell cycle indicating activation of the DNA damage response (DDR), phosphorylation of histone protein H2A, X variant on Ser139 (termed 􀁇H2AX), the transcriptional repressor KAP1 (on Ser824), the checkpoint effector kinase Chk1 (on Ser345) and replication protein A (RPA)

    The results indicated that high-throughput sequencing and a genome-wide analysis of the DNA associated to 􀁇H2AX showed that pyridostatin targets gene bodies containing clusters of sequences that are able to adopt a G-quadruplex conformation. Cellular labeling of a pyridostatin analog to visualize the localization of the small molecule revealed a notable overlap with a human DNA helicase that is known to have G-quadruplex–binding properties, lending support to the existence of functional alternative DNA structures in human cells

    It was concluded that the proto-oncogene SRC is a target for pyridostatin, providing additional support for the druggability of certain cancer genes by G-quadruplex–binding small molecules. The targeting of specific DNA loci to alter the expression of particular genes has been particularly challenging owing to the difficulty in identifying well defined druggable sites.

    Dianas title appears very enthusiastic rather than the original. Her figure is very depictive and funny. She has done a great job in getting a good synopsis. However, the paper is very lengthy and too descriptive.

  9. Maria
    March 15, 2012 at 12:25

    I think this is the most impressive work that I have read this year, ones again Balasubramanian and colleagues amaze me with their contributions to the GQ field.

    In this particular article the authors enlighten the long and winding, but quite encouraging, search to validate the biological relevance of G-quadruplexes (GQs) in human cells. Their results also suggest that certain GQ-ligands can actually target putative G-quadruplex forming sequences (PQSs) located in the nucleus. As stated by the authors their results “highlight the potential druggability of G-quadruplex structures and suggest how pyridostatin, as well as other compounds with similar modes of action, could be exploited as tools for genomic studies and for therapeutic benefit”. As expected, an increased in the relative understanding of something always generates even more questions.

    Regarding the design of ligands: besides targeting the global structure and topologies of known PQS like hTelo, cKit1, cKit2 and KRAS and now SRC among others we need to consider what type of mode of action we might “want” to target. For example, pyridostatin could have a relatively planar surface when end-stacking on GQs however it also has some flexibility that might allow it to interact within grooves, loops or GQ-related proteins if necessary. This could be part of what make it work during both transcription and replication, yet it might also interact with putative RNA GQs that were not discussed in here. In addition, even though I would like to see the behavior of a more rigid ligand like telomestatin, in general I still believed that a potential ligand should have some aromatic surface. However, the key to modulate loci selectivity and targeting methodology I think that relies in the wise design/selection of multifunctional side chains

    Regarding the techniques that we could use to screen among a library of possible compounds, we have to consider that doing the battery of experiments described on this article might not be cost/effective?: I guess that VT-FRET experiments might still be the first and easiest way to start a screening but even though, the more stabilized sequence (in vitro) might not be the one for which the ligand will have preferences in vivo like it happen with pyridostatin. In addition, I wonder how data extracted from more “rigorous” in vitro experiments like TRAP–LIG assay, telomeric restriction fragment (TRF) length assay, cell Viability assays among others, compare with conclusions extracted from the type of experiments discuss in this article by Balasubramanian and colleagues. I guess that to validate/compare the results obtained from different strategies someone can use the most promising isaindigotone derivative from those reported by Gu and Huang (J. Med. Chem. 2009, 52, 2825–2835) which is already characterized by many techniques but that all different from those used by Balasubramanian and colleagues. They can also test some of the triarylpyridines (Chem. Commun., 2008, 1467–1469.) that they have tested in the past since some of their examples have preferential association for particular GQ topologies versus other GQs (discrimination between different GQs).

    It’s good to know that our research aims towards a now well validated target that is “GQ structures in vivo” whether are telomeric or not. At least I personally want to see where our derivatives would go?, which loci did they prefer? If they kill cancer cells, how that’s possible? Again, this is a very nice and extremely relevant article in the GQ field, from now on is “the” article to be cited regarding the biological relevance of GQs in human cells. Diana’s synopsis was really good, reading it first help me to get the whole idea first so I didn’t lose track of the story while reading the details described in the narrative of the article. Her figure was pretty with a nice perspective.

  10. Loruhama
    March 15, 2012 at 12:44

    In this article, Balasubramanian and Jackson present their work in analyzing the effect pyridostatin has on a cell. Pyridostatin has been known to be a G-quadruplex binder, and the authors here emphasize the fact that this molecule induces damage on the DNA of a cell, preventing its replication. The authors explain that the protein γH2AX could be used to verify the damage done to the DNA, because this protein indicated double stranded brakes in the DNA. To be able to determine where pyridostatin was binding to, they used a fluorescent pyridostatin analog. These studies revealed that pyridostatin was localized mostly in putative GQs. They also performed ChIp sequencing to determine the DNA sequence that pyridostatin binded to.

    This article was very interesting, but it was a little difficult to keep up with it because of the way that they present their data. Also I’m not used to reading papers that focus so much on biology so that gave me a hard time to. I also agree with Diana in the fact that their data is very redundant. As for Diana’s synopsis, it was very helpful to understand the main idea of the paper. The picture is fun and creative.

  11. Berti
    March 15, 2012 at 13:45

    Shankar Balasubramanian is one of the top researchers in the GQ area who has been working with quadruplexes since 1999 when he published: “Inhibition of Human Telomerase by PNA- Cationic Peptide Conjugates”, J. G. Harrison, C. Frier, R. Laurant, K. D. Raney and S. Balasubramanian. Bioorg. Med. Chem. Lett., 9, 1273-1278 (1999). In 2011 he design and tested a potent, nontoxic and selective GQ-DNA intercalator called Pyridostatin. Now in this Nature Chemical Biology his team of researchers collaborates with Stephen P. Jackson group to study how this drug interacts with the GQ-DNA regions of human genes. For doing this they used a plethora of experiments – circular dichroism, 1H-NMR, western blot, ChIP-seq- satisfying a broader audience (chemist and biologist) and concluding that “…pyridostatin promotes growth arrest in human cancer cells…”
    This is great article, it has an excellent experimental procedure with all the controls, clear figures and narrative, big news for the GQ research community. Although, my lack of knowledge in some terminology used makes the reading slower. The result that surprises me the most was the effect that attaching the alkyne to pyridostatin (for fluorescent labeling studies) has in the drug binding.
    As a future work, it will be interesting to see pyridostatin mechanism of action. Will it be by breaking the DNA or by intercalation and physical disruption?
    Diana did a great job in her synopsis and picture. They definitely help me in the understanding of the article. I like the violet color of the picture and the integration of the detective on it. Some concepts, specially the corresponding to biomolecular assays were reviewed together with the posted link of the ChIP-seq method.

  12. Loruhama
    March 15, 2012 at 22:41

    In this article, Parkinson and Neidle present a series of tretrasubstituded naphthalene diimide-base (ND) compounds that act as stabilizers for telomeric G-Quadruplexes. As a consequence, these molecules can be used to inhibit the activity of the protein Telomerase and also hPTO1, which is another telomeric protein. These ND molecules form complexes with the telomeric GQs in a 1:1 ratio. Some of their studies suggested an all-parallel topology for telomeric quadruplexes when completed to these types of molecules. They also did FRET melting studies, which led them to understand that the BMSG-SH-3 had a better fit than the BMSG-SH-4 because it could stabilize more the GQ according to these experiments. They also compare this two with BMSG-SH-5 in the sense that the previous two seemed to be better inhibitors.
    I really enjoyed reading this paper. The topic was very interesting and they did a very good discussion of their data. However I do agree with Maxier in the fact that they don’t correlate very well the pictures. As for Maxier’s synopsys, he did a very good job explaining the main points in the paper and giving us his own feedback on the article. I liked the picture, it was very creative.

  13. Maxier A. S.
    March 16, 2012 at 04:26

    This study by Shankar Balasubramanian and Stephen P. Jackson presents a full experimentation of a GQ binder in vivo. The binder used was pyrodostaton that induces cellular arrest. Usually yH2AX appears due to the break so they dyed the binder with Alexa Fluor and overlapping of the helicase with the dye was observed. The ChIP experiment showed the areas in which the pyridostatin showed activity (thanks Diana for the link). It showed that the areas that where affected by the drug where possible GQ segments, specifically the SRC gene.
    To be honest, at the beginning I was very lost reading the paper. I wasn’t sure why they were doing certain experiments. But at the end it all kind of comes together. So it end being a great full procedure of understanding what does this binder does. Yet as everyone else, the biology behind these experiments was very…. intense. I would say that Diana’s synopsis was key for me to understand the paper. And her detective simile to a researcher was kind of cool. So the picture was great.

  14. Y. Melendez
    March 16, 2012 at 04:59

    Balasubramanian et al. analyzed the genomic targets of the Gq binding molecule pyridostatin inside a cell. This was done as a means to investigate whether or not there existed any other type of functional DNA structures in human cells. Through the use of fluorescence tagging, they were able to ascertain the position of the drug within the cell. This was done through the addition a fluorescent dye to the drug through a click. The results suggested that it was in zones that are reputed for being GQ-DNA sites. These are really significant findings for the GQ community, because it’s a very determining find as far as the future of the field goes and its applications.

    I did think that the article was kind of dense, sometimes it got pretty complicated as far as understanding it went, but I think it was a really good article and lots of hard work! As for Diana’s synopsis, I thought it was really really well done, it really simplified reading the article, and it summarized everything in a very simple, but efficient way. The picture is pretty ingenious and fun. I really want to see how this presentation goes.

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