How chromatin changes are preserved after cell division

DNA is found in the nucleus of every cell, woven around proteins called histones. This complex of DNA and proteins found inside the nucleus is called chromatin. In the past, I dedicated a couple of posts to chromatin rearrangements, how they are used by the cell to silence certain genes, and how epigenetic reprogramming has to happen in order for cells to differentiate during development. I'm still learning how these epigenetic mechanisms work, and today I'd like to share with you a couple new readings I've done on the topic.

Chromatin complexes that repress transcription during development are formed by a group of proteins called the Polycomb group (PcG). The proteins in this group form two classes, PRC1 and PRC2. From Wikipedia:

"PRC2 is required for initial targeting of genomic region (PRC Response Elements or PRE) to be silenced, while PRC1 is required for stabilizing this silencing and underlies cellular memory of silenced region after cellular differentiation."

In other words, PCR2 recognizes the current chromatin state and targets the regions to be silenced in order to maintain the same state after cell division. This guarantees that an undifferentiated cell like an embryonic stem cell for example, stays undifferentiated for as long as it's needed.

How does PCR2 distinguish active chromatin (activated genes) from the inactivated one (silenced genes)?

Histones are not static. Imagine these molecules undergoing rearrangements every time they need to change the way they interact with DNA. These changes are called histone modifications and are classified based on the type of histone, amino acid, and position at which they undergo the change. Different histone modifications mark different states of the gene. For example, active genes are usually marked by H3K4me3 and H3K36me2/3, whereas inactive genes are marked by H3K27me3.

In [1], Yuan et al. suggest that active genes are not silenced by PRC2 because, besides having the "active" marks, the chromatin region that contains them is also less compact, with a lower density of nucleosomes and histones H1.

"Once active transcription has ceased upon transcription factor dissociation, either the chromatin-remodeling events or the incorporation of additional histones (including linker histones) would lead to higher nucleosome density, higher H1 content, and more compact chromatin structure, which in turn would convert these nucleosomes from their inert status to ideal substrates of PRC2. Thus, H3K27me3 could be established and lead to further repression of the target genes."

To test their hypothesis, Yuan et al. used a mouse model and the gene CYP26a1 as target, and observed that changes in the local density ("compaction") of the chromatin preceded the establishment of silencing marks.

[1] Wen Yuan, Tong Wu, Hang Fu, Chao Dai, Hui Wu, Nan Liu, Xiang Li, Mo Xu, Zhuqiang Zhang, Tianhui Niu, Zhifu Han, Jijie Chai, Xianghong Jasmine Zhou, Shaorong Gao, & Bing Zhu2 (2012). Dense Chromatin Activates Polycomb Repressive Complex 2 to Regulate H3 Lysine 27 Methylation Science DOI: 10.1126/science.1225237

The mystery of the Delta antigen

Now I feel like I should write a spy-fiction story on the delta antigen... Maybe I will... But for now, here's the real story.

When in the mid '70s a group of patients in Turin, Italy, presented a particularly virulent form of Hepatitis B (HBV), medical researchers thought they had found a new subtype of the virus. Liver biopsies from infected patients revealed a new antigen, which was thought to be a new protein encoded by HBV. There was a mystery to solve, though: why was the new antigen (called Delta) found only in certain HBV infected patients but not others?

A collaboration between the University of Turin and the NIH revealed, thanks to experiments conducted on chimpanzee, that the Delta antigen was indeed a new virus, the Hepatitis D virus (HDV). Why was it only found in HBV-infected patients? Because HDV is a satellite virus, in other words, it can only infect a liver cell if the HBV virus is also present in the cell.

Similarly to viroids, the HDV genome is a circular single-stranded RNA and, even though not as small as a viroid genome, with its ~1700 bases, it's still much smaller than the average viral genome. It utilizes the cellular RNA polymerase in order to replicate, and HBV envelope proteins in order to propagate. HDV and HBV coinfection is rare in the western world, but quite common in sub-Saharan Africa, the Middle East, and the northern part of South America, where it is mostly transmitted among drug users (like the other hepatitis viruses, it is a blood-borne disease).

So, how did HDV develop in relation to HBV? Given the close resemblance that HDV has with viroids, Taylor and Pelchat [1] report as a likely hypothesis that it had originated from a plant virus (plant viruses can be disseminated in human digestive tracts) and infected the liver of an HBV-infected animal. It could also have originated directly from HBV through a random replication error. As the researchers conclude,

"Furthermore, discovery of the widespread occurrence of HDV-like ribozymes and their possible relationship to retrotransposition further demonstrates that some of what appeared to be unique properties of HDV and viroids are barely the tip of a major biological iceberg."

[1] John Taylor, & Martin Pelchat (2012). Origin of hepatitis δ virus Future Medicine DOI: 10.2217/fmb.10.15

It's not a virus, it's a viroid

A virus is a stretch of DNA or RNA, usually a few thousand bases long, enclosed in a protein shell. Once inside the cell, the RNA or DNA from the virus starts producing viral proteins, which are then used for replication.

Now imagine a circular strand of RNA that instead of a few thousand bases comprises a few hundred bases. It doesn't code for proteins, it doesn't come in a shell. And yet it's highly pathogenic and able to reproduce. In plants, that is.

A viroid is essentially a circular strand of RNA, typically between ~250 and ~450 bases long, and it doesn't encode for proteins. As a consequence, it depends entirely on cell proteins in order to replicate and propagate. Currently there are 30 known viroids, all belonging to two families, one that replicates in the cell nucleus, and one in plastids (organelles outside the nucleus) instead. While viral infection is prompted by the proteins the virus codes, it still remains a mystery how non-coding viroids can initiate phenotypic changes in their hosts. These changes are broad in degree and extent, with some viroids inducing no changes at all, and others resulting in severe pathogenicity.

"Potential pathways connecting the first viroid-host interaction that through one or more cross-talking signaling cascades ultimately lead to the macroscopic symptoms. Most components of these pathways, including the initial triggering viroid RNA species, are hypothetical [1]."

In [1], Navarro et al. explore various hypothesis as to how viroids initiate infection. One possibility is that viroid-derived RNAs could be targeting host RNA for silencing. They studied one viroid in particular that causes a severe form of albinism in the leaves, stems and fruits of peach seedlings. They deep sequenced healthy and diseased leaves and provided "direct evidence involving RNA silencing in modulation of host gene expression by a viroid."

It turns out, there's a particular human-infecting virus that behaves more like a viroid than a virus, but that story I'll save for next time. :-)

[1] Beatriz Navarroa,, Andreas Giselb,, Maria-Elena Rodioa,, Sonia Delgadoc,, Ricardo Floresc,, & Francesco Di Serio (2012). Viroids: How to infect a host and cause disease without encoding proteins Biochem DOI: 10.1016/j.biochi.2012.02.020

What's that gene for, again?

I'm always skeptical when you hear prepositions such as "gene X has function Y," as often there are very complicated mechanisms nestled between the "gene" and the "function/phenotype." If you've been following me over the past year (yes, I've been blogging for a year already, time flies!), we've learned that between-gene interactions (epistasis), and changes in gene expression (epigenetics) can completely change the picture.

Recent reviews on the use of RNA interference have given me additional reasons to be skeptical.

Gene function in vivo has been studied through a procedure called "gene knockdown," which uses RNA interference (RNAi) to "tune down" the expression of the gene. RNAi has also been used in to mimic human genetic diseases that would otherwise have no somatic equivalent in the animal world, in particular in studies aimed at discovering novel drug targets. By introducing synthetic RNA into the cell, researchers can effectively silence target genes and thus identify their functions within specific cellular processes.

It certainly is a brilliant tool, but there are several issues one needs to keep in mind when using RNAi. The target specificity, for example, is not always perfect, and several off-target effects (down-regulation of genes different from the target ones) have been documented. When this happens, you can no longer be sure of what genes, if not all, caused the observed change in phenotype. Ideally, in order to minimize off-target effects, one should repeat the experiment with different types of RNAi targeting the same gene. Rescuing the loss of function by re-inserting the mRNA (or making it "immune" to the RNAi) would also provide further evidence. However, this is very hard to realize in practice.

It gets more complicated.

Many genes regulate cellular fitness. The change observed change in phenotype, rather than reflect the knockdown gene, could instead be a direct consequence of lower cell proliferation. In addition, we tend to simplify things thinking that the relationship between gene and phenotype is linear, or that the effect from different genes is additive, when in fact such simple mathematical frameworks often don't capture the reality of the biological world. Interactions and non-linearity are difficult to model. Experiments that target multiple genes rank the results in terms of dose-responses, though such results are often contaminated by false positives and knockdown efficiencies.

All this not to say that this is the end of RNAi experiments, rather, that additional thought has to be given when interpreting the results. We still have a long way to go before we can fully encompass the complexity of our genome, and we are taking one baby step at the time.

William G. Kaelin Jr. (2012). Use and Abuse of RNAi to Study Mammalian Gene Function Science, 337 (6093), 421-422 DOI: 10.1126/science.1225787

Olympic fever, olympic medicine

Will you be missing the Olympics now that they are finally over? I will.

If you enjoyed watching the games, you'll also enjoy the NEJM perspective by David Jones [1], which gives a historical overview of olympic medicine and how it has studied, over the years, the limits of the human body.

It's interesting to read how in the 1904 games in St. Louis the marathon winner had taken strychnine sulfate, five eggs, and brandy during the race and still required medical attention afterwards. Fast forward to the 1984 Olympics in Los Angeles: seven cyclist received blood transfusion to enhance their performance, a practice later condemned by the Olympic Committee.

What drives us to push the limits? It's indeed spectacular to see how far the human body can do and what it can achieve, and yet we step back horrified when we hear about performance-enhancing drugs, or when we read about the borderline training young gymnast undergo (somebody went as far as to define it "child abuse"). When does the body stop being a body and when does it start being a machine? Some sports have in the past received harsh criticism for the life-long consequences they carry. Is medicine's role to let us enjoy these beautiful performances while preserving the athlete's health, or is it to keep pushing the envelope and see how far we can go? Where do we draw the line between what is allowed and what is not? If athletes went as far as blood transfusions in 1984, does it mean gene therapy will be the new dare in the next decade? Would you be willing to permanently alter your genes in order to reach your dreams?

[1] David S. Jones (2012). Olympic Medicine New England Journal of Medicine, 367, 289-292

Rainbow

This was last Tuesday, August 7th. Presumably the largest rainbow in a while, it made it to the local paper.

Anderson Overlook

Not much time to read papers lately, I apologize.

So I give you images instead. This is my favorite spot to shoot at sunset this time of the year. The sun hits it just at the right angle, and there's often stormy clouds sweeping through, which make the whole scene more interesting. It's called Anderson Overlook and those formations you see are part of the Bandelier National Monument.

Feel free to tell me which ones are your favorites. The local library has a call for landscape photographs of the area and I'm thinking of submitting one or two from this set.

The beginning of the end. . . Maybe.

"We share a very special moment - it is the moment when an AIDS-free generation is finally in sight." That's what the US president, Barack Obama, said on July 26.

Well, are we?

A colleague a few days ago brought to our attention some stunning figures: according to the CDC, of all HIV infected individuals in the US, only 25% are under treatment and hence have the virus under control. Quite striking if you consider that in Sweden instead 85% of HIV-positive individuals are undergoing treatment. The consequences of such a poor statistic in the US go beyond the lifespan of the single individual: people under antiretroviral therapy have much lower viral loads and therefore a significantly reduced chance of passing the virus to their partners.

I'm not surprised by the CDC numbers, actually. When newly infected, subjects have no symptoms or may feel like they are coming down with the flu. You can live with this virus for ten years without having symptoms. If you don't have health insurance, and you are feeling well, why bother go see a doctor? In the meantime, though, these individuals continue to spread the virus. And the problem doesn't affect the US alone: according to the World Health Organization, less than half of the infected people worldwide are actually receiving treatment.

These were my thoughts as I read the perspective article "The beginning of the end of AIDS?" in NEJM [1]. The authors base their cautious optimism on a few things: mildly positive results on a recent vaccine trial, more effective drugs, and the news of the first patient to ever be cured of HIV. The latter I discussed in this post. The news was indeed exceptional but, unfortunately, gene therapy is not the way to stop this pandemic: 2/3 of people currently living with HIV/AIDS are in sub-saharan Africa, where drugs are still hard to find, let alone extremely costly procedures like gene therapy. And more effective drugs are not going to solve the problem if they remain unaffordable or unavailable to the majority of infected people.

So yes, in the end, it all boils down to funding:

"Global resources have been declining, not growing, in this period of scientific success. This lack of funding is the major point of divergence between optimism and pessimism."

Why invest on HIV?

"Comprehensive economic models predict that making the needed investments in HIV-related efforts will result in cost savings over the long term."

HIV debilitates the immune system. The effects of diseases like tuberculosis, hepatitis and malaria could be reduced if the spread of HIV could be reversed because of the effect the virus has on the immune system. Making antiretroviral treatment available to all infected people is the best strategy: by keeping the viral load under control, drugs effectively lower the rates of mother-to-infant and sexual infections. According to the CDC, about 100-200 infants are born every year in the US with the virus in their body. Adequate treatment during pregnancy and delivery can reduce the rate of mother-to-child transmission to less than 2%.

As we strive to reach out to every infected person on the planet, funding must not stop for research. A vaccine is the most affordable and most effective way to stop the pandemic and we have to keep pushing in that direction. My supervisor gave a talk last week in which she outlined where we are in terms of vaccine research. She opened the talk remembering how the first vaccine was discovered: the English physician Edward Jenner (1749 - 1823) rubbed pus collected from blisters milkmaids received from cowpox on his gardner's eight-year-old son. He then exposed the boy to pox, twice, and noticed that they boy didn't develop the disease. The audience was of course horrified when my supervisor mentioned the sacrifice of the little boy, and yet when she went on describing how long and, most importantly, how much money is needed to develop and test a vaccine, a girl in the audience raised her hand and asked: "Well, maybe you can't take an eight-year-old boy, but wouldn't you be better off testing the vaccine on yourself?"

We can't, of course. The gardner's boy got lucky, but things don't always go well. The field still hasn't forgotten the failure of the Merck vaccine in 2007, the trial that was halted after the experimental vaccine was found to make some subjects more susceptible to infection. The FDA has a set of very strict regulations on vaccines. They need to be stable, in other words, one has to show that they don't change after they've been grown for several generations in cultures. They have to be attenuated, and remain so after several generations. Vaccines are then tested on mice, first, then monkeys, then, years later, on humans in several phases that take time, money, and then more time and more money. And yet what we really cannot afford is to stop pushing the research forward.

"Every country must develop more effective ways to reach key affected populations and to apply the tools that we know work, if we are to make significant advances."

So, Mr. President, I hope you are right. But I also hope you will keep funding our efforts.

[1] Diane Havlir, & Chris Beyrer (2012). The Beginning of the End of AIDS? New England Journal of Medicine : 10.1056/NEJMp1207138