Category Archives: Epigenetics

Bacterial methylomes and antibiotic potentiation

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Cohen et al., Nature Genetics, 2016

Cohen et al., Nature Genetics, 2016

Antibiotics emerged as miracle drugs and “silver bullets” in the early 20th century, revolutionizing medicine and our ability to combat infectious disease while positively impacting health and lifespans on a large scale. This remarkable triumph held steady for many years, and consequently antibiotic research and development diminished as a priority due to the seeming defeat of bacterial infections. However, the selective pressure that came with antibiotic exposure led to the development of bacterial resistance to these compounds, motivating renewed interest in what is now an extremely important public health issue. Mechanisms of resistance are many and ever-evolving, and we know now that it is not a matter of IF bacteria will become resistant to a class of antibiotics, but when. The search for new and potentially exploitable bacterial vulnerabilities, then, becomes a constant enterprise in order for us to keep pace with the bacteria in the antibiotics/resistance arms race.

Cohen et al., Nature Genetics, 2016

Cohen et al., Nature Genetics, 2016

A new study this week in Nature Genetics describes how manipulating the bacterial DNA methylome affects susceptibility to multiple classes of antibiotics. The authors observed that deleting the dam gene, encoding a DNA methyltransferase, from E. coli causes increased susceptibility to sub-lethal doses of the β-lactam antibiotic ampicillin. Dam specifically methylates GATC sites, and deletion of any of the other three DNA methyltransferases found in E. coli had no effect on the level of antibiotic susceptibility. Using SMRT sequencing, the authors saw that genome-wide GATC methylation patterns did not change after exposure to ampicillin, so they sought alternative explanations for the observed phenotype. Continue reading →

Methylation marks tumor suppressors

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Sharp and broad H3K4me3 peak definitions

Sharp and broad H3K4me3 peak definitions{credit}Chen et al. Nat. Genet. 2015{/credit}

Modifications to histones, including methylation and acetylation, are used by cells to regulate gene expression. Though a lot is now known about how different histone marks correlate with transcriptional activation or repression, the “histone code” has not yet been fully elucidated. As we discussed last week, a recent study found that, contrary to expectation, genes that are dynamically regulated during development do not display histone modifications normally associated with active transcription.

A new study published this week in Nature Genetics reports another unexpected epigenetic pattern. Tri-methylation of histone 3 at lysine 4 (H3K4me3), a mark associated with active transcription, is usually present as a sharp, narrow peak at the gene promoter. The authors of the study observed that some genes show a different pattern of H3K4me3: broad, low density methylation spanning up to 10kb along the gene body.

The broad H3K4me3 mark was associated with high gene expression levels and transcriptional stability in this study. The authors also found that cell identity genes and, interestingly, tumor suppressor genes, were enriched for the broad H3K4me3 mark.

broadpeaks_genes

H3K4me3 density at housekeeping genes and tumor suppressor genes. Right panel is a zoomed-in version of the left panel.  {credit}Chen et al. Nat. Genet. 2015{/credit}

Though it is unclear why tumor suppressors specifically would be associated with this mark, a comparison between normal and tumor cells showed that H3K4me3 peaks at tumor suppressor genes became narrower in cancer cells and that this was associated with transcriptional repression. Finally, the authors showed that candidate tumor suppressor genes could be identified by the broad H3K4me3 mark.

We asked one of the study’s lead authors, Wei Li, to tell us a little more about the study:

What was the motivation for your studies?

The general motivation was to make novel discoveries based on existing ‘big data’ in epigenomics. In order to do so, we have had to develop novel bioinformatic tools that will enable us to look at the data from a completely different angle.  In particular in this study, we developed a new tool to quantify the H3K4me3 signal based on its width only. Most previous studies have only focused on its height or total signal, because the majority of genes (>95%) only have narrow (<1 kb) and high H3K4me3 peaks.   This simple method has never been used in epigenomic data analysis before. We further proved that this computer-derived broad H3K4me3 signal alone is sufficient to define both known and novel tumor suppressors and its performance is even better than the human curated KEGG pathway in cancer (a collection of well-curated signaling networks involved in cancer development).

When you first observed broad H3K4me3 peaks, did you expect that it would be such a widespread feature of tumor suppressor genes?

No, it is totally unexpected. Many people in the field (including ourselves) observed broad H3K4me3 peaks long time ago (even in the first histone mark ChIP-seq paper published in 2007), but all ignored them and treated them as potential sequencing artifacts.  My lab used the UCSC genome browser to check epigenetic patterns gene by gene on a daily basis, and we gradually noticed that broad H3K4me3 peaks are consistently observed in different datasets and specific to a small group of genes. To test whether it is an artifact or not, we decided to perform a functional enrichment analysis of genes marked with broad H3K4me3. If nothing is enriched, it must be a sequencing artifact.  Interestingly, we found an unexpectedly strong enrichment in tumor suppressor genes.

Did you consider whether any other classes of genes were enriched in this histone mark?

We used an unbiased data-driven approach (rather than hypothesis driven) to study the genes marked with broad H3K4me3 peaks. It turns out that only cell identity genes and tumor suppressors are enriched. When we removed cell-type specific broad H3K4me3 peaks by epigenomic conservation analysis, tumor suppressors is the only class of genes that are enriched in the conserved broad H3K4me3.

Widespread shortening of H3K4me3 peaks in cancer

Widespread shortening of H3K4me3 peaks in cancer{credit}Chen et al. Nat. Genet. 2015{/credit}

Tumor suppressors are defined by their role in cancer. Why do you think they show a similar pattern of H3K4me3 in normal cells?

A common feature of tumor suppressors is that they are usually highly expressed in normal cells to prevent tumor formation. This is likely why they show a similar pattern of H3K4me3 because broad H3K4me3 is associated with increased transcription elongation and enhancer activity together leading to exceptionally high gene expression in normal cells.

Not all tumor suppressors show the broad H3K4me3 mark. Why do you think this is?

Cancer is always heterogeneous. To my knowledge, there is no single mechanism in the literature that can specifically explain all tumor suppressors. Broad H3K4med3 is not an exception.

 

Developmentally regulated genes break the rules

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A new study published online this week in Nature Genetics reports that a certain class of genes, those with expression restricted to a specific developmental time point, follow a different set of rules than the rest of the genome.

The modifications to histones in promoter and enhancer regions are generally predictive of gene expression. For example, when a promoter is highly methylated at lysine 4 on histone H3 (H3K4me3), its associated gene is generally highly transcribed. Other marks may also be associated with activation, while different marks are associated with gene repression.

Developmentally regulated genes show similar H3K4me3 levels to silent genes, even though they are highly expressed during development.

Developmentally regulated genes show similar H3K4me3 levels to silent genes, even though they are highly expressed during development.{credit}Pérez-Lluch et al. Nat. Genet. doi: 10.1038/ng.3381{/credit}

SÍlvia Pérez-Lluch et al. examined the expression levels and histone modifications for all genes in the Drosophila modENCODE data set and identified a surprising pattern. Genes that were restricted in their expression to a specific developmental timepoint (called “developmentally regulated genes”) lacked epigenetic marks of active transcription, even when they were highly expressed. The authors confirmed the same pattern using modENCODE data for the netmatode C. elegans. 

Developmentally regulated genes  showed  expression levels during their actively transcribed period that were similar to those of  genes that are expressed stably throughout development. Another pattern identified by the authors was that strong histone marking is also associated with transcriptional stability. Comparable expression and chromatin modification data to that of the fly and worm aren’t yet available for mammals across multiple developmental timepoints. However, using data from ENCODE, the authors were able to show that mammalian cells showed a similar trend with regards to transcriptional stability.

We asked the lead authors of the study,  SÍlvia Pérez-Lluch, Montserrat Corominas and Roderic Guigo to give us a little insight into the history of this study and where they see this research going in the future:

When you began this study, what were your expectations? Did you expect to find that active chromatin marks were missing from so many actively transcribed genes?

We did not. Actually, our initial aim was not to investigate the relationship between chromatin marking and transcription, but the role of histone modifications in the regulation of splicing. We designed our initial experiments to compare levels of histone modifications in exons that were differentially included between Eye-antenna and Wing imaginal discs (EID and WID)—our hypothesis at that time being that the levels of some histone modifications would correlate with differential exon inclusion between these two tissues. But the results were quite frustrating, since we did find, in general, very low levels of marking in exons that were differentially included between WID and EID. This was initially very disappointing to us.  However, we also found, more generally, that many genes that were differentially expressed between WID and EID had also very low levels of a number of histone modifications typically associated to active transcription—even genes with very high expression levels. Since many such genes are likely to be regulated during development, this led us to hypothesize that lack of active histone modifications could be a general feature of developmentally regulated genes. This seemed an implausible hypothesis, going against the current models of the relationship between chromatin marking and transcription. Nevertheless, we turned to modENCODE data to further test it. The results were so strikingly consistent with our model that we “forgot” about our initial aim, and we focused our efforts instead into gathering additional supporting evidence. Understandably, our results were initially met with skepticism—the concern being that lack of chromatin marking could be a technical artifact derived from developmentally regulated genes having restricted expression patterns, and therefore making histone modifications difficult to detect using current technologies. Thus, a substantial amount of our work has been directed to address this concern.

Why do you think this pattern had not been observed before?

We are actually not the first to observe transcription with apparent lack of histone modifications. There have been a few reports of genes being transcribed in the absence of some histone modifications. Our main contribution is to show that this phenomenon is more widespread that generally assumed, and that it characterizes specifically genes that are regulated during development (at least in fly and worm). Why has this not been observed before? Mostly because data containing estimates of gene expression and histone modification along a sufficiently large number of developmental time points were not available before the modENCODE project. Then, we used a very simple, but effective measure to identify genes regulated during development, the coefficient of variation of gene expression. In summary, to make this observation you need both the data and the right approach to look at it

Your study showed that the link with transcriptional stability is also present in mammalian cells. If the association between chromatin marks and developmental regulation also holds in mammals, what, if any, do you think are the implications for biomedical research?

This is difficult to answer. Our initial results suggest that the model could be also applicable to mammals, but the data to test it are not yet available. Here we need to emphasize the importance of well-designed large-scale data production projects that monitor genome activity (transcription, chromatin structure, 3-dimensional genome organization, transcription factor binding, etc.) in a systematic and consistent way. We also want to emphasize that, at this point, our research is very basic. However, one could speculate that if our model holds in mammals, it could contribute to design better-informed approaches to manipulate/modulate expression levels of genes. Extrapolated to mammals, our results suggest that transcription factors play a comparatively more important role than histone modifications in the regulation of tissue specific genes. It has been shown that, in humans, tissue specific genes are more likely to be involved in diseases.

Are you able to speculate as to why developmentally regulated genes use a different epigenetic program compared to other genes?

What we call developmentally regulated genes correspond to genes with variable expression along time, which are often expressed only at a particular time point. Since development is a continuous process, one could speculate that rapid activation and de-activation of genes that are specific to a particular time point is more likely to occur without the need of modifying histone residues in chromatin.

What do you see as the most important next steps in this area?

Maybe the most important issue is to further challenge the model by investigating additional systems—in particular, mammalian systems—including differentiation processes, and additional histone modifications. The ultimate test of the model would come, however, from single-cell analysis, that is, from monitoring whether gene transcription does occur without histone modifications within the same cell. This is currently not possible given available technologies, but it may be feasible in the near future. It would be also important to investigate the role of distal enhancers, and of 3D chromatin structure, in the expression of developmentally regulated genes. Furthermore, we need to dig into the mechanism, by analyzing, for instance, how different classes of genes respond to perturbations of histone modification systems.

 

NIH Common Fund song & video contests

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“We want to know whether our future baby’s health is based on genes or the environment.” This is a concern shared by a lot of would-be parents for sure, and is the question posed to Dr. M. Elizabeth Ross at the beginning of this short video. The video, made by the labs of Dr. Ross and Dr. Christopher E. Mason at Weill Cornell Medical College is part of a competition sponsored by the NIH to commemorate the 10th anniversary of the Common Fund.

Logo for the NIH Common Fund 10-Year Commemoration Song & Video Competitions

The video takes a tongue-in-cheek approach to explaining an NIH-funded project investigating the role of epigenetic changes in birth defects. The contest ends tomorrow, May 9, and the results will be determined by the number of “Likes” the videos receive on YouTube.

To vote for this or any of the other amazing videos (and songs), check out the contest page here.

 

Cancer: what’s Down syndrome got to do with it?

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A Wright's stained bone marrow aspirate smear of patient with precursor B-cell acute lymphoblastic leukemia.

A Wright’s stained bone marrow aspirate smear of patient with precursor B-cell acute lymphoblastic leukemia. {credit}VashiDonsk via Wikipedia{/credit}

Trisomy 21 (having 3 copies of chromosome 21) is most well known as the cause of Down syndrome. But as you can imagine, having an entire extra copy of a chromosome has other negative consequences as well. For one, people with Down syndrome are 20 times more  likely than the average person to develop a severe form of leukemia, B cell acute lymphoblastic leukemia (B-ALL). Two recent studies have helped further our understanding of the molecular disturbances that take place in trisomy 21.

In Nature, Audrey Letourneau et al. took advantage of a rare situation to identify the genes that are misregulated in Down syndrome. The researchers profiled the transcriptomes of identical twins that differed in one crucial aspect: one twin had 3 copies of chromosome 21, while the other had a normal complement of chromosomes. (The samples were collected post-mortem from the fetuses, with the permission of the parents). This approach allowed the researchers to avoid any noise from irrelevant differences, since the genes of both twins would be identical. (Read the article in The Scientist about this study here).

Not surprisingly, they found that trisomy 21 causes gene regulation problems on all chromosomes. Misregulated genes are organized along the chromosomes in domains, and these domains were defined by changes to the chromatin methylation patterns. Importantly for future research efforts, they also showed that the corresponding genomic regions in the mouse model for trisomy 21 were similarly modified compared to control mice.

In a second paper published online this week by Andrew Lane et al. in Nature Genetics looked specifically at the relationship between Down syndrome and B-ALL.  The authors identified two genomic events as the drivers behind Down syndrome-related B cell acute lymphoblastic leukemia (B-ALL): overexpression of the nucleosome remodeling protein HMGN1 and changes in histone methylation marks. (You can read the Dana-Farber Cancer Institute’s press release about the study here).

Through a very meticulous set of experiments, they first show that just having an extra copy of a small region of chromosome 21 (or in this case, the corresponding mouse chromosome, 16) with 31 genes is sufficient for giving B cell precursors the ability to self renew indefinitely—the first step to cancer formation. From there, they identify and confirm a single driver gene on chromosome 21, HMGN1, as being expressed at unusually high levels. This high expression of HMGN1 causes a decrease in one type of methylation (H3K27me3), leading to overexpression of genes usually carrying both H3K27me3 and another histone mark, H3K4me3.

Trisomy 21 karyotype. All the normal chromosomes + 1.

Trisomy 21 karyotype. All the normal chromosomes + 1. {credit}Wikipedia{/credit}

Interestingly, the authors of Letourneau et al. mention HMGN1 as a good candidate for regulating the genome-wide chromatin modifications they found. The accompanying News & Views article by Benjamin Pope and David Gilbert note that HMNG1 should be a target of future study in Down syndrome. Looks like the authors of Lane et al. got the message far in advance!

So a pattern emerges: changes in chromatin methylation patterns are a key event in trisomy 21 overall and in Down syndrome-associated B-ALL specifically. Dr. Lane, lead author of the paper in Nature Genetics  wonders “Could this [chromatin modifications] be a unifying theme for phenotypes (not only cancer) associated with DS?” As I mentioned earlier, having 3 copies of this chromosome is bad for a number of reasons: higher risk of B-ALL and testicular cancer, vision and hearing problems, thyroid issues, higher risk of type I diabetes, gastrointestinal issues, low or no fertility and the more widely known neurocognitive isssues. Future studies on epigenetic changes in Down syndrome, and the regualtion of HMNG1, should be able to unravel the mechanisms underlying these different aspects of Down syndrome.

Outside of Down syndrome, these 2 studies may also lead to a better understanding of (and hopefully new treatments for) cancers caused by epigenetic changes. As the article by Lane et al. showed, changes to the chromatin landscape allowed B cell precursors to make that first step toward leukemia. By understanding how this happens, we can start to find ways to prevent it.