The heritability of fears

Cyborg © EEG

As many of you know, one of my favorite topics here on the blog is epigenetic inheritance, i.e. the mechanisms that regulate changes in gene expression that can be passed from one generation to the next. Epigenetics has revolutionized the way we look at genetic inheritance: Darwin had taught us that the only way the environment can shape the genome of a species is through natural selection. While this is certainly still true, today we also know that:

1) Most of the mutations we see in a population have reached fixation through random drift -- the constant reshuffling from one generation to the next -- not selection.

2) The environment can induce changes in one generation that may indeed be passed on to the next generation not through actual changes in the DNA but, rather, in the way the DNA is "packaged" inside the cell nucleus (for a great explanation on how this work, see my colleague Karissa Sanbomatsu's TED talk).

 In a Nature Neuroscience paper [1], authors Dias and Ressler explored the following premise in a mouse model:

"An important, but often ignored, factor that influences adult nervous systems is exposure of parents to salient environmental stimuli before the conception of their offspring. Such information transfer would be an efficient way for parents to ‘inform’ their offspring about the importance of specific environmental features that they are likely to encounter in their future environments. However, this would necessitate the transgenerational inheritance of environmental information via the germ line by offspring not even conceived at the time."

The researchers used olfactory fear as the stimulus mostly because it's one of the best understood mechanisms, both at the neurological and the molecular biology levels. Of course, a caveat would be that humans, besides being very different from mouse models, they've evolutionarily replaced olfactory stimuli with visual ones.

The researchers used odor-naive male mice and targeted an odorant receptor (M71) whose expression in the olfactory sensory neurons has been shown to be activated by acetophenone. It is important to note that the experiment did not induce any change in the actual DNA of the mice. What they did, instead, was use acetophenone to activate the receptor so it would be expressed inside these special neurons.

As I explained in older posts, DNA is wrapped around "spools" called histones. Cells produce proteins and activate receptors depending on what genes are on the outer surface of the "histone yarn", while hidden parts of the DNA remain unexpressed (as if that gene didn't exist). A molecule like acetophenone can induce changes inside olfactory sensory neurons that cause the histones to move and expose the gene that encodes the M71 odorant receptor. Once this happens, the receptor is "activated."

 Since the mice are initially odor naive, their M71 receptor is inactivated (the gene is not expressed) prior to the exposure to acetophenone. After the receptor activation, these male mice were mated with odor naive females. So, genetically speaking, the offsprings had no reason to have the M71 receptor activated, since neither parent had it activated at birth. Yet the offsprings of the mice stimulated with acetophenone, despite not being previously exposed to any of the odors with which they were tested, were able to detect acetophenone at lower concentrations than the offsprings of mice stimulated with another molecule (propanole).

Not all offsprings were behaviorally tested. Some of the offsprings were kept naive to any exposure so that their neuroanatomy could be tested separately without risking the results to be affected by the behavioral tests. When they looked at the offsprings of the acetophenone exposed mice, the researchers found an increase in the M71 glomerular area together with a significant increase in the numbers of M71-activated olfactory sensory neurons in the main olfactory epithelium.

So, how these epigenetic changes get inherited? To address the question, Dias and Ressler examined the sperm of the acetophenone exposed mice. This part of the paper gets a little technical, but the interesting idea is that they did find molecular changes in the sperm DNA around the Olfr151 gene, which encodes the M71 receptor. They found that the 3' end of Olfr151 was significantly less methylated in the acetophenone induce mice. At the same time, they

"did not observe any histone-mediated epigenetic signatures around the M71 locus when chromatin was immunoprecipitated with antibodies that recognize histone modifications that either permit or repress to transcription."

The authors conclude:

"In summary, we have begun to explore an under-appreciated influence on adult behavior—ancestral experience before conception. From a translational perspective, our results allow us to appreciate how the experiences of a parent, before even conceiving offspring, markedly influence both structure and function in the nervous system of subsequent generations. Such a phenomenon may contribute to the etiology and potential intergenerational transmission of risk for neuropsychiatric disorders, such as phobias, anxiety and post-traumatic stress disorder."

[1] Dias, B., & Ressler, K. (2013). Parental olfactory experience influences behavior and neural structure in subsequent generations Nature Neuroscience, 17 (1), 89-96 DOI: 10.1038/nn.3594




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The encyclopedia of DNA - Part I

The raw numbers of the human genome: three billion base pairs, of which roughly 1% fall into the 20,000 genes in our genome. So, what's all the extra stuff for?

Typing the whole human genome, in 2001, was only the beginning. The next step in disentangling the puzzle was to assign biochemical functions to those three billion base pairs.

"The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions" [1].

Let's start with a bit of a refresher.

Regulatory regions: these are regions in the genome that regulate gene transcription. Thanks to these regulatory sequences, skin cells only express "skin" genes, brain cells express "brain" genes, and so on. Promoters, for example, are regulatory sequences found immediately before the start of the gene, on the same strand, and they initiate the transcription of the gene. There are other regions, called enhancer, which also promote transcription. However, contrary to promoters, enhancers need not be near the gene. They don't even need to be on the same chromosome, and some enhancers have been found in introns, regions of a gene that are removed prior to making mRNA.

Transcription factors: I talked a little bit about them last week. These are proteins that can either promote or block the recruitment of RNA polymerase, and therefore either activate or silence a gene.

And, finally you can review the concepts of chromatin structure and histone modification in a few previous posts.

All these concepts are useful to understand that there's a lot, and I mean A LOT going on, between genes and phenotype. Genes are only the starting point. You can't just look at genes alone in order to try and infer a phenotype.

Started in 2003, the aim of ENCODE was to annotate all functional regions of the genome, where by "functional" they don't just mean encoding proteins, but also presenting some biochemical signature such as protein binding or a specific chromatin structure. The latest findings published in Nature: over 700,000 promoter regions and nearly 400,000 enhancer regions that regulate gene expression.

You can see the complications and layers to this: while we have one unique genome, which is identical in all nucleated cells, once you start looking for function, you have to look at the whole genome and chromatin structure and RNA transcripts of all cell lines, as each cell line will have its own activated and silenced genes, its own chromatin signatures, and so on ... whew, that's A LOT!

So far the ENCODE Project Consortium has integrated the data from 1,640 experiments involving 147 different cell types. They saw that

"The vast majority (80.4%) of the human genome participates in at least one biochemical RNA- and/or chromatin-associated event in at least one cell type."

Many more cell lines are yet to be explored, and yet these initial results already shed light into puzzling questions, like, for example: why do nearly 90% of SNPs found in whole genome disease association studies fall outside genes?

"Single nucleotide polymorphisms (SNPs) associated with disease by GWAS are enriched within non-coding functional elements, with a majority residing in or near ENCODE-defined regions that are out- side of protein-coding genes. In many cases, the disease phenotypes can be associated with a specific cell type or transcription factor."

I can't tell you how excited I am about these results, as I started blogging a little over one year ago raising exactly the point that junk DNA should NOT be called junk DNA.

I'm coming down with the flu (how do you explain to your kids NOT to cough in your face when they have a bug? Sigh), so this will be all for this time. But I've got all the Nature papers printed out and will be talking more about them in the next few weeks. A lot of new (and exciting) stuff to learn!

[1] The ENCODE Project Consortium (2012). An integrated encyclopedia of DNA elements in the human genome Nature DOI: 10.1038/nature11247

Transcription factories for gene expression: the hard working units of the nucleus

You've probably heard it many times already: if you could stretch out the DNA contained in any one nucleated cell in your body, it would be 2 meters (~6 feet) long. Now imagine packing this 2-meter long molecule into a sphere whose diameter is of the order of a few micrometers, roughly one millionth smaller than a meter. Yes, it's going to be packed in there, yet those genes have to be accessible to the "workers" that come in and perform daily tasks such as gene transcription, replication, and DNA repair. Clearly, which genes are accessible and which aren't is going to play a major role in the cell's life and development.

The chromatin, the ensemble of DNA and proteins inside the nucleus, is dynamically regulated. For gene expression, active genes relocate from chromosome regions and cluster into subnuclear compartments called "transcription factories for gene expression."

As you know, transcription is one of the fundamental steps in the making of proteins: the enzyme RNA polymerase II creates a complementary strand of RNA (a precursor of mRNA) from the active gene. The mRNA is then synthesized and translated into the protein's amino acid sequence. The concept of transcription factories comes from the observation that specific regions in the nucleus are highly enriched in RNA polymerase II, and those are the regions from which new RNA transcripts emerge. A second observation is that distant loci, often on different chromosomes, can interact during regulation through long-range regulatory contacts.

"Increasing numbers of examples suggest that regulatory DNA elements also seem capable of undergoing functional contacts with genes located on other chromosomes. [...] By contrast, temporarily inactive alleles are positioned away from transcription factories, suggesting that genes migrate to these subnuclear sites in order to be transcribed. Crucially, the number of transcription factories per cell is severely limited compared to the number of expressed genes, compelling genes to share the same transcription factory [1]."

The above figure is a schematic of a transcription factory: active genes from different chromosomes are recruited from the chromatin. As transcription proceeds and new RNAs are formed, the templates are reeled through the factory bringing downstream nearby genes. Transcripts generated in a transcription factory that are in close proximity have a greater chance to undergo trans-splicing, in other words, the two transcripts are joined into one even though they originated from different RNA polymerases. The resulting joint RNA is called chimeric RNA. A few studies have observed proteins generated from chimeric RNAs.

In addition to trans-splicing, close proximity in a transcription factory increases the chances of translocation, i.e. one genomic region being moved to a different locus.

"It is puzzling that a genome conformation that increases the risk of potentially grave translocations can evolutionarily persist. We speculate that three- dimensional gene clustering of transcribed loci must elicit evolutionary advantages that outweigh the dangers of translocations."

As Schoenfelder et al. conclude,

"A major challenge will be to decipher the relation between these genome conformation changes and the numerous epigenetic alterations of the genome, allowing their integration into a comprehensive picture of the spatial and functional organization of the nucleus."

[1] Schoenfelder, Stefan, et al. (2010). The transcriptional interactome: gene expression in 3D. Current Opinion in Genetics DOI: 10.1016/j.gde.2010.02.002

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

Is epigenetics new? Not to a "smart" influenza virus!

I was browsing the latest papers on Science when I read:

"By mimicking epigenetic regulation in human cells, one flu strain suppresses the expression of antiviral genes [1]."

Wow. Epigenetics and viruses? I had to read that paper!

I've discussed many times how gene expression in cells can be altered through epigenetic changes. The figure below, also taken from [1], shows one of the most common mechanisms by which cells alter gene expression: inside the nucleus, DNA is wound around proteins called histones. The addition of a methyl group (shown in blue in panel b), can alter the transcription of the gene.

In the figure, H3 represents the histone tail. This tail has two amino acids, lysine and arginine, which can be modified in a way that alters the interaction between DNA and the histones. These cause changes in the topology of the chromatin (the way the DNA is packaged inside the nucleus), allowing for certain regions rather than others to be accessed for transcription. If I just gave you a headache, think of chromatin as a tight yarn and you want to poke your finger inside to reach certain threads. The histone tail is like a "lever" that you can use to gain or lose access to the inner parts of the yarn. That's how the cell activates or deactivates genes.

"The ability of histone tails to guide gene function indicates the possibility of targeted control of gene expression by artificial or naturally occurring molecules that can structurally and/or functionally mimic the histone tail [2]."

The influenza A H3N2 subtype has a protein, NS1, that is not vital to viral reproduction but is known to suppress the host's response to the viral infection. In fact, without NS1, the viral infection is significantly mitigated. In a recent Nature paper, Marazzi et al. [2] found that NS1 carries a sequence that resembles the histone H3 tail. This mimicry supports viral infection by halting the transcription of genes essential to counteract the infection.

"We have shown that H3N2 influenza A virus interferes with host gene expression by exploiting the very basic principles of the epigenetic control of gene regulation. By mimicking the histone H3K4 sequence, which has a key role in positive regulation of gene transcription, the influenza virus gains access to histone-interacting transcriptional regulators that govern inducible antiviral gene expression."

Of course, these findings are very intriguing and they raise the question of whether this mechanism is novel to the H3N2 strain or not, and, also, what would happen if a particularly virulent strain like the avian flu would suddenly develop this mechanism as well. At the same time, this opens up new research on ways to attenuate viral infections by targeting the NS1 protein.

[1] Krasnoselsky, A., & Katze, M. (2012). Virology: Influenza's tale of tails Nature DOI: 10.1038/nature11034

[2] Marazzi, I., Ho, J., Kim, J., Manicassamy, B., Dewell, S., Albrecht, R., Seibert, C., Schaefer, U., Jeffrey, K., Prinjha, R., Lee, K., García-Sastre, A., Roeder, R., & Tarakhovsky, A. (2012). Suppression of the antiviral response by an influenza histone mimic Nature, 483 (7390), 428-433 DOI: 10.1038/nature10892

How nucleosomes "protect" our DNA

Did you know that not all mutations happen at an equal rate? There are several kinds of mutations: substitutions, insertions, deletions, etc. Insertions and deletions happen when bits of DNA are either inserted or deleted, whereas substitutions happen when the overall length of the DNA locus doesn't change, but a base is substituted for another. As you all know, we have 4 nucleotides (A, C, G, and T), however, not all possible changes are equally likely. The most frequent substitutions are As with Gs and Cs with Ts.

Mutations happen because of errors in DNA replications or because of DNA lesions. These are chemical processes that are more or less likely depending on the circumstances. For example, DNA is "stronger" when it's a double helix, although occasionally the bonds between the two helices can locally denature, opening up a chance for a mutation to happen.

In all nucleated cells DNA is packaged inside the nucleus in units called nucleosomes: threads of DNA (~147 base pairs) wrap around "spools" formed by 8 protein units called histones. When the DNA is packed into nucleosomes it is more resistant and less prone to mutations. At the same time, chromatin, the assembly of all nucleosomes inside the nucleus, is hardly ever static. See this post where I discuss how nucleosomes are reassembled in order to promote the expression of certain genes versus others (a phenomenon called "chromatin remodeling"). A new study [1] published in the latest issue of Science investigates how the structure and assembly of DNA inside the cells affects the likelihood of certain mutations versus others. They found that nucleosomes act as regulators for substitution mutations, protecting DNA from damage. For example, compared to other DNA states, nucleosomal DNA undergoes 50% less C -> T mutations.

"Furthermore, the rates of G -> T and A -> T mutations were also about two-fold suppressed by nucleosomes. On the basis of these results, we conclude that nucleosome-dependent mutation spectra affect eukaryotic genome structure and evolution and may have implications for understanding the origin of mutations in cancers and in induced pluripotent stem cells."

Without getting into too many technical details, Chen et al. looked at the initial nucleosome profile from two replicates of the yeast Saccharomyces cerevisiae strain Y55, and then tracked subsequent mutations. They also looked at SNPs (single-nucleotide polymorphisms) in the germline of the Japanese killifish medaka. Germline cells are cells that give rise to oocytes and spermatocytes, hence mutations in this line are of evolutionary importance since they get carried on to subsequent generations.

We have revealed that nucleosomes, the most abundant eukaryotic protein-DNA complexes, likely function as a major regulator of substitution mutations in eukaryotes. Binding of proteins to DNA to suppress DNA breathing or to exclude endogenous mutagens may be how cells protect their DNA. However, DNA repair, which often works with varied efficiency between nucleosomal DNA and naked DNA, may also shape the base-specific mutation spectrum."

Chen, X., Chen, Z., Chen, H., Su, Z., Yang, J., Lin, F., Shi, S., & He, X. (2012). Nucleosomes Suppress Spontaneous Mutations Base-Specifically in Eukaryotes Science, 335 (6073), 1235-1238 DOI: 10.1126/science.1217580

Photo: light reflections (or is it refractions?) on a soap bubble. Shutter speed 1/125, focal length 100mm, F-stop f5, ISO speed 100.

Large intergenic noncoding RNAs affect gene expression

I learned this amazing fact from a talk I went to last week: currently, somewhere between 70% and 90% of DNA is estimated to be transcribed into RNA but not translated into proteins. So, the question is: if it's not making proteins, what's all this non-coding RNA doing?

In mammalians in particular, more than a thousand large (over 5 kb) intergenic noncoding RNAs (lincRNA) have been identified [1] and, by looking at expression patterns, researchers were able to see that they are involved in many different biological processes. They are evolutionary conserved across species, indicating that they are indeed functional, yet very little is known of their function. Two 2009 papers [1,2] investigated whether lincRNA are involved in the establishment of chromatin states by creating "genome-wide chromatin-state maps."

We need a refresher here. I discussed chromatin (the package of DNA and proteins inside the nucleus) in this post. The structure and topology of the chromatin changes from cell line to cell line and also during a cell's life, allowing for different genes to be activated or deactivated as needed (for example during cell differentiation). These modifications in the way the DNA is packaged are called chromatin states and are key to understand how and which genes are expressed inside the cell. In particular, there exists a whole family of proteins, called chromatin-modifying complexes, that modify the structure of chromatin to promote or inhibit access genes.

in [1], Guttman et al. looked at a particular genome domain called K4-K36 in genome-wide chromatin-state maps in 4 mouse cell lines. This chromatin signature marks actively transcribed genes, hence, they were able to find lincRNAs "by identifying K4-K36 structures that reside outside protein-coding gene loci."

"These lincRNAs show similar expression levels as protein-coding genes, but lack any protein-coding capacity. Importantly, lincRNAs show significant evolutionary conservation relative to neutral sequences, providing strong evidence that they have been functional in the mammalian lineage [2]."

In [2], Khalil et al. extended the results found in [1] by mapping the K4-K36 domain to 6 human cell types. They found 1,703 new human lincRNA genes and estimated the total number of human lincRNAs to be roughly 4,500. Of all newly discovered lincRNAs, a substantial fraction was found to be associated with PCR2, one of the chromatin-modifying complexes I mentioned above.

"Collectively, these results suggest that many lincRNAs collaborate with chromatin-modifying proteins to repress gene expression at specific loci. [...] Our results suggest an intriguing hypothesis that lincRNAs bind to chromatin-modifying complexes to guide them to specific locations in the genome. [...] Under our model, differentially expressed lincRNAs could bind to these complexes and help establish cell type specific epigenetic states."

The specific experiments conducted by Khalil et al. identified associations with chromatin-modifying complexes that have a repressive role, but the researchers suggest that, with different experiments, one could find additional lincRNA that instead are associated with activating chromatin-modifying complexes.

[1] Guttman, M., Amit, I., Garber, M., French, C., Lin, M., Feldser, D., Huarte, M., Zuk, O., Carey, B., Cassady, J., Cabili, M., Jaenisch, R., Mikkelsen, T., Jacks, T., Hacohen, N., Bernstein, B., Kellis, M., Regev, A., Rinn, J., & Lander, E. (2009). Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals Nature, 458 (7235), 223-227 DOI: 10.1038/nature07672

[2] Khalil, A., Guttman, M., Huarte, M., Garber, M., Raj, A., Rivea Morales, D., Thomas, K., Presser, A., Bernstein, B., van Oudenaarden, A., Regev, A., Lander, E., & Rinn, J. (2009). Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression Proceedings of the National Academy of Sciences, 106 (28), 11667-11672 DOI: 10.1073/pnas.0904715106

Epigenetic reprogramming: how cells start afresh

Last week I talked about the chromatin, the complex of DNA and proteins that resides inside the nucleus. There were two key points to that post: (1) the topology inside of the chromatin, or, in other words, how the chromosomes are arranged inside the nucleus, is correlated to which genes are active and which aren't; (2) these changes in the chromatin that allow for gene expression and gene silencing can be inherited, though how it's still a mystery.

I admit I left that second point a bit vague last week. So, with the help of a fantastic review I found on PubMed [1], today I'd like to develop the topic further.

The rearrangements of the chromosomes within the chromatin determine what are known as epigenetic marks:

"Epigenetic marks are covalent modifications of the DNA (DNA methylation) or post-translational modifications of the histone proteins (histone modifications) that make up the chromatin into which our DNA is packaged. [1]"

Different cells in the body present different epigenetic marks depending on which genes are expressed and which are silent. Within a specific cell line, epigenetic marks are conserved as cells divide, thus maintaining the differentiated state of the cell. For example, skin cells will divide in skin cells and not change into brain cells, right?

This is true for all cells in the body except one very special set: the germline cells. If you think about it, it makes perfect sense: germ cells give rise to an embryo, and hence have to remain undifferentiated. Therefore, all epigenetic marks must be reset in order to enable a completely new undifferentiated state, a process called epigenetic reprogramming.

"It is almost twenty years since the discovery of the biological importance of germline DNA methylation in the context of imprinted genes, and ten years since the identification of the key enzymes responsible for de novo DNA methylation in mammals. Even so, what specifies why specific DNA sequences become epigenetically distinguished in germ cells is still only partially understood."

During developmental epigenetic reprogramming, primordial germ cells emerge with their own epigenetic marks and, as these cells migrate and proliferate, the marks are gradually lost (DNA methylation is globally erased):



It's interesting to see how the new marks are established in an asymmetric fashion for males and females. In the male embryo, de novo methylation takes place and the new marks are established and completed by birth. In the female embryo, the process is arrested in the oocytes and resumed at puberty. In the event of a fertilized oocyte, the marks are erased again, as illustrated by the blue and red line descending again in the above figure.

Smallwood and Kelsey explain the various phases of the above processes in great detail. Interestingly,

"DNA methylation is distributed throughout the genome, at repetitive elements and single-copy sequences. With the recent development of genome-wide methylation profiling techniques employing next-generation sequencing, the full pattern of DNA methylation in gametes, and how it is laid down during germ-cell development, is beginning to emerge. [...]Despite the advances in the identification of key factors in DNA methylation in the germline, many questions remain over mechanism – in particular, how a select number of imprinted gDMRs and CGIs are specified for DNA methylation. The development of deep-sequencing technologies has opened new horizons, and it is now possi- ble to profile DNA methylation on a genome-wide scale in very small amounts of genomic DNA, providing an unparalleled opportunity to shed new light on mechanisms of de novo DNA methylation in germ cells [13,81]. Because the interaction of DNMT3 proteins with nucleosomes is regulated by several histone modifications (at least in vitro) it is now imperative that such capabilities are matched by the development of chromatin immunoprecipitation sequencing (ChIP-Seq) protocols to profile histone modifications in vivo in limited amounts of starting material; this would undoubtedly represent an important advance in the field of epigenetic reprogramming."

I asked my dad, a developmental biologist from the University of Pisa, what his thoughts were on the matter, and this is what he had to say:

"The conclusion to be drawn from these latest findings is thus as follows. So far we have been looking at single epigenetic changes and asked the question what does each one of them mean in relation to the phenotypic effects envisioned on an organismic scale. Needless to say that we have not gone very far by pursuing this simple-minded approach. The newly emerging evidence is pointing to another direction. Taken together, the epigenetic markers of chromatin imprinting, histone acetylation and base methylation should perhaps be considered as systemic modifications rather than simple one-to-one cause-effects relationships. By this I mean to say that the nuclear context in which such modifications occur is as important as any other macromolecular co-factor sustaining their interaction with the phenotypic counterparts. Perhaps by knowing how epigenetic markers are changed on a genomic scale it would be possible in the future to understand how they relate to one another and how altogether have provided living creatures with an adequate responding repertoire to adapt to ever changing environments during evolution."

[1] Smallwood SA, & Kelsey G (2011). De novo DNA methylation: a germ cell perspective. Trends in genetics : TIG PMID: 22019337

Understanding the cell nucleus in order to unravel the mystery of epigenetic heritability

The above image is the striking view of the surface of a cell nucleus (in pink). The dark crater represents a hole in the nucleus and offers a peek inside: the granular consistency that you see there are the chromosomes, bundled together in what may appear a random distribution but, in reality, is nothing but random:

"In all eukaryotic species analyzed so far, spatial genome arrangements are nonrandom: chromosomes or genomic loci occupy preferential positions with respect to each other and/or to nuclear landmarks [1]."

The nucleus contains a combination of DNA and proteins (mostly histones) called chromatin. Histones can be thought of spools around which the DNA wraps, forming a structure called nucleosome. Proteins in the chromatin can be silenced or activated, thus allowing differentiated cells to express only the genes necessary to their specific function. The budding yeast Saccharomyces cerevisiae was the first eukaryote cell to have its entire genome sequenced and, due to its relatively compact size (16 small chromosomes), it has been studied extensively to understand the structure of the cellular nucleus. For example, one of the largest protein complexes on the nuclear envelope is the nuclear pore complex, or NPC, which modulates the exchange of components between the nucleus and the cytoplasm. Several genes are relocated to the NPC when activated, and, as Zimmer and Fabre note [1],

"The region close to the nuclear envelope thus emerges as a mosaic, with the vicinity of NPCs representing zones favorable to transcription, whereas the zones between NPCs are more repressive."

These spatial arrangements are not static but they undergo re-arrangements (through complicated chemical alterations like cytosine methylation and/or post-translational modification of the histone amino acids). The extent of packaging of the nucleosome affects gene expression, however, to this day, little is known on what determines this delicate spatial arrangement.

And here's the intriguing bit: the re-arrangements the chromatin undergoes are generally reversible. And yet there's a level of these modifications that not only remains unmodified, it becomes inherited [2]:

"Chromatin modifications are often termed epigenetic marks; however, an unresolved issue in the field is the relationship between these modifications, including those established during transcription, and epigenetic inheritance (that is, the stability of these alterations during cell divisions and development). It seems that most, if not all, histone modifications are reversible, so it remains to be determined how epigenetic persistence of chromatin states is achieved, and which modifications are heritable."

These are the transgenerational epigenetic modifications I have discussed here and here. It's a real puzzle because heritability happens through the germ line cells, but in this cell line transcription only happens de-novo after fertilization. So at what level and how are epigenetic changes inherited? In [2], Berger reviews the various types of chromatin modifications and concludes with a nice analogy:

"Language is defined by the Webster dictionary as systematic means of communicating ideas using conventionalized signs or marks having understood meanings. This definition can be used to describe the complexity of the relationship between epigenetic marks and the biological processes they influence. As scientists, it falls to us to learn and understand this language a task that we have only begun to undertake."

EDIT: as I was preparing this post, I found this article on Scientific American, which talks about untangling the 3D human genome, and how the topology inside the nucleus determines which genes are on and off. There's a neat video, if you scroll to the bottom of the article.

[1] [1] Zimmer, C., & Fabre, E. (2011). Principles of chromosomal organization: lessons from yeast The Journal of Cell Biology, 192 (5), 723-733 DOI: 10.1083/jcb.201010058

[2] Berger, S. (2007). The complex language of chromatin regulation during transcription Nature, 447 (7143), 407-412 DOI: 10.1038/nature05915