AN INTERVIEW WITH THE EXPERTS
- Top of page
- EPIGENETIC MODIFICATIONS
- DEVELOPMENTAL GENE REGULATION
- LINEAGE RESTRICTION
- TERMINAL DIFFERENTIATION
- AN INTERVIEW WITH THE EXPERTS
Developmental Dynamics: Describe your research.
Giacomo Cavalli (Fig. 1): We are interested in patterning mechanisms that involve the function of Polycomb group (PcG) and trithorax group (trxG) proteins. These epigenetic components were originally discovered as regulators of Hox gene expression. Through this function, they play a master role in the specification of the anteroposterior axis of the body plan. In addition, they regulate a variety of other genes that play important roles in developmental patterning and regulation of cell differentiation and proliferation. One important feature of PcG and trxG proteins is that they are able to maintain the memory of developmental decisions through cell division.
Figure 1. Noel Buckley, Professor, Molecular Neurobiology, King's College London, London, U.K. (Left), and Giacomo Cavalli, Group Leader, Institute of Human Genetics-CNRS, Montpellier, France (Right).
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We are studying the molecular mechanisms of PcG/trxG-dependent epigenetic inheritance of chromatin states, and we are trying to understand the global regulatory logic behind the function of these proteins. We have recently found that PcG proteins have the ability to induce the association of some of their target genes in the three-dimensional space of the cell nucleus. Now we would like to understand how widespread are these contacts and what is their influence on genome regulation.
Noel Buckley (Fig. 1): We have looked at transcriptional regulation in neurons for several years. The idea of “epigenetic regulation” is one that has taken hold relatively recently and has great resonance within neurobiology because of the potential to provide an understanding of the molecular underpinnings of the sorts of long-term changes that are the hallmark of neuronal plasticity and memory. Having said that, caution must be used to distinguish “epigenetic changes” from simple readily reversible histone modifications—even though the former may depend upon the latter. This is a well-recognized caveat and one that many, including Bryan Turner, have been careful to highlight.
Our own work has focused upon changes in histone modifications that accompany the transition from pluripotent ES cells, through multipotent neural stem cells to differentiated neurons, particularly in relation to the neuronal transcription factor, REST. More recently, we have begun to examine chromatin modifications at pan-neuronal and subtype specific genes as neuronal differentiation unfolds.
How did you become interested in epigenetics?
G.C.: Toward the end of the 1980s, it started to become clear in yeast that chromatin was not just a dull way to condense DNA in order to pack it into the nucleus, but rather a functional substrate of genomic regulatory processes. I had the privilege to join in the chromatin field during these years and to witness crucial advances showing that epigenetic regulation of chromatin is at the heart of genome function and contributes important information that can be passed on to the cellular progeny along with the DNA sequence. This is not only true in yeast and Drosophila, our model organism, but also in plants and vertebrates.
N.B.: We took up the theme of epigenetics relatively recently—more a slow embrace rather than an impassioned charge. The drive largely came in parallel with our burgeoning interest in neural stem cells. First, there was an interest in identifying transcription factors that determined or regulated neuronal phenotype. Second was a frustration borne from the dearth of suitable tractable, meaningful cellular systems to carry out these studies—there was no neural equivalent to the myoblast cell lines and hematopoietic stem cells that underpinned much of our current knowledge of hematopoiesis and myogenesis. This barrier has now been broken, and there are several robust neural stem cell models that are amenable to dissecting neurogenesis in a similar manner. The final thread of this trinity was the realization that many long-term changes induced by transcription factors were mediated by means of changes in histone modifications and/or DNA methylation. So, a rather indirect route brought us to epigenetics.
What papers have influenced you the most?
G.C.: One striking paper by Shiv Grewal and Amar Klar (Grewal and Klar,1996) showed that alternative chromatin states of the Schizosaccharomyces pombe mating type locus could be passed on to the majority of the mitotic and meiotic cellular progeny. This was the first clear demonstration that epigenetic regulation can affect heredity, making the point that genes are more than their primary DNA sequence. A second seminal paper in the field is that of Rea et al., identifying the first histone methyltransferase enzymes, which turned out to be proteins previously known for their role in heterochromatin formation (Rea et al.,2000). This study paved the way to the identification of many histone methylases involved in chromatin assembly and inheritance, including the widely conserved Enhancer of zeste protein of the PcG as well as histone methylase proteins of the trxG. More than a single paper, I would like to mention as a third event in epigenetics a flurry of papers published independently by several groups in 2006, all of them mapping the genomic distribution of PcG proteins in flies and mammals (Boyer et al.,2006; Bracken et al.,2006; Lee et al.,2006; Negre et al.,2006; Schwartz et al.,2006; Squazzo et al.,2006; Tolhuis et al.,2006). Together, these studies have a huge impact since they show that PcG proteins regulate in a coordinated fashion a number of conserved transcriptional pathways of fundamental importance for the development of the body plan. They also mark the beginning of a new phase in genome-wide location studies, where DNA chips of the latest generation, tiling through the large genomes of vertebrates, are coupled to chromatin immunoprecipitation to yield very accurate protein distribution maps. This robust technology will produce massive amounts of information in the next few years.
N.B.: As a neurobiologist I remain intrigued by the molecular and cellular basis of mood, perception, and cognition. Some time ago, a colleague pointed out a study that had discovered a significant increase in the incidence of adult schizophrenia in the offspring of mothers who had undergone prolonged starvation in the Chinese famine of 1959–61 in the province of Anhui (St. Clair et al.,2005). It is thankfully rare that we have access to studies such as these on the scale necessary to have sufficient statistical power to allow robust conclusions to be drawn, and although not an ounce of mechanism can be inferred, the influence of epigenetics on something as profound as human behavior can be clearly inferred.
Although, not strictly epigenetics, ChIPchip studies (first pioneered by Rick Young in yeast) have shown on a global scale how changes in histone modifications can be mapped over the whole genome (Ren et al.,2000). This is an immensely powerful technology and allows individual histone marks to be overlaid on to features of the genomic landscape, and this landscape, in turn, can be viewed at different developmental time points. There are now many of these papers, but I would pick two papers from Eric Lander's and Mark Groudine's groups since they were the first to show large-scale correlation between histone methylation, and acetylation and gene activity (Schubeler et al.,2004; Bernstein et al.,2005). I was also taken with Mandy Fisher's work (Azuara et al.,2006) and her demonstration that these marks can coexist in pluripotent stem cells — an idea echoed by Eric Lander later that same year (Bernstein et al.,2006). Both of these studies get away from the idea of individual histone marks as the definitive feature of the epigenome.
Relatively few epigenetically regulated developmental decisions have been identified (e.g., X inactivation, imprinting, maintenance of Hox silencing by PcG, control of neuronal genes by REST). Why are they so difficult to find?
N.B.: We have to first deal with the old chestnut of what we mean by “epigenetics.” There is a semantic and a scientific aspect to this and unfortunately, one clouds the other, especially in neurobiology. A textbook definition of epigenetics usually goes along the lines “reversible heritable changes in gene function or other cell phenotype that occur without a change in DNA sequence.” Neurons are terminally differentiated postmitotic cells, so any change in gene function is not heritable at either the cellular or organismal level. Epigenetic theories of neural development, therefore, embrace the implicit assumption that any “epigenetic change” must also occur in the germ cells in order for the change in gene function to be heritable. Changes in chromatin that are restricted to neurons cannot lead to heritable changes. Nevertheless, I want to expand this argument by reference to schizophrenia (Perkins et al.,2005; Sharma,2005), a devastating psychosis that effects around 1% of the population. Schizophrenia is a neurodevelopmental disorder, at least in the mind of many neurobiologists, and since large-scale population studies such as those used in the Dutch and Chinese famines show a robust epigenetic component to inheritance (see reply to third question), then we must accept that there is an epigenetic component to development—in this case, the development of behavior.
The hard bit is the last part of the question. Except for a few studies showing changes in DNA methylation at schizophrenia risk genes, there is nothing to hint at the exact nature of these heritable changes. This I think is the principle difficulty in identifying epigenetic mechanisms in development—demonstration of the phenomenon of epigenetic inheritance and provision of a direct molecular correlate. All too often, the twin aspects are not robustly linked.
G.C.: Noel is right, there has been a drift in the semantic definition of what is epigenetics, shifting from the developmentalist viewpoint of Waddington—the ensemble of the processes that use the information of the genotype to bring the phenotype into being—to a sharper and DNA-centered definition focused on mechanisms of inheritance not relying on the primary DNA sequence. This second definition was going against the mainstream view following which ALL inheritance was dependent on DNA. Although fascinating as a concept, this put the people working in epigenetics in the trenches. Every work they were delivering “had to” demonstrate that there was such a thing as inheritance after DNA. In my opinion, this preoccupation has prevented many attempts to even identify developmental processes regulated by epigenetic components. Because everybody was trying to decrypt mechanisms, they took phenomena that were overtly regulated epigenetically. This is typically the case of the processes you mention.
In fact, it is likely that many processes are regulated by “classical” transcription factors as well as epigenetic components. Moreover, many processes regulated by epigenetic factors may not have a strong heritable component. Luckily the groundwork of convincing the scientific community that epigenetic factors do regulate genes and can convey inheritance is now done, and this idea starts getting across even to the larger public. This brings development and also cognition and behavior at the center of interest of people involved in epigenetics, and also facilitates access to epigenetic research to those who were not doing it before. I am sure that, in the next years, we will watch discoveries of every sort of process being regulated, in part, by known epigenetic components.
Coming back to semantics, however, this is not without consequences. If we don't need a demonstration of DNA sequence-independent inheritance to admit the implication of epigenetics in a certain process, then are there really “epigenetic” and “nonepigenetic” regulators? In an age when every transcriptional regulator is being found forming chromatin regulatory complexes, is there a fundamental difference between epigenetic stars like Polycomb or heterochromatin proteins and any other transcription factor? The only one I can imagine is that some epigenetic processes involve inheritance, while others don't. In this view, epigenetic regulation would be a very general concept and epigenetic inheritance would be restricted to a part of these processes.
Do you think that many developmental decisions are epigenetically regulated?
N.B.: It is hard to believe otherwise. Any developmental change is accompanied by wholesale changes in gene expression with entire batteries of genes activated or silenced. Changes in histone acetylation and methylation accompany all these events and appear to be stably maintained in the differentiated state. Phenomenologically, this is epigenetics. Again, as in my earlier comments, the missing aspect is often the mechanistic link. Further proof comes from gene knockout studies that show the impact of chromatin modifying activities on developmental decisions. There are many examples, including the role of chromatin remodeling activities such as Brg1 in myogenesis (de la Serna et al.,2001) and neurogenesis (Eroglu et al.,2006). Before being hoist upon my own petard, I accept that this does not in itself constitute proof-positive of epigenetics, but it is a very strong indicator.
G.C.: I fully agree with Noel. When you look at the list of genes bound and repressed by PcG proteins in mouse and human cells (Boyer et al.,2006; Lee et al.,2006), and when you further consider that many of these genes are derepressed upon ES cell differentiation, while they actually become permanently silenced by DNA methylation upon cancer development (Schlesinger et al.,2006; Widschwendter et al.,2006), how can one sensibly doubt that epigenetics regulates important developmental processes?
Both REST and PcG repress/silence genes are involved in neuronal differentiation. Why do neuronal genes need their own repressor/silencer?
N.B.: This is one of the hardest outstanding questions and usually sits beside the equally vexed question of what governs whether a particular gene falls under REST (or PcG) regulation? The reason for coupling these two questions is that it unifies the idea that we need to provide an explanation for why particular genes become regulated by any individual transcription factor. In the case of REST, many targets, but by no means all, are neuronal genes. Equally important is the corollary that, by no means, are all neuronal target genes regulated by REST. Inspection of the 1,300 or so RE1 sites in the human genome (Johnson et al.,2006) provides no insight into any linkage among the targets, other than a bias toward genes associated with neuronal development or function.
One speculation that provides insight, if not explanation, is our recent demonstration that many REST binding sites seemed to have been deposited into 5′UTRs by means of LINE2 retrotransposons (Johnson et al.,2006). In other words, the “selection” of genes may be random. Deposition of a LINE2 element into a 5′UTR usually decreases the rate of transcription so deposition of a LINE2 element that carries a transcription factor binding site, confers regulated repression. Maybe this is an advantage in allowing batteries of genes to be regulated whilst in the continued presence of an activator. Simply switching off the activator may be more expensive in terms of alternative molecular compensatory mechanisms that would be necessary to keep essential genes turned on. Such fine-tuning of gene expression may be more necessary in the vertebrate nervous system where cell number and cell–cell interaction surpass any other biological system.
To address the latter part of the question, PcG, is normally (but not always) associated with silenced genes (stem cells provide an interesting exception) whereas our studies (Belyaev et al.,2004; Bruce et al.,2004; Greenway et al.,2006) show that REST is normally associated with active genes, i.e., employment of REST can maintain repression of an otherwise active gene, whereas in the differentiated state, PcG normally acts to establish and/or maintain silence.
G.C.: Noel is making a strong case for the study of these questions from an evolutionary perspective. We often have a biased view of the processes we are studying, thinking that they are “mature,” i.e., close to their endpoint evolution. There is a temptation to think that if something is as we see it today there must be a very good reason (strong selective pressure) to maintain it. If we understand how certain regulatory circuits have evolved, we come closer to the understanding of whether “chance” or “necessity” assigned certain regulators to certain processes. This approach is going to become more and more important as genomics progresses and many organisms, including nonmodel species are sequenced and studied.
Coming to the specific question, maybe that PcG proteins do regulate a lot of neuronal genes, but the nervous system might require its own dedicated set of epigenetic factors as an additional layer of regulation.
Do you think other tissue-specific gene programs (e.g. muscle) have specialized repressors/silencers?
N.B.: It is interesting that they haven't been discovered, even in the light of complete sequencing of several vertebrate genomes. As alluded to earlier, regulation of gene expression in the nervous system may require finer controls than other tissues because of the scale of the cellular heterogeneity. One further point to bear in mind is that REST is an evolutionary newcomer on the block—it is only found in vertebrates—again coincident with a rapid expansion in brain size and neuronal diversity. Maybe not a completely compelling argument, but an interesting coincidence.
G.C.: I am at a loss here, on the one hand I share Noel's feeling that the nervous system is a very “epigenetic object” with lots of cross-talk between stable cell fates and plasticity, which is the realm of action of epigenetic regulators. On the other hand, I would be very cautious not to make a prediction in this infant field; we may learn soon that muscle development is epigenetically regulated. For instance, a recent paper (Mal,2006) showing a regulatory association between SUV39H1 and MYOD makes me feel very excited about the amount of hidden regulatory potential in tissue development. I hope to read beautiful stories about these subjects in the future to help make up my mind.
Both REST and PcG can occupy actively transcribed loci. How do you explain this?
N.B.: This comes back to the idea of whether you think REST and PcG are repressors or silencers. In most adult tissue and differentiated cells, we find REST present at actively transcribed loci, and abrogation of REST function leads to derepression, indicating that REST acts to regulate levels of transcription rather than to simply silence transcription. This derepression is accompanied by reciprocal increases in histone H3K9 acetylation and decreases in H3K9 dimethylation at the REST binding site (Belyaev et al.,2004; Greenway et al.,2006).
The idea of singular histone modifications offering a read-out of gene activity is losing ground, and it is likely that no singular mark will have such predictive or causative power. This is especially evident in studies of stem cells where much of the cells chromatin appears to carry a “bivalent” or mixed signature comprising both “active” and repressed' marks (Azuara et al.,2006; Bernstein et al.,2006). Also, remember that in the REST−/− embryo, very few target genes were precociously activated (Chen et al.,1998). This of course could be due to absence of appropriate activators. Since embryonic lethality occurred before neurogenesis, then in vivo testing of these ideas awaits production of a conditional REST knockout.
G.C.: Noel is reminding us of a very important point, many regulatory processes result from a balance between putative activation and repression, with activators and repressors coexisting on their target rather than excluding one another. For instance, PcG proteins have to leave with their trxG partners on their targets and the papers cited above by Noel, as well as a fly study by Muller and coworkers (Papp and Muller,2006) really suggest that this is not just a mix of active and silent cells, but rather a true presence of bivalent marks on one and the same chromatin region.
The recent discoveries of the LSD1 and GASC1 histone demethylases suggest histone methylation is more dynamic than previously thought. How might this affect the interpretation of “silencing” histone methylation marks?
G.C.: The discovery of histone demethylases was very important, although in a way expected, since histone methylation patterns were earlier found to vary rather rapidly following certain treatments. Nonetheless, one should keep in mind that methyl groups have an average half-life of about two orders of magnitude longer (approaching the half-life of histones themselves) compared to other histone marks like acetylation. Therefore, methyl marks are likely to be maintained stably on the chromatin template of most genes, making histone methylation a good candidate modification for transmission of epigenetic inheritance. Histone demethylases are probably able to “reset” this memory when needed, and it will be important to dissect when and how is this reset brought upon.
N.B.: I think Giacomo's idea of “resetting” is key. Just because demethylases exist does not mean that all methyl marks will be erased. It simply lends more adaptability to allow reactivation of genes previously silenced. This could be particularly important in the recruitment, expansion, and differentiation of tissue-specific stem cells in response to damage in the adult where silenced genes may need to be reactivated (or conversely, active genes needed for maintenance of the multipotent state may need to be silenced). Identification of the transcription factors that target the demethylases will provide insight into when and where demethylation is employed.
Will it be important to distinguish stably methylated genes from ones that are subject to demethylation?
G.C.: I predict that, thanks to the explosion of high-throughput epigenetic mapping technology, we will actually “see” some of these demethylation events “on the fly.” Most importantly, we will learn about the genome-wide distribution of histone demethylases, and this might help understanding the whole issue. It is early to say whether there will be specific signatures identifying genes that are reversibly methylated as opposed to genes that are not. Even the most stable regulatory gene states are likely to be reprogrammable under extreme circumstances. However, it is likely that different genes (or classes of genes) can be differentially reprogrammed during development or in response to external stimuli.
N.B.: I certainly concur with the power of ChIPchip studies to provide genome-wide views of epigenetic changes. Considering that an oocyte nucleus can reprogram a somatic cell to derive a whole organism then it is clear that all marks are erasable.
Silencing of at least some PcG targets is mediated by pairing-sensitive silencing. Do you think other epigenetic silencers use this strategy?
G.C.: Long-distance pairing can be driven also by heterochromatin. Indeed, the first ever reported case of long-distance repressory chromosomal associations were published by the Henikoff and the Sedat lab 10 years ago and involved heterochromatic regions (Csink and Henikoff,1996; Dernburg et al.,1996). It is a common observation that heterochromatic loci cluster at the so-called chromocenters in both insects and vertebrate cells, and this clustering may affect silencing. Other epigenetics silencing systems may employ similar strategies and, indeed, long-range chromosomal associations are also linked to gene activation, as the work of several labs is recently showing (Spilianakis et al.,2005; Lomvardas et al.,2006).
N.B.: The notion of long distance interactions, both in cis and in trans is extremely important both for silencing and activation since it offers a mechanism to allow coordinate regulation across multiple genes—exactly the sort of mechanism you would expect to see during development and differentiation. The paper by Lomvardas et al. cited by Giacomo is a great example of the power of chromosome capture conformation (3C) to unravel these interactions. In passing I should point out that we have no evidence that REST undertakes such interactions, although I accept that absence of evidence is not evidence of absence. The one report in the literature by Lunyak et al. (Lunyak et al.,2002) that purports to show that REST can silence an entire locus is unfortunately based upon an incorrectly annotated gene order (Belyaev et al.,2004).
What exciting ideas are emerging in the field?
G.C.: One new idea is related to nuclear compartmentalization. The established view of “genes” is that they are regulated by cis-elements located somewhere close to the transcription start site or within “reasonable” (1–100 kb) distances from it. However, the mounting evidence for chromosomal contacts involving loci from different chromosomes or from different parts of the same chromosome raises the exciting possibility that gene regulation uses these contacts.
Another new concept is that noncoding RNAs play important regulatory roles in the genome, affecting all steps of gene regulation, from chromatin structure to stability and trafficking of the mRNA and to regulation of RNA translation. Many different classes of noncoding RNAs exist, many of them playing a role in developmental regulation. It is thus important to analyze each of them, and the initial studies are very promising.
Finally, the flood of genome-wide data raises the need to put a huge effort in understanding the logic of gene-regulatory circuits. For instance, a glance at the PcG target genes reveals that they often include multiple genes involved in the same pathways. Frequently, these genes code for transcriptional regulators. In the case of transactivators, one may speculate that PcG proteins secure a tight block of certain pathways by simultaneously shutting down multiple genes at different steps of the regulatory cascade. But what is the logic of silencing several transcriptional repressors along multiple steps of a pathway? Superficially, shutting down the expression of a tanscriptional repressor as well as of its own target genes does not make much sense. Clearly, one needs to understand how these events are regulated both spatially and temporally.
N.B.: Giacomo is absolutely right. We have produced detailed linear maps of multiple vertebrate genomes, and the application of increasingly sophisticated bioinformatics and cross-genome comparisons is overlaying detailed annotations of regulatory sites. The discoveries of long-range interactions, nuclear compartmentalization and noncoding RNAs all impose new dimensions of complexity onto this linear map. I think, as always, those of us working on development in higher eukaryotes will be keeping a close eye on the work of our colleagues in yeast genetics particularly in relation to building regulatory networks (Lee et al.,2002; Wyrick and Young,2002).
What important questions remain to be answered?
G.C.: Concerning nuclear architecture, a critical implication of the existence of long-range regulatory chromosomal contacts is that a phenotype may be induced from very remote parts of the genome. Therefore, it will be important to map long-range chromosome contacts systematically and to understand how frequently they affect gene regulation. It will also be crucial to study whether these contacts are the cause or the effect of regulation of gene transcription.
Another unanswered question concerns the mechanisms of epigenetic inheritance through DNA replication and mitosis (or meiosis). Indeed, chromosomes undergo major reorganization events during these two phases of the cell cycle. How can a chromatin state survive these events is not known and, largely, not even studied, because of the lack of appropriate approaches. Much of current effort in molecular biology aims to develop tools in order to work on the scale of limited number of molecules (ideally single molecules). This may allow tackling the issue of chromatin inheritance at the molecular scale in next few years.
Finally, a grand challenge for the future is to understand how the different gene regulatory “tools” are harnessed in a coordinated way during development. Orchestration has always been an issue in developmental biology, but certainly the new discoveries give a mind-boggling perspective of the combinatorial complexity that can be reached. Everything, from transcriptional output to gene product's location, can be dynamically regulated in a fine-tuned way by different processes. The other side of the coin is that there are also many risks for molecular mistakes that can potentially damage the developmental process. We will thus need to analyze how these complex regulatory processes can be coordinated in order to combine developmental plasticity with robustness.
N.B.: All of this is true. Development has often been perceived as a balance between external signals and intrinsic programs, a description that finds its reflection in some classic embryological terms such as “competent” and “determined.” The interplay between intercellular signals and wholesale remodeling of the genome that occurs as differentiation and development proceed will increasingly occupy our experiments—whether such changes are defined as epigenetic or otherwise. At the moment, most researchers focus on either the chromatin or on the signaling events, but these must converge if we are to offer comprehensive molecular correlates of “fate” and “potential.” Again, I think this interplay will be at its most complex in the nervous system where the complexity of signals and diversity of cell type is greater than with any other tissue.