Adapted from an original article by Jennifer Altman in the July-August 2011 issue of Alzheimer Actualités, a newsletter published in French by the Ipsen Foundation. The Alzforum editors acknowledge the Foundation’s generosity in making this summary freely available in English.
9 December 2011. The one-gene, one-protein dogma has been successfully used to identify mutant genes and dysfunctional proteins associated with a multitude of inherited conditions, including Alzheimer’s disease (AD). But this view of the genome now seems simplistic. It has become increasingly apparent that genes are embedded in a network of molecules, known as the epigenome, that regulates gene expression to meet varying demands on the cell and the organism. At the annual IPSEN colloquium in the Neurosciences, researchers from leading laboratories explored the epigenetic links between nervous system function and behavior in health and disease, and the new therapeutic directions that may open up as a result of this new knowledge. The meeting, held in Paris on 18 April 2011, was organized by Paolo Sassone-Corsi of the University of California, Irvine, and Yves Christen, Fondation IPSEN, Paris, France.
Although the molecular and cellular exploration of epigenetic mechanisms has only recently begun, Jean-Pierre Changeux, Institut Pasteur, Paris, France, traced the concept back to C.H. Waddington, professor of Animal Genetics at the University of Edinburgh until 1975, who in 1942 defined epigenetics as the interaction of genes with their surroundings to produce a phenotype, that is, the precise form of the adult body. Waddington envisaged an "epigenetic landscape," a slope formed of bifurcating valleys, down which a ball rolls. The exact shape of the ridges and valleys, representing a combination of genetic and environmental influences that differs for each cell type or organism, determines where the ball ends up.
Today, the term "epigenetics" has a variety of applications: Peter Becker, Ludwig-Maximilians-Universität München, Munich, Germany, and Adrian Bird, University of Edinburgh, U.K., used it to denote the regulation of cellular differentiation in developmental biology, while Shelley Berger and Ted Abel, University of Pennsylvania School of Medicine, Philadelphia; Sassone-Corsi; and David Sweatt, University of Alabama at Birmingham, all applied it to the organism’s accommodation to the environment in adult life. Changeux extended this use of the term to include the processes modeling the human brain to fit its social and cultural environment. An exciting development, discussed by Michael Meaney, McGill University, Montréal, Canada, and Isabelle Mansuy, University of Zurich, Switzerland, is that at least some environmentally driven epigenetic changes may be passed on to later generations—giving a fresh twist to the theories of inheritance and, especially, evolution.
At the heart of epigenetics are the mechanisms that regulate the transcription of genes in response to the needs of the cell and the whole organism. The identification of the molecules involved in these communication and regulation processes is key to understanding how the epigenome works.
As cells differentiate into various types, the subset of genes being transcribed narrows to just those required for the specialized functions performed by that cell, be it a neuron or a skin cell. Which genes are actively transcribed and which remain silent rely on a combination of molecular mechanisms: regulation of the way the DNA ribbon is packed into the cell nucleus, a process known as chromatin remodeling, and DNA methylation, which in most cases preventing genes from being transcribed. Both mechanisms depend on specific enzymes and regulators that are recruited to the DNA in response to the activation of complex signaling pathways in the cytoplasm and nucleus. In mature cells, similar mechanisms increase or decrease the activity of genes, or switch on silent genes, in response to metabolic demands or experience.
Packing It in and Shutting It Up
The way the DNA is packed into the nucleus determines which stretches of the molecule, and, hence, which genes, are exposed to the transcription machinery. Each human cell contains over two meters of DNA, representing about 20,000 genes, fitted into a nucleus about 10 μm in diameter. Becker reminded the audience that this is achieved in an orderly fashion by the winding of the DNA ribbon around a series of nucleosome cores made up of histone proteins. Nucleosomes are further packaged into the dense material called chromatin. Transcriptionally inactive stretches of DNA are packed very tight, but where genes are active, the DNA ribbon is spooled out and becomes free of the histones.
Molecular machines known as chromatin remodelers orchestrate this condensation and relaxation of the chromatin structure, Becker continued. They bind to a “pioneer adaptor factor” inserted into the DNA ribbon between two closely arrayed nucleosomes; once attached to the DNA, this complex slides the nucleosomes apart. During differentiation, remodelers help to establish and maintain the integrity of chromatin, allowing the genome flexibility to respond to developmental, metabolic, and environmental signals. One family of developmental remodelers, the chromatin accessibility complexes, or CHRACs, are involved in the spacing and higher-order packing of nucleosomes: By regulating nucleosome spacing, CHRACs may lay the foundation for repressing genes that the differentiated cell does not need.
Chromatin plasticity and the availability of DNA for transcription are also regulated by changes in the shapes of the nucleosomes around which the DNA winds. The core of each nucleosome consists of four globular histone molecules (H2A, H2B, H3, and H4), each of which has tails that stick out of the core. The conformation of the histones and their packing in the nucleosome are modified by attaching acetyl, methyl, or other groups to the amino acids that make up the tails and to a few amino acids in the globular part of the histone. Berger and Anne Schaeffer, Rockefeller University, New York, each noted that both the nature of the group and where it attaches determine whether the neighboring chromatin is relaxed or condensed. Attaching the side chains requires a range of enzymes; for example, histone acetyltransferases add acetyl groups, while histone deacetylases remove them.
Aging cells have more relaxed chromatin structure, accompanied by increased histone, than younger cells. In yeast cells, Berger showed that aging is slowed down by Sir2, a histone deacetylase belonging to the sirtuin family (sir = silencing information regulator), which removes the acetyl group from a lysine in the tail of histone H4. Sir2 is antagonized by an acetylase, Sas2. These enzymes may be particularly important in aging yeast because they are concentrated at the ends of the chromosomes, the telomeres, which unravel and wear away as cells age. Human homologs of Sir2 and Sas2 are known, and similar mechanisms in human aging are being studied. The Sir2 homolog, SirT1, may reduce tau pathology (see ARF related news story) and seems to promote learning and memory (see ARF related news story and ARF news story)
Genes in differentiated cells can also be silenced by double methylation of a lysine on the tail of histone H3 by the enzyme histone lysine methyltransferase, GLP/G9a, finds Schaeffer. When GLP/G9a was experimentally inactivated in adult neurons in culture, many non-neuronal genes were activated, and transcription of neuronal genes involved in serotonin and dopamine synthesis and function ramped up. In mice, switching off GLP/G9a in postnatal neurons led to impaired learning, memory, and environmental adaptation, all symptoms of the rare and severe 9q34 mental retardation syndrome seen in humans who have a mutation in GLP.
A third way to silence genes is the direct attachment of methyl groups to sequences in the promoter region on one strand of the DNA. These CpG islands, named for their runs of cytosine and guanine bases bound by strong phosphodiester bonds, are found near transcription start sites in the promoters of many mammalian genes. Methyl groups attached to these CpGs attract repressor complexes that prevent transcription—about 70 percent of CpGs in the human genome are methylated. Bird noted that the methyl-CpG-binding protein 2 (MECP2) is an important repressor, binding directly to methylated CpG islands and acting as an anchor for co-repressors, such as Sin3a, and histone deacetylases 1 and 2. The actions of MECP2 ran like a refrain through the meeting.
Bird and Lisa Monteggia, University of Texas Southwestern Medical Center at Dallas, both reviewed mutations in the MECP2 gene that render MECP2 non-functional. MECP2 mutations are the most common cause of Rett syndrome, a severe developmental disorder on the autistic spectrum seen in about 1:15,000 girls (see ARF related news story). In the brain, MECP2 is found predominantly in mature neurons, where it is believed to repress gene transcription, Monteggia noted, although large changes in gene expression have not been seen in neurons lacking MECP2. Bird demonstrated that in neurons, MECP2 seems to substitute for about half the H1 histones that secure the turns of the DNA ribbon around the nucleosome core in most other cells. As a result, loss of MECP2 disrupts epigenetic control in mature neurons, resulting in twice as much histone acetylation and altered transcription.
The entraining of metabolic pathways to the cycle of day and night is fundamental to biological function. Sassone-Corsi explained that this circadian clock requires the rhythmic activation and shutting down of about 15 percent of the cell’s genes, so it is not surprising to find that epigenetic mechanisms are involved. In mammals, the diurnal fluctuation of light intensity drives changes in the chromatin in "clock central," the suprachiasmatic nucleus in the hypothalamus. The chromatin is remodeled through modifications of histone tails, which act as metabolic sensors. CLOCK, a transcription factor integral to the process, stimulates histone acetylation, which is balanced by the histone deacetylase SIRT1. The CLOCK-stimulated acetylation is driven indirectly by another transcription factor, MLL1, a methyl transferase originally discovered through its role in leukemia. It adds three methyl groups to histone H3 in a circadian fashion. MLL1 also associates with CLOCK, but only at specific times in the daily cycle, activating the acetylation of other sites on histone H3 and promoting gene expression. What drives MLL1 awaits further investigation.
Synapses and Memory
The epigenetic programs that maintain the dynamic state of the synaptic network, both during its establishment and as it is modified by experience throughout life, are slowly becoming apparent—as is the age-related decline in epigenetic mechanisms associated with memory storage (see ARF related news story). Connectivity patterns laid down between neurons during postnatal development are remodeled in response to an animal’s experience and tailored throughout life. Learning reinforces transmission at synapses that are regularly used, while those that are rarely active lose potency.
One essential factor for synapse function seems to be the repressor MECP2, which recognizes epigenetic markers. Although MECP2 has targets throughout the genome, Monteggia showed that in neurons, it has very precise and specific actions. Neurons from mice lacking the Mecp2 gene do not show large changes in gene expression or altered neuron morphology, but careful electrophysiological analysis reveals loss of synaptic transmission specifically at excitatory synapses. Synaptic stimulation seems to weaken MECP2 repression of genes that code for proteins involved in excitatory transmission, though these target genes are yet to be identified. Extending this to people with Rett syndrome, who lack MECP2, Monteggia considers that depressed excitatory transmission will alter the establishment of synaptic connectivity during the development of the nervous system.
Epigenetic mechanisms that control cellular differentiation may also be at work in learning and memory: Sweatt thinks they may determine specific changes in synaptic function and connectivity. He finds that in the hippocampus, DNA methylation is required for synaptic plasticity, memory formation, and spatial memory. Several methylated genes code for proteins participating in synapse stabilization, including the synaptic growth factor BDNF, and calcineurin, a protein phosphatase that helps regulate the production of several neurotransmitters. Consistent with the hippocampus as a staging post in laying down memories, the changes in DNA methylation disappear within a day of training. Longer-term storage requires stabilized patterns of synaptic connectivity in the cortex. In the anterior cingulate cortex, a shift in the methylation of the calcineurin gene promoter is essential for stabilizing synapses and for the maintenance of memories over a long period. Such a change may be an epigenetic "mark" that identifies a neuron made receptive by experience. (See also ARF related news story for related work on long-term cortical storage.)
The transition from short-term, labile memory to long-term, stable storage requires new protein synthesis, which results from activation of genes in response to transmission at specific synapses. Abel identified histone acetylation as one mechanism that promotes this transition. Acetylation is mediated by CREB, a transcription factor that acts as a molecular switch. CREB binds to Cre, the cyclic AMP response element, in gene promoters, and, in response to neuronal activation, is phosphorylated by various kinases. Phosphorylated CREB forms a complex with the CREB-binding protein CBP, which stimulates histone acetylation, relaxing the chromatin. The CREB/CBP complex can then bind to gene promoters and stimulate the basal gene transcription machinery. Mice lacking CBP have several memory deficits: They fail to associate electric shocks with a particular place or to recognize novel objects. These deficits can be reversed by inhibiting histone deacetylases, which prevent chromatin relaxation. The CBP complex targets certain genes, including members of the nuclear hormone receptor family; one function of these is enhancing the production of BDNF.
Social and Cultural Experience
Looking up from the minutiae of synaptic mechanisms to the connectivity of the brain as a whole, Changeux discussed the epigenetic mechanisms at work in creating and maintaining brain plasticity. It is startling to realize that, while mice and humans have approximately the same number of genes, mice have only 40 million neurons, whereas humans have 50-100 billion—an illustration that genes provide just a blueprint for the developing nervous system. The environment, working through epigenetic mechanisms, becomes particularly important in shaping this basic plan during the long postnatal period in humans. Varying extra-genetic social and cultural influences create individual differences and, on a higher level, provide for cultural heritage. The learning of new skills can take over established circuits, even in adults.
The environment in which children are raised also affects their health and well-being. According to Meaney and Mansuy, children who suffer neglect or abuse tend to develop both physical and mental health problems as adults. Similarly, Meaney finds that when rat pups do not get adequate maternal care, they have higher stress responses than those with more attentive mothers, although this can be reversed if deprived pups are placed with attentive mothers and vice versa. He reported that the number of receptors for the glucocorticoid hormone in the hippocampus is reduced in poorly mothered offspring, and that epigenetic regulation seems to be central to this effect. He went on to show that maternal care stimulates serotonin release, which triggers an intracellular pathway involving the histone acetyltransferase CBP, which in this pathway binds to the promoter of the glucocorticoid receptor gene. The result is reduced DNA methylation and increased histone acetylation, allowing increased binding of a specific transcription factor. Meaney did not say which one. Glucocorticoid receptors are also reduced in human victims of childhood abuse who have committed suicide.
So, can nurture become nature? Evidence is accumulating for the inheritance of some stable epigenetic changes in gene transcription that result from individual experience (see ARF related news story). Meaney went on to show that poorly nurtured rat pups themselves pay less attention to their offspring, and that epigenetic markers in the genes coding for the estrogen receptor, the "nurturing" hormone oxytocin, and the neuromodulator dopamine all seem to be transmitted to the offspring. In a study carried out by Mansuy’s group, baby mice that were exposed to unpredictable maternal deprivation and stressed mothers showed signs of depression, impulsive behavior, and impaired social skills; these characteristics were also displayed by their offspring in the following two generations. The possibility that these negative effects of early stress were transmitted solely through nurture is ruled out by mating each generation of stressed males with normally reared females.
One of the signals involved in coping with and adaptation to stress is corticotrophin releasing hormone, mediated by type 2 receptors in the hypothalamus. Mansuy reported that second- and third-generation offspring of the stressed male mice have fewer of these receptors, which seems to be the result of altered methylation of CpG islands in the promoter of the receptor gene in both the father’s sperm and the brains of their offspring. Genes such as Mecp2 are also affected, hinting at more widespread modifications in gene transcription.
Epigenetics and Pathology
Clearly, epigenetic mechanisms can play it both ways: ensuring the stable, long-term patterns of gene transcription required by differentiated cells, and at the same time, allowing dynamic changes, such as the circadian fluctuations of protein production. Perhaps not surprisingly, alterations in the epigenetic control of transcription are now being associated with both normal aging (ARF related news story) and
pathological states, such as AD (ARF related news story; see also an overview of research into epigenetics and AD). Because some epigenetic processes promote plasticity, there is hope that they can be harnessed to treat pathological changes. Li-Huei Tsai, Massachusetts Institute of Technology, demonstrated that epigenetic mechanisms might reverse cognitive impairments and neurodegeneration in the inducible CK-p25 mouse, which displays all the pathological features of AD. Chemical inhibition of histone deacetylation restored learning and longer-term memory storage in these animals, and synaptic density also improved. Tsai’s group is now searching for more effective small-molecule histone deacetylase inhibitors. Although the ability of these inhibitors to improve cognitive function was discussed at this meeting, the evidence comes only from animal models; other data indicate that their use as therapeutics may not be straightforward (see ARF related news story). However,
companies are pursuing this strategy in clinical trials (see ARF related news story).
Use of addictive drugs also leads to stable changes in synaptic transmission in some brain circuits—changes that are, essentially, a form of memory, explained Eric Nestler, Mount Sinai School of Medicine, New York. His research group is investigating cocaine-induced alterations in gene transcription in the nucleus accumbens, a brain area affected by addictive drugs. A three-pronged mapping process is used to identify, first, genes that are being transcribed when exposed to cocaine; second, which of these show changes in acetylation and methylation; and third, the genes that are regulated by two transcription factors known to mediate aspects of addictive behavior. One surprising discovery is that cocaine causes the relaxation of large parts of the chromatin in nucleus accumbens neurons by repressing methylation of histone H3, leading to a permissive state of gene regulation. The histone deacetylase SIRT1, also important in circadian cycles, is heavily involved, regulating the genes for several transcription factors, including MECP2 and G9a, both discussed above for their involvement in dendritic growth and induction, and in the stabilization of synapses. The molecular circuitry is clearly complex, but the occurrence of similar mechanisms in different contexts should facilitate unraveling it and help open up novel ways of treating drug addiction.
A proof of principle that some epigenetic changes may be reversible was presented by Bird, based on work on a mouse model of Rett syndrome. Because the human MECP2 gene is on the X chromosome, Rett syndrome affects only those girls who have one mutated gene, with a normal gene on the other X chromosome. Boys with an MECP2 mutation, because they have only one X chromosome, rarely survive birth. Mice with inactivated Mecp2 genes develop severe Rett-like behavior, but advanced symptoms were fully or partially reversed when the gene was reactivated. Although such gene manipulation is unlikely to be feasible in humans, the experiment demonstrates that loss of MECP2 function in mice does not result in irrevocable brain damage, and so manipulations to increase the effectiveness of the normal gene on the other X chromosome may be fruitful.
Unlike Rett syndrome, other disorders on the autistic spectrum do not have clear-cut genetics origins. Thomas Bourgeron, Institut Pasteur, Paris, France, reported that the mutations identified so far in autism mostly affect synaptic stability and sleep-wake cycles. It seems likely, however, that the behavioral variability seen in autistic disorders is the result of more than one mutation in each affected individual. A variant in the gene coding for the synaptic scaffold protein SHANK2 has been traced through several generations in some families, but other mutations seem to be spontaneous. Disturbed sleep patterns may mean that autistic children do not have adequate opportunity to consolidate the information they receive when awake, and so suffer from synaptic overload. Although epigenetic regulation has not yet been investigated in these conditions, the information on synaptic plasticity and circadian rhythms should provide leads to mechanisms worth investigating.
Given the infinite variation among humans—even identical twins are never 100 percent alike (see ARF related news story)—it is hardly surprising that epigenetic regulation is turning out to be so complex, but its study so fruitful. Nestler emphasized that elucidation of the concerted epigenetic changes in networked pathways are essential for a proper understanding, but this will take time. Ultimately, a picture may emerge that provides, on the one hand, a more complete explanation of our humanity, and on the other, a map that will help with precision targeting of new therapies.—Jennifer Altman.
Jennifer Altman is a freelance science writer in Todmorden, U.K.