10 Dec 2005

Take a break from Aβ and tau, and consider something completely different: Is it nature or nurture? Societies have mulled this over ever since the concept crept into our collective consciousness. Today, we know there is no simple answer and that human biology and behavior is rarely, if ever, explained by either phenomenon alone. Epigenetics, the study of changes to the genome that, unlike mutations, do not alter the DNA sequence, has exposed just how inextricably linked nature and nurture truly are. It is not surprising then, that the theme served as an epicurean delight at the 35th Annual Conference of the Society for Neuroscience in Washington, D.C., last month, in the form of a symposium of uniformly high quality.

Epigenetics is of interest to a growing number of biologists and clinicians. It offers an explanation for such disparate phenomena as twin discordance (see ARF related news story) and atypical social bonding (see Fries et al., 2005). At the molecular level, it likely plays crucial roles in neurotransmission (Stadler et al., 2005) and learning and memory in mammals (see ARF related news story). It reveals potential mechanisms underlying various neurologic diseases, including schizophrenia (see Dong et al., 2005) and Rett syndrome (see ARF related news story), and it even suggests a means of treatment (see Sharma, 2005). In fact, researchers are currently developing small molecules that perturb methylation and acetylation, two of the most consequential epigenetic changes, in the hope that they may be turned into drugs to treat a variety of diseases, including schizophrenia (see Tremolizzo et al., 2005), and neurodegenerative disorders, such as Huntington disease (see ARF related news story).

To fully understand epigenetics, one must appreciate why it exists. At the Washington symposium, co-chairs Jonathan Pollock and Christine Colvis from the National Institute on Drug Abuse, Bethesda, Maryland, reminded the audience that epigenetics has its roots in adaptation. As organisms become more complex with longer generational times, it becomes harder and harder for them to adapt through natural selection, Pollock suggested. In short, where genetic change cannot keep pace with environmental change, complex organisms have evolved other means to adapt. Specialized tissues such as neurons have arisen to allow organisms to react rapidly to external influences. But that requires that specific subsets of genes be turned on and off in specialized cells. This is where chromatin remodeling enters the picture; epigenetic change, therefore, is a key element of adaptation based on genetic reprogramming.

It is easy to envisage how the apparatus for genetic reprogramming, once it had evolved to facilitate tissue differentiation in multicellular organisms, was co-opted for more immediate benefit, such as the response to environmental stimuli. Frances Champagne, University of Cambridge, England, reported on how something as simple and innocuous as maternal care can begin the process of chromatin remodeling right out of the womb. Work from Champagne’s and other laboratories reveals that rat pups who get licked and groomed the most turn out to do better in tests of learning and memory, such as the Morris water maze. They also have higher levels of both N-methyl-D-aspartate (NMDA) glutamate receptors and synaptophysin, a synaptic marker, in their brain, suggesting molecular adaptation. Well-groomed pups have higher levels of brain glucocorticoid receptor and appear less anxious than pups that get licked less. Notably, long-time Champagne collaborator Michael Meaney and colleagues at Douglas Hospital Research Center, Montreal, Canada, confirmed in 1999 that this pup behavior is epigenetic rather than inherited. If pups born to so-called “low licking/grooming” mothers are instead reared by “high licking/grooming” mothers, they will behave just like their adopted siblings (see Francis et al., 1999).

Champagne and colleagues have since probed epigenetic changes that underlie behavioral differences in rat pups. She found, for example, that the promoter region of the glucocorticoid receptor (GR) has 17 potential methylation sites and that pups born to mothers that lick less have much higher levels of GR promoter methylation, particularly at one specific methylation site, number 16. These methylation differences are not apparent at birth, but gradually emerge after 5 to 6 days of postnatal care. It turns out that these methylation patterns have molecular consequences. They prevent the binding of transcription factors, such as nerve growth factor-inducible protein A (NGFI-A), Champagne revealed.

These epigenetic changes need not be permanent; this is important when one considers epigenetics in terms of disease pathology or drug addiction. Champagne reported that when rats born to low licking/grooming mothers are given the histone deacetylase (HDAC) inhibitor trichostatin A, then by adulthood their methylation pattern changes to that of rats brought up by high licking/grooming mothers.

Acetylation of histones and methylation of DNA are almost as intertwined as the strands of nucleic acids themselves. Champagne showed that hypermethylation of the GR promoter correlates with hypoacetylation of histone H3. Because the HDAC inhibitor promotes acetylation of the H3 histone, demethylation of the GR promoter ensues, followed by increased expression (see Weaver et al., 2004). Indeed, Champagne showed that infusion of trichostatin A into adult rats leads to greater binding of NGFI-A to the GR promoter. She also revealed that directly stimulating methylation by administering methionine, a precursor to the methyl donor S-adenosyl-methionine, can achieve a similar reversal of maternal programming. Some of this work just appeared in the November 23 Journal of Neuroscience (see Weaver et al., 2005).

Epigenetics and Drug Abuse
In a related vein, Arvind Kumar, from the University of Texas Southwestern Medical Center, Dallas, reported how chronic and acute exposure to drugs of abuse can cause epigenetic changes around a variety of promoters. The long-term effects of drug addiction have been explored for some years. It has become apparent that they can lead to changes in gene expression in the brain, particularly in the striatum, where many of the neurons take part in reward pathways that can lead to addiction. But exactly how those changes occur remains mysterious.

Kumar’s work centers on epigenetic changes in rodent striatum in response to either chronic or acute cocaine administration. While acute cocaine induces expression of the transcription factor cFos, chronic exposure to the drug desensitizes cFos levels back to normal and instead leads to the accumulation of a truncated form of FosB (δFosB). Using chromatin immunoprecipitation experiments, or ChIP, Kumar probes the relation between cocaine and covalent modifications—phosphorylation, acetylation, and phosphoacetylation—of histones on target promoter regions in rats.

He reported that acute administration of cocaine leads to a fivefold increase in phosphoacetylation of histone H3 on the cFos promoter, but no change in newly acetylated histone H3. Histone H4 acetylation does increase about threefold, however. Chronic administration of the drug, on the other hand, leads to increased acetylation of histone H3 around the FosB promoter, Kumar reported, but no changes in histone H4. The ChIPs failed to detect any changes in histone acetylation patterns on promoters that do not respond to the drug, such as those for β-tubulin or tyrosine hydroxylase, suggesting that the phosphorylation and acetylation changes are specific to cocaine-induced promoters.

Kumar has also looked at changes occurring around other gene promoters. Expression of the protein kinase Cdk5 (see ARF related SfN story), for example, is unaffected by acute cocaine but is induced by chronic exposure. Would the chromatin pattern mimic those seen for FosB? Kumar found that not only was acetylated histone H3 increased on the Cdk5 promoter, but that δFosB also bound to it under chronic cocaine administration, suggesting that δFosB may play a direct role in cocaine addiction, a possibility that has been hotly debated.

And like Champagne, Kumar and colleagues have modified the addiction pattern using deacetylase inhibitors. Kumar reported that both trichostatin A and sodium butyrate, a competitive inhibitor of HDACs, enhanced histone H3 phosphoacetylation in response to the drug when administered to rats, and that the animals scored much higher on a test that measures the reward component of their response to cocaine. Overexpression of histone deacetylase, on the other hand, dramatically decreased scores in this behavioral test, confirming the importance of histone acetylation in cocaine addiction. Most of this work was just published in the October Neuron (see Kumar et al., 2005). Kumar and colleagues are now using “ChIP on chip” assays, probing chromatin immunoprecipitation samples with DNA promoter chips, or microarrays, in a genome-wide search for promoters that are hyper- and hypoacetylated after chronic cocaine exposure. So far, nearly 85 promoters have been isolated that have increased acetylated H3 and H4 histones following chronic cocaine administration, and 48 promoters where acetylation of H3 and H4 decreased, he reported.

Richard Goodman, Oregon Health Sciences University, Portland, reported his ChIP experiments to find genes that bind to CREB, or cyclic AMP response element (CRE) binding protein. Though it has been known for many years that CREB binds to CRE, one of the quintessential promoter elements, there are still aspects of CRE/CREB biology that are poorly understood. As Goodman pointed out, in PC 12 cells there is a CRE sequence in the promoter for the somatostatin gene, yet neither CREB nor CREB binding protein (CBP) bind to it. This goes to show that CRE binding is not constitutive, Goodman suggested. Instead, it may depend on how the DNA is programmed in individual cells.

Goodman and colleagues use a sophisticated screening method called SACO, or serial amplification of chromosome occupancy, designed to be unbiased, to identify regions of DNA that bind CREB. SACO combines a ChIP experiment with serial analysis of gene expression, or SAGE (see ARF related news story), to identify DNA in chromatin segments pulled down by CREB antibodies. Using this approach, the researchers have identified thousands of potential genes regulated by CREB (see Impey et al., 2004).

In his SfN presentation, Goodman focused on one of these regions, which happens to harbor a non-coding sequence. In fact, about 20 percent of the sequences identified by the SACO experiment did not code for protein, said Goodman. One of the identified sequences lies upstream of a microRNA (miRNA) sequence called miR132. This microRNA is expressed in neurons and its CRE element does bind to CREB. In fact, expression of miR132 increases dramatically when cells are treated with brain-derived growth factor—the response is even greater than that seen for cFos.

So what does miR132 do? Goodman reported that the microRNA strongly induces neurite outgrowth and that antisense miR132 has the opposite effect. By using a prediction algorithm (MIRANDA, see Enright et al., 2003), he and colleagues identified a putative binding site in the 3’untranslated region (UTR) of the gene encoding the GTPase-activating protein, p250GAP. Goodman reported that miR132 inhibits expression of p250GAP and that that, in turn, promotes neurite outgrowth. This is an example of a CREB-regulated miRNA that regulates growth of neurites by responding to extrinsic trophic cues. The work appeared last month in PNAS (see Vo et al., 2005).

As for epigenetics, miR132 and other miRNAs identified in the SACO screen may also bind to other 3’UTRs, hinted Goodman, most notably that for methyl-CpG binding protein 2 (MeCP2), which is mutated in Rett syndrome. Though MeCP2 was initially identified as a transcriptional repressor that binds to methylated DNA, researchers recently showed that it is involved in RNA splicing, as well (see ARF related news story). Because the MeCP2 gene has a very large 3’UTR that possibly binds up to three miRNAs identified in the SACO screen, CREB signaling may mediate RNA splicing and play an important role in the regulation of methylation-linked transcriptional repression.

Epigenetics in Long-Term Memory
Last but not least, Mark Mayford, Scripps Research Institute, La Jolla, California, also addressed CREB signaling in his studies of epigenetic mechanisms in memory formation. Mayford described experiments to address the role of nuclear calcium signaling in the CREB-mediated induction of protein synthesis that is required for long-term memory.

Using the inducible tetracycline promoter system, Mayford and colleagues generated mice that express calmodulin binding protein (CaMBP) only in the nucleus of neurons in forebrain—the system allows expression to be turned off by adding doxycycline to the diet. Mayford checked the system by inducing seizures in adult mice. In control animals, seizures lead to activation of CREB (by phosphorylation on serine 133) in the CA1 neurons of the hippocampus, but this is blocked in the transgenic animals.

In most electrophysiological tests, the CaMBP mice behave just as controls. Excitatory postsynaptic potentials and paired-pulse facilitations are normal, for example. But they do behave differently in learning and memory tests. When CaMBP is on, latency increases in the Morris water maze and the mice show no preference for the quadrant where the hidden platform is located. If the calmodulin inhibitor is off, then the mice do just as well as control animals. Fear conditioning and novel object recognition tests revealed that mice expressing CaMBP have normal short-term memory but impaired long-term memory, Mayford reported.

Epigenetics entered this line of study when Mayford asked what targets Ca2+/calmodulin might activate to facilitate long-term memory. One likely target, Mayford suggested, is CBP, or CREB binding protein. CBP has histone acetyltransferase (HAT) activity and HATs can activate transcription by unraveling the chromatin to expose promoter elements. They have also been implicated many times in learning and memory (see ARF related conference story; ARF related news story. When Mayford and colleagues expressed a dominant-negative CBP, rendered HAT-less by a point mutation, then spatial memory was impaired. Long-term memory declines, while short-term memory is unaltered. Furthermore, trichostatin A, the deacetylase inhibitor used by Champagne and Kumar above, can rescue animals expressing the dominant-negative CBP, showing once again that histone acetylation and chromatin modification play a key role in behavioral response to the environment. For more detailed reading on the symposium, see a monograph prepared in advance by the presenters (Colvis et al., 2005).—Tom Fagan.

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References

News Citations

1. Painting a Better Picture of Twin Discordance 13 Jul 2005
2. For Better Memory, Try Keeping Your HAT On… 25 Jun 2004
3. Multitasking Rett Protein Shines Spotlight on RNA Splicing in Neurologic and Psychiatric Disease 28 Oct 2005
4. Drugs Slow Neurodegeneration in Fly Model of Huntington's 22 Nov 2002
5. SfN: P25 at Synapses—A Bite Peps Up, A Binge Crashes the System 9 Dec 2005
6. SAGE Genie Manages Gene Expression Data 22 Nov 2002
7. New Orleans: Symposium Probes Why Synapses Are Suffering 25 Nov 2003

Paper Citations

1. Fries AB, Ziegler TE, Kurian JR, Jacoris S, Pollak SD. Early experience in humans is associated with changes in neuropeptides critical for regulating social behavior. Proc Natl Acad Sci U S A. 2005 Nov 22;102(47):17237-40. PubMed.
2. Stadler F, Kolb G, Rubusch L, Baker SP, Jones EG, Akbarian S. Histone methylation at gene promoters is associated with developmental regulation and region-specific expression of ionotropic and metabotropic glutamate receptors in human brain. J Neurochem. 2005 Jul;94(2):324-36. PubMed.
3. Dong E, Agis-Balboa RC, Simonini MV, Grayson DR, Costa E, Guidotti A. Reelin and glutamic acid decarboxylase67 promoter remodeling in an epigenetic methionine-induced mouse model of schizophrenia. Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12578-83. PubMed.
4. Sharma RP. Schizophrenia, epigenetics and ligand-activated nuclear receptors: a framework for chromatin therapeutics. Schizophr Res. 2005 Jan 1;72(2-3):79-90. PubMed.
5. Basu J, Basu S, Bhattacharyya S. Ego functions in relation to stressful life events and indices of psychopathology in paranoid schizophrenia. Psychol Rep. 2004 Dec;95(3 Pt 2):1248-52. PubMed.
6. Francis D, Diorio J, Liu D, Meaney MJ. Nongenomic transmission across generations of maternal behavior and stress responses in the rat. Science. 1999 Nov 5;286(5442):1155-8. PubMed.
7. Weaver IC, Cervoni N, Champagne FA, D'Alessio AC, Sharma S, Seckl JR, Dymov S, Szyf M, Meaney MJ. Epigenetic programming by maternal behavior. Nat Neurosci. 2004 Aug;7(8):847-54. PubMed.
8. Weaver IC, Champagne FA, Brown SE, Dymov S, Sharma S, Meaney MJ, Szyf M. Reversal of maternal programming of stress responses in adult offspring through methyl supplementation: altering epigenetic marking later in life. J Neurosci. 2005 Nov 23;25(47):11045-54. PubMed.
9. Kumar A, Choi KH, Renthal W, Tsankova NM, Theobald DE, Truong HT, Russo SJ, LaPlant Q, Sasaki TS, Whistler KN, Neve RL, Self DW, Nestler EJ. Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum. Neuron. 2005 Oct 20;48(2):303-14. PubMed.
10. Impey S, McCorkle SR, Cha-Molstad H, Dwyer JM, Yochum GS, Boss JM, McWeeney S, Dunn JJ, Mandel G, Goodman RH. Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell. 2004 Dec 29;119(7):1041-54. PubMed.
11. Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS. MicroRNA targets in Drosophila. Genome Biol. 2003;5(1):R1. PubMed.
12. Vo N, Klein ME, Varlamova O, Keller DM, Yamamoto T, Goodman RH, Impey S. A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis. Proc Natl Acad Sci U S A. 2005 Nov 8;102(45):16426-31. PubMed.
13. Colvis CM, Pollock JD, Goodman RH, Impey S, Dunn J, Mandel G, Champagne FA, Mayford M, Korzus E, Kumar A, Renthal W, Theobald DE, Nestler EJ. Epigenetic mechanisms and gene networks in the nervous system. J Neurosci. 2005 Nov 9;25(45):10379-89. PubMed.

Further Reading

Papers

1. Colvis CM, Pollock JD, Goodman RH, Impey S, Dunn J, Mandel G, Champagne FA, Mayford M, Korzus E, Kumar A, Renthal W, Theobald DE, Nestler EJ. Epigenetic mechanisms and gene networks in the nervous system. J Neurosci. 2005 Nov 9;25(45):10379-89. PubMed.