Monday, April 02, 2007

Epigenetics in Memory, II   posted by amnestic @ 4/02/2007 09:09:00 PM

Epigenetics. What is it? Let's ask some reputable sources.

From the glossary of a review in the current Nature Reviews Genetics:

Epigenetic - Refers to mitotically or meiotically heritable changes in gene expression that do not involve a change in DNA sequence.

From the Cell epigenetics review issue a month and a half ago:

Epigenetics, in a broad sense, is a bridge between genotype and phenotype-a phenomenon that changes the final outcome of a locus or chromosome without changing the underlying DNA sequence. ... More specifically, epigenetics may be defined as the study of any potentially stable and, ideally, heritable change in gene expression or cellular phenotype that occurs without changes in Watson-Crick base-pairing of DNA.

Both definitions suggest that, to be epigenetic, something must 1) be heritable and 2) not be attributable to DNA sequence. Imagine if you took that "ideally" very liberally and dropped the heritability requirement. Now a change in gene function must be attributable to something besides DNA sequence. At that point epigenetics becomes gene regulation. Living cells are constantly performing gene regulation in myriad ways. If we loosen the definition enough, epigenetic could mean signal transduction, regulation of protein synthesis and degradation, and any and all transcriptional modulation. Definitions can and do change. One thing I'd like to do in this post is generate discussion as to the most useful way to use the term epigenetics, so that we can communicate most efficiently about the various biological topics now gathering under its umbrella.

Here is a third definition from David Sweatt and Jonathan Levenson:

A third definition posits that epigenetics is the mechanism for the stable maintenance of gene expression that involves physically 'marking' DNA or its associated proteins. This allows genotypically identical cells (such as all cells in an individual human) to be phenotypically distinct (for example, a neuron is phenotypically distinct from a liver cell). The molecular and physical basis for this type of change in DNA or chromatin structure is the focus of this review. By this definition, the regulation of chromatin structure is equivalent to epigenetics.

In this definition, they are equating a mechanism for epigenetics with epigenetics. Now we have the same term at two levels of analysis. If you're a Hofstadter fan you can probably think of a lot of mischief to stir up when you run into this situation, but that's not the real problem. The problem stems from the fact that we already know that epigenetics isn't restricted to the regulation of chromatin structure, so in simplifying the term to one set of mechanisms we are discarding information and excluding mechanisms that we may not have even discovered yet. A lot of the time, Sweatt and Levenson use the term 'epigenetic mechanisms' to mean regulation of histone modification and DNA methylation. If we mean chromatin structure, why not say that? Why do Sweatt and company trade in 'chromatin modification' for 'epigenetics'? It's not an entirely arbitrary thing to do. The best understood mechanisms for epigenetic phenomena are DNA methylation and histone modification, hands down, and if you search Pubmed for epigenetics you will have a very difficult time finding an article that doesn't involve some combination of these chromatin 'marks'.

The most-studied instance of epigenetics is probably imprinting. It has something for everyone, from an exciting battle-of-the-sexes evolutionary explanation to a cellular mechanism veiled in mystery. Very briefly, imprinting occurs when, for instance, you get the exact same DNA sequence for Gene A from mom and dad (you get two of each gene in case you forgot), but only the Dad copy of Gene A is active. The Mom copy is imprinted and thus silenced. Some percentage of human genes are imprinted. I think it's less than ten percent, can't remember. How is the gene expressed differently if the DNA sequence is the same? Well, during egg-ogenesis Mom added a methyl group (CH3, not a very reactive group, just makes the molecule bulkier) to a bunch of the cytosines in and around Gene A, thus changing them to 5-methyl-cytosines. This is called DNA methylation. In this example the gene is maternally imprinted, but don't get it twisted. Dads can imprint too. DNA methylation can affect gene expression directly by hiding sequences that would normally be recognized by regulatory proteins or indirectly as there are proteins that recognize methylated DNA and can recruit chromatin modification complexes to that locus. By the way, if any of this is terribly interesting to you please go check out one or two of the billion reviews on this stuff as I am not an expert in this area and I'd hate to lead you astray.

DNA is often packed up by wrapping it around proteins called histones like you wrap the string around your yo-yo before you put it in the drawer. Unwrapping DNA from the histones allows other proteins (like RNA polymerase) access to the DNA and generally means an upregulation of that gene's activity. You can attach or detach acetyl groups or methyl groups (or phosphate groups or ubiquitin groups or SUMO groups, histones must look like Katamari Damacy) to the histones to make them more or less attracted to the DNA. Histone acetylation (by HATs) and deacetylation (HDACs, histone deacetylases) are core mechanisms for switching a section of chromosome between transcriptionally active and inactive states. There is a complex relationship between DNA methylation and histone modifications. As a basic scheme, DNA methylation is more often associated with silent genes wrapped tightly around deacetylated histones. The two occur independently though. Many transcriptional activators play their part by dragging some histone modification machinery to the gene they are interested in, so histones are dynamically modified in many cases of transcriptional regulation, whereas DNA methylation has been thought to be a more stable, long-lasting modification.

So, yes. Very often 'epigenetic mechanism' refers to DNA or histone modification, but there are other ways of passing information between generations of organisms or cells without touching the DNA sequence. Yeast can transmit information across generations in the form of prions, infectious protein conformations. Think of Ice-9 in Cat's Cradle. When prions touch other normally folded proteins of the same type, they can transfom the normal protein. So a protein with the exact same amino acid sequence can fold up two different ways, one of which (the prion form) can be passed on to the offspring of the prion-infected yeast. Thus, a phenotypic determinant is passed across generations with no help from the DNA sequence. Also, last year a French group reported an instance of epigenetics in which accumulation of some abnormal RNA molecules in mouse sperm is responsible for inheritance of a specific coat pattern. All that is to say is that there are lots of ways to transmit information across generations and cell divisions.

I think the thing about chromatin modification that was initially appealing to Sweatt and colleagues in terms of memory was its apparent stability. Research into cell differentiation, the process by which your basic stem cell produces the diverse array of cell-types required, has implicated chromatin modification as a strong influence. The idea is that to make, for instance, a neuron, you have to permanently set up a certain transcriptional program to make things like neurotransmitter receptors, so you go ahead and permanently turn on those genes. In some sense you could extend cell differentiation to a highly refined level so that the cell types aren't broad classes like neuron and skin cell, but rather 'neurons storing memory A' and 'neurons storing memory B'. Following this train of thought or something like it, they began to look for signs of chromatin modification in response to learning. They discovered that after a hippocampus-dependent form of fear conditioning, histones in the hippocampus are modified. Acetylation and phosphorylation are high one hour after training, but return to baseline by 24 hours. Given that we know that many genes are upregulated in response to fear conditioning and that transcription factors that activate genes have a history of modifying histones it would have been quite a surprise if we hadn't seen any changes. At this point, I began to get a little unhappy with the papers coming out of the Sweatt lab because I felt like the histone-modification story wasn't telling us much that we didn't know.

So far, the epigenetics in memory story has been presented in a theoretical framework emphasizing the stability of chromatin modifications and the persistence of long-term memories. Just so I don't mischaracterize, here is a quote from 2006:

One question that has eluded neuroscientists is, How can the brain store information over the lifetime of an organism in the face of molecular turnover? This question is especially relevant for understanding a complex process such as cognition, which relies heavily on the ability to store and recall information for periods longer than the half-lives of most of the molecules utilized in these processes. Chromatin is the one structure that remains relatively constant in almost every cell of a metazoan. It is not surprising that many recent studies in the nervous system indicate that from invertebrates to mammals, chromatin is a dynamic structure that integrates potentially hundreds of signals from the cell surface and effects a coordinated and appropriate transcriptional response. More important, chromatin is perhaps the only structure in a neuron capable of such higher-level signal integration and information storage that is not continually turned over.

To rephrase: Memories are persistent. Therefore the molecular mechanisms for memory are persistent. Most molecules don't last, so they can't do the job. Chromatin structure lasts, so it is a good candidate for long-term memory storage.

Here are my various criticisms of this line of thinking:

1) You can't have your cake and eat it too. Is chromatin a dynamic structure changing in the face of new stimuli or is it a solid, stable basis for long-term information storage? Once a cell's epigenome has been modified to store a memory does it somehow get write-protected? It seems to me that there are some chromatin modifications that are highly stable (i.e. the ones that control cell differentiation) that should be very unresponsive to the day-to-day vagaries of cell signaling, otherwise you would have neurons de-differentiating all willy-nilly. Another separate set of chromatin modifications must respond to cellular stimuli and allow dynamic gene expression to fit the needs of the cell in the current situation.

2) A cell only has one epigenome, but encodes more than one memory. That's the wonderful thing about synapses; they allow multiple excitability configurations. An average neuron has thousands of synapses. Memory 1 can light up synapses X, Y, and Z and cause the cell to fire a burst of action potentials whereas Memory 2 lights up a different set of synapses causing the cell to fire at a slower, more consistent rate. Maybe when synapse X loses its ability to excite the cell you forget a little bit and the burst of action potentials is weaker. That's one scenario. The point is illustrated empirically in studies like Vazdarjanova and Guzowski, (2004), which shows that 35% of cells in the CA1 region of the hippocampus light up in response to a single experience, and there is some 15% overlap between cell populations responding to two different contexts. The point of that report is actually to show that representations are relatively non-overlapping, but they aren't so sparse that you can afford a cell per experience. Is there some way to propose chromatin modification as a long-term memory mechanism that doesn't make this sparsity requirement?

3) The molecular turnover problem was never a problem. Sure molecules turnover, so the memory can't be housed in a single individual molecule. Dendritic spines (structures forming excitatory synapses), on the other hand, can remain stable for as long as we can record them. All of the molecular constituents of the spine turn over, but the structure remains. All of your skin cells die and are replaced, but your body remains intact. It's not an issue. The only way that molecular turnover would be an issue is some critical mass of the molecules in a structure turned over at once. I can imagine some mechanisms involving the post-synaptic scaffolding and structurally induced biochemical states, but the mechanism doesn't matter for putting the molecular turnover issue to bed. We have observed neuronal structures remaining intact for long periods of time. Apparently there are parts of the cell that are stable besides the chromatin.

I can maybe think of a way out of #2, but #3 seems very clear and very much in conflict with the theoretical framework for this work. So when the new Miller and Sweatt came out I was thinking, "This is just the histone papers again with DNA methylation." I skimmed it, saw some of the experiments I expected, and gave it a general thumbs down. Now that I've given it a few reads I think the paper has some relevant and really novel findings, a major one being that DNA methylation is regulatable in nondividing cells.

The enzymes that methylate DNA are called DNA methyltransferases (DNMTs). They most commonly add the methyl group to the C in the sequence, CG. Remember that DNA is double-stranded so you get a CG - GC quartet of nucleotides at these preferential methylation sites. There are two major classes of DNMT in mammals: de novo and maintenance. Maintenance DNMTs make sure that if a portion of the genome was methylated in a dividing cell, say a neuronal precursor cell, then that portion will be methylated in the daughter cells produced by the division. During DNA replication just prior to cell division you have double stranded DNA containing one of the original strands (with methylated CGs) and one newly synthesized strand. A maintenance DNMT recognizes the dsDNA with only a methylated CG facing an unmethylated GC and makes them match. De novo DNMTs can make new methylation sites where there was no indication of methylation to begin with. Their major role is during the 'reset' of methylation states that occurs during gametogenesis and in the preimplantation embryo, and they have been considered relatively quiet in the fully developed adult.

The first thing Miller and Sweatt did was to show that the level of de novo DNMT mRNA is increased after fear conditioning. They then injected DNMT inhibitors into the hippocampus after fear conditioning and blocked long-term memory consolidation. I initially thought this experiment was suspect because I didn't understand that DNA methylation is not expected to be part of a cell's normal function. I associated DNA methylation with histone deacetylation and my understanding is that histone acetylation states are very commonly altered to tweak this or that gene's level up or down. In reality, people have hardly tested the effects of DNMT inhibitors in non-dividing cells. The literature is full of attempts to selectively target cancer cells with DNMT inhibitors because they have a habit of dividing too much. You would still like a more specific manipulation, a way of inhibiting DNMTs at specific genomic loci would be nice, but the behavioral control experiments in this paper go a long way toward showing that the drugs are not simply killing off neurons. The inhibitors are only effective at a time point shortly after training, defining a maximum window of some 6 hours in which relevant genes are demethylated and remethylated or vice versa in response to behavioral training.

They next showed regulation of methylation at genes that are positively and negatively associated with memory formation. Protein phosphatase 1, a 'molecular constraint on learning and memory', is transiently methylated leading to reduced levels, whereas reelin, which has been associated with synaptic plasticity shows reduced methylation levels and increased mRNA expression. This second finding is pretty important. The identity of the mammalian demethylating enzyme is unclear leading some to question if an active demethylase exists at all. Demethylation in dividing cells can be achieved by simply being lazy about methylating new DNA. According to Hermann et al. in 2004, the 'best evidence so far for active demethylation in vivo" was the rapidity of sperm chromatin demethylation after fertilization. I'd say reduced methylation at the reelin locus in non-dividing cells within an hour after behavioral training is quite strong evidence. In fact, this window of very rapid demethylation at a specific locus suggests a strategy for investigating the biochemistry of mammalian demethylation. It should be possible to trap any proteins that are associated with the reelin promoter in the hour or so after training using techniques like chromatin immunoprecipitation.

Finally, they emphasize that the methylation changes are transient. Everything returns to baseline by 24 hours after conditioning. This very obviously contradicts the idea of DNA methylation as a memory storage tool. For some reason, the discussion section still won't let it go though. Look at this:

This attribute is, however, consistent with the role of the hippocampus as a structure contributing to memory consolidation but not memory storage. It will be interesting to determine if the Crick/Holliday mechanism plays a role in perpetuating long-term changes in adult neurons in the cortex, at known sites of long-term memory storage.

The hippocampus' role in various types of memory may or may not be temporary. This is a broadly debated issue and in my humble opinion, the folks making the case for a long-term role for the hippocampus are doing a fine job. Regardless, no one suggests that the hippocampus is only involved for less than a day. So there must at the very least be a mechanism besides DNA methylation that helps maintain the memory for a week or so.

I spent a lot of time looking into ways to specifically affect DNA methylation at certain loci. I found a paper where they made chimeric DNMTs with a custom DNA-binding domain and inserted the recognition sequence for that domain into the area to be methylated. To achieve this in vivo with any sort of temporal specificity would require the combination of at least three transgenes either through traditional methods or injection of viral vectors. The temporal specificity (within 6 hours) would still not really be on point, so I guess this isn't the solution.

So now we know that some genes are regulated by methylation state in response to learning. The obvious next question is which genes. How does the cell decide which genes to turn up and which ones to turn down? Is there a signature pattern of CGs that indicates memory repressor vs memory enhancer genes? Do transcription factors that are already implicated in memory-related gene expression recruit DNA demethylase activity?

A broader question remains. Why should mRNAs get expressed in response to learning anyway? Why involve the nucleus when the action is out at the synapses? I'm convinced that its important, but for my money, local protein synthesis from pre-existing RNAs seems a more specific response to changes in input patterns. Does anyone reading have an explanation for the need to produce new RNAs so far from the locus of plasticity?

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