Thursday, February 26, 2009

Epigenetics and gene structures   posted by p-ter @ 2/26/2009 07:40:00 PM

Following up on this previous post on epigentics, I thought I'd point to a couple nice examples of using epigenetic information to obtain insight into basic biology.

The first is, I think it's fair to say, a landmark paper identifying a set of over a thousand likely functional non-coding RNAs in mouse cell lines. The approach used here was epigenetic: the authors generated genome-wide maps of chromatin modifications known to mark promoters and transcribed regions, and screened out all the regions of the genome already known to be transcriptionally active. This left them with a set of putatively functional transcripts, which tended to be highly evolutionarily conserved (indicating function), and many of which they confirmed via other means to be novel long non-coding RNAs.

The second is a nice paper demonstrating that one of the same epigentic marks used above to identify transcribed regions is present, in humans, mice, and nemotodes, preferentially on exons (rather than on the entirety of the transcribed region). As this mark is present only in genes that are being transcribed, the authors conclude that it is placed in conjunction with transcription, and likely in conjunction with splicing. They speculate about the role that this mark could play in gene regulation, but in general, this paper raises many more interesting questions than can currently be answered.

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Tuesday, February 24, 2009

Epigenetics in the NY Times   posted by p-ter @ 2/24/2009 07:50:00 PM

Not news to many readers, I'm sure, but Nicholas Wade has a nice article on epigenetics and gene regulation. Some people in the article complain about the lack of a focused investment by the government in this area. I found this a little odd--isn't quite a bit of large-scale work being done by the ENCODE project?

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Sunday, May 27, 2007

Everything and nothing   posted by amnestic @ 5/27/2007 10:47:00 AM

Redefinition / turnin' your play into a tragedy / exhibit level degree on the mic / passionately - Kweli

Nature has an Insight section up ostensibly about epigenetics starting with an article by Adrian Bird suggesting a re-definition. It's free. I think the term is useful to the extent that you can predict something about a phenomenon or mechanism by knowing that it is 'epigenetic'. Bird's suggestion is to make the term less useful and more inclusive. The last section is labeled 'Refining a definition' when in fact he is doing just the opposite:
...there might be a place for a view of epigenetics that keeps the sense of the prevailing usages but avoids the constraints imposed by stringently requiring heritability. The following could be a unifying definition of epigenetic events: the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states. This definition is inclusive of chromosomal marks, because transient modifications associated with both DNA repair or cell-cycle phases and stable changes maintained across multiple cell generations qualify.

He wants to allow transience and yet use epigenetics to explain stable phenomena:
A growing idea is that functional states of neurons, which can be stable for many years, involve epigenetic phenomena, but these states will not be transmitted to daughter cells because almost all neurons never divide.
Without such epigenetic mechanisms, hard-won changes in genetic programming could be dissipated and lost;
With this refinement, epigenetics is everything and nothing. The only thing you can infer about an epigenetic event is that it doesn't change the DNA sequence. Bird wants to claim that a unifying trait is that epigenetics is 'responsive' rather than 'proactive'. I don't understand. If you're going to introduce new terms, why not choose to bring into to broader use the distinction between meiotically and mitotically heritable or force people to be specific about which chromatin modification they are referring to instead of saying 'epigenetic modifications'? Responsive and proactive are more loosely defined concepts destined to muddy waters and lead away from insight.


Thursday, April 26, 2007

Epigenetics News on 'epigenetics'   posted by amnestic @ 4/26/2007 03:16:00 PM

Trevor at Epigenetics News follows up on our recent discussions about the term's increasing popularity. He's cites several good reasons why we might expect to see the term more often, and one reason that may not be all that good:
With all of these high profile and highly funded areas becoming closely associated with epigenetics, is it any surprise that more researchers are finding ways to include their focus as part of "epigenetics"?

If a large enough portion of researchers stretch the word to apply to their topic of interest eventually 'epigenetics' will just mean 'biology'.

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Sunday, April 15, 2007

On the use of the word 'epigenetic'   posted by amnestic @ 4/15/2007 10:27:00 PM

It appears that I'm not the only one musing on the meaning of 'epigenetic'. This timely essay from Mark Ptashne in Current Biology, takes a hard line, even calling into question the connection between histone modifications and epigenetics.
As a glance at the literature will reveal, however, histone modifications - acetylation, phosphorylation, methylation, and so on - are now often explicitly called 'epigenetic modifications'. This despite the fact that, so far as I am aware, no histone modification has been shown to be heritable.

This section might be relevant if you are looking to 'epigenetic mechanisms' for the stability and persistence necessary for a long-term memory mechanism:
Why might one be pre-disposed to misuse the term epigenetic? The term is sometimes used in the context of "maintaining stable states of gene expression", as though some 'locked in' mechanism, involving histone and/or DNA modifications, were required for stable states of gene expression in eukaryotes. But, in the first instance, there would seem to be no such special requirement: lambda lysogens are essentially infinitely stable in the absence of the specific signal that inactivates the repressor, and this system - of course - involves no histones.


Tuesday, April 03, 2007

Note to self: avoid vinclozolin   posted by p-ter @ 4/03/2007 06:04:00 PM

Speaking of epigenetics, here's a rather interesting phenomenon:
Environmental contamination by endocrine-disrupting chemicals (EDC) can have epigenetic effects (by DNA methylation) on the germ line and promote disease across subsequent generations. In natural populations, both sexes may encounter affected as well as unaffected individuals during the breeding season, and any diminution in attractiveness could compromise reproductive success. Here we examine mate preference in male and female rats whose progenitors had been treated with the antiandrogenic fungicide vinclozolin. This effect is sex-specific, and we demonstrate that females three generations removed from the exposure discriminate and prefer males who do not have a history of exposure, whereas similarly epigenetically imprinted males do not exhibit such a preference. The observations suggest that the consequences of EDCs are not just transgenerational but can be "transpopulational", because in many mammalian species, males are the dispersing sex. This result indicates that epigenetic transgenerational inheritance of EDC action represents an unappreciated force in sexual selection. Our observations provide direct experimental evidence for a role of epigenetics as a determinant factor in evolution.


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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Sunday, March 25, 2007

Nature Reviews Epigenetics Focus   posted by amnestic @ 3/25/2007 09:51:00 PM

I'm working on a follow-up to my knee-jerk negative reaction to Miller and Sweatt last week. Surveying the field, I found that Nature Reviews Genetics just dropped an issue focusing in epigenetics with a complementary website.

At least a couple of the reviews spotlight our recently acquired ability to assay DNA methylation states on a genome-wide scale. I'm reading the Jirtle and Skinner piece now. Go check it out and report back.


Friday, March 16, 2007

Epigenetics in memory   posted by amnestic @ 3/16/2007 08:13:00 AM

There's a new article by Miller and Sweatt in Neuron claiming that DNA methylation is a step in memory formation. They show methylation and demethylation of particular genes (reelin and PP1) following fear conditioning, and they show that inhibition of DNA methyl transferases (the name says it all) during the memory consolidation can disrupt memory. I hope now that they've started naming genes at which the methylation matters they'll study something more specific. The idea of inhibiting all DNA methylation in a cell for any length of time seems too blunt an instrument. The cells responsible for that memory could've just keeled over or radicaly changed because some cell cycle regulator got turned up too high for instance. They do show that animals can form a new, strong memory a couple days after administration of the drug, but in some cases, animals with a hippocampal lesion can perform these tasks. The nervous system can achieve learning (especially learning as important as conditioned fear) through many means, so seemingly normal behavior after an insult isn't that strong a control.

The scope of the processes disrupted by a DNMT inhibitor is indicated by this sentence from Miller and Sweatt:
Many developmentally important processes utilize this "prima donna" of epigenetics (Scarano et al., 2005 and Santos et al., 2005), including gene imprinting, cell differentiation, X chromosome inactivation, and long-term transcriptional regulation (Bestor et al., 1988 and Okano et al., 1998).

Sorry to be such a naysayer. It is an interesting hypothesis. It is likely the case that DNA methylation is regulated during memory formation. If we just think that memory will require transcriptional regulation then probably some DNA modifications will have to be done and undone. Cis-regulatory sequences that control a gene's level of activation can act through recruitment of histone acetyl transferases and other chromatin modifiers, so a precedent exists. But I don't think there is any special connection between epigenetics and memory. Memory requires cells to be cells and work properly, so it will require transcriptional regulation and DNA/chromatin modification. For any further connection, I think we have to start naming names.

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Friday, February 23, 2007

Epigenetics: the hippest field on the block   posted by p-ter @ 2/23/2007 07:15:00 PM

The current issue of Cell is dedicated to the rapidly changing field of epigenetics, loosely defined at the ensemble of chromatin and DNA modifications that structure the genome and control gene expression. The only review I've worked my way through yet is this one: Timescales of Genetic and Epigenetic Inheritance.
According to classical evolutionary theory, phenotypic variation originates from random mutations that are independent of selective pressure. However, recent findings suggest that organisms have evolved mechanisms to influence the timing or genomic location of heritable variability. Hypervariable contingency loci and epigenetic switches increase the variability of specific phenotypes; error-prone DNA replicases produce bursts of variability in times of stress. Interestingly, these mechanisms seem to tune the variability of a given phenotype to match the variability of the acting selective pressure. Although these observations do not undermine Darwin's theory, they suggest that selection and variability are less independent than once thought.
The authors limit themselves largely to bacteria and yeast, but they document a great deal of evidence that parameters like the mutation rate change in response to external stimuli, perhaps in almost a "directed" fashion. Some work in primates has shown a correlation between the amount of mutation raining down on a population and the amount of beneficial mutations that arise. Could "directed" mutagenesis be an explanation for this?

The main points from the paper are summarized in the figure below; essentially, epigenetic inheritance is a way for a population to respond to environmental changes that occur on time scales shorter than those needed for genetic evolution.

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