More epigenetics, EN-2 and autism... the plot thickens

I don't mind admitting that I was to some extent 'winging it' with my previous post on epigenetics and Engrailed-2 (EN-2) as a consequence of the findings reported by Jill James and colleagues [1] with autism in mind. Although an avid follower of the science of epigenetics when (cautiously) applied to autism, I am by no means any authority on the subject matter particularly when it comes to the nitty-gritty details. You can probably therefore expect similar things in my latest discussions on yet more work from this research group which appeared recently [2].

I have only one rule. Everybody fights, no one quits.

And so, with that pinch of salt at the ready...

The final conclusion made in the most recent James article boils down to the suggestion that "persistent postnatal overexpression of EN-2 suggests that the closing of this programed developmental window may have been missed in some individuals with autism because of epigenetic abnormalities". That being said I think we have quite a way to come before we can substantiate this finding particularly when the main protagonist in the latest article is something called 5-hydroxymethylcytosine (5-hmC) and results which show "that elevated 5-hmC in the EN-2 promoter is associated with a significant decrease in repressive MeCP2 and histone H3K27me3 that appear to over-ride 5-mC hypermethylation". The H3K27me3 bit comes from their previous findings by the way.

To most readers that probably sounds as complicated as it first did to me so I will try and explain more.

EN-2 as I've talked about in that post on the previous James work, has been linked to cases of autism as per the example of the study by Wang and colleagues [3] linking mutations in this gene to cases of autism. The idea being that mice bred without the gene (the homeobox domain of EN2) show some of the [mouse] signs and symptoms of autism alongside issues with the cerebellum and a reduction in the number of Purkinje cells which have been previously noted in cases of autism [4]. The previous James results in this area reported on hypermethylation of the EN-2 promoter region which would normally equate as gene silencing in epigenetic terms, in line with the more structural genomic issues seen in autism that I've just talked about. But, and it is an important point, when they looked at EN-2 expression and protein levels - function and products of the gene - they actually found that levels were increased in their autism samples despite the methylation mark and its 'stop talking' properties. They noted on that occasion that "transcriptional upregulation by other epigenetic mechanisms predominated over the repressive tendencies of DNA cytosine methylation".

Their latest foray into this area sought to further clarify just what might be going on specifically with EN-2 gene-specific DNA hypermethylation previously reported. To do this they focused on both measuring 5-hmC and also 5-methylcytosine (5-mC) among other things based on the same tissue samples (post-mortem cerebellum samples) detailed in their previous study. 5-hmC is apparently an oxidation product of 5-mC mediated via something called TETs.

What they found, far from answering the question of a discrepancy between epigenetic gene silencing of EN-2 but increased gene function and products, actually makes the whole thing a lot more complicated. So they observed "a significant increase in both 5-mC and 5-hmC in the autism cerebellum relative to the control samples". Further that there was "a significant increase in 5-hmC content within the upstream EN-2 promoter region" and "a highly significant positive correlation... was found between 5-hmC content and EN-2 gene expression in the 5’ promoter CpG island in autism but not in control samples". They note that: "that 5-hmC accumulation is mechanistically related to gene upregulation" something which I think ties into other work hinting at the demethylating role for 5-hmC [5].

Insofar as my mention of MeCP2 and histone H3K27me3 from the latest and previous James reports, I can't really say too much more aside from noting again: "reduced MeCP2-mediated gene repression may have contributed to persistent EN-2 gene overexpression in the autism samples". Actually the authors speculate that MeCP2 binding and histone H3K27 trimethylation might work together in a "repressive" manner but when reduced as they were "may contribute to aberrant overexpression of EN-2 in the autism cerebellum" as per their findings.

I have to say that I struggled with getting my head around these findings and I'd quite understand if readers also struggled with my interpretation of them ("If you can't explain something to a six-year-old/granny, you really don't understand it yourself"). I understand that we don't all walk around with our genes stuck in the 'on' or 'off' position and that particularly during foetal and the early post-natal periods, genes are being switched on and off at a surprising rate for many, many different important reasons. I also understand that DNA methylation is an important part of the whole genes switched on or off thing but not the only way that this process can happen as per the authors mention of chromatin and some previous text in this area [6]. With my very limited knowledge of this area, I am however not yet convinced that we have the full story here; specifically in terms of why the original finding of hypermethylation of the EN-2 promotor region (gene silencing) yet increased expression and protein levels were reported. I wonder if indeed we might be able to learn more from a two-hit approach whereby hypermethylation of only one gene allele leaves the other still working?

Just before I finish I'd like to also draw your attention to another paper which has started to ask similar questions about 5-hmC and might be contrasted with the recent James paper. Zhubi and colleagues [7] (open-access here) looked at 5-hmC with a couple of other potentially important genes linked to cases of autism (RELN and GAD1) in mind. They reported: "a significant increase in TET1 expression and an enrichment in the level of 5-hmC... at the promoters of GAD1 and RELN in ASD when compared with CON [controls]". Further that their data are: "consistent with the hypothesis that an increase of 5-hmC (relative to 5-mC) at specific gene domains enhances the binding of MeCP2 to 5-hmC and reduces expression of the corresponding target genes in ASD [autism spectrum disorder] cerebella".

The plot thickens...

So then to some music... Hey Jude.

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[1] James SJ. et al. Complex epigenetic regulation of Engrailed-2 (EN-2) homeobox gene in the autism cerebellum. Translational Psychiatry. 2013; 3: e232.

[2] James SJ. et al. Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum. Translational Psychiatry. 2014. 7 October.

[3] Wang L. et al. Association of the ENGRAILED 2 (EN2) gene with autism in Chinese Han population. Am J Med Genet B Neuropsychiatr Genet. 2008 Jun 5;147B(4):434-8.

[4] Fatemi SH. et al. Purkinje cell size is reduced in cerebellum of patients with autism. Cell Mol Neurobiol. 2002 Apr;22(2):171-5.

[5] Dahl C. et al. Advances in DNA methylation: 5-hydroxymethylcytosine revisited. Clin Chim Acta. 2011 May 12;412(11-12):831-6.

[6] Lasalle JM. et al. Autism genes keep turning up chromatin. OA Autism. 2013 Jun 19;1(2):14.

[7] Zhubi A. et al. Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum. Transl Psychiatry. 2014 Jan 21;4:e349.

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James SJ, Shpyleva S, Melnyk S, Pavliv O, & Pogribny IP (2014). Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum. Translational psychiatry, 4 PMID: 25290267

Epigenetics, EN-2 and the 'autism brain'

A paper by Jill James and colleagues* (open-access) caught my eye recently. Centred on Engrailed-2 (EN-2), a gene with more than a passing relationship to cases of autism (see here), James et al report results based on analysis of a small number of post-mortem cerebellar samples with a particular focus on an epigenetic evaluation.

Cerebellum @ Wikipedia  

What is epigenetics? Well, I've written before about some of the basic concepts involved (see here) and how despite not everyone being enamoured with the rise and rise of the science, the discipline of epigenomics adds quite a distinctive layer to the functioning of a persons genome.

The basic tenet: your DNA might not necessarily be your destiny and that subtle changes to the epigenome can influence the expression of certain genes or not. Certainly in areas such as cancer medicine, epigenetics is starting to make some real waves (see here).

With autism in mind, epigenetics is also starting to make an impact on the scientific literature and promises so much more. I'm taken for example, back to some previous work looking at prefrontal cortex neurons** with autism in mind which concluded that there might be more to see in this area at least for some cases of autism.

Anyhow....

• James and colleagues focused on cerebellar samples because (a) the cerebellum has been a real area of interest to autism research, and (b) EN-2 is "highly expressed in Purkinje cells"; reaffirming some interesting observations noted about Purkinje cells in the cerebellum of people with autism***.
• They analysed 26 samples from 13 people with autism and 13 asymptomatic controls. Details of how participants died and other details are provided in the paper, bearing in mind the various discussions on how post-mortem brain samples from those deceased who had autism are subject to various confounders including how they died and the role of any comorbidity. Incidentally, some of the autism samples originated from the same place which had that very unfortunate freezer malfunction last year (see here).
• Various methods and techniques were used to assess the details of epigenetic functions focused on methylation. I can't and won't pretend to understand all of them but interestingly as well as looking at EN-2 promoter region methylation, global methylation and "the methylation status of histones H3K27 (associated with gene silencing) and histone H3 lysine 4 (H3K4; associated with gene activation)" was also included (see here), part of the histone code.
• Results: some interesting ones such as the finding of hypermethylation of DNA extracted from autism cerebellum samples, which contrasts sharply with the DNA hypomethylation of immune cells noted by some of the authorship group on another occasion****. The authors speculate that this could be indicative of "tissue-specific" DNA methylation in autism; also noting that short of looking at brain samples - which is neither desirable or feasible for the living - we can't conclude too much from "peripheral cell DNA methylation patterns". This should make for some interesting future discussions I reckon.
• Alongside this global hypermethylation, James reports hypermethylation of the EN-2 promoter region. This, alongside sustained gene expression of EN-2 and greater levels of EN-2 protein in the autism samples. Similarly when looking at the methylation of histones (H3K27 and H3K4), the histone H3K27 which is linked to gene suppression was decreased and the histone H3K4 linked to gene activation, was increased (albeit not significantly).
• Assuming that I've understood this all correctly, the suggestion is that epigenetic issues with the histones involved in gene suppression or gene activation (via methylation) were congruent with a pattern of "sustained EN-2" gene over-expression which might tie into the loss of Purkinje cells***** noted in the cerebellum of some people with autism. At least I think so.

It all makes for some really rather interesting findings. That for example, the modification of histones ties into the levels of gene expression and importantly gene protein levels is really exciting and perhaps a valuable addition to the notion that mutation in the form of SNPs are the only influencing variable on gene function. Indeed that an epigenetic process might affect the timing of gene activation/suppression at critical periods of development is also an important point bearing in mind that we don't all walk around with all our genes permanently stuck in the 'on' position.

One also starts to wonder about not just the availability of methyl groups in this process but also the functioning of things like the DNA methyltransferase enzyme family (adding methyl groups) and indeed the demethylase enzymes (removing methyl groups) and the circumstances of their control at certain periods of development. Indeed methylation is only one facet of histone modification, as per the acetlyation and deacteylation of histone which potentially brings us back to things like the valproate connection being made to cases of autism (see here). It's all quite complicated.

Perhaps just as important are the implications of hypermethylation and those histone modifications to other genes tied into things like neuronal development and immune function in conditions like autism. Noting for example the Saxena paper (covered here) and their linking quite a few of the autism-related genes to things like immune function, James and colleagues make mention of one demethylase, JMJD3 (see here) and its potential link to the "IL-6 gene promoter" with regards to processes such as neuroinflammation. Certainly one has to ponder how deep the rabbit hole goes.

OK, coming back down to earth, caution is required in that this was a relatively small scale study which is again, always going to be confounded by the use of post-mortem brain samples and factors such as cause of death and the important point that autism is a behavioural label and that link of possible heightened comorbidity. Added to the fact that the focus was on one particular gene - one of quite a few - with some apparent connection to autism, the results should be viewed as preliminary at best.

That being said, we have a template now for expanding this area of work to cover other candidate genes in different tissues, to start working on those all-important rodent models. Then, with some degree of caution and assuming a strong connection is made, looking at the various factors which might potentially influence and moderate such epigenetic issues - including sex differences****** (open-access) - bearing mind the golden concept of phenotypes.

This could be something quite big...

Speaking of big (famous), they don't come much bigger than this lady....

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* James SJ. et al. Complex epigenetic regulation of Engrailed-2 (EN-2) homeobox gene in the autism cerebellum. Translational Psychiatry. 2013: 3; e232.

** Shulha HP. et al. Epigenetic signatures of autism: trimethylated H3K4 landscapes in prefrontal neurons. Arch Gen Psychiatry. 2012; 69: 314-324.

*** Fatemi SH. et al. Purkinje cell size is reduced in cerebellum of patients with autism. Cell Mol Neurobiol. 2002; 22: 171-175.

**** Melnyk S. et al. Metabolic imbalance associated with methylation dysregulation and oxidative damage in children with autism. J Autism Dev Disord. 2012; 42: 367-377.

***** Baader SL. et al. Ectopic overexpression of engrailed-2 in cerebellar Purkinje cells causes restricted cell loss and retarded external germinal layer development at lobule junctions. J Neurosci. 1998; 18: 1763-1773.

****** McCarthy MM. et al. The epigenetics of sex differences in the brain. J Neurosci. 2009; 29: 12815-12823.

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James SJ, Shpyleva S, Melnyk S, Pavliv O, & Pogribny IP (2013). Complex epigenetic regulation of Engrailed-2 (EN-2) homeobox gene in the autism cerebellum. Translational psychiatry, 3 PMID: 23423141

Epigenetics 101 and autism

Just relaxing @ Paul Whiteley

Ladies and Gentlemen. In the red corner, several million/billion years in the making, the current champion, genetics. In the blue corner, weighing in at several billion/trillion pounds, the challenger, environment.

[OK fellas, a good clean fight].

Ladies and Gentlemen... lets get ready to rumble!

Dramatic opening to post finished as I offer one of my more descriptive entries on the emerging field of epigenetics and some of the research already carried out with specific regards to autism spectrum conditions. I admit that I am sailing on the waves of current interest in this field with this post, being brought to it by the news that we may be seeing a lot more of the word 'epigenetics' in autism research circles soon as per this announcement on funding for Prof. Margaret Daniele Fallin based at Johns Hopkins Bloomberg School of Public Health and an accompanying 'what is epigenetics' post by Autism Speaks. The news that poverty might also have an epigenetic angle also took my interest (take note Politicians the world over).

Epigenetics has been mentioned before on this blog; normally posting a link to this piece in Time magazine about why your DNA is not necessarily your destiny. In short, epigenetics is the study of changes to gene activity without alterations to the genetic code passed down at least one generation. Epigenetics is quite an intellectually satisfying approach for lots of reasons; primarily perhaps because instead of pitting genes against environment in some kind of grudge match boxing contest, it actually suggests that our environment - our diet, our various stresses and exposures right from our earliest days - can affect gene activity by switching genes on or off. Synergy in action.

Another interesting article on epigenetics recently appeared on the Scientific American blog. I don't want to plagiarise what is a very good article, but there are some interesting data and concepts discussed which I do want to mention.

• I can't pretend to be an expert in this area so I won't try. Instead I refer you to quite a good introduction which presents the terms chromatin (the stuff of chromosomes), the DNA-protein mix nicely packaged up to fit into a cell nucleus, and histone, the scaffold around which DNA is wrapped to form nucleosomes.
• There are various ways that histone can be 'modified' including acetylation, methylation and phosphorylation. Such modifications have onward effects which have been described in a 'histone code' which seems to be growing all the time.
• The concept of Lamarckism is gaining in popularity as a consequence of the epigenetics tide. Lamarckism basically states that certain characteristics acquired by a parent can be passed to offspring. So coupled to epigenetics, parental (or grandparental) nutrition for example, might alter gene expression which then gets passed down to successive generations. I'm thinking Barker hypothesis and thin-fat bodies.

I've talked before about genetics on this blog and how whichever way you look at it, genes, mutations, etc. we are all very much a product of mutation and the emerging view is that genetics in relation to autism is a very, very, very complicated thing. I would hasten to add that concepts of Lamarckism are not to be viewed as another 'blame game' idea given that exposure events are likely to be multiple and complex and, as per the wartime famine studies related to the Barker hypothesis, events are not always likely to be under our control.

Very interesting, I (hope I) hear you say. But what about autism?

Well, epigenetics is obviously quite a new area for autism simply because a quick search of PubMed (26/01/12) only reveals 37 entries for the words 'autism and epigenetics'. That's not however to say that there isn't some interesting data already available to look at as I hope I will show.

This overview paper by N. Carolyn Schanen* (full-text) is as good a start as any. It is quite a long paper and not exactly easy to follow unless you are a molecular biologist, but nevertheless it offers some interesting discussions not least forecasting where we are today in terms of lacking any significant, universal genetic markers for autism.

This paper by Mehler and Purpura** (full-text) also has some interesting discussions about epigenetics. Appreciating that there is some degree of speculation in the text on their theory "of a functionally impaired locus coeruleus-noradrenergic (LC-NA) system", I find myself also drawn to their ideas on fever potentially affecting the presentation of symptoms in some cases of autism as per other research among the literature and the possible involvement of antipyretics in some cases.

Finally, this paper by Grafodatskaya and colleagues*** I think sets the tone for where genetics research might be heading in autism research with epigenetics at the helm. Noting that the authorship list includes Peter Szatmari who commented on the twins study published last year (the game changer!), it is difficult to argue against the notion that genes and genetic research is in the midst of a revolution. A revolution where the boxing match between nature and environment might just be replaced by an altogether more understanding relationship.

Cue the candle-lit dinner and (b)romantic music... (for my non-UK audience, read more about the background to this song here).

* Schanen NC. Epigenetics of autism spectrum disorders. Human Molecular Genetics. 2006; 15: R138-R150.

** Mehler MF. & Purpura DP. Autism, fever, epigenetics and the locus coeruleus. Brain Research Reviews. 2009; 59: 388-392.

*** Grafodatskaya D. et al. Autism spectrum disorders and epigenetics. Journal of the American Academy of Child and Adolescent Psychiatry. 2010; 49: 794-809.