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epigenetics[1] and non-coding RNAs. I encourage everyone to check out the new Cell and find “the first genome-wide high-resolution mapping of DNA methylation and the first systematic analysis of the role of DNA methylation in regulating gene expression for any organism” and a new microRNA target prediction method (rna22) that makes big (perhaps too radical?) predictions about the percentage of mRNAs regulated by this pathway.

I haven’t even had time to read the methylation paper yet, it looks like tiling arrays are used and this will be the third instance of that technique I’ve come across in the past week. Basically, this is an array that contains little chunks of sequence making up all of the non-repetitive parts of the genome. You can then wash some sample over it and see which chunks of sequence on the array get sample stuck to them through hybridization. Looks like in this paper they pulled out all the methylated vs. unmethylated DNA and hybridized that to the tiling array. Scanning the paper I note that when methylation occurs within the coding portion of a gene it is likely to be expressed whereas when methylation occurs in the promoter region it is likely to be controlled in a tissue-specific manner. Also, they present some evidence downplaying the role of microRNAs in transcriptional regulation through guided methylation, which was getting some buzz a couple or three months ago. BTW, this study was performed in a plant genome; Arabidopsis thaliana to be exact.

On the other hand, microRNAs in translational regulation are still getting played up as a major force. I won’t pretend to understand all of the pros and cons of rna22′s algorithm, but they do some false positive and sensitivity analysis and predict that it will find 1 false-positive binding site per 10,000 nucleotides and will discover 83% of real binding sites. With those rates in mind, consider the number of binding sites their algorithm predicts in the human genome. Conventional wisdom is that microRNAs are most likely to bind to the 3′ untranslated region of mRNAs, so that is where you should look for binding sites. 92.3% of 3′ UTRs in the human genome contain one or more “target islands” according to rna22. Even better, 99% of coding sequences are in the human genome do the same. That result almost seems outlandish to me, but I really have no expertise to evaluate it from.

Whenever the Schratt et al. paper came out earlier this year I was totally hyped on it. One microRNA (miR-134) was found to control LIMK-1 expression and thus dendritic spine morphogenesis. The translational repression of LIMK-1 was released in response to brain-derived neurotrophic factor (BDNF, associated with LTP and memory and all that jazz). I thought, “Maybe miR-134 has multiple synapse-related targets that are co-upregulated by release from microRNA inhibition in the face of synaptic activity.” I was thinking in the 50-100 range. This paper predicts 2318 targets for miR-134. There are not that many dendritically localized RNAs, so my little theory is at best incomplete.

Finally, everything you knew about microRNA-target hybridization is wrong. Many heuristic-based approaches have focused on the “seed region” of miRNAs for target recognition. The idea is that it is particularly important for the first 7 or so nucleotides of a miRNA to match its target sequence and bind effectively. This paper says good seed-binding can still lead to crappy translational repression, and poor seed-binding (weird base-pairs or nucleotides with no binding partner at all) can still lead to strong repression. So everything right is wrong again and we can all go back to the drawing board, but at least now we have IBM on our side.

[1]I’m a little concerned that people won’t understand the connection between epigenetics and DNA methylation. It seems like lately when I see the term it refers to any sort of heritability that can’t be directly attributed to DNA sequence. When I first learned the term it was primarily in relation to the molecular modifications that can occur around the DNA. For instance, DNA can be methylated and histones (the proteins that DNA wraps around to condense) can be acetylated or methylated or phosphorylated. People were very concerned with the methylation states of chromosomes and how they were modified by paternal and maternal imprinting. Of late, it seems that there is increasing focus on potential environmental effects on germline genome which are epigenetic in the broad sense, but may or may not be in the DNA methylation sense. Maybe I’m the only one who finds this distinction necessary/troublesome.

11 Comments

  1. When I first learned the term it was primarily in relation to the molecular modifications that can occur around the DNA 
     
    that’s what I’m referring to when I say epigenetics.

  2. Could you, maybe in a separate post, lay out what the informed layperson (i.e. not a chemist) should take away from all this at this moment in the developing research story? 
     
    For example your concern that people will assume epigenetics only means non-coding inheritaance whereas to you it seems to primarily mean tissue-specific expression. Have I got that right? What consequences do you see (sociodynamically, I mean) from these new insights? Could scientists eventually learn from this how to make cells sit up and play tricks? For example take somatic cells and make them express as stem cells?

  3. I’ll let coffee mug talk about the consequences, but epigenetics is the study of modifications to DNA (or the proteins that package the DNA). When I think epigenetics, I mostly think methylation. methylation of the DNA around a gene leads controls its expression. differential methylation of DNA in different tissues, then, leads to tissue-specific expression.  
     
    something that interests me is whether, should different sperm have different methylation profiles (as they do), does this information get transmitted to the next generation, and what role does it play in the generation of phenotypes?  
     
    both questions, for me, fall into the realm of epigenetics, and both are important.

  4. Dick - 
    I’ll try to give you an idea of what I am talking about, but my thinking isn’t entriely crystal clear, so bear with me. It seems like there are three partially overlapping phenomena that people refer to as ‘epigenetic’. 
     
    1) inherited gene activity states that do not depend on the DNA sequence. for instance, there are some genes that dads would like expressed in developing embryos and that moms would not like expressed and vice versa. if a moms do not like the gene they will methylate the heck out of the DNA in and around its sequence particularly in eggs. so any chromosome that comes from a female will have that gene turned off, so at best a developing embryo can only get a single dose of that gene from its dad.  
     
    i am paraphrasing this, but my paraphrase may be less clear than the original passage: 
    Another example of epigenetic inheritance, discovered about 15 years ago in mammals, is parental imprinting. In parental imprinting, certain autosomal genes have seemingly unusual inheritance patterns. For example, the mouse Igf2 gene is expressed in a mouse only if it was inherited from the mouse?s father. It is said to be maternally imprinted, inasmuch as a copy of the gene derived from the mother is inactive. Conversely, the mouse H19 gene is expressed only if it was inherited from the mother; H19 is paternally imprinted. The consequence of parental imprinting is that imprinted genes are expressed as if they were hemizygous, even though there are two copies of each of these autosomal genes in each cell. Furthermore, when these genes are examined at the molecular level, no changes in their DNA sequences are observed. Rather, the only changes that are seen are extra methyl (?CH3) groups present on certain bases of the DNA of the imprinted genes. Occasional bases of the DNA of most higher organisms are methylated (an exception being Drosophila). These methyl groups are enzymatically added and removed, through the action of special methylases and demethylases. The level of methylation generally correlates with the transcriptional state of a gene: active genes are less methylated than inactive genes. However, whether altered levels of DNA methylation cause epigenetic changes in gene activity or whether altered methylation levels arise as a consequence of such changes is unknown. 
    the news from that Cell paper i cited is that it matters which segment of the DNA sequence is methylated. methylation in the actual coding sequence of the gene seems to be associated with the gene being in an active state (not silenced), whereas when the methylation occurs in the regulatory region where factors that modulate the gene’s activity level usually bind (the promoter) the gene is likely to be silenced. reading this, the simplistic prediction is that you are better off looking in the promoter region of gene’s whose activity level seems determined by which parent they were inherited from rather than gene sequence. 
     
    the ‘epi’ in epigenetics seems to mean ‘above genetics’. people refer to these methylation states and a couple other chemical modifications that happen either on the DNA itself or really close as the ‘epigenome’, so that would be epigenetics in the physical molecular sense. but the -genetics in this case could also be referring to the genetics in the more classical, mendelian sense. epi would be the study of heredity unpredicted by the old school. when you can draw the same pedigree diagram, but for some reason when the final product is different depending on something besides which genes are there. 
     
    the first example i mentioned can fit both definitions, but people sometimes divorce the two and seem only to mean it in the molecular sense or the heredity sense: 
     
    i’m going to continue this in another comment…

  5. 2) gene activity states that do not depend on the DNA sequence. 
     
    here’s the example i am familiar with. maybe this has only happened in the field of learning and memory: 
    Epigenetic mechanisms: a common theme in vertebrate and invertebrate memory formation 
    J.M. Levenson and J.D. Sweatt 
    In this review we address the idea that conservation of epigenetic mechanisms for information storage represents a unifying model in biology, with epigenetic mechanisms being utilized for cellular memory at levels from behavioral memory to development to cellular differentiation. Epigenetic mechanisms typically involve alterations in chromatin structure, which in turn regulate gene expression. An emerging idea is that the regulation of chromatin structure through histone acetylation and DNA methylation may mediate long-lasting behavioral change in the context of learning and memory. We find this idea fascinating because similar mechanisms are used for triggering and storing long-term ?memory? at the cellular level, for example when cells differentiate. An additional intriguing aspect of the hypothesis of a role for epigenetic mechanisms in information storage is that lifelong behavioral memory storage may involve lasting changes in the physical, three-dimensional structure of DNA itself. 
    When I read this I feel like I really must be missing something because these guys have basically written this same review twice and Nature Reviews thought it was a good idea, but to me it looks like they could have said: “We think that genes have to be regulated. Probably after memories are formed genes in neurons are regulated.” The problem is that this is nothing special and I’m not sure it is ‘epigenetic’. Gene regulation happens all the time in response to all sorts of environmental stimuli. Most often, the mediator between stimulus and genome is a transcription factor (TF). TFs often have buddies called co-activators or co-repressors, that help them physically control a gene’s activity state. For instance: 
    A transcription factor-binding domain of the coactivator CBP is essential for long-term memory and the expression of specific target genes  
    Marcelo A. Wood, Michelle A. Attner, Ana M.M. Oliveira, Paul K. Brindle, and Ted Abel  
    Transcriptional activation is a key process required for long-term memory formation. Recently, the transcriptional coactivator CREB-binding protein (CBP) was shown to be critical for hippocampus-dependent long-term memory and hippocampal synaptic plasticity. As a coactivator with intrinsic histone acetyltransferase activity, CBP interacts with numerous transcription factors and contains multiple functional domains…… 
    Histone acetylation is one of three or four major ‘epigenetic modifications’ in Levenson and Sweatt’s view, but it looks to me like it should be called chromatin modification, drop the ?misuse? of the term ‘epigenetic’, and stop acting like they have discovered something magical by using a new term to describe the fact that genes are regulated in response to experience. Not that the study of histone modification in relation to memory isn’t cool! So there is someone taking it too far on the molecular side and leaving out the heritable portion. 
     
    aaaaaaand..

  6. 3) heritable stuff that doesn’t depend on DNA sequence that also doesn’t necessarily depend on DNA methylation or chromatin modificaiton or any change really necessarily close to the genome. 
     
    this was the one i was concerned about people getting mixed up with because i have come across it in the news. friends have asked me what i think about ‘epigenetics’ and they are referring to this smoking and obesity study (news article): 
    Scientific evidence suggests that this environmentally triggered gene expression, or epigenetic imprint, might have repercussions far beyond the immediate host body. This could explain why underweight babies born to malnourished Dutch women during World War II grew up to give birth to underweight children decades after the war and food rationing had ended. Recent studies have demonstrated that the sons of men who began smoking before reaching puberty were more prone to obesity. Clearly, the epigenetic change that took place in both original groups of parents had lastingly adverse effects on subsequent generations. “The trick,” says Szyf, “is to be able to control what you activate and deactivate.” 
    … 
    In a country propping up an increasingly fragile health care system, epigenetics may force politicians to rethink their economics. Although the field is still developing, evidence is being accumulated that points to the fact that a variety of diverse social and environmental factors such as nutrition, pollutants, housing and childcare will have a significant impact on the health of Canadians now and in the generations to come. “If we really want to strengthen our economy and the health and performance of the individuals within that economy, then we need to focus on early development and the quality of family life,” says Meaney.I’m not sure what Meaney studies, but I think it might be developmental biology instead of epigenetics. It seems that in this instance the term ‘epigenetic’ is simply referring to any correlation between some factor in the parents’ environment and the offspring outcome. This article is politicizing the term and paying lip service to the molecular definition while divorcing the concept. i can’t find the smoking and obesity study right now, but i think i may have seen it in the past and i’m not sure they hypothesized any explicit mechanism for their effect. did they measure the obese children’s methylation state at the ‘smoking parental imprinting site’ or something? 
     
    i’m not very good at sociodynamics, so if anybody else wants to step up to the plate for that be my guest. 
     
    as far as making the cells sit up and play tricks and converting them to stem cells, that does seem to be a focus in this area of research.  
    Epigenetic reprogramming in mammals. 
     
    * Morgan HD, 
    * Santos F, 
    * Green K, 
    * Dean W, 
    * Reik W. 
     
    Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge, UK. 
     
    Epigenetic marking systems confer stability of gene expression during mammalian development. Genome-wide epigenetic reprogramming occurs at stages when developmental potency of cells changes. At fertilization, the paternal genome exchanges protamines for histones, undergoes DNA demethylation, and acquires histone modifications, whereas the maternal genome appears epigenetically more static. During preimplantation development, there is passive DNA demethylation and further reorganization of histone modifications. In blastocysts, embryonic and extraembryonic lineages first show different epigenetic marks. This epigenetic reprogramming is likely to be needed for totipotency, correct initiation of embryonic gene expression, and early lineage development in the embryo. Comparative work demonstrates reprogramming in all mammalian species analysed, but the extent and timing varies, consistent with notable differences between species during preimplantation development. Parental imprinting marks originate in sperm and oocytes and are generally protected from this genome-wide reprogramming. Early primordial germ cells possess imprinting marks similar to those of somatic cells. However, rapid DNA demethylation after midgestation erases these parental imprints, in preparation for sex-specific de novo methylation during gametogenesis. Aberrant reprogramming of somatic epigenetic marks after somatic cell nuclear transfer leads to epigenetic defects in cloned embryos and stem cells. Links between epigenetic marking systems appear to be developmentally regulated contributing to plasticity. A number of activities that confer epigenetic marks are firmly established, while for those that remove marks, particularly methylation, some interesting candidates have emerged recently which need thorough testing in vivo. A mechanistic understanding of reprogramming will be crucial for medical applications of stem cell technology.

  7. Coffee Mug, you should make those excellent comments into a post. 
     
    Here are some additional thoughts? 
     
    Epigenetics could be caused by passing a molecular compound. This could be a maternal mRNA passed into the egg cell. Or it could be something passed from the mother to the fetus in the womb. Or it could be something in the mother?s diet that affects methylation in the baby. 
     
    There are also stochastic variations in gene silencing. This is a fuzzy area. The methylation of DNA, the acetylation state of histones, and the location of the DNA in the nucleus could act to stabilize the cell state. I.e., genes that are off stay off and genes that are on stay on. It isn?t always clear whether methylation turned the gene off or whether being off led to methylation. (Likewise for histone acetylation.) 
     
    (In your post you referred to microRNA?s. I?ve read about siRNA in mustard plants causing targeted methylization. The siRNA mechanism used some of the same molecular machinery as miRNA?s but I thought the processes differed somewhat. Do researchers consider siRNA to be a type of microRNA?)

  8. Fly- 
    there was another case of passing a molecular compound coming from miRNAs in the sperm.. i haven’t read it. abstract: 
    http://www.nature.com/nature/journal/v441/n7092/abs/nature04674.html 
    Paramutation is a heritable epigenetic modification induced in plants by cross-talk between allelic loci. Here we report a similar modification of the mouse Kit gene in the progeny of heterozygotes with the null mutant Kittm1Alf (a lacZ insertion). In spite of a homozygous wild-type genotype, their offspring maintain, to a variable extent, the white spots characteristic of Kit mutant animals. Efficiently inherited from either male or female parents, the modified phenotype results from a decrease in Kit messenger RNA levels with the accumulation of non-polyadenylated RNA molecules of abnormal sizes. Sustained transcriptional activity at the postmeiotic stages?at which time the gene is normally silent?leads to the accumulation of RNA in spermatozoa. Microinjection into fertilized eggs either of total RNA from Kittm1Alf/+ heterozygotes or of Kit-specific microRNAs induced a heritable white tail phenotype. Our results identify an unexpected mode of epigenetic inheritance associated with the zygotic transfer of RNA molecules. 
     
    no miRNAs and siRNAs are still ostensibly different.. one of the major differences is that miRNAs are transcribed from endogenous loci in a sterotypical precursor form- pri-miRNAs. i described it in more detail in my RNAi fundamentals post.. 
     
    the microRNA reference was to a particular section in the Cell Arabidopsis paper.. 
     
    Previous evidence suggested that microRNAs might recruit DNA methylation enzymes to their target genes (Bao et al., 2004). However, we found that annealing sites in microRNA target genes were methylated at a level slightly below the genome average (22 of 136, ?16.2%) (see Figure S4 for PHB as an example). In addition, we found that only one (MIR416a) of the 103 microRNA precursor genes was methylated. For trans-acting siRNAs (tasiRNAs) (Allen et al., 2005; Peragine et al., 2004; Vazquez et al., 2004), we found that 2 of the 5 tasiRNA-generating loci (TAS1b and TAS3) and 7 of 9 tasiRNA target sites contained methylation. However, bisulfite sequencing of the methylated tasiRNA target sites in ARF3 revealed CG methylation but an absence of non-CG methylation, which is a hallmark of RNA-directed DNA methylation (Figure S8) (Chan et al., 2005). Furthermore, DNA methylation persisted in the dcl2 dcl3 dcl4 triple mutant that lacks detectable tasiRNAs (Henderson et al., 2006). Overall, these results do not support a general role for miRNAs or tasiRNAs in the active targeting of DNA methylation. 
     
    don’t ask me how tasiRNA works.. this is the first time i’ve seen it.. i’ve heard of rasiRNA and that is supposed to use some of the same machinery as siRNA, but haven’t had time to catch up on that either..

  9. Dick- 
     
    I think that the main lesson to be learned from the field of epigenetics, for the “informed layperson” as you put it, is that not all heritable information is encoded in the DNA sequence. Since epigenetic information can be altered by environmental inputs (metabolite concentrations, hormone levels, etc.) but the actual sequence of DNA bases cannot, epigenetics provides a mechanism by which the record of an environmental state can potentially be retained through many cell divisions and even organism generations.  
     
    In addition, while the genetic code is very simple and has been “cracked” decades ago, enabling us to predict protein sequences from gene sequences with confidence, the epigenetic code is far less well understood, and the number of combinatorial interactions that can potentially be observed is much greater. It will take a number of additional experiments to get to the point where we can sequence someone’s DNA (even in a manner that reveals methylation) and say “genes X, Y, and Z are important to your phenotype but genes A, B, and C aren’t”. The parent of origin, cell type, and environmental conditions can all control whether or not a gene will be active.

  10. Thanks for clearing up my confusion on miRNA and RNA induced methylation in Arabidopsis. 
     
    I?m surprised that the mouse sperm carried enough miRNA to have an affect on the fertilized egg.

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