Epigenomics and Synthetic Biology: 2015 in Review

Despite how slow it feels doing it, every year science propels at a startling rate. Here are some paradigm shifts that really impacted my perceptions. Some are a selection of my contributions to EpiGenie and Epibeat, while the rest are written by others from places such as The Scientist and MIT Tech Review.

Epigenomics and Synthetic Biology Breakthroughs:

  1. The labs of Alexander Meissner, J. Keith Joung, and John Rinn brought the non-controversial paper: CRISPR used in human embryonic stem cells to show fundamental difference between mice and men.
  2. Michael Skinner shows that Epigenetics drives genetics straight into evolution
  3. Sperm miRNA Drives Intergenerational Stress Response
  4. A ‘new’ epigenetic mark: 6mA Makes the Grade as a Eukaryotic Epigenetic Mark
  5. DNA Methylation Helps Muscles Remember
  6. The Brain’s Circular RNAs
  7. Epigenome Editing with CRISPR-dCas9, TALEs, and Zinc Fingers
  8. Obesity Alters Sperm Epigenome
  9. CRISPR Gets Creative with Histone Acetylation
  10. Antidepressant Exerts Epigenetic Changes
  11. Move over Optogenetics, here comes Magnetogenetics: Discovery of long-sought biological compass claimed
  12. 5fC is Stable in Mammalian Brains
  13. CRISPR Inversion Untangles How CTCF Controls Chromatin Looping
  14. Epigenetic Clock Goes from Analog to iWatch
  15. New Biotech?! Viral Elements Horizontally Transfer Parasite’s Genes into Non-Standard Host Genomes
  16. RNAi pesticide: ‘Deep inside its labs, Monsanto is learning how to modify crops by spraying them with RNA rather than tinkering with their genes
  17. CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function at Protocadherins
  18. CRISPR-Display: For the lncRNA Enthusiast that has Everything
  19. CTCF Tucks Genes Into Their Lamina Associated Beds
  20. Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligonucleotides
  21. A DNMT1 (protein) and miRNA complex inactivating the catalytic region
  22. For that question you hear a million times: RNA doesn’t correlate with protein? Huh?
  23. Using Synbio to make narcotics: Brewing Bad
  24. Early Maternal Alcohol Consumption Alters Hippocampal DNA Methylation, Gene Expression and Volume in a Mouse Model
  25. Spray Your Way Free of Cystic Fibrosis with a Gene Editing Nasal Spray
  26. Sex On the Brain: DNA Methylation Defines Gender
  27. New Insights into Puzzling Placental DNA Methylation Domains
  28. 5hmC: a Helping Hand in Drug Addiction
  29. Optogenetic CRISPR/Cas9
  30. Evolving a bigger brain with human DNA
  31. Computed Chromatin Conformation Verfied by CRISPR [Awesome Video]
  32. A Reporter System to Trace Dynamic Changes of DNA Methylation at Single-Cell Resolution

Epigenomic Controversy

Sometimes big ideas need pilots. These pilots need more verification before public exposure but aren’t always uninterpretable, however they should be read with caution in regards to over-interpreting. Small sample sizes aren’t always applicable to a diverse general population and could be noisy but these experiments may start some interesting thoughts and still reveal underlying biology. But sometimes press releases aren’t handled properly.

  1. A paper on Intergenerational Holocaust Effects on Stress Signalling lead to this popular press and then this interesting response.
  2. A talk at ASHG about Epigenomics of Homosexuality got made into popular press and was followed by this critique and subsequent rebuttal.

CRISPR Craze & Crisis

There was  a gene editing summit that called together the leaders of the field. Interestingly it represented a wide variety of the thought spectrum. They came to a conclusion I think most can get behind: don’t start engineering babies clinically, but let the basic research into editing embryos (that don’t go to term) go strong.

Also of interest was the tweeting and blogging of Paul Knoepfler via IPSCell that showed off some of the academic communicative power of social media.

Gene drives have caught my attention as well since they can hijack evolution. They have a lot of potential for disease, with Malaria being the trailblazer. However, they also conjure up images of a dystopia where there’s a new bioterrorism tool in town, capable of specific genocide with just a few genome edited agents needed to ‘infiltrate’ the population.

I’ve also been thinking alot about the designer baby CRISPR CRISIS, it seems that enough of prominet researchers in genomics dismiss the notion of it, since single genes rule very few traits, but I’m still quite worried about myostatin. The condition works in humans, is famous in cows, and has been done in pigssheep, and dogs. It’s just a single gene that can easily make your child athlete of the century or a soldier in a superhuman army. And of course, something about this old Spiderman comic strip reflects on the Stan Lee’s judgement of the current designer baby CRISPR CRISIS:

CVbG-8hWsAA4i4f.jpg-large

 

Genome Editing Human Embryos

It seems that the ethical buzz was coming from a paper by a unknown Chinese group not involved with any of the genome editing pioneers. They took the unviable leftovers from In Vitro Fertilization (IVF) and then genome edited these human ’embyros’.

Interestingly, the success was quite poor. There was:

  • A Low Editing Rate
  • Toxicity
  • Rampant Off-Target effects

This in stark contrast to the use of CRISPR/Cas9 in dozens of animals ranging the entire tree of life. Ultimately, it doesn’t appear to be human limitation, as mammals including monkeys have been done much more successfully, but rather a result of poor experimental design, as these effects can be almost entirely attenuated by good guide RNA design and it seems that they didn’t considers the different chromatin states of embroynic stem cells that would influence off-target effects.

This was probably due to rushing the design in order to claim to be the first to alter human embryos, as opposed to the much more informative, well done, and ethically appeasing altering of Human Embryonic Stem Cells (HESCs) that showed off human CRISPR/Cas9 genome editing can be done properly in germ-line cells, with all its perks, and lead to breakthrough at the basic level in addition to all the clinical potential of genome editing technologies.

This speculation is apparent as there was a large outcry when the Chinese authors tried the ‘high impact’ journals and it seems they settled on a much lesser known open access that has additional concerns with the peer review process, mainly that it took one day, instead of 6 months to a year of the purgatory that is usually is.

Ultimately, it seems this was rushed for fame of unknown researchers and unknown journal, rather than science. But it’s still a Pubmed indexed journal with an impact factor and published by Springer. It is a bit of a shame as the Pandora’s box of CRISPR in human embryos needed to be opened quite slowly and carefully. The parts are all relatively easily accessible and not restricted, which is what has lead to spectacular pace of CRISPR/Cas9 genome editing development. While the field has its leaders developing CRISPR for the clinic the proper way, the technology it is now at the place where it can be picked up by many more who may not just be interested in the somatic line. But here we are now, waiting to see if genome editing technology will change the world, by curing inherited human disease or being used to design sci-fi nightmares. Either way, human inheritance has entered the designer era.

Sources:

  1. The Primary Publication
  2. Nature News
  3. Stem Cell Assays

Biology of Aging Research Proposal

1) Background:

Although normal aging is characterized by a number changes, none are as central to the identity of a person as their memories1. While there has been a large

amount of research into age related diseases of the brain, there is relatively little known about the molecular mechanisms behind how a healthy brain ages. Currently, aging is viewed as risk factor, rather than a cause, for a number of neurodegenerative diseases1. An understanding of the mechanisms behind why some people show relatively little age-related cognitive decline (ARCD), and others show significant and disabling ARCD, will provide great insight for both the basic and clinical sciences.

Initial studies into normal cognitive aging have shown that as we age gene expression patterns in the brain change; particularly genes involved in synaptic plasticity and vesicular transport show decreased expression after age 402. However, the casual link between aging and gene expression patterns has remained unknown until recently. More recent studies have begun to suggest that epigenetic mechanisms are the missing casual link3, 4. Modern epigenetics can be defined as “a mitotically (or meiotically) inheritable change in gene expression, independent of an alteration in DNA sequence5”. Epigenetic mechanisms encompass a number of molecular marks, such as histone modifications, DNA methylation, and microRNAs.

Epigenetic mechanisms
Epigenetic mechanisms (Photo credit: Wikipedia)

Most molecular research into ARCD has examined mouse models1, 6, 7, which allow for unprecedented observations and interventions, and have focused on the hippocampus, as it is involved in the consolidation of memory from the short-term to the long-term. At this time most studies have chosen to solely examine histone acetylation as the dynamic response to memory consolidation8, 9. These studies have shown that there is an age related failure in establishing histone acetylation in “learning-regulated genes” during memory consolidation. Furthermore, many studies have shown that treatment with histone deacytlase inhibitors (HDACs) can attenuate ARCD and related biomarkers8, 10, 11

While a narrowing of focus into histone acetylation has provided great insight, there is also emerging evidence suggesting that histone modifications act in concert with a number of other epigenetic factors12, 13. Indeed, DNA methyltransferase (DMNT) inhibitors have also been shown to interfere with memory consolidation14. Furthermore, many studies have also narrowed their focus into a relatively small amount candidate genes and/or single pathway being affected as a result of epigenetic modifications8, 15. However, in most cases the epigenome rarely works alone and typically affects gene expression on a much larger scale7, 16. In this research I hypothesize that memory consolidation involves epigenomic changes on a genome-wide scale in the hippocampus and that the dysregulation of these epigenetic marks contributes to ARCD.

 

2) Specific Objectives:

Specific Objective 1: Evaluate the epigenomic and transcriptomic changes associated with ARCD.

Specific Objective 2: Evaluate the epigenomic and transcriptomic changes associated with interventions that attenuate ARCD.

3) Proposed Research:

Evaluate the epigenomic and transcriptomic changes associated with ARCD.

To evaluate the epigenomic and transcriptomic changes associated with ARCD, I will use a C57BL/6J mouse model6. This model will allow for the collection of a

Workflow overview of the MeDIP procedure. MeDI...
Workflow overview of the MeDIP procedure. MeDIP procedure is followed by array-hybridization (A) or high-throughput/next generation sequencing (B) (Photo credit: Wikipedia)

significant amount (n=10) of hippocampal samples (CA1, CA3, and Dentate Gyrus). These samples will be collected from mice at the ages of 3, 8, and 16 months, which represent young, middle-aged, and old mice respectively6. The use of these 3 age groups will be sufficient for experimentation, as previous research has shown significant memory impairment in 16 month-old mice, with the 8 month-old mice showing an intermediate phenotype8. Samples will be obtained both before and at multiple time points during memory consolidation as initiated by fear conditioning17 (10 min, 30 min, 60 min, and 1d after)8. The different regions of the hippocampus will then be sequenced using next-generation sequencing technologies, including methylated DNA immunoprecipitation paired with sequencing (MeDIP-seq), chromatin immunoprecipitation paired with sequencing (ChIP-Seq), and RNA-Seq methodologies. These technologies will allow for an assessment of repressive epigenetics marks, such as DNA methylation (5-methylcytosine) and H4 lysine-27 trimethylation (H4K27me3). Additional Chip-Seq will also examine the activating marks of H4 lysine-12 acetylation (H4K12ac) and H4 lysine-3 tri-methylation (H4K3me3). Finally, micoRNA and gene expression patterns can also be assessed at a genome wide level by RNA-seq. Bioinformatic analysis will then be done to examine regions of the genome that show overlapping and unique responses of the (epi)genome upon memory consolidation across all age groups (Figure 1). Results from top candidates will be confirmed via sodium bisulfite sequencing, respective qPCR technologies (immunoprecipitation or reverse transcription based), and western blotting to confirm whether the observed alterations in gene expression and/or epigenetic marks translate to the protein level.

It should be noted that the histone modifications chosen only represent a fraction of those that occur, and while the ones chosen are the most extensively studied in this context, it is possible that there are more informative marks. If any of the marks are found to be non-informative in the preliminary experiments, alternative histone modifications will then be examined in a large-scale screening assay based off western blotting.

Evaluate the epigenomic and transcriptomic changes associated with interventions that affect ARCD.

The evaluation of epigenomic and transcriptomic changes associated with interventions that attenuate ARCD will be done using a similar experimental design to

English: Schematic showing how antisense DNA p...
English: Schematic showing how antisense DNA prevents protein translation. (Photo credit: Wikipedia)

the previous objective, but will focus on time points and regions of interest. The main difference will be the addition of two new groups; mice that serve as a matched-control for the methodology of intervention (i.e: vehicle injection) and those that have received the intervention (Figure 1). The interventions used will include HDAC2 inhibitors, which have received much attention in prior research11. Furthermore, given that DMNT inhibitors interfere with memory consolidation14, methyl-donor rich diets, such as those with choline18, will also be examined. Novel interventions, such as RNAi for transcripts of interest and environmental enrichment19 will also be examined.

It should be noted that there is a large amount of variation in the methodologies of the interventions mentioned. If during preliminary experiments an intervention does not attenuate ARCD, additional experiments will be carried out to optimize the conditions for each intervention (i.e.: dosage and timing of exposure).

Image

4) Expected Findings and Overall Significance of Proposed Research:

The proposed research will provide an unprecedented resolution of the dynamic and transient epigenetic and transcriptomic changes occurring upon memory consolidation. The results are expected to reveal a large number of previously unobserved interactions between the epigenome, genome, and transcriptome due to the relatively unbiased nature of sequencing technologies. The use of mice at different ages will allow for the identification of novel regions that become deregulated with age and contribute to ARCD. The integration of multiple data sets by bioinformatic analysis will also provide novel insight into how epigenetic mechanisms co-regulate changes in gene expression and allow for cross-communication between relatively distinct regions of the genome and/or pathways during memory consolidation. Furthermore, the examination of interventions that attenuate ARCD will also provide a number of novel insights into clinical treatment for ARCD. The use of HDAC inhibitors will allow for an examination of the most commonly used intervention11, and has the potential to reveal the mechanisms behind many of the potential negative side effects associated with a drug of such global nature.  The use of RNAi will allow for assessment of the effect in changes the dosage of individual candidate genes. Finally dietary choline supplementation and environmental enrichment serve as noninvasive interventions, with the potential for positive global epigenetic changes18, 19. These interventions, both established and novel, will confirm and reveal much about the inner workings of the aging brain and also further our understanding of the effectiveness of treatments for ARCD.

5) References:

1. Barnes, C. A. Secrets of aging: What does a normally aging brain look like? F1000 Biol. Rep. 3, 22-22. Epub 2011 Oct 3 (2011).

2. Lu, T. et al. Gene regulation and DNA damage in the ageing human brain. Nature 429, 883-891 (2004).

3. Fischer, A., Sananbenesi, F., Wang, X., Dobbin, M. & Tsai, L. H. Recovery of learning and memory is associated with chromatin remodelling. Nature 447, 178-182 (2007).

4. Graff, J. & Tsai, L. H. Histone acetylation: molecular mnemonics on the chromatin. Nat. Rev. Neurosci. 14, 97-111 (2013).

5. Berger, S. L., Kouzarides, T., Shiekhattar, R. & Shilatifard, A. An operational definition of epigenetics. Genes Dev. 23, 781-783 (2009).

6. Jucker, M. & Ingram, D. K. Murine models of brain aging and age-related neurodegenerative diseases. Behav. Brain Res. 85, 1-26 (1997).

7. Sweatt, J. D. Neuroscience. Epigenetics and cognitive aging. Science 328, 701-702 (2010).

8. Peleg, S. et al. Altered histone acetylation is associated with age-dependent memory impairment in mice. Science 328, 753-756 (2010).

9. Bousiges, O. et al. Detection of Histone Acetylation Levels in the Dorsal Hippocampus Reveals Early Tagging on Specific Residues of H2B and H4 Histones in Response to Learning. PLoS One 8, e57816 (2013).

10. Graff, J. et al. An epigenetic blockade of cognitive functions in the neurodegenerating brain. Nature 483, 222-226 (2012).

11. Day, J. J. & Sweatt, J. D. Epigenetic treatments for cognitive impairments. Neuropsychopharmacology 37, 247-260 (2012).

12. Miller, C. A., Campbell, S. L. & Sweatt, J. D. DNA methylation and histone acetylation work in concert to regulate memory formation and synaptic plasticity. Neurobiol. Learn. Mem. 89, 599-603 (2008).

13. Castellano, J. F. et al. Age-related memory impairment is associated with disrupted multivariate epigenetic coordination in the hippocampus. PLoS One 7, e33249 (2012).

14. Miller, C. A. & Sweatt, J. D. Covalent modification of DNA regulates memory formation. Neuron 53, 857-869 (2007).

15. Salih, D. A. et al. FoxO6 regulates memory consolidation and synaptic function. Genes Dev. 26, 2780-2801 (2012).

16. Bird, A. Perceptions of epigenetics. Nature 447, 396-398 (2007).

17. Fischer, A., Sananbenesi, F., Wang, X., Dobbin, M. & Tsai, L. H. Recovery of learning and memory is associated with chromatin remodelling. Nature 447, 178-182 (2007).

18. Zeisel, S. H. & da Costa, K. A. Choline: an essential nutrient for public health. Nutr. Rev. 67, 615-623 (2009).

19. Pang, T. Y. & Hannan, A. J. Enhancement of cognitive function in models of brain disease through environmental enrichment and physical activity. Neuropharmacology 64, 515-528 (2013).

 

You Are What You Eat?!

Diet has long been known to alter gene expression.1 These alterations were previously thought to only depend on the genome of the mammal consuming the food. Any differences in gene expression were though to only depend on genetic variation in the consumer population. However, recent research has discovered a direct route for the plant genome to interact with the mammalian genome via epigenetic mechanisms.2 Ultimately, this cross-kingdom interaction holds great potential for understanding inherited molecular variation.

Changes in gene expression across cells are accomplished by a number of cellular communication mechanisms including microvesicles (MVs). MVs are small vesicles that are shed by almost all cell types under normal and pathological conditions. 3 MVs are known to selectively interact with target cells and mediate intercellular communication. The intercellular communication is carried out by the transport of bioactive lipids, mRNA, and protein between cells. MVs have recently been shown to contain miRNAs.4, 5 miRNAs are small, noncoding RNA molecules that mediate post transcriptional silencing and affect ~30% of protein coding genes.6 miRNAs are known to regulate critical biological processes and alterations in miRNAs are linked to disease.7 Zhang et al. have shown that endogenous miRNAs are present in human plasma where miRNAs can be selectively packaged into MVs and actively delivered into recipient cells. Intriguingly, Zhang et al. established that exogenous miRNAs act in a manner similar to endogoneous miRNAs in that they both can regulate gene expression and cell function. Through the aforementioned research, Zhang et al.  have shown that exogenous miRNAs are packaged into MVs and provide a novel method for intercellular communication.

In their latest research, Zhang et al. expanded on their previous work by discovering a novel cross-kingdom interaction between plant (rice) miRNAs and the mammals (humans, mice, and calves) that have consumed the plant miRNAs.2 Three important aspects of the cross-kingdom interaction were examined. First, the research showed that plant miRNAs are present in mammalian serum and MV’s. Second, the research showed plant miRNAs in mammals are of plant origin.  Third, the research showed plant miRNAs consumed by mammals are capable of altering mammalian gene and protein expression. Zhang et al. established the three important aspects of the cross-kingdom interaction by observing that rice mir-168a is capable of reducing LDLRAP1 transcript levels. LDLRAP1 is a receptor for Low Density Lipoprotein (LDL), also known as “bad cholesterol”, and  is responsible for removing LDL particles from the circulatory system. Zhang et al. fed mice mir-168a, which was associated with a down-regulation of LDLRAP1, and increased LDL levels. Additionally, the authors observed that consumption of mammalian miRNA has a similar affect on its target gene. The discovery of the cross-kingdom interaction creates a world of possibilities in the understanding of mammalian and plant genetics.

The cross-kingdom interaction sheds new light on the intimate association between mammals and plants at the level of inherited molecular variation. The potential for variation lies in the fact that plants encode thousands of miRNAs with only six nucleotides of perfect complementary being sufficient to promote RNA silencing in mammals. 8, 9 Furthermore, miRNAs can act as switch and fine-tuner of gene expression. 6 An individual miRNA may also have up to a few hundred different target mRNAs. 10 The properties of miRNAs suggest that plant miRNAs have the potential to regulate many genes in mammals. If allelic variants were found in plant miRNAs and/or their target mammalian genes, it would suggest the possibility of co-evolution between local plant and mammalian populations. If co-evolution were to occur, it would create the possibility of regional “epigenetic linkage units” in which an allele for a plant miRNA may be “epigenetically linked” to an allele of a mammalian target gene. Indeed, one can imagine humans caring for a plant population that provides a benefit. Mammals would be indirectly receiving epigenetic information through the environment and thus “inherit” different alleles from plants through the local environment. This process of “inheritance” is similar in principle to horizontal gene transfer; the main difference being that the transfer would be indirect as it occurs through the environment. In other words, the cross-kingdom effect could create regional biological systems where the plant miRNAs parallel as “plasmids” to the mammalian genome, and the environment parallels the “cytoplasm”. Regional biological systems would then be created where plant and mammalian members with mutually beneficial alleles form an “epigenetic linkage unit” with a regional fitness advantage. The biological system would blur the lines between genetics and environment, which is a common theme to epigenetic phenomenon. The cross-kingdom interaction would then have the potential to drive evolutionary forces. Therefore, the cross-kingdom interaction brings about a paradigm shift in our understanding of inherited molecular variation, suggesting that inherited molecular diversity is shaped by the interplay between regional mammalian and plant genomes. A greater understanding of the interplay between the mammalian and plant genomes will expand our knowledge on inherited molecular variation.

When interpreting the cross-kingdom interaction, one must not get carried away as there are many other environmental conditions to consider. Maternal behaviour and chemical exposure are amongst the wealth of environmental conditions known to modify gene expression via epigenetic mechanisms.11-13 Given the great amount of environmental “noise”, the cross-kingdom interaction must be examined to determine if it is of any significance amongst others factors or just “lost in the noise”. Only when the significance of the cross-kingdom interaction is established can it hold any ground to the understanding of inherited molecular variation as there must be a fitness advantage in order for the “epigenetic linkage units” to be selected for. Despite its limitations, the cross-kingdom interaction gives life to a potential paradigm shift and warrants further examination in the quest to better understand inherited molecular variation. Ultimately, you may find out that not only do your own genes determine what you are, but so do the genes of what you eat.

Figure 1

Plant and human populations in regional environmental/biological systems with distinct “epigenetic linkage units”. Colours in humans and plants are used to identify distinct alleles with the same colour between species indicating a beneficial interaction. Additionally, the colours in the regional environments indicate a cross-kingdom interaction that is best suited for that environment.

References

1. Wolff, G.L., Kodell, R.L., Moore, S.R., Cooney, C.A. (1998). Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice. FASEB J. 12, 949-957.

2. Zhang, L., Hou, D., Chen, X., Li, D., Zhu, L., Zhang, Y., Li, J., Bian, Z., Liang, X., Cai, X. et al. (2011). Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res.

3. Denzer, K., van Eijk, M., Kleijmeer, M.J., Jakobson, E., de Groot, C., Geuze, H.J. (2000). Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. J. Immunol. 165, 1259-1265.

4. Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J.J., Lotvall, J.O. (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654-659.

5. Skog, J., Wurdinger, T., van Rijn, S., Meijer, D.H., Gainche, L., Sena-Esteves, M., Curry, W.T.,Jr, Carter, B.S., Krichevsky, A.M., Breakefield, X.O. (2008). Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470-1476.

6. Mukherji, S., Ebert, M.S., Zheng, G.X., Tsang, J.S., Sharp, P.A., van Oudenaarden, A. (2011). MicroRNAs can generate thresholds in target gene expression. Nat. Genet. 43, 854-859.

7. Zhang, Y., Liu, D., Chen, X., Li, J., Li, L., Bian, Z., Sun, F., Lu, J., Yin, Y., Cai, X. et al. (2010). Secreted monocytic miR-150 enhances targeted endothelial cell migration. Mol. Cell 39, 133-144.

8. Rajagopalan, R., Vaucheret, H., Trejo, J., Bartel, D.P. (2006). A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20, 3407-3425.

9. Lewis, B.P., Burge, C.B., Bartel, D.P. (2005). Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15-20.

10. Bartel, D.P. (2009). MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233.

11. Anway, M.D., Cupp, A.S., Uzumcu, M., Skinner, M.K. (2005). Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308, 1466-1469.

12. Vandegehuchte, M.B., Lemiere, F., Vanhaecke, L., Vanden Berghe, W., Janssen, C.R. (2010). Direct and transgenerational impact on Daphnia magna of chemicals with a known effect on DNA methylation. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 151, 278-285.

13. Weaver, I.C.G., Cervoni, N., Champagne, F.A., D’Alessio, A.C., Sharma, S., Seckl, J.R., Dymov, S., Szyf, M., Meaney, M.J. (2004). Epigenetic programming by maternal behavior. Nat. Neurosci. 7, 847-854.