Monthly Archives: February 2012

Electrical signalling and eye induction

A new paper in Development shows that bioelectric signals initiate the development of the eye in the African clawed frog, Xenopus laevis.

Embryonic induction, the process by which one tissue organises the patterning of neighbouring tissues, is a key concept in understanding animal development. The prototypic example of induction is Spemann’s organiser: a tissue that when grafted to the opposite side of the amphibian embryo can induce the development of ectopic body axes. Induction is generally mediated by secreted inducing molecules: either small molecules like Retinoic acid, or polypeptides such as members of the Wnt or BMP families. However, has the application of the technical innovations of molecular biology biased our perceptions of development to the detriment of other, more biophysical, mechanisms?

Using a voltage-sensitive dye, Pai et al. detected bilateral patches of hyperpolarisation in the anterior neural field of Xenopus embryos. Electrophysiological measurement confirmed that these cells maintained a ~10 mV transmembrane potential difference (Vmem) with their neighbours. By marking these hyperpolarised cell clusters in a fixation resistant manner and then sectioning the embryos, it was found that the cluster cells contribute to both the lens and the retinal layers of the eyes.

To disrupt the potential activity of hyperpolarisation in eye development, the researchers expressed constitutively active ion channels in cells giving rise to the anterior neural plate. They found that nearly 50% of the resulting embryos showed eye defects, ranging from absence to incomplete development. The loss of hyperpolarised cluster Vmem  also disrupted the expression of two transcription factors important for eye patterning, Pax6 and Rx1. Interestingly, interfering with Pax6 activity (by injection of a dominant negative construct) caused the loss of the hyperpolarisation signal. Therefore, the hyperpolarised cluster Vmem was necessary for proper development of the eye and acts in a positive feedback loop with Pax6.

Pai et al. went on to perform gain of function experiments, in which they asked whether modulation of Vmem could induce ectopic eyes. Injection of mRNAs encoding dominant negative ion channels (the main one used was an ATP-sensitive potassium channel) into the early embryo had widespread effects. In a quarter of cases the endogenous eyes showed defects, while in 20% of cases ectopic eye tissue was found in other parts of the embryo. In 7.5% of cases well formed ectopic eyes were induced. These ectopic inductions occurred in many different locations, including the gut, mesoderm, and the tail. Manipulation of Vmem was also shown to be able to cause focal ectopic expression of the eye marker genes pax6 and rx1.

Presumably, the gain of function experiments work by the local production of Vmems due to mosaicism in distribution of the dominant negative mRNAs. I was slightly surprised that such differences weren’t caused by the constitutively active ion channel experiments as well.  The fact that similar results are found with other ion channels, show that it is the biophysical character Vmem itself that is responsible for this inductive capability, and not a specific species of ion or gene product.

Although the idea that voltage gradients and electrical fields have roles in development is not new, this paper brings to the fore their potential importance. It demonstrates that restricted hyperpolarisation can act as an instructive signal necessary for patterning in vertebrate embryos. Further work is needed to dissect this pathway: Do the hyperpolarised cell clusters induce eye specification in neighbouring cells? or does the Vmem signal act in an autocrine manner? How does the Vmem signal fit into other signalling pathways involved in eye induction and patterning? Bioelectric effects are no doubt important for developmental patterning and morphogenesis. The use of voltage sensitive dyes and other neuroscience-derived techniques will help to test the extent of their roles in development in coming years.

Pai, V., Aw, S., Shomrat, T., Lemire, J., & Levin, M. (2011). Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis Development, 139 (2), 313-323 DOI: 10.1242/dev.073759


On Retrons

the secondary structure of msDNA Ec73. The 76 nt RNA (in box), is joined to a 73nt ssDNA. Note the 2'-5 phosphodiester bond connecting the two molecules at the branching guanosine.

Retrons are an understudied type of prokaryotic retroelement responsible for the synthesis of an enigmatic species of small extra-chromosomal satellite DNA termed multicopy single-stranded DNA (msDNA). msDNAs are actually composed of both a single-stranded (ss) DNA and a ssRNA. The 5′ end of the msDNA is covalently bonded to an internal guanosine residue of the msRNA by a unique 2′-5′ phosphodiester bond, whilst the 3′ ends of the molecules are joined by a small stretch of base-pairing. msDNAs are therefore a sort of looped hybrid molecule, but extensive internal base pairing creates various stem-loop/hairpin secondary structures (see figure). The retron, (ie. the genetic loci encoding the msRNA and msDNA molecules (msr and msd) and the gene encoding the reverse transcriptase (ret) responsible for the synthesis of msDNA) is transcribed as an operon.

Retrons are present in a wide variety of eubacterial, and some archaeal, genomes. A recent study identified 97 different retron-like reverse transcriptase genes within bacteria, however their distribution is sporadic. For instance, seven distinct retron elements have been found amongst E. coli strains, but only 15% of natural E. coli isolates produce msDNAs. Based on their sporadic occurrence and analysis of codon usage, retrons have been suggested to be a recent addition to the E. coli genome.

A major exception to the sporadic distribution found in most bacteria is within the myxobacteria, where all ten genera include msDNA-producing species. Myxobacterial retrons form a phylogenetically related group. These features, as well as sequence divergence, suggest that the common ancestor of the extant myxobacteria contained a retron as much as 150 million years ago, which has been vertically transmitted.

Retrons have not been shown to be mobile genetic elements, although the presence of reverse transcriptase does suggest this possibility. A clue to their propagation is the association of many of them with prophage sequences, suggesting their spread could be associated with bacteriophage. However, as with many observations about retrons, there are plenty of exceptions.

Organisation of a retron operon. note the inverse orientations and short overlap of msr and msd.

msDNA is essentially a cDNA produced from a short region of an mRNA template. During msDNA synthesis, an RNA template derived from the operon mRNA and composed of msr and msd, is folded into a specific secondary structure due to flanking inverted repeat sequences. The msd sequence is then reverse transcribed by the retron reverse transcriptase, using the 2’OH group of the ‘branching’ guanosine residue as a primer. The lagging RNA template strand is then degraded by RNaseH activity (probably host cell derived), leaving the msDNA covalently bonded at it’s 5′ end and base paired to the msRNA at their 3′ ends.

No function has been unequivocally attributed to msDNA. Mutating retron ret genes to prevent synthesis of E. coli or myxococcal msDNAs produces no detectable effects. Overexpression of certain E. coli msDNAs has been shown to increase mutation rate. msDNAs generally form hairpin structures by complementary base pairing of inverted repeat sequences (see figure). However, in many msDNA hairpins the base pairing is imperfect. It appears that the overexpression associated mutation rate phenotype is due to mismatch containing msDNAs sequestering the mismatch repair enzyme MutS. Overexpression of msDNAs without mismatch-containing hairpins does not cause similar effects. It is possible that msDNA could be regulating MutS availability by this titration mechanism in normal conditions or as part of a stress response. However, the overexpression experiments lead to msDNA concentrations far beyond normal physiological levels, so can yield no more than a hint of normal function.

In conclusion, the lacunae in our understanding of retrons and msDNA, are far more striking than the known facts. Are retrons parasitic elements? or do msDNAs have physiological roles in their host cells? Are retrons mobile elements? Just what does msDNA do? Judging from the literature, interest in retrons peaked around 1990, and recent years have been very fallow. I do hope that funding agencies and researchers keep pursuing the answers to these questions and don’t let them remain as an interesting oddity in the literature.

Lampson, B., Inouye, M., & Inouye, S. (2005). Retrons, msDNA, and the bacterial genome Cytogenetic and Genome Research, 110 (1-4), 491-499 DOI: 10.1159/000084982

Simon, D., & Zimmerly, S. (2008). A diversity of uncharacterized reverse transcriptases in bacteria Nucleic Acids Research, 36 (22), 7219-7229 DOI: 10.1093/nar/gkn867

Amyloid-like oligomers and long-term memory.

A new paper in Cell, shows that the formation of amyloid-like oligomers of a RNA-binding protein in synaptic membranes is necessary for the persistence of memories in Drosophila.

If memory is the maintenance of learning associated changes in synaptic efficacy and number, and these synaptic modifications are associated with changes in protein composition, how do they persist for years when individual protein molecules are turned over in days?

Cytoplasmic polyadenylation element-binding proteins (CPEBs) are a family of RNA-binding proteins that have been shown to regulate activity-dependent protein synthesis at synapses. A Drosophila CPEB, Orb2, has been shown to be required for the long-term persistence of memory (Keleman et al.). Previous studies on a CPEB from the sea slug Aplysia, had shown that it had prion-like properties, in that it could exist in two conformational states: a monomeric form and a self-perpetuating multimeric form with amyloid-like properties. Synaptic stimulation leads to the conversion of Aplysia CPEB into the multimeric form, and the formation of a self-sustaining synaptic mark. Blocking the activity of multimeric ApCPEB inhibited the long-term facilitation of a synaptic response (Si et al.).  Based on these Aplysia studies, Majumdar et al. hypothesised that Orb2’s role in Drosophila long-term memory could also be mediated by activity-dependent formation of amyloid-like oligomers.

Purifying Orb2 from Drosophila head extracts, Majumdar et al. show that it either exists in a monomeric form, or in oligomers consisting of between two and six Orb2 molecules. Orb2 oligomers are resistant to denaturation by heat or detergents, showing that they have amyloid-like characteristics. The oligomeric form of Orb2 appears to be localised to synaptic membranes, whilst the monomeric form is mainly found in cell bodies. Stimulation of neurons involved in memory formation increased the level of oligomeric Orb2.

Majumdar et al. go on to show that there are two different isoforms of Orb2 involved in the formation of Orb2 oligomers, Orb2A and Orb2B. Although the Orb2 oligomers found in vivo were mainly composed of Orb2B, the researchers found Orb2 oligomerisation requires Orb2A. Both isoforms contain a glutamine rich unstructured prion-like domain that acts as a substrate for oligomerisation, but a short 8 amino acid N-terminal domain of Orb2A catalyses the oligomerisation reaction. Orb2A appears to regulate the formation of amyloid-like Orb2 oligomers.

The previous study (Keleman et al.) that showed that Orb2 was involved in long-term memory stabilisation had used a technique that reduced the Orb2 concentration. By generating flies that carried a single point mutation in the Orb2A N-terminal domain, specifically impairing Orb2 oligomerisation, Majumdar et al. went on to show Orb2 oligomerisation is necessary for long-term memory persistence.

This work is exciting on two different fronts. It shows a mechanism for the generation of stable synaptic marks by the formation of amyloid-like oligomers. This could be a more widespread phenomenon underlying memory formation and persistence. More broadly, this study yields insight into potential physiological roles for amyloid formation and prion-like mechanisms.

Insoluble amyloid-like aggregations are a common pathological characteristic in human neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease. In the case of prion diseases such as Creutzfeld-Jacob disease, amyloidogenic forms of Prion-Protein propagate the disease by seeding the conformational changes to the non-pathogenic form. The authors of the paper do not call the Orb2 oligomers prions, as the mechanisms underlying oligomerisation are not understood. However, in the case of ApCPEB, multimerisation was shown to be self-sustaining, and hence prion-like.

Is there a link between the preponderance of amyloid associated neurodegenerative diseases and the role of amyloid-like oligomers in memory? It is probably unwise to extrapolate too far from these results. However, one could postulate that if this is a widespread mechanism of memory formation, amyloid associated diseases could be more likely to affect the brain. Likewise, if amyloid-like protein multimerisation has normal physiological roles in the brain, it could be a more permissive environment for amyloid formation than other tissues.

Majumdar, A., Cesario, W., White-Grindley, E., Jiang, H., Ren, F., Khan, M., Li, L., Choi, E., Kannan, K., Guo, F., Unruh, J., Slaughter, B., & Si, K. (2012). Critical Role of Amyloid-like Oligomers of Drosophila Orb2 in the Persistence of Memory Cell, 148 (3), 515-529 DOI: 10.1016/j.cell.2012.01.004

Si, K., Choi, Y., White-Grindley, E., Majumdar, A., & Kandel, E. (2010). Aplysia CPEB Can Form Prion-like Multimers in Sensory Neurons that Contribute to Long-Term Facilitation Cell, 140 (3), 421-435 DOI: 10.1016/j.cell.2010.01.008

Keleman, K., Krüttner, S., Alenius, M., & Dickson, B. (2007). Function of the Drosophila CPEB protein Orb2 in long-term courtship memory Nature Neuroscience, 10 (12), 1587-1593 DOI: 10.1038/nn1996

small silencing RNAs. I: Piwi-interacting RNAs.

Three major classes of small RNAs involved in gene silencing have been found in animals: microRNAs (miRNAs), small-interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). miRNAs are involved in the regulation of mRNA stability and translation, whilst the main purpose of the siRNA and piRNA pathways appears to be the defense of the cell and genome from viruses and transposable elements. Unlike the other two systems that are ubiquitously active, the piRNA pathway is generally only active in germline cells, the most important locus of defense against transposons.

A common feature of all three pathways is the formation of RNA-induced silencing complexes (RISCs), composed of a small RNA bound to an Argonaute family protein. The small RNA guides RISC to specific target RNAs, resulting in target silencing (generally by the Argonaute protein ‘slicing’ the cognate RNA). A key stage in the miRNA and siRNA silencing pathways is the recognition of double stranded RNAs, and their cleavage by Dicer proteins. This is not a feature of the piRNA system. Another difference is that piRNAs range from 22nt to 30nt in length, whilst siRNAs and miRNAs are 21 or 22-24nt long respectively. When piRNAs were first discovered they were called repeat-associated small-interfering RNAs (rasiRNAs). However, as they are not always associated with repeat sequences and as they bind a specific clade of Argonaute proteins, the PIWI family, they were subsequently renamed.

The piRNA system in Drosophila

A Drosophila melanogaster egg chamber. The large nurse cell nuclei are visible in the upper half, whilst the follicle cells cover the oocyte in the lower half.

The piRNA transposon silencing system has been most comprehensively analysed during oogenesis in the fruitfly, Drosophila melanogaster. Within a Drosophila egg chamber, the germline cells (fifteen nurse cells and the oocyte) share a common syncytial cytoplasm. They are surrounded by a layer of somatic follicle cells, which exchange developmental signals and nutrients with the germline cells. The Drosophila genome harbours over a hundred transposon families, including representatives of all three major classes (LTR and non-LTR retrotransposons, and DNA elements). Some retrotransposons, such as the gypsy family, form viral particles that have been shown to be able to invade the germline from the follicle cells via cellular transport vesicles. Therefore the germline is under threat from transposable elements primarily from within, but also from the somatic follicle cells. Two different variants of the piRNA system function in the germline and the somatic follicle cells: a more complicated system involving three PIWI family Argonaute proteins and a piRNA amplification system functions in the germline, whilst a simpler system involving only one PIWI protein works in the follicle cells to silence a more limited repertoire of retrotransposons.

The piRNA pathway in somatic follicle cells

Approximately 70% of somatic piRNAs map to transposons or transposon fragments. Of these 90% are antisense to active transposons. Mapping piRNAs to genomic sequence has yielded a great insight into genomic structure and the piRNA system of transposon control: piRNAs are derived from large clusters of densely packed, inactive transposon copies and fragments. piRNA clusters are a conserved feature of piRNA biology. They generally span dozens to hundreds of kilobases and are located in the heterochromatin associated with centromeres or telomeres. In the case of Drosophila somatic follicle cells two piRNA-clusters dominate: The flamenco locus and cluster 20A. Follicle cell piRNAs from these clusters are derived from one DNA strand, meaning that transcription is unidirectional. In flamenco and cluster 20A, the transposon fragments are generally oriented antisense to the direction of transcription, explaining the strong antisense bias of somatic follicle cell piRNAs. A P-element insertion at the beginning of the flamenco cluster blocks piRNA production from the whole 180kb cluster, suggesting that the formation of long single stranded transcripts of piRNA clusters is a necessary stage of piRNA biogenesis. However, the mechanisms of piRNA generation are not clear. It appears likely that the long piRNA precursor transcripts are stochastically cut into smaller fragments. Piwi then selectively binds fragments with a 5′ uridine (75% of Piwi-bound piRNAs have a 5′ uridine residue), and the pre-piRNAs are then 3′ trimmed to generate the final piRNA.

The germline piRNA pathway and ping-pong amplification.

In addition to Piwi, Drosophila ovarian germline cells express two related PIWI family Argonaute proteins: Aubergine (Aub) and AGO3. Unlike Piwi, which is localised to the nucleus, Aub and AGO3 are associated with an electron-dense peri-nuclear region of cytoplasm called nuage. Most importantly, they act together in a sophisticated piRNA amplification loop that is dependent on target expression, termed the ping-pong cycle. In a simplified version: Aub complexed with an antisense piRNA targets and slices a sense transcript of an active transposon, resulting in the production of novel sense piRNA species which are loaded onto AGO3. The AGO3-piRNA complexes then cleave complementary piRNA cluster transcripts, resulting in the production of novel antisense piRNA to be complexed with Aub. The ping-pong cycle results in the amplification of sets of antisense and sense piRNAs that are 10nt out of register with each other, suggesting the site of Aub slicer activity and providing a useful signal that shows that ping-pong amplification has occurred.

In the germline, more piRNA clusters are active, representing a larger spectrum of transposons. They are also expressed bi-directionally. An outstanding question is why this doesn’t trigger ping-pong amplification? The most likely reason is that the processes of piRNA biogenesis and transposon silencing are tightly localised and regulated. The roles of other proteins in these processes are starting to be understood. Proteins containing Tudor domains appear to be very important in the localisation and function of Aub and AGO3 in the nuage.

Many other intriguing aspects of piRNA biology are yet to be understood. Although the bulk of piRNAs are directed against transposons, some are involved in the regulation of cellular mRNAs. These piRNAs are derived from mRNAs rather than cluster transcripts: Are these transcripts marked in some way to be processed into piRNAs? The links between the primary piRNA biogenesis pathway and the ping-pong amplification system are also poorly understood. An interesting aspect of the piRNA system active in mouse spermatogenesis, is that the nucleus localised mouse PIWI family protein MIWI2 has been implicated in guiding de novo DNA methylation at transposon loci. Is this a more widespread phenomenon?

The piRNA system has been likened to an acquired immune response and works together with the (more acute response) siRNA pathway in transposon silencing. Future posts will discuss the other small RNA systems, and go further into piRNA biology.

Senti, K., & Brennecke, J. (2010). The piRNA pathway: a fly’s perspective on the guardian of the genome Trends in Genetics, 26 (12), 499-509 DOI: 10.1016/j.tig.2010.08.007

Khurana, J., & Theurkauf, W. (2010). piRNAs, transposon silencing, and Drosophila germline development The Journal of Cell Biology, 191 (5), 905-913 DOI: 10.1083/jcb.201006034

of further interest: piRNAs in the brain: epigenetics and memory

The New Mol Biol Carnival is out

The new Carnival of Molecular Biology is out hosted by The Ocelloid. It calls itself number 18, but I think secretly it’s number 19. The post I submitted has been favourably commented on by Mauro Mandioli of the The Aphid Room, and I’d like to take this opportunity to say that his carnival post on karyotype variation in aphids is fascinating. Apparently, chromosome number within a clonal population of the peach potato aphid, can vary between sister individuals, and even within individual embryos. I look forward to finding out more on aphid molecular biology from his blog.

A novel gene transfer agent from Bartonella

A gene transfer agent (GTA) that preferentially packages host adaptability genes has been discovered in the pathogenic bacterial genus Bartonella.

Bartonella are vector borne, facultative intracellular bacteria that infect erythrocytes and endothelial cells in various mammalian species. Berglund et al. sequenced the genome of the rodent infecting species Bartonella grahamii, and compared it with various other sequenced Bartonella genomes. Rodent associated Bartonella species had a larger amount of imported genes than other species. Most of these horizontally transferred genes were either bacteriophage derived, or encoded proteins involved in secretion or transport systems. A large proportion of the imported genes were located in a 461kb segment of the genome that the authors termed the chromosomal high plasticity zone.

The chromosomal high plasticity zone was present in all Bartonella species, but was most expanded in the rodent associated species. It showed similarities to auxiliary replicons (ie large extra-chromosomal elements) from related species.  Genes encoding type IV and type V secretion systems, that mediate bacterial invasion of erythrocytes and endothelial cells, were ancestrally located in the chromosomal high plasticity zone.

Bacteriophage particles were observed in Bartonella cultures. When these phage were isolated it was found that they contained proteins encoded at a cluster of phage genes in the high plasticity zone (phage cluster II), and packaged 14kb strands of DNA. By hybridisation of the phage encapsulated DNA against microarrays covering the bacterial chromosome, it was found that the phage contained DNA from the entire bacterial genome but that DNA from the high plasticity zone was overrepresented. The peak of the hybridisation signal was located at another cluster of phage genes within the high plasticity zone (phage cluster III).

It appears that auxiliary DNA replication from a phage derived origin of replication within the high plasticity zone is amplifying the Bartonella genome. The products of this ‘run-off’ replication are then being packaged into phage-like gene transfer agents. This is the first time a coupling of run-off replication and GTAs has been demonstrated. Previously discovered GTAs have packaged chromosomal DNA randomly, but in this instance the packaged DNA is biased towards DNA from the high plasticity zone, which is enriched for host adaptability genes.

The genes of the Bartonella GTA didn’t show any similarity to the RcGTA-like agents that are found in many other alpha-proteobacteria. Interestingly, Bartonella GTA-like clusters were identified in some other Rhizobiales species that also possessed RcGTA-like clusters. It would be interesting to enquire as to whether the GTAs in these species are also associated with run-off replication.

The authors suggest that the linkage of run-off replication and the GTA is selectively advantageous, in that not only is the rapid spread of new genes facilitated, but so is gene diversification by recombination.

 Berglund, E., Frank, A., Calteau, A., Vinnere Pettersson, O., Granberg, F., Eriksson, A., Näslund, K., Holmberg, M., Lindroos, H., & Andersson, S. (2009). Run-Off Replication of Host-Adaptability Genes Is Associated with Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella grahamii PLoS Genetics, 5 (7) DOI: 10.1371/journal.pgen.1000546

On Transposable Elements and Regulatory Evolution

Transposable elements (TEs), generally considered molecular parasites on the genome, are increasingly being linked to the evolution of new biological functions. TEs have been shown to be a source of novel genes and exons, the ‘arms race’ between them and their hosts has been a driving force in the evolution of epigenetic silencing mechanisms, and they have been shown to serve as cis-acting regulatory elements for host genes. This last role, as regulatory elements has potentially wide ramifications: TE mobilisation could cause changes to the expression of co-regulated suites of genes. Recently, the emergence of novel TEs and their mobilisation has been argued to be a causative factor underlying such ‘punctuated equilibria’ evolutionary phenomena as the Cambrian explosion and the rapid speciation of cichlid fishes. Two new papers analysing mammalian genomic evolution further link transposable elements with the spread of regulatory elements through the genome, and the evolution of novel characters.

CTCF binding sites.

CTCF (CCCTC-binding factor) is a DNA-binding protein with such a diverse and exciting array of potential roles attributed to it that it has been called a ‘master weaver of the genome’. It acts as an insulator, dividing different chromatin domains, and is therefore important for transcriptional activation and repression. This role appears to be linked to the formation of long distance chromosomal loops, and hence to the global organisation of the chromosomes within the nucleus. Schmidt et al. used ChIP-seq to define all the CTCF binding events in liver cells from five eutherian mammals (human, macaque, mouse, rat, and dog) and a marsupial (opossum). Using this data they defined a core DNA sequence motif that CTCF commonly binds, as well as sets of CTCF binding events that are conserved between the various species. In some lineages certain CTCF bound DNA sequence motifs were overrepresented. These overrepresented ‘motif-words’ were often embedded within lineage specific SINE repeats (short interspersed nuclear elements, non-autonomous non-LTR retrotransposons). For instance, mice and rats share about 2000 CTCF binding events that are associated with B2 SINES, mice have a further 5,300 B2 associated binding events and rats a further 1,200. Enrichments of CTCF binding events associated with lineage specific SINEs also occurred in the canine and opossum genomes (on a lesser scale). Surprisingly however, no similar TE associated enrichment occurred in the primate lineage. Looking at CTCF binding events that were conserved between multiple mammals, Schmidt et al. were also able to find over 100 binding events that were associated with fossilised ancestral transposable sequences.

Overall, this data shows that CTCF binding has expanded via retrotransposition in multiple mammalian lineages and that this is an ancient mechanism of regulatory evolution. CTCF binds a long DNA sequence motif (33/34bp) that is less likely to be generated by random point mutations than the smaller motifs more commonly bound by transcription factors. This is one reason why CTCF binding site expansion should be more associated with TEs than other regulatory sequence motifs. Another suggestion that the authors make to explain this association is that CTCF binding may protect TEs from repressive DNA or chromatin modifications.

Transposons and the evolution of pregnancy

During mammalian pregnancy, endometrial stromal cells (ESCs) differentiate in response to progesterone and signalling via the cAMP second messenger pathway, to produce a vascularised placenta that can accommodate implantation (a process termed decidualisation). The enhancer that drives expression of Prolactin in response to progesterone/cAMP signalling in ESCs is derived from a MER20 transposon (a hAT-Charlie family DNA transposon). Lynch et al. have found a strong association between MER20 elements and genes that are differentially expressed in mammalian ESCs and genes that are responsive to progesterone/cAMP signalling.

Analysing MER20s that are located close to stromally regulated genes, they found that, based on their association with CpG islands and various histone modifications, they often had regulatory potential. They then tested whether 21 randomly chosen MER20s bound various transcription factors and insulator proteins. 14 MER20s bound a suite of 5 different insulator proteins (including CTCF), whilst 5 different transcription factors important for ESC development bound together in 4 cases. This suggested that MER20s could be classified into ‘insulator’ and ‘enhancer-repressor’ types. Using a reporter gene assay in various cell types, they then showed that the majority of these MER20s acted as regulatory elements in response to progesterone/cAMP signalling specifically in ESCs.

This data indicates that the rewiring of the gene regulatory network of ESCs during the evolution of pregnancy was partly mediated by MER20 transposition events. In this case, MER20s contain sequences for regulatory assemblies of transcription factors responsive to specific signalling pathways, and hence have acted as cell type specific regulatory elements.

These two papers, as well as an increasing number of other studies, show that TEs are important agents of gene regulatory network evolution. The findings of Lynch et al. especially confirm the perspicacity of the discoverer of transposable elements, Barbara McClintock in terming them ‘controlling elements’.

See also: Retrotransposons as regulatory elements

Lynch, V., Leclerc, R., May, G., & Wagner, G. (2011). Transposon-mediated rewiring of gene regulatory networks contributed to the evolution of pregnancy in mammals Nature Genetics, 43 (11), 1154-1159 DOI: 10.1038/ng.917

Schmidt, D., Schwalie, P., Wilson, M., Ballester, B., Gonçalves, A., Kutter, C., Brown, G., Marshall, A., Flicek, P., & Odom, D. (2012). Waves of Retrotransposon Expansion Remodel Genome Organization and CTCF Binding in Multiple Mammalian Lineages Cell, 148 (1-2), 335-348 DOI: 10.1016/j.cell.2011.11.058

Zeh, D., Zeh, J., & Ishida, Y. (2009). Transposable elements and an epigenetic basis for punctuated equilibria BioEssays, 31 (7), 715-726 DOI: 10.1002/bies.200900026

Phillips, J., & Corces, V. (2009). CTCF: Master Weaver of the Genome Cell, 137 (7), 1194-1211 DOI: 10.1016/j.cell.2009.06.001