Category Archives: Chromatin

The Transposon/piRNA/Chromatin Nexus

Close observation of chromatin states at piRNA-silenced genomic loci demonstrates the power of transposons to change native gene expression.

As reviewed in an earlier post, the Drosophila Piwi/piRNA transposon silencing pathway can be divided into two facets; a complex pathway operating in the germline centred on the Piwi-family argonautes Aubergine and AGO3 localised in peri-nuclear nuage, and a linear pathway operational in the somatic follicle cells. In this linear pathway, piRNAs derived from uni-directional piRNA clusters such as flamenco target Piwi to mediate silencing of a limited subset of retrotransposons. Unlike Aub and AGO3, Piwi is localised to the nucleus, leading to speculation that rather than silencing transposons post-transcriptionally by ‘slicing’ their transcripts, it may act at the transcriptional level. There are many precedents in other organisms for argonautes mediating transcriptional silencing via interactions with chromatin modification and DNA methylation pathways. However, whether one of these silencing modes is employed by Drosophila Piwi was unresolved. A new paper from the lab of Julius Brennecke, generally analysing the linear piRNA pathway active in a cell line derived from the somatic follicle cells surrounding the oocyte (OSC cells) includes important findings for a number of aspects of Piwi-mediated transposon silencing leading to insights on the wider genomic ecology of transposon insertions.

In the first section of the paper, Sienski et al. demonstrate that Maelstrom (Mael), a protein containing putative RNA and DNA binding domains, expressed in both cytoplasm and nuclei and previously implicated in a number of Piwi-pathway effects, acts downstream of Piwi to effect TE silencing. Silencing requires the nuclear localisation of both Piwi and Mael. Further, mutation of the residues necessary for ‘slicer’ activity in Piwi did not de-repress TEs, suggesting a different mechanism for Piwi-mediated silencing.

Sienski et al. go on to marshal three different high-throughput techniques to show that Piwi mediates gene silencing at the transcriptional level. Knocking down (KD) the expression of Piwi pathway factors (piwi, mael) in OSC cells they determined the set of repressed transposable elements (TEs) by comparing RNA levels (RNA-seq). Changes in the steady-state RNA levels were highly correlated with transcription rate as monitored by RNA polymerase II occupancy (ChIP-seq) and levels of nascent RNAs (GRO-seq). Judging by how closely correlated derepression of TEs was to transcription rate, it seems unlikely that the linear piRNA pathway active in follicle cells acts post-transcriptionally at all.

Reasoning that Piwi-mediated transcriptional gene silencing may involve chromatin modification, Sienski et al. profiled the distribution of the repressive histone mark H3K9me3 in OSCs after piwi or mael knockdown. H3K9me3 levels at transposable elements known to be repressed by the piRNA pathway were significantly reduced in the absence of Piwi (and to a lesser extent Mael). This data was from across the genome irrespective of whether the TE was inserted into heterochromatic or euchromatic regions. To negate general effects associated with heterochromatin, the authors looked more closely at TE insertions within euchromatic regions.

Approximate sketch of the patterns of RNA pol II occupancy (ie Transcription), and H3K9me3 at the mdg1 locus after piwi or mael knockdown and normally in control.

Approximate sketch of the patterns of RNA pol II occupancy (ie Transcription), and H3K9me3 at the mdg1 locus after piwi or mael knockdown and normally in control.

At a specific euchromatic insertion of the retrotransposon mdg1, they observed that upon either piwi KD or mael KD, transcription downstream of the insertion strongly increased. However, although this transcriptional bleeding into the surrounding area was similar upon TE derepression due to either piwi KD or mael KD, the pattern of H3K9me3 was very different. Normally this mdg1 insertion displays H3K9me3 in the surrounding 12kb, peaking at the insertion site. This was strongly reduced in piwi KD cells, but in mael KD, H3K9me3 was moderately reduced at the insertion site but had actually spread further downstream (see figure). Similar patterns were observed at nearly all euchromatic mdg1 insertions, as well as other TEs known to be targeted by the linear piRNA pathway active in OSC cells.

Strikingly, most euchromatic H3K9me3 peaks were sensitive to piwi knockdown, whilst 88% of H3K9me3 peaks were found within 5Kb of TE insertions. Piwi-mediated transposon silencing therefore seems to be the main trigger for H3K9 trimethylation in euchromatin.

This transposon silencing mechanism appears to have a major impact on native genes upon TE insertion in their vicinity. An insertion of the retrotransposon gypsy into the first intron of the expanded (ex) gene serves as paradigm for these effects. In OSC cells, the gypsy insertion triggered H3K9me3 spreading into the surrounding 10-12Kb. In control cells RNA polymerase II occupancy was observable at the ex transcription start site (TSS) but weak. Upon piwi or mael knockdown, transcription from the ex TSS was massively increased. As in the earlier mdg1 example, H3K9me3 levels were greatly reduced upon piwi KD but not in mael KD cells. Sienski et al. observed similar effects on the transcription of 28 more genes with nearby TE insertions in OSC cells.

This data has a number of ramifications speaking of a complex interplay between transcription, the establishment and maintenance of repressive chromatin states and the Piwi pathway. Firstly, H3K9me3 considered a transcriptionally repressive histone mark is compatible with transcription. In fact, based on it’s pattern in mael KD cells, the authors propose that downstream transcriptional bleeding leads to the spread of H3K9me3. Further, although H3K9me3 has an integral role in Piwi-mediated silencing, it is not the final silencing mark. H3K9 trimethylation is downstream of Piwi action, but is either upstream or acts in parallel to Mael, which mediates an unknown silencing step crucial to Piwi transcriptional gene silencing.

Importantly, this paper has demonstrated the impact that TE insertion and subsequent piRNA pathway transcriptional repression can have on native gene expression. There are two different modes in which the inactivation of Piwi-mediated TE silencing can lead to the transcriptional activation of these loci. Firstly, the spreading of repressive chromatin marks at transposons can suppress RNA polymerase II access to the genes promoter. Alleviation of TE repression hence leads to (re-)activation of gene expression. Conversely, as TEs (especially the long terminal repeats of some retrotransposons) can serve as promoters, the loss of their repressed chromatin state upon piRNA pathway loss, can activate transcription of downstream regions. Although both these modes lead to transcriptional activation after Piwi pathway loss, they demonstrate that transposon insertion can either activate or repress transcription within relatively extensive genomic surroundings. This underscores the scope for transposons to act as regulatory elements, or to produce new chimerical transcripts and hence potential new genes.

These experiments were mainly performed in one cell type that only partially reflects the activity of what is already a subset of piwi/piRNA action during Drosophila oogenesis.  Piwi and Mael are also active in the nurse cells and oocyte, and this paper suggests that they have similar roles within the context of the expanded piRNA pathways active in the germline. It will be interesting to integrate this nuclear-localised transcriptional-silencing aspect of piRNA silencing into the context of ping-pong amplification and bi-directional piRNA cluster transcripts. Further, do these Piwi-mediated chromatin effects in the germline impact on the transcriptional status of TEs and genes later in somatic development? And if not, do other systems have equivalent activity?

This paper underlines again the importance of the arms race between mobile genetic elements and genomic immune systems such as the piRNA pathway on the wider genomic regulatory context. This contest is being observed to have shaped so many aspects of genome organisation throughout evolution that it sometimes becomes hard to differentiate parasitism from regulation. It is clear however, that to understand the evolutionary impact of mobile elements we must also understand the import of the various epigenetic mechanisms controlling their spread. The minutiae of these mechanisms with regard to their targets, plasticity, adaptability, heritability – often different from organism to organism – has major evolutionary significance. Evolution works differently depending on these mechanisms.

Sienski, G., Dönertas, D., & Brennecke, J. (2012). Transcriptional Silencing of Transposons by Piwi and Maelstrom and Its Impact on Chromatin State and Gene Expression Cell, 151 (5), 964-980 DOI: 10.1016/j.cell.2012.10.040

Breaking Neuronal Symmetry by Chromatin Memories

The asymmetric fates of two bilaterally symmetrical neurons are determined by a two-step activation program at a miRNA locus. Very low levels of transcription ‘prime’ the locus many cell generations before the final fate determination is imposed by a bilateral ‘boost’.

Animal nervous systems are generally bilaterally symmetrical anatomically, whilst displaying many functionally important left-right asymmetries. How is asymmetry imposed on a bilaterally symmetrical ground plan? The nematode C. elegans with its invariant cell lineage and tractable genetics offers a powerful model system in which to tackle this issue. Cochella and Hobert have published an elegant new paper describing how a distinct chromatin state at a microRNA locus serves as a molecular mark encoding a memory of a cell’s ancestry in an asymmetric lineage.  After many cell generations, this mark engenders a different response to terminal differentiation from its’ bilaterally symmetric partner cell.

The bilaterally symmetrical pair of gustatory neurons ASEL(eft) and ASER(ight) express different repertoires of chemoreceptors. This functional asymmetry is underpinned by the differential expression of two transcription factors. DIE-1 is expressed in ASEL and COG-1 in ASER. Together with a microRNA, lsy-6, which represses COG-1 expression, they form a bistable feedback loop responsible for determining the asymmetric fates of ASE neurons. Loss of any of these three factors results in conversion of one ASE to the other. However, the asymmetric expression of die-1 and cog-1 only occurs within the post-mitotic neurons themselves. How then is this asymmetry established?

A schematic representation of the features of asymmetric ASE specification. Note the original asymmetry in the lineages is determined at the 4 cell stage, tbx-37/38 expression in the great-granddaughters of ABa, and lsy-6 in the loop in ASEL.

A schematic representation of the features of asymmetric ASE specification. Note the original asymmetry in the lineages is determined at the 4 cell stage, tbx-37/38 expression in the great-granddaughters of ABa, and lsy-6 in the loop in ASEL.

The two ASEs are derived from different cell lineages that diverge at the 4-cell stage. The two daughters of the 2-cell stage blastomere AB, ABa and ABp, differentiate from each other due to signalling from one of the other 4-cell stage blastomeres to ABp. This signalling event represses the expression in the ABp lineage of a pair of redundant transcription factors, TBX-37 and TBX-38, which are transiently expressed in the 8 great-granddaughter cells derived from ABa. The expression of these TBX proteins is crucial to the asymmetric fate specification of ASE neurons as in tbx-37/38 double mutants ASEL is converted into ASER. However, TBX-37 and 38 are only expressed in the lineage giving rise to ASEL six cell generations before it’s birth. This large gap between the different stages of asymmetric ASE determination lead researchers to postulate the existence of a ‘memory mark’ linking TBX-37/38 action to the expression of the asymmetry defining feedback loop.

During this hiatus between TBX-37/38 expression and terminal ASE determination, the lineages giving rise to the two neurons become symmetric. A number of left/right pairs of neuronal precursors expressing the proneural gene hlh-14 develop from the two lineages, but only the pair of ASE mother cells express the ‘terminal selector’ transcription factor CHE-1. CHE-1 drives the expression of many ASE-expressed genes and activates expression of the asymmetric loop components, lsy-6, die-1, and cog-1. It is at this point that the TBX-37/38-dependent memory mark must integrate into the bilateral activity of CHE-1 generating the asymmetric expression of the loop components.

To try to discover the nature of the memory mark Cochella and Hobert performed a detailed analysis of the expression of the loop components. lsy-6 was suggested to act upstream of die-1 and cog-1 by genetic experiments, and the researchers found that it was the first of the loop components to be expressed.  It is expressed asymmetrically from the start in the ASEL mother cell. Deletion of lsy-6 results in conversion of ASEL to ASER. A construct of lsy-6 in combination with 932 bp of upstream sequence is able to rescue this effect, but sometimes leads to the conversion of ASER to ASEL. This suggested that the ‘upstream element’ construct drove ectopic expression in ASER as well as ASEL. Indeed, the upstream element contains CHE-1 binding motifs causing expression in both the ASE neurons. Cochella and Hobert therefore assayed other lsy-6 surrounding sequence for cis-regulatory information limiting its expression to ASEL. In fact, a construct including the upstream element, lsy-6, and 300 bp of downstream sequence completely rescued lsy-6 null alleles, eliminated ectopic ASER conversion, and was expressed identically to the endogenous miRNA. Normal lsy-6 expression is therefore regulated by both the upstream and downstream elements.

When the downstream element was used alone to drive expression of a reporter gene, it produced a very different pattern. Expression started early in a few ABa-derived blastomeres, one cell division after the expression of tbx-37/38, continuing in the progenitive lineage of ASEL until its’ birth.It never drove expression in ABp derived lineages. Expression from the downstream element was completely lost in tbx-37/38 double mutants, whilst mis-expression of TBX-37/38 in ABp derived cells lead to ectopic expression of the downstream element reporter. The downstream element contains a predicted binding-site for T-box proteins, directly linking the lineage–dependent expression of tbx-37/38 with theasymmetry-defining loop.

The expression pattern driven by the downstream reporter suggested that lsy-6 may be expressed far earlier than previously observed. The researchers therefore used a very sensitive technique to image potential lsy-6 transcripts. This showed that a few lsy-6 RNAs were present in cells in the lineage giving rise to ASEL five generations before strong expression is observed in the ASEL mother cell.

Broadly therefore, lsy-6 expression occurs in two phases; a very low level of activation early, dependent on the downstream element, and a second upstream element-dependent higher level of expression in the ASEL mother cell. However, deletion of the downstream element within large genomic constructs abrogated expression at all stages, and failed to rescue lsy-6 null alleles. This contrasted with earlier observations in which the upstream element alone could drive expression and rescue. The difference between these observations suggested  that, within a normal genomic context, the upstream element can only function in combination with the downstream element.

The authors therefore posited a model in which early downstream element/tbx-37/38– dependent transcription may ‘prime’ the locus in some way, rendering it competent to respond to the later transcriptional ‘boost’ mediated by CHE-1 acting on the upstream element.

Cochella and Hobert tested their model by substituting priming via tbx-37/38/downstream element for priming via ectopic CHE-1. In worms with the downstream element deleted, they drove early expression of CHE-1 from a heat-shock promoter approximately 4 cell generations before its’ normal time of expression. This caused low levels of lsy-6 transcription, rescuing the priming phase and allowing later ASEL expression and determination. Priming is therefore not dependent on a specific transcription factor acting on the downstream element, rather as long as low levels of transcription occur at the locus, it is primed.

This suggested that the memory mark causing the different response of the lsy-6 locus may be a lineage-specific transcription-dependent chromatin state. Using a cunning technique to visualise the level of chromatin compaction on transgenic arrays containing the lsy-6 locus, they observed chromatin decompaction of thelocus in the ASEL progenitive lineage 1 cell division after tbx-37/38 expression. Chromatin decompaction is associated with active genes; in the absence of early transcription the locus becomes compacted and refractory to CHE-1 activation later. In tbx-37/38 double mutants this lineage-specific decompaction was never observed, nor was it seen when the downstream element was deleted.

The memory mark is therefore chromatin decompaction at a miRNA locus linked to very low levels of transcription imposed within a cell lineage at an early stage of development. This primed state relays asymmetric information into an otherwise bilaterally symmetrical developmental program, facilitating diversification of neuronal cell fates. The timing of the priming mechanism fits in with earlier evidence that C. elegans embryos are relatively developmentally plastic until the 64-128 cell stage when developmental genes become compacted and refractory to ectopic activation.

Although I find this paper very elegant and convincing, I do have a few qualms about the most crucial experiment: the early ectopic activation by CHE-1. It seems like a slightly dirty experiment and I think I would’ve preferred to see ectopic induction of lsy-6 transcription via an unrelated mechanism. Perhaps experiments such as these would’ve had their own problems and my doubts are unfounded. I would also have liked to see the compaction assay performed with the ectopic CHE-1 induced activation.

The demonstration of a chromatin-based lineage specific prepattern facilitating differential responses to more generic inputs later in embryogenesis has wide implications, not just for asymmetries in the worm nervous system, but for the way we understand development in many animals. Firstly a technical point; to visualise early lsy-6 transcription the authors had to use a very labour intensive and hi-tech form of in situ hybridisation. The transcription they found, of just a few individual RNA molecules per cell, had massive developmental significance. Generally the techniques used to judge expression in developmental studies is nowhere near as sensitive, implying that we may be missing a lot of important information. Secondly, a more general point; A cell or tissues’ ‘competence’ to respond to developmental signalling, a concept derived from experimental embryology, and perhaps disdained in more genetical perspectives is relevant here. Molecular memories encoded by chromatin states may be a very widespread mode for imposing pre-pattern or developmental competence during embryogenesis. It seems to me that these types of understandings can begin to blend together the two different meanings of epigenetics; namely the derivation of the word by Waddington from epigenesis (meaning the increase in complexity during development), with the more current usage of epigenetics as describing a diverse collection of non-genetic inherited information.

Cochella, L., & Hobert, O. (2012). Embryonic Priming of a miRNA Locus Predetermines Postmitotic Neuronal Left/Right Asymmetry in C. elegans Cell, 151 (6), 1229-1242 DOI: 10.1016/j.cell.2012.10.049

The Heterodox Dinokaryon

The nuclei of dinoflagellates display a highly derived organisation; chromosomes are permanently condensed and seem to lack histone proteins. A new study in Current Biology links the emergence of these characters to the importation of a novel family of nuclear proteins originating in giant viruses.

A Haeckel print of various Dinoflagellates

Dinoflagellates are a diverse and successful phylum of protists.  Many are photosynthetic with a major role in the oceans’ primary production, whilst others have symbiotic, parasitic or predatory lifestyles. Their nuclei are highly unusual. Whereas in all other eukaryotes chromosomes only condense during mitosis, dinoflagellate chromosomes display a permanently condensed, liquid crystalline form. This ‘cholesteric’ structure produces a banded appearance in electron micrographs. Another key dinoflagellate heterodoxy is the absence (or at least undetectability) of histone proteins and the nucleosomal organisation of chromatin. These differences are so radical that dinoflagellates were suggested to represent an intermediate ‘mesokaryotic’ stage between prokarya and eukarya. Molecular phylogenetics has since clarified that they are in fact a sister clade to apicomplexan protists, leaving no doubt that that the dinoflagellate nuclear organisation – the dinokaryon – is derived from standard eukaryotic ancestors. Other atypical features of the dinokaryon include very high DNA content and the replacement of as much as 70% of the base thymine with the rare base 5-hydoxymethyluracil.  However, there is some variability in the occurrence of these features. For instance the chromosome banding patterns are not always evident and some dinoflagellate species’ chromosomes can be decondensed at certain stages of their lifecycles.

A dinoflagellate nucleus. Note the condensed chromosomes with characteristic banding pattern (not Blastodinium sp.).

To investigate the emergence of these dinokaryotic characteristics during the early evolution of the dinoflagellates, Gornik et al. investigated the nuclei of two early-branching members of the lineage.  Perkinsus marinus represents the closest known lineage not included within the dinoflagellates proper, whilst Hematodinium sp. branches basally within the clade. In line with their expectations the genome of P. marinus is organised into nucleosomal units, whilst that of Hematodinium sp. is not and appears to be 80 times larger. The P. marinus genome contains sequences for the 4 core histones as well as the linker histone H1, all of which were prominently detectable as protein in extracts from nuclei. Genome sequence is not available for Hematodinium sp., however transcriptomic sequencing revealed the presence of the four core histones as well as a number of variants. Unlike the histone genes of P. marinus the sequences were quite divergent from the highly conserved eukaryotic norm, however the core ‘histone-fold’ regions were relatively well preserved, as were key residues that serve as sites for post-translational modification.  Histone genes have been found in other dinoflagellate genomes recently, but histone protein expression had not previously been detected. Gornik et al could identify histone H2A protein in nuclear extracts from Hematotinium sp. However, whereas in P. marinus and other eukaryotes, histone proteins are the dominant species in such extracts, in Hematodinium sp a single 30kDa species dominated.

When this band was extracted and the protein identified by mass spectrometry, it was found to correspond to a novel family of proteins, at least 4 of which were expressed in Hematodinium sp., whilst 13 were found in the transcriptome. This family of proteins only appears to be present in dinoflagellates; no homologues were found in other eukaryotic groups or in prokaryotes. However database searching did reveal homology with a protein of unknown function widely found encoded in the genomes of phycodnaviruses, a family of giant viruses infecting algae. Gornik et al. therefore named these proteins Dinoflagellate/Viral NucleoProteins (DVNPs).

Like histones and many other DNA-binding proteins, DVNPs are highly basic proteins. They are relatively variable in their N-terminal regions, with higher conservation in a core region, which may potentially include a DNA-binding helix-turn-helix motif. Biochemical experiments demonstrated that DVNPs have a high affinity for DNA and are post-translationally modified at various residues by phosphorylation.

The phycodnaviridae are members of the nucleocytoplasmic large DNA viruses (NCLDVs), a monophyletic clade of giant viruses that encode much more of their replication apparatus than is typical of viruses. They are predicted to have emerged more than 2 billion years ago, predating the first dinoflagellates by more than a billion years. As most phycodnaviruses include DVNP orthologues dinoflagellates must have acquired DVNPs from the phycodnaviruses early in their evolution. As yet there is no information on the roles of DVNPs in the phycodnaviridae, but the fact that both taxa have expanded genomes suggests a possible similar function. Do DVNPs allow such efficient DNA packing that the costs of genome expansion are somehow minimised?

The DVNPs are not the first family of putative histone-replacement proteins discovered in dinoflagellates. Later-branching taxa express ‘histone-like proteins’ (HLPs), probably related to the bacterial DNA-binding protein HU, and shown to be able to bend DNA in vitro. HLPs are not found in Hematodinium sp. or other early-branching dinoflagellates, whereas DVNPs are found in combination with HLPs in later-branching taxa. DVNPs therefore seem to be associated with the core dinokaryotic characteristics of permanently condensed chromosomes and expanded genome size, whilst the presence of HLPs correlates with other characters such as the chromosome banding patterns observed in later-branching taxa.

The observation that dinoflagellates do in fact encode and express divergent histones at low levels raises the question of what their roles could be if they are not primarily responsible for the bulk packing of DNA? Linked to this is the broad question of how DVNPs and HLPs act to condense dinoflagellate chromosomes. Considering the vast quantity of research attempting to understand the biology of eukaryotic chromosomes, it is rather daunting to find a whole new way of doing things; how do transcription and replication mechanisms work in the context of permanently condensed chromosomes? How does this link in with genome expansion? I don’t know how much dinoflagellate genomic data is available, but I imagine that a finished genome sequence would be of great use. Perhaps though, I’d prefer instead to prioritise biochemical and structural studies of these various proteins actions on DNA.

Gornik, S., Ford, K., Mulhern, T., Bacic, A., McFadden, G., & Waller, R. (2012). Loss of Nucleosomal DNA Condensation Coincides with Appearance of a Novel Nuclear Protein in Dinoflagellates Current Biology DOI: 10.1016/j.cub.2012.10.036

Opening up the RNA-chromatin network

In eukaryotic nuclei, DNA is coiled around histone proteins to form nucleosomes. The pattern by which nucleosomes are compacted into higher-order structures determines the accessibility of chromatin and hence it’s transcriptional activity. Many different factors, including the linker histone H1, histone modifications, chromatin remodelling enzymes and non-histone proteins play important roles in structuring chromatin. Various classes of RNA have also been implicated in regulating the higher-order structure of chromatin. Among many examples; Argonaute associated small silencing RNAs are known to sometimes exert their inhibitory effects by directing histone modifications or DNA methylation and lncRNAs have been shown to serve as cis-acting scaffolds coordinating the action of histone-modifying enzymes. It’s been known for decades that RNA makes up a proportion of chromatin, but exactly what types of RNAs and what their roles are is not yet clear. A new paper in Molecular Cell (Schubert et al.) sets out to answer these questions, characterising chromatin-associated RNAs in Drosophila and finding an important role for RNA in regulating chromatin compaction and accessibility.

Schubert et al. found that RNAs were involved in maintaining the accessibility of chromatin using an assay in which chromatin is digested by a nuclease (DNase). This digestion creates a ladder of DNA of different sizes on a gel, ranging from single nucleosomal fragments to far larger pieces. The extent of digestion is dependent on the level of chromatin condensation; the more compacted the nucleosomal structure, the more refractory it will be to DNase digestion. The researchers found that incubation of chromatin with an RNase prior to DNase treatment resulted in more compacted chromatin; DNase digestion was less efficient. Using different RNases and inhibitors they discovered that the RNA population involved was single-stranded and synthesised by RNA polymerase II.

Similar results were found when the authors used density-gradient centrifugation to isolate chromatin. They found a fraction of RNA associated with chromatin (caRNA) that when digested, resulted in chromatin becoming more compacted and shifting to higher density fractions. Interestingly, this RNA-dependent chromatin condensation effect is reversible; when the compacted chromatin was extracted and incubated with fresh cellular extracts, it reopens, again migrating in lighter fractions, re-associated with RNA. This caRNA-dependent chromatin accessibility is also dependent on chromatin-associated proteins, as it could not be rescued under denaturing conditions.

Using mass spectrometry, the authors identified 59 proteins that had lower affinity for chromatin after removal of RNA. One of these was the highly abundant chromatin decondensation factor 31 (Df31). A study that I have long intended to write about (Filion et al. 2010) used the binding of Df31 and 52 other chromatin associated proteins to determine that rather than dividing Drosophila chromatin into two types: transcriptionally repressed heterochromatin and active euchromatin, we should instead think in terms of five different classes which they colour coded. Green, blue and black chromatin are broadly transcriptionally repressed, whilst red and yellow are euchromatic and more transcriptionally active. Df31 is found bound to these red and yellow types of more open chromatin. Schubert et al found that Df31 chromatin binding is stabilised by caRNAs. Df31 binds histone H3 in the absence of RNA, but its affinity is substantially enhanced by the addition of RNA. RNAi knockdown of Df31 causes a fraction of genomic DNA to be more compacted.

Deep sequencing of the pool of caRNAs revealed that they were enriched for non-coding RNAs, especially a class termed small nucleolar RNAs (snoRNAs). snoRNAs are known to guide the modification of bases in ribosomal, transfer and messenger RNAs. They have also been implicated in RNA editing and splicing. Schubert et al. found that 30 of the 186 snoRNAs expressed in Drosophila embryos were found associated with chromatin. Using fluorescent in situ hybridisation, they showed that two of the most highly enriched snoRNAs could be visualised binding to the interbands of Drosophila polytene chromosomes (ie. euchromatin). These two snoRNAs were able to ‘rescue’ compacted chromatin in the density-gradient experiments, and were shown to directly interact with Df31.

The authors also found that extracts of human cells could rescue RNase treated compacted chromatin, and that snoRNAs are found associated with chromatin in human cells.

Schubert et al have therefore characterised a novel conserved role for snoRNAs mediating the accessibility of higher-order chromatin structures. As none of the protein components known to complex with snoRNAs in snoRNPs were identified in the mass spectrometry experiments, it appears that snoRNAs form distinct ribonucleoprotein complexes to mediate this chromatin associated role. Df31 is one such important interactor, linking chromatin-associated snoRNAs and histone proteins within nucleosomes to maintain accessibility of red and yellow chromatin. However, the knockdown of Df31 resulted in far more limited and localised chromatin compaction than RNase treatment, suggesting that the role of caRNAs goes beyond the maintenance of open euchromatin, but also to regulating heterochromatic accessibility. Seeing as 58 other chromatin proteins, as well as many other snoRNAs and other caRNAs, were implicated in RNA-associated chromatin accessibility functions, this initial study has just revealed the tip of an iceberg. Just how this RNA-chromatin network functions to maintain accessibility requires a lot of work. As with many of the best studies, the light shone serves to partially illuminate the scale of our ignorance.

Schubert T, Pusch MC, Diermeier S, Benes V, Kremmer E, Imhof A, & Längst G (2012). Df31 Protein and snoRNAs Maintain Accessible Higher-Order Structures of Chromatin. Molecular cell PMID: 23022379

Filion GJ, van Bemmel JG, Braunschweig U, Talhout W, Kind J, Ward LD, Brugman W, de Castro IJ, Kerkhoven RM, Bussemaker HJ, & van Steensel B (2010). Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell, 143 (2), 212-24 PMID: 20888037

The CSR-1 siRNA pathway gets more enigmatic

A recent paper forces a reappraisal of the role of CSR-1 its associated 22G-RNAs, and demonstrates a positive regulatory role for this RNAi pathway in C. elegans.

As described in a previous post, depletion of the Argonaute protein CSR-1, or the proteins responsible for the biogenesis of the endo-siRNAs with which its complexes (the RdRP EGO-1, and the helicase DRH-3), results in defective mitotic chromosome segregation and sterility. To explain these findings Claycomb et al. proposed that the CSR-1 22G-RNA pathway acted to organise the proper compaction of the holocentric chromosomes of C. elegans, and the assembly of the kinetochores necessary for their proper segregation. (I strongly recommend reading the earlier post describing this paper’s findings).

Claycomb et al. had found that expression of most genes targeted by CSR-1 associated 22G-RNAs was not significantly altered in csr-1 mutants. Avgousti et al. went back over the same data and found that, although this was true in the main, expression of most of the genes encoding histone proteins was downregulated in csr-1 mutants. It had previously been shown that downregulation of just one histone gene could cause chromosome segregation and sterility phenotypes in worms. This lead Avgousti et al. to hypothesise that the defects seen in csr-1, ego-1 and drh-3 mutants may be caused by defective histone production, rather than the model proposed by Claycomb et al.

Histone proteins make up the core of the nucleosome and are multiply encoded in all eukaryotic genomes. Histone mRNAs are processed in a special way; generally their 3’UTRs are not polyadenylated; instead, downstream of a conserved stem-loop structure, a histone specific sequence (HDE) is recognised and cleaved by the U7 snRNA (an important splicing factor). Both HDE sequences and the U7 snRNA are not present in C. elegans. Avgousti et al therefore tested whether this key histone mRNA processing stage was instead being mediated by CSR-1 and its associated endo-siRNAs in worms.

Using a synthetic oligonucleotide identical to the region of the 3’UTR downstream of the stem-loop from the histone 2A pre-mRNA, they demonstrated that CSR-1 directly binds histone mRNAs. This binding was abrogated upon RNAi depletion of the RdRP EGO-1, showing that CSR-1 binding was dependent on the 22G-RNAs generated by EGO-1. Avgousti et al. also demonstrated that upon knockdown of CSR-1 or EGO-1, or in drh-3 mutants, unprocessed histone pre-mRNAs accumulate, whilst processed histone mRNAs and proteins are depleted.

The strongest evidence supporting the hypothesis that defective histone mRNA processing causes the defects seen in csr-1 mutants was a series of transgenic rescue experiments. Histone overexpression from transgenes, designed to not require 3’UTR mRNA cleavage, was able to counteract the effects of csr-1 or ego-1 RNAi knockdown, whereas transgenes that did required 3’UTR processing could not.

It seems likely therefore that in C. elegans the 3’UTR cleavage of histone pre-mRNAs is performed by CSR-1/22G-RNA complexes. CSR-1 has been shown to possess endonuclease ‘slicer’ activity, but although a likely candidate, it is too early to say whether it directly performs the cleavage or recruits other factors to perform the reaction. I think this paper blows a large hole in the model proposed by Claycomb et al. to explain the role of CSR-1 22G-RNAs; suggesting that the observed chromosome segregation defects are indirectly caused by a failure to produce adequate histones, rather than a failure to direct the organisation of mitotic chromosomes. However, the hypothesis certainly requires further and more subtle experiments. The paper also further muddies the waters on the question of just what the CSR-1 22G-RNA system is doing in most cases. The recognition of histone mRNA 3’UTRs can only account for a very small proportion of this endo-siRNA population. As discussed in other posts the CSR-1 22G-RNA system is the prime candidate to be an epigenetic licensing anti-silencing pathway. Do the two different CSR-1 isoforms perform two different functions; one licensing transcription and the other replacing the U7 snRNA splicing apparatus? Is this pre-mRNA splicing role confined to histone mRNAs? An important first of this paper is the demonstration of a positive role in regulating gene expression for an RNAi system. Generally, the various RNAi pathways negatively regulate gene expression; either resulting in slicing and degradation of transcripts, directing silencing chromatin modifications etc. In this case mRNA processing by the CSR-1 endo-siRNA system leads to proper expression of histones at key periods of rapid cell division (eg. early embryogenesis). Personally, I’m looking forward to more contentious interpretations of this pathway from the research groups involved!

Avgousti DC, Palani S, Sherman Y, & Grishok A (2012). CSR-1 RNAi pathway positively regulates histone expression in C. elegans. The EMBO journal PMID: 22863779

Claycomb JM, Batista PJ, Pang KM, Gu W, Vasale JJ, van Wolfswinkel JC, Chaves DA, Shirayama M, Mitani S, Ketting RF, Conte D Jr, & Mello CC (2009). The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation. Cell, 139 (1), 123-34 PMID: 19804758

Interacting small RNA pathways in worms 5: Global Genome Surveillance

As discussed previously, in C. elegans more than 15,000 21U-RNAs are expressed from two large clusters on chromosome IV. As very few of these piRNAs exhibit perfect sequence complementarity with other endogenous sequences within in the C. elegans genome, it’s been difficult to deduce the targets and functions of this system. New studies from the labs of Eric Miska and Craig Mello have significantly advanced our understandings of piRNAs in C. elegans. Bagijn et al. and Lee et al. have confirmed a previous supposition that 21U-RNAs can base-pair with imperfectly complementary sequences. This less stringent base-pairing opens up the entire transcriptome to possible piRNA-mediated regulation. Bagijn et al, Ashe et al, and Shirayama et al. have further characterised the effector pathways downstream of PRG-1/piRNA targeting. Together these papers outline a finely tuned genomic surveillance mechanism capable of discerning self and non-self transcripts.

 Targets

Previous studies had shown that 21U-RNAs act upstream of 22G-RNAs to repress the activity of Tc3 transposons. Lee et al. therefore investigated whether the expression of 22G-RNAs was altered in worms mutant for the worm Piwi protein PRG-1. When all 22G-RNAs were considered together, they couldn’t observe a correlation between PRG-1 activity and 22G-RNAs. However, 22G-RNAs can be divided into four different pathways defined by the different AGOs with which they complex. When those associated with 2 different ‘Eri’ pathways, and the CSR-1 22G-RNAs were discounted, Lee et al. observed that WAGO-associated 22G-RNAs tended to be depleted in prg-1 mutants. mRNAs targeted by WAGO 22G-RNAs showed a tendency towards upregulation in prg-1 mutants, consistent with a repressive role for these small RNAs.

22G-RNAs are not evenly distributed on their target mRNAs. Instead, hotspots occur where 22G-RNA species are far more common than at other points on the same mRNA. Lee et al. postulated that these hotspots could be regions where PRG-1/21U-RNA complexes recruit RNA-dependent RNA polymerases to generate 22G-RNAs. If this is as widespread a phenomenon as suggested by the depletion of 22G-RNAs in prg-1 mutants, imperfect base-pairing of 21U-RNAs to their targets must be occurring, as only 29 WAGO targets show perfect complementarity to 21U-RNAs. Lee et al. therefore asked whether 22G-RNAs are enriched in mRNA coding regions with the potential for energetically favourable but imperfect base pairing to 21U-RNAs. Basing their parameters on the imperfect base-pairing observed for miRNA target interactions (in which strong base-pairing in a ‘seed’ region of nucleotides 2-8, facilitates less perfect pairing between the rest of the sequences), Lee et al. determined the level of 22G-RNAs within a 100nt window around potential 21U-RNA binding sites in wild-type and prg-1 mutant worms.  Allowing 2 mismatches and 1 G:U wobble pair outside the seed region, and at most 1 G:U pair within the seed (parameters that meant that more than 50% of genes contained potential 21U-RNA binding sites), the researchers found that 22G-RNAs mapping to within ± 50nt of the 21U-RNA binding sites on WAGO mRNA targets, were 3 fold enriched in wildtype worms relative to the prg-1 mutants. A smaller enrichment of 1.4 fold was also observed for CSR-1 mRNA targets. The level of 22G-RNA enrichment correlated with the expression levels of 21U-RNAs. Lee et al. confirmed the importance of pairing within the seed region by comparing this data with another set of potential 21U-RNA targets with similar total mismatches but poor seed pairing, which showed little 22G-RNA enrichment.

Bagijn et al performed slightly different analyses, which came to similar conclusions. They identified 681,746 potential binding sites for 16,003 21U-RNAs if 3 mismatches are allowed (not biased away from seed region). They then looked to see what proportions of these sites 22G-RNAs also mapped to, finding 1.6, 1.4, 1.5 and 1.2 fold 22G-RNA enrichments relative to matched control sequences, at 0, 1, 2 and 3 mismatch sites respectively. As with Lee et al. the levels of 22G-RNAs correlated with 21U-RNA abundance.

These analyses suggest that 21U-RNAs are targeting a large proportion of the germline transcriptome, including both protein-coding genes and transposons. In relation to the repression of Tc3 (discussed in the previous post), Bagijn et al. identified three piRNAs with imperfect complementarity to sequences in the TIRs, perhaps clearing up some of the ambiguities from previous studies. By using their data on 22G-RNA enrichments at potential 21U-RNA binding sites, they were able to rank the likelihood of piRNA regulation for transcripts. Six of eleven candidates that showed strong reductions in 22G-RNAs in prg-1 mutants also showed statistically significant transcriptional upregulation (including both transposons and protein-coding genes).

piRNA sensors

Most of the papers under discussion used transgenic ‘sensor’ lines to dissect the mechanisms of PRG-1/21U-RNA mediated silencing. Bagijn et al generated a ‘piRNA sensor’ in which a GFP – histone H2B fusion gene with a sequence complementary to a known 21U-RNA is inserted into the C. elegans genome. In the control sensor line (in which the reverse complement of the 21U-RNA is included instead), the GFP reporter protein is expressed in germline nuclei. In the piRNA sensor line the transgene is silenced. This silencing is dependent on PRG-1, as the sensor was desilenced in prg-1 mutants. Bagijn et al detected 22G-RNAs mapping to the piRNA sensor mRNA close to the piRNA target site, that weren’t present in the control sensor or in prg-1 mutants. As levels of both the piRNA sensor pre-mRNA and mRNA were raised in prg-1 mutants, it appears that silencing occurs at the level of transcription, and possibly post-transcriptionally as well.

By crossing the piRNA sensor line into various mutant worm strains, Bagijn et al showed that the silencing involved many of the 22G-RNA pathway components discussed previously, including the helicase DRH-3, the RdRPs EGO-1 and RRF-1, and various WAGOs. As has been discussed previously, many of the WAGO proteins have overlapping functions and hence display partial redundancy when impaired. However, wago-9 (also known as hrde-1) appears to be an especially important AGO in the silencing process; the sole single WAGO mutant to cause desilencing phenotypes. All these 22G-RNA components act downstream of PRG-1, as 21U-RNAs are still present in these mutants while 22G-RNAs fail to accumulate. In prg-1 mutants both classes of small RNAs are affected.

Although in other animals Piwi proteins are known to act by slicing it’s targets, transgenes encoding PRG-1 with a mutated endonuclease motif could rescue the desilencing observed in prg-1 mutants. By mutating the piRNA target sequence in the sensor, Bagijn et al. then showed that 2 mismatches are tolerated for piRNA mediated silencing to occur. The residues changed could be anywhere in the sequence including at positions 10 and 11 – the normal site for Piwi slicing activity.

Lee et al. undertook very similar experiments to Bagijn et al, generating a piRNA sensor line and confirming that mismatches with the piRNA target site were tolerated, including at the normal slicing site. However there is a crucial difference between the two sets of experiments using piRNA sensors. In Bagijn et al’s study, PRG-1 was required continuously for silencing to be maintained through the generations. That is to say that an already silenced sensor would be desilenced when crossed into a prg-1 mutant line. Lee et al’s piRNA sensor only required PRG-1 for silencing to be initiated, but not for it to be maintained. When Lee et al’s silenced sensor was outcrossed into prg-1 mutants, GFP expression was not activated, whereas if the transgene was introduced into prg-1 mutants it was not silenced. This important disparity may well be caused by differences in the (broadly similar) compositions of the sensor transgenes, and will be discussed later.

Long-term heritability.

The PRG-1-dependent silencing of the piRNA sensors is incredibly long lived, being transmissable through many generations. This type of epigenetically heritable effect has been previously observed with various RNAi paradigms in C. elegans, with disagreement over whether epigenetic transmission is primarily through inheritance of small RNAs, or via chromatin modifications, or both (see these earlier posts: 1, 2 ). Ashe et al. generated another transgenic sensor line to monitor the heritability of dsRNA-induced RNAi. They found that both the piRNA-mediated silencing pathway and the heritable RNAi silencing pathway converge on a common group of nuclear factors responsible for 22G-RNA mediated silencing. These include two proteins of unknown function, NRDE-1 and NRDE-2, known to mediate nuclear RNAi via interactions with nascent transcripts; the heterochromatin protein 1 orthologue, HPL-2; the nuclear AGO WAGO-9; and a SET-domain protein, SET-25, thought to be a methyltransferase responsible for histone H3 lysine-9 trimethylation (a repressive chromatin modification). This collection of downstream effectors suggests that the heritability of both RNAi and piRNA mediated silencing rests on epigenetically stable chromatin marks.

However, if a silenced piRNA sensor strain is crossed with a strain expressing a independent GFP transgene, a dominant silencing of both transgenes occurs. This trans­-acting silencing effect is most likely mediated by secondary siRNAs (ie 22G-RNAs). When small RNA populations are assayed after the induction of RNAi silencing, a large diversity of small RNAs with little 5’ bias (ie Dicer products) were found to target the sensor. However, by the 4th generation, targeting small RNAs had clarified themselves into antisense 22G-RNAs. These 22G-RNAs appeared to be generated de novo in each generation. It therefore appears likely that epigenetic silencing is achieved by the inheritance of both small RNAs and chromatin marks, is reaffirmed in each generation, and acts at both transcriptional and post-transcriptional levels.

RNAe 

In the previously discussed papers, transgenes that were engineered to contain 21U-RNA binding sites were actively silenced in C. elegans. Shirayama et al. describe how transgenes containing non-endogenous genes can be silenced even in the absence of perfect piRNA recognition sequences. They found that when transgene fusions of gfp  and endogenous genes were inserted into the genome, they were occasionally completely silenced, whilst sometimes exactly the same construct, inserted into the same genomic site, was expressed. When a silent line was crossed to an expressing line, the transgene was invariably silenced in 100% of progeny. This dominant trans-acting silencing was heritable through many generations.  Shirayama et al termed this phenomenon RNA-induced epigenetic silencing (RNAe)(rather forestalling there own explanation of it’s causes).

By analysing levels of pre-mRNAs and mRNAs in silenced and active lines, Shirayama et al. found that silencing occurs at both the transcriptional and post-transcriptional levels. Silenced alleles were activated when crossed into lines mutant for various factors involved in repressive chromatin formation (the polycomb group proteins MES-3 + 4, and heterochromatin protein HPL-2). Whilst ChIP experiments demonstrated that silenced transgenes were enriched for histone H3K9 trimethylation. Hence RNAe involves transcriptional silencing at the level of chromatin formation.

The trans- acting nature of RNAe suggested a small RNA component. Crossing silenced alleles into lines mutant for factors known to function in the WAGO 22G-RNA pathway (rde-3,+mut-7) resulted in desilencing. Similarly transgenes were desilenced in worms mutant for the nuclear AGO encoding wago-9; effects which could be enhanced by mutations in additional wago genes. Sequencing of small RNAs from silenced worm gonads revealed a strong accumulation of 22G-RNAs targeted against gfp. The gfp genes were always combined with endogenous genes in the transgene constructs. Interestingly, 22G-RNAs were not produced against this part of the transgene, suggesting a mechanism protecting against the silencing of endogenous genes.

When a silenced transgene is crossed into prg-1 mutants it does not become reactivated, however when Shirayama et al performed transgenesis directly into prg-1 mutants silencing failed to occur. This demonstrates that PRG-1 is required for the initiation of RNAe and not for it’s maintenance (in agreement with the findings of Ashe et al and Lee et al). Although Shirayama et al did not find the exact piRNA recognition sites triggering this silencing, they did apparently identify a number of candidate piRNAs, the recognition sites of which displayed heightened expression of 22G-RNAs. It seems therefore that the recognition of foreign nucleic acids and their suppression via RNAe is the primary function of the PRG-1/21U-RNA system.

Discussion 

All of these studies are in general agreement about the basic dynamics of piRNA silencing in C. elegans. piRNA target recognition is mismatch tolerant. PRG-1 does not act by endonuclease activity. Instead, upon recognition it recruits a RdRP complex to generate 22G-RNAs against sequence adjacent to the 21U-RNA binding site. These 22G-RNAs complex with WAGOs (especially WAGO-1 and WAGO-9) that effect genetic silencing at both the level of repressive chromatin and post-transcriptionally. PRG-1/21U-RNA recognition triggers a self-sustaining WAGO-22G-RNA dependent silencing.

However, there is still a question about the targets of this system. With a few mismatches tolerated the known repertoire of 21U-RNAs can target the whole genome. Obviously, it’s not all repressed, so just what is happening? The computational analyses of Bagijn et al suggested that protein-coding mRNA sequences showed signs of bias against potential piRNA recognition sequences, whilst transposon and pseudogene sequences did not.  They also found evidence to suggest that transposon insertions between the conserved 21U-RNA promoter motif and the 21U-RNA gene itself generate new transposon-targeting piRNAs. These findings lead to a model of C. elegans piRNA biology with similarities to that observed in Drosophila; piRNA clusters rich in transposon sequence, generating piRNAs primarily used to repress transposon mobilisation in the germline, with endogenous genes being selected to avoid piRNA mediated repression. Lee et al. did find similar trends but were not convinced that the numbers were strong enough, instead emphasising that many of the findings in these experiments suggest the existence of an anti-silencing pathway.

 Anti-Silencing

When Shirayama et al. used dsRNA to silence gfp containing transgenes that hadn’t been silenced by RNAe, they were surprised to find the silencing induced was inherited very stably, whereas RNAi against gfp transgenic lines that had been produced by earlier methods never silenced as heritably. They then crossed these earlier transgenic lines to silenced gfp lines to test whether they were susceptible to trans-silencing. Instead they found that these lines would dominantly activate gfp expression. This suggests the presence of a dominant trans-acting mechanism competing against the trans-acting silencing mechanism.

Another line of evidence suggesting the existence of an anti-silencing pathway is the difference in the requirement for prg-1 for silencing between the piRNA sensors of Bagijn et al and Lee et al.  In the Bagijn et al sensor the 21U-RNA target site was flanked by endogenous sequences that are known targets of CSR-1 22G-RNAs. In contrast, in Lee et al’s sensor the piRNA recognition site was surrounded by sequences not targeted by this small RNA population.

A likely explanation for why the silencing of the Bagijn et al sensor required prg-1 in each generation, was that a CSR-1 based anti-silencing pathway counteracts the silencing induced by PRG-1 triggered WAGO mediated silencing. Likewise, CSR-1 22G-RNAs could be the trans-acting anti-silencing agents suggested in Shirayama et al’s experiments. Shirayama et al’s fusion transgenes were more or less susceptible to silencing depending on the endogenous gene fused to gfp, and as noted earlier, in general silencing associated 22G-RNAs were only enriched for the gfp genes and not against the endogenous sequences in the transgenes.

It therefore seems that an anti-silencing pathway exists that licenses ‘self’ transcripts, protecting them from WAGO 22G-RNA silencing. The CSR-1 pathway is the perfect candidate for this activity, as its’ associated 22G-RNAs are known to target thousands of germline expressed transcripts without inducing silencing. Further dissection of these pathways is obviously necessary to prove CSR-1’s role, and whether the anti-silencing pathway also acts downstream of PRG-1, or just antagonistically to it.

The Miska lab agrees about the existence of the licensing pathway, however they suggest that some of the results indicate the existence of another mechanism. Ashe et al. reported that their piRNA sensor could be stably silenced in a prg-1 independent manner, but only if it had been present in a heterozygous state for multiple generations. This is evidence of a mechanism for detecting unpaired chromatin during meiosis. Shirayama et al’s findings too are indicative of a mechanism for detecting unpaired alleles. This could have significant implications evolutionarily, as it may facilitate the phenotypic expression of recessive traits.

One can describe this self and non-self recognition system as a type of acquired genetic immune system. Although, as yet, the details are only understood in outline, it appears to be a exquisitely finely tuned, and effective system. Until recently no viruses were known to infect C. elegans. This system must be part of the reason why. There are still major questions about to what extent transposons are controlled by the piRNA system in C. elegans and how much of its’ functionality is devoted to the regulation of endogenous genes. As is evident from this series of posts, C. elegans has so many different AGOs, and so many different facets to it’s RNAi systems (germline + soma RNAi, nuclear + cytoplasmic RNAi, RNAe etc) that it can be very difficult to dissect them from each other and then be able to see them in the round. Perhaps the most interesting aspect of this work is the light shed on piRNA and RNAi systems in vertebrates and Drosophila. Secondary siRNA systems have not been found in these organisms, so this system of genetic immunity does not work in the same way in these other clades. The 21U-RNA triggering 22G-RNA generation mechanism has been likened to the amplification of secondary piRNAs by the ping-pong amplification system in Drosophila (a system missing in C. elegans). However, the most fascinating aspect of this work could be the light shed on vertebrate piRNA systems. Hundreds of thousands of ‘pachytene’ piRNAs, without known targets, are expressed in the mammalian germline. Do these piRNAs also tolerate mismatches when binding? And do they also mediate a genome surveillance system capable of detecting non-self transcripts? or unmatched chromatin? Are mammalian transcripts marked in some way to avoid this silencing? No doubt these C. elegans studies will invigorate research in mammalian piRNAs, and no doubt I’ll revisit it soon.

See also: Epigenetic Licensing of a Sex Determination Gene
The CSR-1 siRNA pathway gets more enigmatic

Bagijn MP, Goldstein LD, Sapetschnig A, Weick EM, Bouasker S, Lehrbach NJ, Simard MJ, & Miska EA (2012). Function, targets, and evolution of Caenorhabditis elegans piRNAs. Science (New York, N.Y.), 337 (6094), 574-8 PMID: 22700655

Lee HC, Gu W, Shirayama M, Youngman E, Conte D Jr, & Mello CC (2012). C. elegans piRNAs Mediate the Genome-wide Surveillance of Germline Transcripts. Cell, 150 (1), 78-87 PMID: 22738724

Ashe A, Sapetschnig A, Weick EM, Mitchell J, Bagijn MP, Cording AC, Doebley AL, Goldstein LD, Lehrbach NJ, Le Pen J, Pintacuda G, Sakaguchi A, Sarkies P, Ahmed S, & Miska EA (2012). piRNAs Can Trigger a Multigenerational Epigenetic Memory in the Germline of C. elegans. Cell, 150 (1), 88-99 PMID: 22738725

Shirayama M, Seth M, Lee HC, Gu W, Ishidate T, Conte D Jr, & Mello CC (2012). piRNAs Initiate an Epigenetic Memory of Nonself RNA in the C. elegans Germline. Cell, 150 (1), 65-77 PMID: 22738726

On Genome Topology 2: The Fractal Globule

As a follow-up to my last post on the use of Hi-C to discover highly self-interacting genomic ‘topological domains’, I wanted to discuss a very interesting aspect of the original paper describing Hi-C. As well as finding a division of the genome into two chromatin compartments, Lieberman-Aiden et al. used their Hi-C data to compare and contrast two models of the topology of chromatin folding within the nucleus.

In this first description of Hi-C, Leberman-Aiden divided their genome-wide contact matrix into 1Mb regions (ie.10 times less definition than the Dixon et al study). They found that, at this level of resolution, the genome can be partitioned into two varieties of spatial compartment, termed A and B. Greater interaction occurs within each compartment than across compartments. Compartment A displays a more open form of chromatin, with a high gene density and high levels of gene expression. Compartment B shows a more densely packed, closed chromatin state. Although the authors do not equate these compartments to euchromatin and heterochromatin, they sound distinctly similar to this old cytogenetic division.

In the later section of the paper, Lieberman-Aiden et al. discuss how their Hi-C data can be used to test models of the three dimensional folding of chromatin. The ‘Equilibrium globule’ model has been used to describe polymers in a poor solvent at equilibrium. In it chromatin is pictured as being in a densely knotted configuration. The ‘Fractal Globule’ model describes polymers self-organising into long-lived, non-equilibrium conformations:

“This highly compact state is formed by an unentangled polymer when it crumples into a series of small globules in a “beads-on-a-string” configuration. These beads serve as monomers in subsequent rounds of spontaneous crumpling until only a single globule-of-globules-of-globules remains. The resulting structure resembles a Peano curve, a continuous fractal trajectory that densely fills 3D space without crossing itself”

(C) Top: An unfolded polymer chain, 4000 monomers (4.8 Mb) long. Coloration corresponds to distance from one endpoint, ranging from blue to cyan, green, yellow, orange, and red. Middle: An equilibrium globule. The structure is highly entangled; loci that are nearby along the contour (similar color) need not be nearby in 3D. Bottom: A fractal globule. Nearby loci along the contour tend to be nearby in 3D, leading to monochromatic blocks both on the surface and in cross-section. The structure lacks knots. (D) Genome architecture at three scales. Top: Two compartments, corresponding to open and closed chromatin, spatially partition the genome. Chromosomes (blue, cyan, green) occupy distinct territories. Middle: Individual chromosomes weave back-and-forth between the open and closed chromatin compartments. Bottom: At the scale of single megabases, the chromosome consists of a series of fractal globules.

When the intrachromasomal contact probability is plotted against genomic distance a power law scaling is observed between ~500kb and ~7Mb. This scaling figure (s1.08) is much closer to that predicted for the fractal globule model (s-1) than that for the equilibrium globule (s-3/2). Likewise, data on the 3D distance between pairs of loci from 3D-FISH is in agreement with a fractal globule topology.

It therefore seems that, at the scale of several megabases, chromatin is organised in these knot-free conformations of globules within globules, allowing unfolding and refolding, whilst also enabling maximally dense packing. I must admit that I don’t have too much insight into the meaning of this; but frankly fractals are cool, and I love the idea of crumpling into globules of globules!

Lieberman-Aiden, E., van Berkum, N., Williams, L., Imakaev, M., Ragoczy, T., Telling, A., Amit, I., Lajoie, B., Sabo, P., Dorschner, M., Sandstrom, R., Bernstein, B., Bender, M., Groudine, M., Gnirke, A., Stamatoyannopoulos, J., Mirny, L., Lander, E., & Dekker, J. (2009). Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome Science, 326 (5950), 289-293 DOI: 10.1126/science.1181369