Short hairpin RNA analysis of epigenetic antiretroviral mechanisms in mouse embryos reveals different mechanisms and levels of activity across lineages
The integration of retroviral material into the genome causes many problems for adult organisms, but it can be especially dangerous for organisms during the embryo stage, as the mutagenic risks associated with retroviral reactivation are particularly significant at this point in development. As a consequence, the stem cells that make up the embryo are equipped with the epigenetic mechanisms to silence retroviral genes to limit or prevent this damage, although differences between the exact methods used by the various stem cell types have remained unclear.
The first author of a recent paper on this subject, Professor Michael Golding (Texas A & M University, TX, USA), has succeeded in shedding light on the various epigenetic mechanisms by which embryonic stem cells cope with integrated retroviral material. Alerted to the potential for varying mechanisms of gene silencing among the stem cells that make up the embryo by observing the way in which X-chromosome inactivation occurred among these cell types, Professor Golding and his group studied the response of the different types of embryonic stem cells to retroviral reactivation using a mouse model.
Mammalian embryos have three different lineages of stem cells, each giving rise to a different ‘component‘: embryonic stem (ES) cells form the fetus, trophodecterm stem (TS) cells form the placenta and the extraembryonic endoderm stem cells (XEN) form the yolk sac. Professor Golding‘s group examined the variation in response to retroviral attack by infecting mouse embryo stem cells with mouse leukemia virus and tracking viral activity.
The results showed differing levels of response to the retrovirus across the lineages. The TS cells appeared passive, in that viral activity remained constant over time in these cells. The ES cells were moderately active, showing a constant decline in viral activity, while the XEN cells mounted an extremely aggressive response, causing viral activity to be silenced extremely rapidly. On this, Professor Golding commented, “the three cell lineages of early mammalian embryo have vastly different viral silencing strategies as well as different capacities to suppress retroviral activity”.
The group made use of a short hairpin RNA library to analyze the specific epigenetic modifiers used by each stem cell lineage, and discovered that, as their paper puts it, “Multiple chromatin remodeling and polycomb repressor complex proteins act to modulate integrated, as well as endogenous, retroviral element silencing…”.
The differing response to the retrovirus between different stem cell lineages was attributed to “a subset of factors displaying differential effects between stem cell types”.
In terms of the further implications of this research, Professor Golding was enthusiastic: “This discovery that all three stem cell types of the early embryo utilize slightly different mechanisms to control gene expression has profound implications for how we diagnose and treat fetal diseases”.
Sources: Golding MC, Zhang L, Mann MRW: Multiple epigenetic modifiers induce aggressive viral extinction in extraembryonic endoderm stem cells. Cell Stem Cell 6(5), 457–467 (2010); Texas A & M University Press Release: http://tamunews.tamu.edu/2010/06/24/researcher-explains-how-embryo-fights-retroviral-infection/
Pulmonary arterial hypertension (PAH), one of several subtypes of pulmonary hypertension, is a potentially life-threatening disease associated with the constriction of blood vessels within and around the lungs. There are multiple different potential causes for this disease, one of which has been established as a genetic mutation, although this only accounts for a relatively small number of patients.
A group of Chicago-based researchers, led by Professor Stephen Archer (University of Chicago, IL, USA), have been investigating the potential genetic causes of PAH using a rat model. Their findings indicate that the reduced expression and activity of SOD2, which leads to the mechanical cause of the PAH – “excessive proliferation and impaired apoptosis of pulmonary artery smooth muscle cells…” explain the authors in their recent study, has an epigenetic basis.
Having established that SOD2 was the likely culprit, but failing to discover any direct genetic mutation, Professor Archer‘s group employed bisulfite sequencing to examine the methylation status of the SOD2 gene, and observed the selective hypermethylation of a CpG island in an enhancer region of intron 2 and another present in the promoter. Experimental manipulation of this hypermethylation via a DNA methyltransferase inhibitor returned SOD2 expression levels back to normal and alleviated the PAH-like symptoms experienced by the rat models, identifying a possible target for the potential use of similar therapies in humans with this form of PAH.
Given the mechanism uncovered by this research, there may be implications beyond the focus of PAH. As study author Dr Jalees Rehman (University of Chicago) commented, “What would be a very interesting question to ask is whether this same thing happens in other blood vessels, where smooth muscle cells also cause disease”.
Sources: Archer SL, Marsboom G, Kim GH et al.: Epigenetic attenuation of mitochondrial superoxide dismutase 2 (SOD2) in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target. Circulation121(24), 2661–2671 (2010); University of Chicago Medical Center Press Release: www.uchospitals.edu/news/2010/20100608-epigenetics.html
Bladder cancer is a serious, relatively common disease that often necessitates the removal of all or part of the bladder in order to eliminate the tumor. The epigenetic mechanisms of histone modifications and DNA methylation are known to play important roles in the initiation and progression of bladder cancer, and so the recent demonstration by a group of Danish researchers of a new twist on a diagnostic test for the epigenetic signs of bladder cancer is valuable progress in this area.
The group, led by Professor Per Guldberg (Danish Cancer Society Institute of Cancer Biology, Copenhagen, Denmark), claims to have developed “…a simple and cost-effective approach to custom-designed multiplex ligation-dependent probe amplication”, to quote their recent paper.
Although it has been possible to screen for the methylation patterns characteristic of bladder cancer for some time via methylation specific (MS)-MLPA, the challenge of synthesizing a single long probe, and the lack of commercial ‘kits‘ for this screening have limited the practicality of the method. In order to avoid these problems, Professor Guldberg‘s group has adapted the original assay to use several shorter, more easily synthesized probes that can be joined together for analysis at a later stage.
While testing this new assay, the group examined both tumor and urine samples, and found that tumor markers in an individual‘s urine were usually identical to those of the tumor sample, a finding which may allow for a nonsurgical approach to this sort of assay in future.
Sources: Serizawa RR, Ralfkiær U, Dahl C et al.: Custom-designed MLPA using multiple short synthetic probes: application to methylation analysis of five promoter CpG islands in tumor and urine specimens from patients with bladder cancer. J. Mol. Diagn. (2010) (Epub ahead of print).
It has been known for some time that microRNAs (miRNAs) are capable of binding to other RNAs and interfering with their function. In the case of mRNA, this can result in gene silencing. What was not considered was the potential relevance of the reverse process; RNAs complementary to the miRNAs targeting a particular gene binding to those miRNAs and thus preventing gene silencing.
A recent study from a group led by Professor Pier Paolo Pandolfi (Beth Israel Deaconess Medical Center, MA, USA) has investigated this possibility, and discovered that noncoding RNA (ncRNA) and pseudogene-based RNA sequences similar to the mRNA of a particular gene can limit the ability of miRNAs to regulate that mRNA by competing to bind with the silencing miRNAs. RNAs capable of such interaction have been termed ‘competitive endogenous RNAs‘ (ceRNA) by the study group.
miRNAs are short RNA sequences (22 nucleotides on average) involved in post-transcriptional gene regulation via their tendency to bind to other complementary strands of RNA in the cell, preventing further biological activity from those targets for as long as they remain bound. They are rendered somewhat specific by the need for complementary sequences on their targets, but a given miRNA may still be able to bind to hundreds of different mRNA sequences.
Pseudogenes, duplicates of existing genes inactivated and modified by past mutations, by definition do not produce the functional final products of the gene they are related to, but many can and do produce mRNA that can be very similar to that of the actual gene. This mRNA, then, makes an excellent competitor for the attention of gene-silencing miRNAs, which may often be unable to distinguish between the two.
This discovery of ceRNA activity reveals not only an additional layer of gene regulation, but also a new potential biological activity of a large number of previously disregarded pseudogenes and ncRNAs. As Professor Pandolfi elaborated: “Because this new function does not depend on the blueprint that RNAs harbor in their protein-encoding nucleotide sequence, the discovery additionally holds true for the thousands of noncoding RNA molecules in the cell”.
Although the study itself focused on the PTEN tumor suppressor gene and its pseudogene PTENP1, the principles of ceRNA action can be theoretically extrapolated to any RNA molecule, according to Professor Pandolfi: “…now that we know what to listen for, we can computationally predict which RNA can act as a ceRNA and what that ceRNA can do.”
Although this discovery is perhaps too recent for the therapeutic implications to be properly assessed, there is bound to be an eventual application of this new knowledge. Professor Pandolfi concluded, “…we now know the function of an estimated 30,000 new entities, offering a novel dimension by which cellular and tumor biology can be regulated, and effectively doubling the size of the functional genome.”
Sources: Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, Pandolfi PP: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465, 1033–1038 (2010); Beth Israel Deaconess Medical Center Press Release: www.bidmc.org/News/InResearch/2010/June/Pandolfi.aspx