Emerging role of RNA•DNA hybrids in<i>C9orf72</i>-linked neurodegeneration

Wang, Jiou; Haeusler, Aaron R.; Simko, Eric A J

doi:10.1080/15384101.2014.995490

Cited by 26 publications

(21 citation statements)

References 100 publications

(111 reference statements)

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“…These studies suggest that RNA-DNA hybrids or R-loops are key mediators of epigenetic repression [39]. Recent discoveries in other repeat expansion disorders, including Fragile X Syndrome and Freidriech’s Ataxia support this reasoning [21, 34].…”

Section: Discussionmentioning

confidence: 99%

A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

Esanov

Cabrera

Andrade

et al. 2017

Mol Neurodegeneration

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BackgroundAmyotrophic Lateral Sclerosis (ALS) is a fatal and progressive neurodegenerative disorder with identified genetic causes representing a significant minority of all cases. A GGGGCC hexanucleotide repeat expansion (HRE) mutation within the C9ORF72 gene has recently been identified as the most frequent known cause of ALS. The expansion leads to partial heterochromatinization of the locus, yet mutant RNAs and dipeptide repeat proteins (DPRs) are still produced in sufficient quantities to confer neurotoxicity. The levels of these toxic HRE products positively correlate with cellular toxicity and phenotypic severity across multiple disease models. Moreover, the degree of epigenetic repression inversely correlates with some facets of clinical presentation in C9-ALS patients. Recently, bacterial artificial chromosomes (BAC) have been used to generate transgenic mice that harbor the HRE mutation, complementing other relevant model systems such as patient-derived induced pluripotent stem cells (iPSCs). While epigenetic features of the HRE have been investigated in various model systems and post-mortem tissues, epigenetic dysregulation at the expanded locus in C9-BAC mice remains unexplored.Methods and ResultsHere, we sought to determine whether clinically relevant epigenetic perturbations caused by the HRE are mirrored in a C9-BAC mouse model. We used complementary DNA methylation assessment and immunoprecipitation methods to demonstrate that epigenetic aberrations caused by the HRE, such as DNA and histone methylation, are recapitulated in the C9-BAC mice. Strikingly, we found that cytosine hypermethylation within the promoter region of the human transgene occurred in a subset of C9-BAC mice similar to what is observed in patient populations. Moreover, we show that partial heterochromatinization of the C9 HRE occurs during the first weeks of the mouse lifespan, indicating age-dependent epigenetic repression. Using iPSC neurons, we found that preventing R-loop formation did not impede heterochromatinization of the HRE.ConclusionsTaken together, these observations provide further insight into mechanism and developmental time-course of epigenetic perturbations conferred by the C9ORF72 HRE. Finally, we suggest that epigenetic repression of the C9ORF72 HRE and nearby gene promoter could impede or delay motor neuron degeneration in C9-BAC mouse models of ALS/FTD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-017-0185-9) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

Esanov

Cabrera

Andrade

et al. 2017

Mol Neurodegeneration

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“…Alterations in nuclear R-loop levels are associated with disruption of transcription, DNA repair, and other key genomic processes 5-9 . Identification of changes in R-loop abundance and distribution in different cell types could inform us on mechanisms that lead to cell type-specific pathology [10][11][12][13][14][15] .However, efforts to study the regulatory functions of R-loops have been hindered because of the sub-optimal methods used to enrich for and recover these chromatin structures. Therefore, there is a critical need to develop new methods that will allow for enhanced and systematic discovery of R-loops.Currently, two distinct strategies are used to map the distribution of R-loops.…”

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confidence: 99%

Mapping native R-loops genome-wide using a targeted nuclease approach

Yan

Shields

Bonasio

et al. 2018

Preprint

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R-loops are three-stranded DNA:RNA hybrids that are pervasive in the eukaryotic and prokaryotic genomes and have been implicated in a variety of nuclear processes, including transcription, replication, DNA repair, and chromosome segregation. While R-loops may have physiological roles, the formation of stable, aberrant R-loops has been observed in disease, particularly neurological disorders and cancer. Despite the importance of these structures, methods to assess their distribution in the genome invariably rely on affinity purification, which requires large amounts of input material, is plagued by high level of noise, and is poorly suited to capture dynamic and unstable R-loops. Here, we present a new method that leverages the affinity of RNase H for DNA:RNA hybrids to target micrococcal nuclease to genomic sites that contain R-loops, which are subsequently cleaved, released, and sequenced. Our R-loop mapping method, MapR, is as specific as existing techniques, less prone to recover non-specific repetitive sequences, and more sensitive, allowing for genome-wide coverage with low input material and read numbers, in a fraction of the time.Main R-loops are three-stranded nucleic acid structures that contain a DNA:RNA hybrid and a displaced single strand of DNA 1 . R-loops are dynamic structures whose levels are tightly controlled across the genome 2-4 . Alterations in nuclear R-loop levels are associated with disruption of transcription, DNA repair, and other key genomic processes 5-9 . Identification of changes in R-loop abundance and distribution in different cell types could inform us on mechanisms that lead to cell type-specific pathology [10][11][12][13][14][15] .However, efforts to study the regulatory functions of R-loops have been hindered because of the sub-optimal methods used to enrich for and recover these chromatin structures. Therefore, there is a critical need to develop new methods that will allow for enhanced and systematic discovery of R-loops.Currently, two distinct strategies are used to map the distribution of R-loops. The predominant strategy relies on the immunoprecipitation of chromatin containing R-loops using a monoclonal antibody, S9.6, thought to be specific for DNA:RNA hybrids 16 . DNA:RNA immunoprecipitation (DRIP) and all its variants 17-20 (bis-DRIP, S1-DRIP and RDIP), were foundational to the study of genome-wide R-loop localization, but share similar disadvantages: 1) they prepare chromatin for immunoprecipitation using harsh physical and biochemical treatments (high temperatures, strong detergents, sonication and/or prolonged enzymatic digestion of chromatin) in the absence of fixation, which might disrupt less stable R-loops before they can be detected, and 2) they rely on the S9.6 antibody whose strict specificity for DNA:RNA hybrids remains a subject of debate (e.g. it might also bind dsRNA 21 ). The second, more recent, strategy to map R-loops takes advantage of the natural affinity of RNase H for DNA:RNA hybrids. RNase H is an enzyme that degrades the RNA strand of DNA:RNA het...

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“…Again, consistent with the sequence of the C9 hexanucleotide, DNA methylation has also been found to be associated with the repeat expansion [107–111]. Since bidirectional transcription also occurs at the expansion [112–116] and that R loops are enriched at genes producing antisense RNA [5,12], it is possible that R loops reduce C9 expression through the regulation of bidirectional transcription and chromatin modification (reviewed in Ref. [117]).…”

Section: R-loop Connections To Neurological Disordersmentioning

confidence: 89%

R Loops and Links to Human Disease

Richard

Manley

2017

Journal of Molecular Biology

164

133

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Aberrant R-loop structures are increasingly being realized as an important contributor to human disease. R loops, which are mainly co-transcriptional, abundant RNA/DNA hybrids, form naturally and can indeed be beneficial for transcription regulation at certain loci. However, their unwanted persistence elsewhere or in particular situations can lead to DNA double-strand breaks, chromosome rearrangements, and hypermutation, which are all sources of genomic instability. Mutations in genes involved in R-loop resolution or mutations leading to R-loop formation at specific genes affect the normal physiology of the cell. We discuss here the examples of diseases for which a link with R loops has been described, as well as how disease-causing mutations might participate in the development and/or progression of diseases that include repeat-associated conditions, other neurological disorders, and cancers.

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Emerging role of RNA•DNA hybrids inC9orf72-linked neurodegeneration

Cited by 26 publications

References 100 publications

A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

Mapping native R-loops genome-wide using a targeted nuclease approach

R Loops and Links to Human Disease

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