scholarly journals RNA strand invasion activity of the Polycomb complex PRC2

2019 ◽  
Author(s):  
Célia Alecki ◽  
Victoria Chiwara ◽  
Lionel A. Sanz ◽  
Daniel Grau ◽  
Osvaldo Arias Pérez ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Epigenetic Regulation ◽  
Dna Sequences ◽  
Polycomb Group ◽  
Epigenetic Mechanism ◽  
Biological Information ◽  
Loop Formation ◽  
Strand Invasion ◽  
Nucleic Acid Structures

AbstractEpigenetic regulation is conveyed through information encoded by specific chromatin features. Non-canonical nucleic acid structures could in principle also convey biological information but their role(s) in epigenetic regulation is not known. Polycomb Group (PcG) proteins form memory of transient transcriptional repression events that is necessary for development. In Drosophila, PcG proteins are recruited to specific DNA sequences, Polycomb Response Elements (PREs). PREs are switchable memory elements that can exist in repressed, active, or unengaged states 1,2. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. Biochemically, PcG protein complexes modify chromatin to maintain gene repression 1,3,4. However, PcG proteins also interact with both RNA and DNA, and RNA is implicated in the targeting of PcG function. We find that R-loops, three-stranded nucleic acid structures formed when an RNA hybridizes to its complementary DNA and displaces the other DNA strand 5, form at many PREs in Drosophila embryos, and correlate with the repressive state. R-loops are recognized by the PcG complex PRC1 in vitro. Unexpectedly, we find that the PcG complex PRC2 has RNA strand invasion activity, which can drive formation of RNA-DNA hybrids, the key component of R-loops. Our results suggest a new mechanism for targeting PcG function through R-loop formation by PRC2 and recognition by PRC1. More generally, our findings suggest formation and recognition 6 of non-canonical nucleic acid structures as an epigenetic mechanism.

scholarly journals R-Loop Tracker: Web Access-Based Tool for R-Loop Detection and Analysis in Genomic DNA Sequences

2021 ◽  
Vol 22 (23) ◽  
pp. 12857
Author(s):  
Václav Brázda ◽  
Jan Havlík ◽  
Jan Kolomazník ◽  
Oldřich Trenz ◽  
Jiří Šťastný
Keyword(s):  
Nucleic Acid ◽  
Dna Sequences ◽  
In Silico ◽  
Genomic Dna ◽  
Loop Formation ◽  
Loop Detection ◽  
Genome Features ◽  
Web Access ◽  
Nucleic Acid Structures ◽  
The Web

R-loops are common non-B nucleic acid structures formed by a three-stranded nucleic acid composed of an RNA–DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. Because the aberrant R-loop formation leads to increased mutagenesis, hyper-recombination, rearrangements, and transcription-replication collisions, it is regarded as important in human diseases. Therefore, its prevalence and distribution in genomes are studied intensively. However, in silico tools for R-loop prediction are limited, and therefore, we have developed the R-loop tracker tool, which was implemented as a part of the DNA Analyser web server. This new tool is focused upon (1) prediction of R-loops in genomic DNA without length and sequence limitations; (2) integration of R-loop tracker results with other tools for nucleic acids analyses, including Genome Browser; (3) internal cross-evaluation of in silico results with experimental data, where available; (4) easy export and correlation analyses with other genome features and markers; and (5) enhanced visualization outputs. Our new R-loop tracker tool is freely accessible on the web pages of DNA Analyser tools, and its implementation on the web-based server allows effective analyses not only for DNA segments but also for full chromosomes and genomes.

scholarly journals Differential Contributions of DNA-Binding Proteins to Polycomb Response Element Activity at the Drosophila giant Gene

Genetics ◽  
2020 ◽  
Vol 214 (3) ◽  
pp. 623-634
Author(s):  
Elnaz Ghotbi ◽  
Kristina Lackey ◽  
Vicki Wong ◽  
Katie T. Thompson ◽  
Evan G. Caston ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Target Genes ◽  
Dna Binding Proteins ◽  
Polycomb Group ◽  
Link Type ◽  
Element Activity

Polycomb-group (PcG) proteins are evolutionarily conserved epigenetic regulators whose primary function is to maintain the transcriptional repression of target genes. Recruitment of Drosophila melanogaster PcG proteins to target genes requires the presence of one or more Polycomb Response Elements (PREs). The functions or necessity for more than one PRE at a gene are not clear and individual PREs at some loci may have distinct regulatory roles. Various combinations of sequence-specific DNA-binding proteins are present at a given PRE, but only Pleiohomeotic (Pho) is present at all strong PREs. The giant (gt) locus has two PREs, a proximal PRE1 and a distal PRE2. During early embryonic development, Pho binds to PRE1 ∼30-min prior to stable binding to PRE2. This observation indicated a possible dependence of PRE2 on PRE1 for PcG recruitment; however, we find here that PRE2 recruits PcG proteins and maintains transcriptional repression independently of Pho binding to PRE1. Pho-like (Phol) is partially redundant with Pho during larval development and binds to the same DNA sequences in vitro. Although binding of Pho to PRE1 is dependent on the presence of consensus Pho-Phol-binding sites, Phol binding is less so and appears to play a minimal role in recruiting other PcG proteins to gt. Another PRE-binding protein, Sp1/Kruppel-like factor, is dependent on the presence of Pho for PRE1 binding. Further, we show that, in addition to silencing gene expression, PcG proteins dampen transcription of an active gene.

Development of aerial and belowground tubers in potato is governed by photoperiod and epigenetic mechanism

2021 ◽  
Author(s):  
Kirtikumar R Kondhare ◽  
Amit Kumar ◽  
Nikita S Patil ◽  
Nilam N Malankar ◽  
Kishan Saha ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Developmental Plasticity ◽  
Integration Site ◽  
Leukemia Virus ◽  
Polycomb Group ◽  
Epigenetic Mechanism ◽  
Growth Responses ◽  
Short Day

Abstract Plants exhibit diverse developmental plasticity and modulate growth responses under various environmental conditions. Potato (Solanum tuberosum), a modified stem and an important food crop, serves as a substantial portion of the world’s subsistence food supply. In the past two decades, crucial molecular signals have been identified that govern the tuberization (potato development) mechanism. Interestingly, microRNA156 overexpression in potato provided the first evidence for induction of profuse aerial stolons and tubers from axillary-meristems under short-day photoperiod. A similar phenotype was noticed for overexpression of epigenetic modifiers - MUTICOPY SUPRESSOR OF IRA1 (StMSI1) or ENAHNCER OF ZESTE 2 (StE[z]2), and knockdown of B-CELL SPECIFIC MOLONEY MURINE LEUKEMIA VIRUS INTEGRATION SITE 1 (StBMI1). This striking phenotype represents a classic example of modulation of plant architecture and developmental plasticity. Differentiation of a stolon to a tuber or a shoot under in vitro or in vivo conditions symbolizes another example of organ level plasticity and dual fate acquisition in potato. Stolon-to-tuber transition is governed by short-day photoperiod, mobile RNAs/proteins, phytohormones, a plethora of small RNAs and their targets. Recent studies show that polycomb group proteins control microRNA156, phytohormone metabolism/transport/signalling, and key tuberization genes through histone modifications to govern tuber development. Our comparative analysis of differentially expressed genes between the overexpression lines of StMSI1, StBEL5 (BEL1-LIKE transcription factor) and POTH15 (POTATO HOMEOBOX 15 transcription factor) revealed >1000 common genes, indicative of a mutual gene regulatory network potentially involved in the formation of aerial and belowground tubers. In this review, in addition to key tuberization factors, we highlight the role of photoperiod and epigenetic mechanism that regulates the development of aerial and belowground tubers in potato.

scholarly journals The effects of molecular crowding and CpG hypermethylation on DNA G-quadruplexes formed by the C9orf72 nucleotide repeat expansion

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kadir. A. Ozcan ◽  
Layla T. Ghaffari ◽  
Aaron R. Haeusler
Keyword(s):  
Nucleic Acid ◽  
Genetic Mutation ◽  
Repeat Expansion ◽  
Molecular Crowding ◽  
Dna Hypermethylation ◽  
Dynamic Simulations ◽  
Cpg Hypermethylation ◽  
Nucleic Acid Structures ◽  
Nucleotide Repeat

AbstractA nucleotide repeat expansion (NRE), (G4C2)n, located in a classically noncoding region of C9orf72 (C9), is the most common genetic mutation associated with ALS/FTD. There is increasing evidence that nucleic acid structures formed by the C9-NRE may both contribute to ALS/FTD, and serve as therapeutic targets, but there is limited characterization of these nucleic acid structures under physiologically and disease relevant conditions. Here we show in vitro that the C9-NRE DNA can form both parallel and antiparallel DNA G-quadruplex (GQ) topological structures and that the structural preference of these DNA GQs can be dependent on the molecular crowding conditions. Additionally, 5-methylcytosine DNA hypermethylation, which is observed in the C9-NRE locus in some patients, has minimal effects on GQ topological preferences. Finally, molecular dynamic simulations of methylated and nonmethylated GQ structures support in vitro data showing that DNA GQ structures formed by the C9-NRE DNA are stable, with structural fluctuations limited to the cytosine-containing loop regions. These findings provide new insight into the structural polymorphic preferences and stability of DNA GQs formed by the C9-NRE in both the methylated and nonmethylated states, as well as reveal important features to guide the development of upstream therapeutic approaches to potentially attenuate C9-NRE-linked diseases.

scholarly journals S phase R-loop formation is restricted by PrimPol-mediated repriming

2018 ◽  
Author(s):  
Saša Šviković ◽  
Alastair Crisp ◽  
Sue Mei Tan-Wong ◽  
Thomas A. Guilliam ◽  
Aidan J. Doherty ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Secondary Structure ◽  
Genetic Instability ◽  
S Phase ◽  
Loop Formation ◽  
Dna Motifs ◽  
G Quadruplex ◽  
Nucleic Acid Structures ◽  
Careful Management

SummaryDuring DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here we show in vivo how R-loop formation drives a short purine-rich repeat, (GAA)10, to become a replication impediment that requires the repriming activity of the primase-polymerase PrimPol for its processive replication. Further, we show that loss of PrimPol results in a significant increase in R-loop formation around the repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R-loops promote the creation of replication blocks at susceptible sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication.

scholarly journals Rdh54/Tid1 Inhibits Rad51-Rad54-Mediated D-loop Formation and Limits D-loop Length

2020 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella Hartono ◽  
Aurèle Piazza ◽  
Vanessa Som ◽  
William Wright ◽  
...  
Keyword(s):  
Binding Sites ◽  
Loop Length ◽  
Loop Formation ◽  
Independent Manner ◽  
Potential Binding ◽  
D Loop ◽  
Strand Invasion ◽  
Type Switch ◽  
Dna Strand

ABSTRACTDisplacement loops (D-loops) are intermediates formed during homologous recombination that play a pivotal role in the fidelity of repair. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54 is also present in somatic cells where its function is less understood. While Rdh54 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54-mediated invasions. Here, we show that Rdh54 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54 uniquely restricts the lengths of Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast, whereas Rdh54 expression is suppressed in diploids. We propose that Rdh54 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54 acts as a physical roadblock to Rad54’s translocation activity, limiting D-loop formation and D-loop length.

scholarly journals Building an RNA-based Toggle Switch using Inhibitory RNA Aptamers

2021 ◽  
Author(s):  
Alicia Climent Catala ◽  
Thomas E Ouldridge ◽  
Guy-Bart V Stan ◽  
Wooli Bae
Keyword(s):  
Nucleic Acid ◽  
Synthetic Biology ◽  
Dna Sequences ◽  
Transcriptional Activity ◽  
Green Light ◽  
Rna Aptamers ◽  
Toggle Switch ◽  
Mutual Inhibition ◽  
T7 Promoter

Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in-vitro RNA-based toggle switch using RNA aptamers ca- pable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and Malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 in- hibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in cell-like con- ditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control ele- ment for nucleic acid networks in synthetic biology applications.

scholarly journals Cross talk between microRNA and epigenetic regulation in adult neurogenesis

2010 ◽  
Vol 189 (1) ◽  
pp. 127-141 ◽  
Author(s):  
Keith E. Szulwach ◽  
Xuekun Li ◽  
Richard D. Smrt ◽  
Yujing Li ◽  
Yuping Luo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Epigenetic Regulation ◽  
Cross Talk ◽  
Adult Neurogenesis ◽  
Cell Biology ◽  
Polycomb Group ◽  
Stem Cell Biology ◽  
Proliferation And Differentiation

Both microRNAs (miRNAs) and epigenetic regulation have important functions in stem cell biology, although the interactions between these two pathways are not well understood. Here, we show that MeCP2, a DNA methyl-CpG–binding protein, can epigenetically regulate specific miRNAs in adult neural stem cells (aNSCs). MeCP2-mediated epigenetic regulation of one such miRNA, miR-137, involves coregulation by Sox2, a core transcription factor in stem cells. miR-137 modulates the proliferation and differentiation of aNSCs in vitro and in vivo. Overexpression of miR-137 promotes the proliferation of aNSCs, whereas a reduction of miR-137 enhances aNSC differentiation. We further show that miR-137 post-transcriptionally represses the expression of Ezh2, a histone methyltransferase and Polycomb group (PcG) protein. The miR-137–mediated repression of Ezh2 feeds back to chromatin, resulting in a global decrease in histone H3 trimethyl lysine 27. Coexpression of Ezh2 can rescue phenotypes associated with miR-137 overexpression. These results demonstrate that cross talk between miRNA and epigenetic regulation contributes to the modulation of adult neurogenesis.

scholarly journals Proximity labeling identifies a repertoire of site-specific R-loop modulators

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Qingqing Yan ◽  
Phillip Wulfridge ◽  
John Doherty ◽  
Jose L. Fernandez-Luna ◽  
Pedro J. Real ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Genome Instability ◽  
Neurological Diseases ◽  
Loop Formation ◽  
Induced Pluripotent ◽  
Nucleic Acid Structures ◽  
Activity Dependent ◽  
Labeling System

AbstractR-loops are three-stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains. One of the most consistently enriched proteins was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP resolves R-loops in vitro and that it is necessary to suppress R-loops in vivo at its genomic targets. Furthermore, deletion of the ADNP homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets, and compromises neuronal differentiation. Notably, patient-derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

scholarly journals Interplay between DNA sequence and negative superhelicity drives R-loop structures

2019 ◽  
Vol 116 (13) ◽  
pp. 6260-6269 ◽  
Author(s):  
Robert Stolz ◽  
Shaheen Sulthana ◽  
Stella R. Hartono ◽  
Maika Malig ◽  
Craig J. Benham ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Energy State ◽  
In Vitro Transcription ◽  
Loop Formation ◽  
Base Pairing ◽  
Theoretical Predictions ◽  
Nucleic Acid Structures ◽  
The Impact

R-loops are abundant three-stranded nucleic-acid structures that formin cisduring transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms—junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.

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