scholarly journals Expanded CAG/CTG repeats resist gene silencing mediated by targeted Epigenome editing

2021 ◽  
Author(s):  
Bin Yang ◽  
Alicia C Borgeaud ◽  
Marcela Buřičová ◽  
Lorène Aeschbach ◽  
Oscar Rodríguez-Lima ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Dna Methyltransferase ◽  
Rational Design ◽  
Repeat Instability ◽  
Efficient Treatment ◽  
Ctg Repeats ◽  
Epigenome Editing ◽  
Molecule Inhibitor ◽  
Gfp Reporter ◽  
Ctg Repeat

Abstract Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Previous studies have implicated the histone deacetylase HDAC5 and the DNA methyltransferase DNMT1 as modulators of repeat instability via mechanisms that are not fully understood. Using PInT, we found no evidence that HDAC5 or DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by targeting HDAC5 and DNMT1. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.

scholarly journals Expanded CAG/CTG Repeats Resist Gene Silencing Mediated by Targeted Epigenome Editing

2018 ◽  
Author(s):  
Bin Yang ◽  
Alicia C. Borgeaud ◽  
Lorène Aeschbach ◽  
Oscar Rodríguez-Lima ◽  
Gustavo A. Ruiz Buendía ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Dna Methyltransferase ◽  
Rational Design ◽  
Repeat Instability ◽  
Efficient Treatment ◽  
Ctg Repeats ◽  
Epigenome Editing ◽  
Molecule Inhibitor ◽  
Gfp Reporter ◽  
Ctg Repeat

AbstractExpanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Using PInT, we found no evidence that the histone deacetylase HDAC5 or the DNA methyltransferase DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by HDAC5 or DNMT1 targeting. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.

scholarly journals Transcriptionally Repressive Chromatin Remodelling and CpG Methylation in the Presence of Expanded CTG-Repeats at the DM1 Locus

2013 ◽  
Vol 2013 ◽  
pp. 1-16 ◽  
Author(s):  
Judith Rixt Brouwer ◽  
Aline Huguet ◽  
Annie Nicole ◽  
Arnold Munnich ◽  
Geneviève Gourdon
Keyword(s):  
Transgenic Mice ◽  
Trinucleotide Repeat ◽  
Cpg Methylation ◽  
Chromatin Remodelling ◽  
Ctcf Binding ◽  
Type I ◽  
Repeat Instability ◽  
Ctg Repeats ◽  
Repressive Chromatin ◽  
Ctg Repeat

An expanded CTG-repeat in the 3′ UTR of theDMPKgene is responsible for myotonic dystrophy type I (DM1). Somatic and intergenerational instability cause the disease to become more severe during life and in subsequent generations. Evidence is accumulating that trinucleotide repeat instability and disease progression involve aberrant chromatin dynamics. We explored the chromatin environment in relation to expanded CTG-repeat tracts in hearts from transgenic mice carrying the DM1 locus with different repeat lengths. Using bisulfite sequencing we detected abundant CpG methylation in the regions flanking the expanded CTG-repeat. CpG methylation was postulated to affect CTCF binding but we found that CTCF binding is not affected by CTG-repeat length in our transgenic mice. We detected significantly decreasedDMPKsense andSIX5transcript expression levels in mice with expanded CTG-repeats. Expression of the DM1 antisense transcript was barely affected by CTG-repeat expansion. In line with altered gene expression, ChIP studies revealed a locally less active chromatin conformation around the expanded CTG-repeat, namely, decreased enrichment of active histone mark H3K9/14Ac and increased H3K9Me3 enrichment (repressive chromatin mark). We also observed binding of PCNA around the repeats, a candidate that could launch chromatin remodelling cascades at expanded repeats, ultimately affecting gene transcription and repeat instability.

scholarly journals Three-dimensional chromatin interactions remain stable upon CAG/CTG repeat expansion

2020 ◽  
Vol 6 (27) ◽  
pp. eaaz4012 ◽  
Author(s):  
Gustavo A. Ruiz Buendía ◽  
Marion Leleu ◽  
Flavia Marzetta ◽  
Ludovica Vanzan ◽  
Jennifer Y. Tan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Three Dimensional ◽  
Cag Repeat ◽  
Ctcf Binding ◽  
Chromatin Interaction ◽  
Chromatin Conformation ◽  
Repeat Instability ◽  
Ctg Repeats ◽  
Chromatin Interactions ◽  
Ctg Repeat

Expanded CAG/CTG repeats underlie 13 neurological disorders, including myotonic dystrophy type 1 (DM1) and Huntington’s disease (HD). Upon expansion, disease loci acquire heterochromatic characteristics, which may provoke changes to chromatin conformation and thereby affect both gene expression and repeat instability. Here, we tested this hypothesis by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD–derived cells. We find that allele sizes ranging from 15 to 1700 repeats displayed similar chromatin interaction profiles. This was true for both loci and for alleles with different DNA methylation levels and CTCF binding. Moreover, the ectopic insertion of an expanded CAG repeat tract did not change the conformation of the surrounding chromatin. We conclude that CAG/CTG repeat expansions are not enough to alter chromatin conformation in cis. Therefore, it is unlikely that changes in chromatin interactions drive repeat instability or changes in gene expression in these disorders.

scholarly journals Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

2017 ◽  
Author(s):  
Loréne Aeschbach ◽  
Vincent Dion
Keyword(s):  
Tandem Repeats ◽  
Neuromuscular Disorders ◽  
Pcr Amplification ◽  
Repeat Instability ◽  
Gold Standard Method ◽  
Ctg Repeats ◽  
Somatic Tissues ◽  
Small Pool ◽  
Carry Over ◽  
Ctg Repeat

AbstractExpanded CAG/CTG repeats underlie the aetiology of 14 neurological and neuromuscular disorders. The size of the repeat tract determines in large part the severity of these disorders with longer tracts causing more severe phenotypes. Expanded CAG/CTG repeats are also unstable in somatic tissues, which is thought to modify disease progression. Routine molecular biology applications involving these repeats, including quantifying their instability, are plagued by low PCR yields. This leads to the need for setting up more PCRs of the same locus, thereby increasing the risk of carry-over contamination. Here we aimed to reduce this risk by pre-treating the samples with a Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCRs. We successfully applied this method to the PCR amplification of expanded CAG/CTG repeats, their sequencing, and their molecular cloning. In addition, we optimized the gold-standard method for measuring repeat instability, small-pool PCR, such that it can be used together with Ung and dUTP-containing PCRs, without compromising data quality. We expect that the protocols herein to be applicable for molecular diagnostics of expanded repeat disorders and to manipulate other tandem repeats.

scholarly journals Epigenetic Changes in Host Ribosomal DNA Promoter Induced by an Asymptomatic Plant Virus Infection

Biology ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 91 ◽  
Author(s):  
Miryam Pérez-Cañamás ◽  
Elizabeth Hevia ◽  
Carmen Hernández
Keyword(s):  
Gene Silencing ◽  
Ribosomal Dna ◽  
Plant Virus ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Methylation Pattern ◽  
Transcriptional Gene Silencing ◽  
Post Transcriptional Gene Silencing ◽  
The Impact

DNA cytosine methylation is one of the main epigenetic mechanisms in higher eukaryotes and is considered to play a key role in transcriptional gene silencing. In plants, cytosine methylation can occur in all sequence contexts (CG, CHG, and CHH), and its levels are controlled by multiple pathways, including de novo methylation, maintenance methylation, and demethylation. Modulation of DNA methylation represents a potentially robust mechanism to adjust gene expression following exposure to different stresses. However, the potential involvement of epigenetics in plant-virus interactions has been scarcely explored, especially with regard to RNA viruses. Here, we studied the impact of a symptomless viral infection on the epigenetic status of the host genome. We focused our attention on the interaction between Nicotiana benthamiana and Pelargonium line pattern virus (PLPV, family Tombusviridae), and analyzed cytosine methylation in the repetitive genomic element corresponding to ribosomal DNA (rDNA). Through a combination of bisulfite sequencing and RT-qPCR, we obtained data showing that PLPV infection gives rise to a reduction in methylation at CG sites of the rDNA promoter. Such a reduction correlated with an increase and decrease, respectively, in the expression levels of some key demethylases and of MET1, the DNA methyltransferase responsible for the maintenance of CG methylation. Hypomethylation of rDNA promoter was associated with a five-fold augmentation of rRNA precursor levels. The PLPV protein p37, reported as a suppressor of post-transcriptional gene silencing, did not lead to the same effects when expressed alone and, thus, it is unlikely to act as suppressor of transcriptional gene silencing. Collectively, the results suggest that PLPV infection as a whole is able to modulate host transcriptional activity through changes in the cytosine methylation pattern arising from misregulation of methyltransferases/demethylases balance.

scholarly journals Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

2016 ◽  
Vol 44 (11) ◽  
pp. 5190-5203 ◽  
Author(s):  
Piera Cilli ◽  
Ilenia Ventura ◽  
Anna Minoprio ◽  
Ettore Meccia ◽  
Alberto Martire ◽  
...  
Keyword(s):  
Dna Glycosylases ◽  
Repeat Instability ◽  
Ctg Repeat

419. Rational design of bifunctional siRNAs that silence genes and recruit the innate immune system to treat ectopic pregnancies

2008 ◽  
Vol 20 (9) ◽  
pp. 99
Author(s):  
S. Tong ◽  
M. P. Gantier ◽  
M. Belhke ◽  
B. R. G. Williams
Keyword(s):  
Immune System ◽  
Gene Silencing ◽  
Rational Design ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Micro Rna ◽  
Peripheral Blood Mononuclear ◽  
Sirna Sequence ◽  
Sense Strand ◽  
Ectopic Pregnancies

RNA interference (RNAi) is a new therapeutic approach, silencing genes to disrupt diseases. However, short interfering siRNAs (molecule used in RNAi) can have off-target effects, activating the immune system through RNA sensing toll-like receptors (TLR) 3, 7 and 8. We have previously proposed that in some diseases (cancers, ectopic pregnancies) it may be useful to enhance the immune response. A novel class of immunostimulatory siRNAs could be developed, silencing genes important to disease and recruiting the immune system to further aid disease clearance. We set out to develop a rational design strategy that enhances immunostimulatory properties to any siRNA sequence but maintains effective gene silencing. We screened a set of siRNAs targeting lamin. All were of the same sequence, except for different immunostimulatory motifs on the 3′ end of the sense strand. We also investigated a different design where we added a small micro-RNA like poly-uridine bulge (potentially immunostimulatory) on the sense strand. We used human peripheral blood mononuclear cells (PBMCs) to test for immunostimulation, and HEK 293-T-cells to test for lamin gene knockdown.Of all strategies tested, the poly-uridine bulge was best. It silenced the lamin gene as effectively as control, but caused a 2–3 fold increase of IFN-α and TNF-α. We verified this approach by adding the poly-uridine bulge onto an siRNA of low immunostimulatory potential targeting GFP. The bulge markedly enhanced immunostimulation in a dose response manner, and did not compromise gene knockdown. The addition of a poly-uridine bulge to siRNAs can increase immunostimulation without affecting gene silencing efficacy. Immunostimulatory siRNAs might be particularly efficacious to treat ectopic pregnancies where there are abundant immune cells, and functional TLR 7/8 in the trophoblast (unpublished observations). We now plan to test this immunostimulatory siRNA approach in an in vivo ectopic pregnancy model using JEG-3 cells xenographted in NOD-SCID mice.

Rational Design of a Robust Antibody-like Small-Molecule Inhibitor Nanoplatform for Enhanced Photoimmunotherapy

2020 ◽  
Vol 12 (36) ◽  
pp. 40085-40093 ◽  
Author(s):  
Qi Shang ◽  
Shiyao Zhou ◽  
Yue Jiang ◽  
Dong Wang ◽  
Jiqian Wang ◽  
...  
Keyword(s):  
Small Molecule ◽  
Rational Design ◽  
Small Molecule Inhibitor ◽  
Molecule Inhibitor

scholarly journals A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation

2016 ◽  
Vol 113 (50) ◽  
pp. 14366-14371 ◽  
Author(s):  
Derek Lessing ◽  
Thomas O. Dial ◽  
Chunyao Wei ◽  
Bernhard Payer ◽  
Lieselot L. G. Carrette ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Small Molecules ◽  
High Throughput ◽  
Dosage Compensation ◽  
Dna Methyltransferase ◽  
Aurora Kinase ◽  
Functional Genes ◽  
X Chromosomes ◽  
Gfp Reporter ◽  
Small Molecule Screen

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by “priming” cells with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

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