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 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 MSH2-Dependent Germinal CTG Repeat Expansions Are Produced Continuously in Spermatogonia from DM1 Transgenic Mice

2004 ◽  
Vol 24 (2) ◽  
pp. 629-637 ◽  
Author(s):  
Cédric Savouret ◽  
Corinne Garcia-Cordier ◽  
Jérôme Megret ◽  
Hein te Riele ◽  
Claudine Junien ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cell Types ◽  
Somatic Tissue ◽  
Kinase Gene ◽  
Ctg Repeats ◽  
Significant Difference ◽  
Genomic Environment ◽  
Repeat Expansions ◽  
Germinal Mosaicism ◽  
Ctg Repeat

ABSTRACT Myotonic dystrophy type 1 is a neuromuscular affection associated with the expansion of an unstable CTG repeat in the DM protein kinase gene. The disease is characterized by somatic tissue-specific mosaicism and very high intergenerational instability with a strong bias towards expansions. We used transgenic mice carrying more than 300 unstable CTG repeats within their large human genomic environment to investigate the dynamics of CTG repeat germinal mosaicism in males. Germinal mosaicism towards expansions was already present in spermatozoa at 7 weeks of age and continued to increase with age, suggesting that expansions are continuously produced throughout life. To determine the precise stage at which germinal expansions occur during spermatogenesis, we sorted and collected the different germ cell types produced during spermatogenesis from males of different ages and analyzed the CTG repeat mosaicism in each fraction. Strong mosaicisms towards expansions were already observed in spermatogonia before meiosis. In transgenic Msh2-deficient mice, germinal instability of the CTG repeats (only contractions) also occurs premeiotically. No significant difference in mosaicism was detected between spermatogonia and spermatozoa, arguing against continued expansions during postmeiotic stages. This indicates that germinal expansions are produced at the beginning of spermatogenesis, in spermatogonia, by a meiosis-independent mechanism involving MSH2.

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

2019 ◽  
Author(s):  
Gustavo A. Ruiz Buendía ◽  
Marion Leleu ◽  
Flavia Marzetta ◽  
Ludovica Vanzan ◽  
Jennifer Y. Tan ◽  
...  
Keyword(s):  
Neurological Disorders ◽  
Three Dimensional ◽  
Repeat Expansion ◽  
Ctcf Binding ◽  
Chromatin Interaction ◽  
Ctg Repeats ◽  
Repeat Tract ◽  
Chromatin Interactions ◽  
Chromatin Folding ◽  
Ctg Repeat

AbstractExpanded CAG/CTG repeats underlie thirteen neurological disorders, including myotonic dystrophy (DM1) and Huntington’s disease (HD). Upon expansion, CAG/CTG repeat loci acquire heterochromatic characteristics. This observation raises the hypothesis that repeat expansion provokes changes to higher order chromatin folding and thereby affects both gene expression in cis and the genetic instability of the repeat tract. Here we tested this hypothesis directly by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD patient-derived cells. Surprisingly, chromatin contacts remain unchanged upon repeat expansion at both loci. This was true for loci with different DNA methylation levels and CTCF binding. Repeat sizes ranging from 15 to 1,700 displayed strikingly similar chromatin interaction profiles. Our findings argue that extensive changes in heterochromatic properties are not enough to alter chromatin folding at expanded CAG/CTG repeat loci. Moreover, the ectopic insertion of an expanded repeat tract did not change three-dimensional chromatin contacts. We conclude that expanded CAG/CTG repeats have little to no effect on chromatin conformation.

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 Increased Instability of Human CTG Repeat Tracts on Yeast Artificial Chromosomes during Gametogenesis

1999 ◽  
Vol 19 (6) ◽  
pp. 4153-4158 ◽  
Author(s):  
Haim Cohen ◽  
Dorothy D. Sears ◽  
Drora Zenvirth ◽  
Philip Hieter ◽  
Giora Simchen
Keyword(s):  
Genetic Analysis ◽  
Trinucleotide Repeat ◽  
Mitotic Division ◽  
Human Diseases ◽  
Ctg Repeats ◽  
Artificial Chromosomes ◽  
Repeat Tract ◽  
Yeast Artificial Chromosomes ◽  
Dmpk Gene ◽  
Ctg Repeat

ABSTRACT Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases. The mechanism and timing of the expansion events are poorly understood, however. We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable. The instability is 6 to 10 times more pronounced in meiosis than during mitotic division. The influence of meiosis on instability is 4.4 times greater when the second YAC with a repeat tract is not present. Most of the changes we observed in trinucleotide repeat tracts are large contractions of 21 to 50 repeats. The orientation of the insert with the repeats has no effect on the frequency and distribution of the contractions. In our experiments, expansions were found almost exclusively during gametogenesis. Genetic analysis of segregating markers among meiotic progeny excluded unequal crossover as the mechanism for instability. These unique patterns have novel implications for possible mechanisms of repeat instability.

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 Expansion of trinucleotide CTG repeats in the TCF4 gene as a marker of fuchs’ endothelial corneal dystrophy

2019 ◽  
Vol 12 (2) ◽  
pp. 11-18
Author(s):  
Sanasar S. Papanyan ◽  
Sergey Yu. Astakhov ◽  
Vladimir D. Nazarov ◽  
Sergey V. Lapin ◽  
Sergey A. Novikov ◽  
...  
Keyword(s):  
Progressive Disease ◽  
Trinucleotide Repeat ◽  
Corneal Edema ◽  
Corneal Dystrophy ◽  
Triplet Repeat ◽  
Triplet Repeats ◽  
Ctg Repeats ◽  
Laboratory Marker ◽  
The Third ◽  
Ctg Repeat

Fuchs’ endothelial corneal dystrophy (FECD) is an inherited severe and progressive disease, characte­rized by endothelial cell density decrease and increasing corneal edema. FECD development may be linked to expanded trinucleotide repeat, CTG, in the third intron of the TCF4 gene. The study focuses on estimating the prevalence of expanded CTG repeat in TCF4 gene in the Russian population, in patients with normal cornea and in patients with FECD (by applying triplet repeat PCR technique and capillary electrophoresis). 51 patients with FECD and 38 patients with normal cornea were examined. The estimation of the number of CTG triplet repeats in TCF4 gene determination is the veracious laboratory marker of FECD.

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