Genome instability | EPFL Graph Search

Genome instability (also genetic instability or genomic instability) refers to a high frequency of mutations within the genome of a cellular lineage. These mutations can include changes in nucleic acid sequences, chromosomal rearrangements or aneuploidy. Genome instability does occur in bacteria. In multicellular organisms genome instability is central to carcinogenesis, and in humans it is also a factor in some neurodegenerative diseases such as amyotrophic lateral sclerosis or the neuromuscular disease myotonic dystrophy. The sources of genome instability have only recently begun to be elucidated. A high frequency of externally caused DNA damage can be one source of genome instability since DNA damage can cause inaccurate translesion DNA synthesis past the damage or errors in repair, leading to mutation. Another source of genome instability may be epigenetic or mutational reductions in expression of DNA repair genes. Because endogenous (metabolically-caused) DNA damage is very frequ

Genomic Instability Profiles at the Single Cell Level in Mouse Colorectal Cancers of Defined Genotypes

Maxim Norkin, Paloma Ordonez Moran

The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with Apclox/lox, LSL-KrasG12D, and Tp53lox/lox targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.

2021

Functional analysis of the telomeric long non-coding RNA TERRA

Patricia Renck Nunes

Telomeres are nucleoprotein structures present in the end of linear chromosomes. In humans, a core complex of 6 proteins cap and protect the chromosome ends from being recognized as double-strand breaks and eliciting unwanted DNA damage and repair responses, which lead to chromosomal end-to-end fusions and genomic instability. In addition to the shelterins, another 200 proteins are detected at telomeres by mass spectrometry. Their functions at telomere are not yet completely understood. Telomeres are transcribed into a long non-coding RNA called TERRA. It is believed that subtelomeric CpG islands act as TERRA promoters. TERRA is suggested to regulate telomerase activity, to participate in the processing of uncapped telomeres, in telomeric heterochromatin formation and in promoting recombination-mediated telomere elongation. TERRA levels are increased in patients affected by the ICF (immunodeficiency, centromeric instability, facial anomalies) syndrome. Since these patients also present very short telomeres, it has been suggested that TERRA regulates telomere length, either by inhibiting telomerase or by promoting access to end-attacking nucleases. We used HCT116 DNMT1 (deletion of exons3-5/exons3-5) DNMT3B (-/-) (DKO) cells as a model for the ICF syndrome to try to comprehend the relationship between TERRA expression and telomere length. In this thesis, we try to understand (1) if there is a direct correlation between TERRA expression and telomere length, (2) what drives TERRA transcription, (3) what are the consequences of TERRA overexpression, and (4) what could cause the short telomere phenotype in DKO cells. We show that not all telomeres overexpress TERRA in DKO cells, but even telomeres with normal TERRA expression are short. By analyzing subtelomeric sequences, we found that the presence and density of subtelomeric CpG islands determine if its transcript will be upregulated in DKO cells or not. We used absolute quantification methods to demonstrate that TERRA is expressed from multiple chromosome ends, disproving a recent proposal that TERRA stems only from one locus. We also discovered that CpG-negative subtelomeres express TERRA in levels similar to CpG-positive ones, suggesting that the presence of subtelomeric CpG-islands is not a requirement for high TERRA expression. We next evaluated the telomere length dynamics in DKO cells. We found that DKO cells show lower telomere elongation rates than the WT. Moreover, DKO cells fail to downregulate TERRA in S-phase, in contrast to WT HCT116 and HeLa cells. The downregulation of TERRA in late S-phase has been proposed to alleviate TERRA-dependent telomerase inhibition and thereby allow the enzyme to extend short telomeres. To better understand TERRA transcription, we performed a siRNA screen to discover new TERRA transcription regulators. We found ZNF148, ZFX, PLAG1 and EGR1 to be new repressors of TERRA transcription. Finally, we established a CRISPR activation system to induce overexpression of endogenous TERRA. Overexpression of 10q and 13q TERRA in HCT116 did not lead to telomere shortening in cis. In HeLa cells, overexpression of multiple TERRA species led to deposition of the heterochromatin marks H3K9me3 and H4K20me3 in the telomeres. These marks were also enriched in subtelomeric sequences in cis. Since TERRA has been shown to interact with SUV39H1 to ensure H3K9me3 deposition, we speculate that TERRA also recruits SUV420H2 to induce H4K20me3 accumulation.

EPFL2019

CSL controls telomere maintenance and genome stability in human dermal fibroblasts

Paris Jafari, Joachim Lingner, Thomas Frédéric Lunardi

Genomic instability is a hallmark of cancer. Whether it also occurs in Cancer Associated Fibroblasts (CAFs) remains to be carefully investigated. Loss of CSL/RBP-J kappa, the effector of canonical NOTCH signaling with intrinsic transcription repressive function, causes conversion of dermal fibroblasts into CAFs. Here, we find that CSL down-modulation triggers DNA damage, telomere loss and chromosome end fusions that also occur in skin Squamous Cell Carcinoma (SCC)-associated CAFs, in which CSL is decreased. Separately from its role in transcription, we show that CSL is part of a multiprotein telomere protective complex, binding directly and with high affinity to telomeric DNA as well as to UPF1 and Ku70/Ku80 proteins and being required for their telomere association. Taken together, the findings point to a central role of CSL in telomere homeostasis with important implications for genomic instability of cancer stromal cells and beyond.

Springer2019

Functional analysis of the telomeric long non-coding RNA TERRA

Patricia Renck Nunes

EPFL2019