Biology subjects

Lazzerini Denchi, E.

Publications and source records attributed to Lazzerini Denchi, E..

6 recordsLinked to original sources

bioRxiv2026

MUTYH activity maintains telomere stability in response to chronic telomeric 8-oxoguanine damage in cancer cells

Telomeres are highly susceptible to oxidative DNA damage, particularly 8-oxoguanine (8-oxoG), which is processed by glycosylase-initiated base excision repair (BER). OGG1 removes 8-oxoG opposite C, and MUTYH removes A misinserted opposite 8-oxoG to prevent mutations. While OGG1 has an established role in telomere protection, the contribution of MUTYH to telomere stability in cancer cells after oxidative DNA damage remains poorly understood. Using a chemoptogenetic system to induce targeted 8-oxoG lesions specifically at telomeres in HeLa cancer cells, we demonstrate that MUTYH is required to prevent telomere shortening, telomere loss, and genomic instability after chronic damage. Yet, telomere damage in MUTYH-deficient cells does not cause sustained DNA damage signaling or reduced cellular proliferation. Whole-genome sequencing further reveals enrichment of G to T transversions within telomeric repeats in MUTYH-deficient cells, consistent with increased mutagenesis due to unrepaired 8-oxoG:A mispairs. Combined loss of MUTYH and OGG1 rescues damage-induced telomere aberrations and genomic instability, implicating BER-generated single-strand break (SSB) intermediates as major contributors to telomere instability. In agreement, exo-FISH and S1-END-seq analyses reveal that repair-proficient cells rapidly accumulate SSB intermediates after damage, which are later resolved, whereas glycosylase-deficient cells exhibit SSBs at later time points. Together, these findings identify MUTYH as a critical guardian of telomere integrity during chronic oxidative stress and provide insight into how defective BER at telomeres contributes to genomic instability in cancer cells, with implications for cancers associated with MUTYH deficiency and mutations.

molecular biology↗

bioRxiv2025

Conserved and Unique Features of Terminal Telomeric Sequences in ALT-Positive Cancer Cells

A significant portion of human cancers utilize a recombination-based pathway, Alternative Lengthening of Telomeres (ALT), to maintain telomere length. Targeting the ALT is of growing interest as a cancer therapy, yet a substantial knowledge gap remains regarding the basic features of telomeres in ALT-positive cells. To address this, we adopted END-seq, an unbiased sequencing-based approach, to define the identity and regulation of the terminal sequences of chromosomes in ALT cells. Our data reveal that the terminal portions of chromosomes in ALT cells contain canonical telomeric sequences with the same 5 terminus bias (-ATC) observed in non-ALT cells. Furthermore, as reported for non-ALT cells, POT1 is required to preserve the precise regulation of the 5 end in cells that maintain telomere length using the ALT pathway. Thus, the regulation of the terminal 5 of chromosomes occurs independently of the mechanism of telomere elongation. Additionally, we employed an S1 endonuclease-based sequencing method to determine the presence and origin of single-stranded regions within ALT telomeres. These data shed light on conserved and unique features of ALT telomeres.

molecular biology↗

bioRxiv2025

Optical Pooled Screening for the Discovery of Regulators of the Alternative Lengthening of Telomeres Pathway

Telomere elongation is essential for the proliferation of cancer cells. Telomere length control is achieved by either the activation of the telomerase enzyme or the recombination-based Alternative Lengthening of Telomeres (ALT) pathway. ALT is active in about 10-15% of human cancers, but its molecular underpinnings remain poorly understood, preventing the discovery of potential novel therapeutic targets. Pooled CRISPR-based functional genomic screens enable the unbiased discovery of molecular factors involved in cancer biology. Recently, Optical Pooled Screens (OPS) have significantly extended the capabilities of pooled functional genomics screens to enable sensitive imaging-based readouts at the single cell level and large scale. To gain a better understanding of the ALT pathway, we developed a novel OPS assay that employs telomeric native DNA FISH (nFISH) as an optical quantitative readout to measure ALT activity. The assay uses standard OPS protocols for library preparation and sequencing. As a critical element, an optimized nFISH protocol is performed before in situ sequencing to maximize the assay performance. We show that the modified nFISH protocol faithfully detects changes in ALT activity upon CRISPR knock-out (KO) of the FANCM and BLM genes which were previously implicated in ALT. Overall, the OPS-nFISH assay is a reliable method that can provide deep insights into the ALT pathway in a high-throughput format.

cell biology↗

bioRxiv2024

Identification of Novel Modulators of the ALT Pathway Through a Native FISH-Based Optical Screen

A significant portion of human cancers utilize a recombination-based pathway, Alternative Lengthening of Telomeres (ALT), to extend telomeres. To gain further insights into this pathway, we developed a high-throughput imaging-based screen named TAILS (Telomeric ALT In situ Localization Screen), to identify genes that either promote or inhibit ALT activity. Screening over 1000 genes implicated in DNA transactions, TAILS revealed both well-established and novel ALT modulators. We have identified new factors that promote ALT, such as the nucleosome-remodeling factor CHD4 and the chromatin reader SGF29, as well as factors that suppress ALT, including the RNA helicases DDX39A/B, the replication factor TIMELESS, and components of the chromatin assembly factor CAF1. Our data indicate that defects in histone deposition significantly contribute to ALT-associated phenotypes. Based on these findings, we demonstrate that pharmacological treatments can be employed to either exacerbate or suppress ALT-associated phenotypes.

molecular biology↗

bioRxiv2020

Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by utilizing the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed Alternative Lengthening of Telomeres (ALT). Cells that employ ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML-Bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We find that PML is required for the ALT mechanism, and that this necessity stems from APBs role in localizing the BLM-TOP3A-RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

cancer biology↗

bioRxiv2018

POT1a depletion in the developing brain leads to p53-dependent neuronal cell death and ataxia.

The processes that control genome stability are essential for the development of the Central Nervous System (CNS) and the prevention of neurological disease. Here we investigated whether activation of a DNA damage response by dysfunctional telomeres affects neurogenesis. More specifically, we analyzed ATR-dependent DNA damage response by depletion of POT1a in neural progenitors. These experiments revealed that POT1a inactivation leads to a partially penetrant lethality, and surviving mice displayed ataxia due to loss of neuronal progenitor cells, and died by 3 weeks of age. Inactivation of p53 was sufficient to completely suppress the lethality associated with POT1a depletion and rescued the neuronal defects characterizing POT1a depleted animals. In contrast, activation of ATM by the depletion of the shelterin protein TRF2 resulted in a fully penetrant lethality that could not be rescued by p53 inactivation (Lobanova et al.). This data reveals that activation of distinct types of DNA damage response pathway give rise to different types of neuropathology. Moreover, our data provides an explanation for the heterogeneity of the neurological defects observed in patients affected by telomere biological disorders.

molecular biology↗