31 research outputs found
Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations
It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5′ splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations
Cell cycle-dependent resolution of DNA double-strand breaks
DNA double strand breaks (DSBs) elicit prompt activation of DNA damage response (DDR), which arrests cell-cycle either in G1/S or G2/M in order to avoid entering S and M phase with damaged DNAs. Since mammalian tissues contain both proliferating and quiescent cells, there might be fundamental difference in DDR between proliferating and quiescent cells (or G0-arrested). To investigate these differences, we studied recruitment of DSB repair factors and resolution of DNA lesions induced at site-specific DSBs in asynchronously proliferating, G0-, or G1-arrested cells. Strikingly, DSBs occurring in G0 quiescent cells are not repaired and maintain a sustained activation of the p53-pathway. Conversely, re-entry into cell cycle of damaged G0-arrested cells, occurs with a delayed clearance of DNA repair factors initially recruited to DSBs, indicating an inefficient repair when compared to DSBs induced in asynchronously proliferating or G1-synchronized cells. Moreover, we found that initial recognition of DSBs and assembly of DSB factors is largely similar in asynchronously proliferating, G0-, or G1-synchronized cells. Our study thereby demonstrates that repair and resolution of DSBs is strongly dependent on the cell-cycle state
Genome-wide mapping of 8-oxo-7,8-dihydro-2'-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells
8-Oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is
one of the major DNA modifications and a potent
pre-mutagenic lesion prone to mispair with 2-
deoxyadenosine (dA). Several thousand residues of
8-oxodG are constitutively generated in the genome
of mammalian cells, but their genomic distribution
has not yet been fully characterized. Here, by
using OxiDIP-Seq, a highly sensitive methodology
that uses immuno-precipitation with efficient anti–
8-oxodG antibodies combined with high-throughput
sequencing, we report the genome-wide distribution
of 8-oxodG in human non-tumorigenic epithelial
breast cells (MCF10A), and mouse embryonic
fibroblasts (MEFs). OxiDIP-Seq revealed sites of 8-
oxodG accumulation overlapping with H2AX ChIPSeq
signals within the gene body of transcribed long
genes, particularly at the DNA replication origins
contained therein. We propose that the presence of
persistent single-stranded DNA, as a consequence
of transcription-replication clashes at these sites, determines
local vulnerability to DNA oxidation and/or
its slow repair. This oxidatively-generated damage,
likely in combination with other kinds of lesion, might
contribute to the formation of DNA double strand
breaks and activation of DNA damage response
In Vivo Functional Platform Targeting Patient-Derived Xenografts Identifies WDR5-Myc Association as a Critical Determinant of Pancreatic Cancer
SummaryCurrent treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1–4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease
An Image-Based Approach to the Evaluation of Oncogene Activation Effects on Cell's Genomic Stability
Optimized Super-Resolution Imaging of Nuclear Sites in an Engineered Leukemia Cell Line
Stimulated emission depletion (STED) microscopy and Separation of Photons by Lifetime Tuning (SPLIT) provide optical super-resolution imaging, at the nanometer scale, on intact single cell nuclei. The resolution that can be achieved is limited by factors such as photobleaching and/or reduction of signal-to-noise, which depend on the conditions of image acquisition.
Here we use image correlation spectroscopy (ICS) to quantify, in an unbiased way, the resolution and signal-to-noise of STED and SPLIT images and determine how they are affected by multiple parameters such as photobleaching, STED power, and number of averages. Imaging conditions optimization is particularly relevant when it comes to analyzing genomic processes, such as DNA replication, occurring within the crowded nuclear environment. As an application, we optimize the imaging conditions on an engineered cell line, U937-PR9, derived \u202
Structured Illumination Microscopy as a Tool to Investigate Oncogene-Induced Alterations in Chromatin Organization
Alterations induced by the PML-RARα oncogene revealed by image cross correlation spectroscopy
The molecular mechanisms that underlie oncogene-induced genomic damage are still poorly understood. To un-derstand how oncogenes affect chromatin architecture, it is important to visualize fundamental processes such as DNA replica-tion and transcription in intact nuclei and quantify the alterations of their spatiotemporal organization induced by oncogenes. Here, we apply superresolution microscopy in combination with image cross correlation spectroscopy to the U937-PR9 cell line, an in vitro model of acute promyelocytic leukemia that allows us to activate the expression of the PML-RARa oncogene and analyze its effects on the spatiotemporal organization of functional nuclear processes. More specifically, we perform Tau-stimulated emission depletion imaging, a superresolution technique based on the concept of separation of photons by life-time tuning. Tau-stimulated emission depletion imaging is combined with a robust image analysis protocol that quickly produces a value of colocalization fraction on several hundreds of single cells and allows observation of cell-to-cell variability. Upon acti-vation of the oncogene, we detect a significant increase in the fraction of transcription sites colocalized with PML/PML-RARa. This increase of colocalization can be ascribed to oncogene-induced disruption of physiological PML bodies and the abnormal occurrence of a relatively large number of PML-RARa microspeckles. We also detect a significant cell-to-cell variability of this increase of colocalization, which can be ascribed, at least in part, to a heterogeneous response of the cells to the activation of the oncogene. These results prove that our method efficiently reveals oncogene-induced alterations in the spatial organization of nuclear processes and suggest that the abnormal localization of PML-RARa could interfere with the transcription machinery, potentially leading to DNA damage and genomic instability.SIGNIFICANCE The molecular mechanisms that underlie oncogene-induced genomic damage are still poorly understood. Here, we combine image cross correlation spectroscopy with state-of-the-art superresolution microscopy to quantify alterations induced by the PML-RARa oncogene in an in vitro model of acute promyelocytic leukemia. We find that activation of the oncogene induces a significant increase in the fraction of transcription sites colocalized with PML/PML-RARa due to disruption of physiological PML bodies into a large number of PML-RARa microspeckles. We also detect a heterogeneous response of the cells to the activation of the oncogene. These results suggest that our image cross correlation spectroscopy-based approach can be useful for characterizing global alterations in the spatial organization of chromatin, in single cells, in response to oncogene activation, or in other triggering events
Sequence-specific double strand breaks trigger P-TEFb-dependent Rpb1-CTD hyperphosphorylation.
Double strand DNA breaks (DSBs) are one of the most challenging forms of DNA damage which, if left unrepaired, can trigger cellular death and can contribute to cancer. A number of studies have been focused on DNA-damage response (DDR) mechanisms, and most of them rely on the induction of DSBs triggered by chemical compounds or radiations. However, genotoxic drugs and radiation treatments of cultured cell lines induce random DSBs throughout the genome, thus heterogeneously across the cell population, leading to variability of the cellular response. To overcome this aspect, we used here a recently described cell-based DSBs system whereby, upon induction of an inducible restriction enzyme, hundreds of site-specific DSBs are generated across the genome. We show here that sequence-specific DSBs are sufficient to activate the positive transcription elongation factor b (P-TEFb), to trigger hyperphosphorylation of the largest RNA polymerase II carboxyl-terminal-domain (Rpb1-CTD) and to induce activation of p53-transcriptional axis resulting in cell cycle arrest