Quantitative detection of rare interphase chromosome breaks and translocations by high-throughput imaging

Burman, Bharat; Misteli, Tom; Pegoraro, Gianluca

doi:10.1186/s13059-015-0718-x

Cited by 21 publications

(17 citation statements)

References 27 publications

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“…Automated analysis of all images was performed based on a modified version of a previously described Acapella 2.6 (PerkinElmer) custom script [30, 33–35]. This custom script performed automated nucleus detection based on the maximal projection of the DAPI image (ex.…”

Section: Methodsmentioning

confidence: 99%

Comparative analysis of 2D and 3D distance measurements to study spatial genome organization

Finn

Pegoraro

Shachar

et al. 2017

Methods

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The spatial organization of eukaryotic genomes is non-random, cell-type specific, and has been linked to cellular function. The investigation of spatial organization has traditionally relied extensively on fluorescence microscopy. The validity of the imaging methods used to probe spatial genome organization often depends on the accuracy and precision of distance measurements. Imaging-based measurements may either use 2 dimensional datasets or 3D datasets including the z-axis information in image stacks. Here we compare the suitability of 2D versus 3D distance measurements in the analysis of various features of spatial genome organization. We find in general good agreement between 2D and 3D analysis with higher convergence of measurements as the interrogated distance increases, especially in flat cells. Overall, 3D distance measurements are more accurate than 2D distances, but are also more prone to noise. In particular, z-stacks are prone to error due to imaging properties such as limited resolution along the z-axis and optical aberrations, and we also find significant deviations from unimodal distance distributions caused by low sampling frequency in z. These deviations can be ameliorated by sampling at much higher frequency in the z-direction. We conclude that 2D distances are preferred for comparative analyses between cells, but 3D distances are preferred when comparing to theoretical models in large samples of cells. In general, 2D distance measurements remain preferable for many applications of analysis of spatial genome organization.

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Section: Methodsmentioning

confidence: 99%

Comparative analysis of 2D and 3D distance measurements to study spatial genome organization

Finn

Pegoraro

Shachar

et al. 2017

Methods

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“…To estimate the frequency of ETO-induced MLL fusions, we developed C-Fusion 3D, a highthroughput imaging-based methodology to probe chromosome breakage and rare translocations in individual cells with high sensitivity. Built upon the rationale of high-throughput break-apart fluorescence in situ hybridization (FISH) (Burman et al, 2015a;Roukos et al, 2013), C-Fusion 3D detects individual cells with chromosome breakage and/or fusions by measuring the extent of spatial separation or colocalization of individual chromosome breaks in 3D (Figure 2A). We found that C-Fusion 3D can detect individual cells with chromosome translocations at frequencies down to 10 -4 (data not shown).…”

Section: Mll Fusions Form Rarely In Hematopoietic Cell Lines and Progmentioning

confidence: 99%

“…FISH spots were identified in all channels in maximal projected images by using the spot detection algorithm C. Distances between all different spots of all different channels were calculated in 2D by using custom-made building blocks, while the z plane having the maximum pixel intensity of each spot was identified. Single cell information was then exported as text files and custom made scripts in R developed based on previous versions (Burman et al, 2015a), were used to calculate the Euclidean distances between different spots in 3D and to calculate frequencies of cells with chromosome breakage and/or translocations. Correction of shifts due to chromatic aberration in z was performed by defining the pairwise shifts in z between all the different channels measured on images of a stained genomic locus by FISH using a single BAC labeled with all different color fluorophores.…”

Section: C-fusion 3dmentioning

confidence: 99%

“…The value of z plane showing the maximum pixel intensity of each spot in every channel was corrected according the identified shift between the different channels in z. To determine the thresholds of physical separation and proximity, which are indicative of chromosome damage and intact loci respectively (Burman et al, 2015a;Burman et al, 2015b), we plotted the distribution of intrachromosomal distances (Green/Red and FarRed/Blue distances) in non-damaged control cells. We established a threshold of separation/proximity 4 pixels (corresponds to 1.2 µm when images were acquired with 2x binning).…”

Section: C-fusion 3dmentioning

confidence: 99%

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Spatial chromosome folding and active transcription drive DNA fragility and formation of oncogenic MLL translocations

Gothe

Bouwman

Gusmao

et al. 2018

Preprint

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How spatial chromosome organization influences genome integrity is still poorly understood.Here we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities, are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CTCF/cohesin bound sites at the bases of chromatin loops and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias, are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hot spots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability, and are key contributors to the occurrence of genome rearrangements that drive cancer. Reimand et al., 2016) and topological constraints during loop extrusion dynamics (Canela et al., 2017).Although physiologically important, TOP2 functions are also inherently risky for a cell, as they involve the formation of a transient DSB to allow the passage of duplex DNA through the break. During this controlled process, the two-TOP2 subunits are covalently linked through their active sites to each 5′-terminus of a DSB via a phosphodiester bond (Pommier et al., 2016).These key intermediates of TOP2 activity, called TOP2 cleavage complexes (TOP2ccs) are normally short-lived, as topoisomerases quickly ligate the DNA ends upon the passage of the duplex DNA. In the presence of nearby DNA lesions or upon treatment with topoisomerase poisons, however, these cleavage complexes can be stabilized on DNA and may contribute to genomic instability by acting as road blocks to DNA-tracking systems that attempt to traverse them (Ashour et al., 2015).The mechanism by which TOP2ccs are converted to DSBs underlies the action of widely used class of anticancer agents that trap TOP2 at the intermediate cleavage complex step (Nitiss, 2009). The TOP2 poison etoposide (ETO), is among the most effective and widely used anticancer agents in the clinic, but treatment with etoposide is associated with the occurrence of therapy-related acute myeloid leukemias (t-AMLs) (Allan and Travis, 2005; Wright and Vaughan, 2014). Approximately one third of t-AML cases are associated with recurrent chromosome translocations between the mixed lineage leukemia gene (MLL) and various potential translocation partners, and amongst them, AF9, AF4, AF6, and ENL, account for approximately 80% of the cases. Sequencing of MLL fusions from patients with t-AML has shown that MLL breakpoints occur in breakpoint cluster regions (BCRs) at restricted genomic positions near exon 12 of the MLL gene, while BCRs within potential translocation partners are ENL fusions two times more frequently than MLL-AF4, MLL-AF6, MLL-AF9 ( Figure 2D), although the frequency of ENL breakage was not signi...

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“…Great strides have been made in the realm of high-content DNA FISH microscopy approaches (hiFISH), which utilize automated image acquisition and analysis tools to interrogate DNA probe libraries. 21,29,30 Potentially, this approach can accurately quantify gene pairing events that occur across larger linear genomic distances and between chromosomes at high-resolution but current highcontent microscopy experiments are still limited to only 4 distinct fluorophores for visualization (one of which must be dedicated to a nuclear stain for automated image analysis). Therefore, current methods are inadequate for the co-visualization of multiple genes in conjunction with co-detection of chromosome territories, sub-nuclear structures and/ or distinct epigenetic marks.…”

Section: Introductionmentioning

confidence: 99%

Spectral imaging to visualize higher-order genomic organization

Sawyer

Shevtsov

Dundr

2016

Nucleus

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A concern in the field of genomics is the proper interpretation of large, high-throughput sequencing datasets. The use of DNA FISH followed by high-content microscopy is a valuable tool for validation and contextualization of frequently occurring gene pairing events at the single-cell level identified by deep sequencing. However, these techniques possess certain limitations. Firstly, they do not permit the study of colocalization of many gene loci simultaneously. Secondly, the direct assessment of the relative position of many clustered gene loci within their respective chromosome territories is impossible. Thus, methods are required to advance the study of higher-order nuclear and cellular organization. Here, we describe a multiplexed DNA FISH technique combined with indirect immunofluorescence to study the relative position of 6 distinct genomic or cellular structures. This can be achieved in a single hybridization step using spectral imaging during image acquisition and linear unmixing. Here, we detail the use of this method to quantify gene pairing between highly expressed spliceosomal genes and compare these data to randomly positioned in silico simulated gene clusters. This is a potentially universally applicable approach for the validation of 3C-based technologies, deep imaging of spatial organization within the nucleus and global cellular organization.

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Quantitative detection of rare interphase chromosome breaks and translocations by high-throughput imaging

Cited by 21 publications

References 27 publications

Comparative analysis of 2D and 3D distance measurements to study spatial genome organization

Comparative analysis of 2D and 3D distance measurements to study spatial genome organization

Spatial chromosome folding and active transcription drive DNA fragility and formation of oncogenic MLL translocations

Spectral imaging to visualize higher-order genomic organization

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