Probing the kinetic landscape of Hox transcription factor–DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy

Papadopoulos, Dimitrios K.; Krmpot, Aleksandar J.; Nikolić, Stanko N.; Krautz, Robert; Terenius, Lars; Tomančák, Pavel; Rigler, Rudolf; Gehring, Walter; Vukojević, Vladana

doi:10.1016/j.mod.2015.09.004

Cited by 16 publications

(20 citation statements)

References 56 publications

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“…4c, d). We next performed Fluorescence Cross-Correlation Spectroscopy (FCCS) 33,34 in live HeLa cells stably co-expressing RFP-PCNA and GFP-DONSON, to measure the degree of co-diffusion of these molecules. Significantly increased co-diffusion of PCNA and DONSON was observed in S-phase PCNA foci, but not in nuclei of non-replicating cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

et al. 2017

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To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication, and protect, repair and restart damaged forks. Here we identify DONSON as a novel fork protection factor, and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilises forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype. In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

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Section: Resultsmentioning

confidence: 99%

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

et al. 2017

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“…Through simultaneous measurement, heterogeneities present between locations become much more obvious due to the high-degree of temporal synchronisation allowing accurate comparison. Simultaneous measurements can be achieved through camera-based (image correlation spectroscopy, ICS) [10] or multi-spot approaches [11] , [12] , [13] . While the spatial awareness of these approaches have allowed the study of a wide range of cellular processes, especially through there further development (e.g.…”

Section: Introductionmentioning

confidence: 99%

Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data

Waithe

Schneider

Chojnacki

et al. 2018

Methods

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“…To answer this question, and to characterize protein mobility in different chromatin regions, several strategies have been proposed that add spatial information to the single-point FCS measurement. Maps of diffusion coefficients have been obtained, for instance, by sequential acquisition of single-point FCS measurements (21, 22), by light-sheet illumination (23, 24) or by parallel acquisition of FCS data at multiple observation volumes (25, 26). Another method that has been used to measure fluctuations at, and between, different points in the nucleus is scanning FCS, which is easily implemented on confocal laser scanning microscopes, but whose temporal resolution is typically limited, by scanning, to the millisecond range (27, 28).…”

Section: Introductionmentioning

confidence: 99%

Measuring mobility in chromatin by intensity sorted FCS

Bona

Mancini

Mazza

et al. 2018

Preprint

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6The architectural organization of chromatin can play an important role in genome regulation 7 by affecting the mobility of molecules within its surroundings via binding interactions and 8 molecular crowding. The diffusion of molecules at specific locations in the nucleus can be 9 studied by Fluorescence Correlation Spectroscopy (FCS), a well-established technique based 10 on the analysis of fluorescence intensity fluctuations detected in a confocal observation 11 volume. However, detecting subtle variations of mobility between different chromatin regions 12 remains challenging with currently-available FCS methods. 13 Here we introduce a method that samples multiple positions by slowly scanning the FCS 14 observation volume across the nucleus. Analyzing the data in short time segments, we 15 preserve the high temporal resolution of single-point FCS while probing different nuclear 16 regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a 17 reference, we efficiently sort the FCS segments into different populations and obtain average 18 correlation functions that are associated to different chromatin regions. This sorting and 19 averaging strategy renders the method statistically robust while preserving the observation of 20 intranuclear variations of mobility. 21Using this approach, we quantified diffusion of monomeric GFP in high versus low 22 chromatin density regions. We found that GFP mobility was reduced in heterochromatin, 23 especially within perinucleolar heterochromatin. Moreover, we found that modulation of 24 chromatin compaction by ATP depletion, or treatment with solution of different osmolarity, 25 differentially-affected the ratio of diffusion in both regions. Then, we used the approach to 26 probe the mobility of estrogen receptor-α (ER) in the vicinity of an integrated multicopy 27 prolactin gene array. Finally, we discussed the coupling of this method with stimulated 28 emission depletion (STED)-FCS, for performing FCS at sub-diffraction spatial scales. 29 30 31 32 33 34 74data at multiple observation volumes (25, 26). Another method that has been used to measure 75 fluctuations at, and between, different points in the nucleus is scanning FCS, which is easily 76 implemented on confocal laser scanning microscopes, but whose temporal resolution is 77 typically limited, by scanning, to the millisecond range (27, 28). Finally, diffusion maps have 78 been recently obtained from the analysis of raster image correlation spectroscopy (RICS) data 79 (29). However, these methods will only provide an accurate description of the mobility 80 properties in different nuclear regions if they are immobile during FCS data acquisition. 81A significant advantage can be gained if the different chromatin regions are identified with 82 the help of a reference marker (30). For instance, the intensity of a fluorescent protein can be 83 used to identify specific sub-nuclear regions or, simply, the intensity of a DNA marker (e.g. 84Hoechst) can be used to identify regions of different ch...

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Probing the kinetic landscape of Hox transcription factor–DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy

Cited by 16 publications

References 56 publications

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data

Measuring mobility in chromatin by intensity sorted FCS

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