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Image-based modeling reveals dynamic redistribution of DNA damage into nuclear sub-domains.

Costes SV, Ponomarev A, Chen JL, Nguyen D, Cucinotta FA, Barcellos-Hoff MH - PLoS Comput. Biol. (2007)

Bottom Line: This deviation from the expected DNA-weighted random pattern can be further characterized by "relative DNA image measurements." This novel imaging approach shows that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent than predicted in regions with lower DNA density.These data suggest that DNA damage-induced foci are restricted to certain regions of the nucleus of human epithelial cells.It is possible that DNA lesions are collected in these nuclear sub-domains for more efficient repair.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America. svcostes@lbl.gov

ABSTRACT
Several proteins involved in the response to DNA double strand breaks (DSB) form microscopically visible nuclear domains, or foci, after exposure to ionizing radiation. Radiation-induced foci (RIF) are believed to be located where DNA damage occurs. To test this assumption, we analyzed the spatial distribution of 53BP1, phosphorylated ATM, and gammaH2AX RIF in cells irradiated with high linear energy transfer (LET) radiation and low LET. Since energy is randomly deposited along high-LET particle paths, RIF along these paths should also be randomly distributed. The probability to induce DSB can be derived from DNA fragment data measured experimentally by pulsed-field gel electrophoresis. We used this probability in Monte Carlo simulations to predict DSB locations in synthetic nuclei geometrically described by a complete set of human chromosomes, taking into account microscope optics from real experiments. As expected, simulations produced DNA-weighted random (Poisson) distributions. In contrast, the distributions of RIF obtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) were non-random. This deviation from the expected DNA-weighted random pattern can be further characterized by "relative DNA image measurements." This novel imaging approach shows that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent than predicted in regions with lower DNA density. The same preferential nuclear location was also measured for RIF induced by 1 Gy of low-LET radiation. This deviation from random behavior was evident only 5 min after irradiation for phosphorylated ATM RIF, while gammaH2AX and 53BP1 RIF showed pronounced deviations up to 30 min after exposure. These data suggest that DNA damage-induced foci are restricted to certain regions of the nucleus of human epithelial cells. It is possible that DNA lesions are collected in these nuclear sub-domains for more efficient repair.

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Related in: MedlinePlus

Illustration of Rdna and Rgrad MeasurementsThree hypothetical foci patterns over the same nucleus are illustrated with their corresponding Rdna and Rgrad values. Upper images (A,C,E) are overlays of the DAPI image with the center of hypothetical foci (in red). Lower images (B,D,F) are overlays of the foci location with the gradient image of DAPI. The gradient operator is often used in imaging as an edge detector. To illustrate this, the green arrow in (C) delineates the contour of the edge of a bright DAPI region. One can see in the corresponding gradient image in (D) that the same contour correlates to a bright gradient region. Rdna measures the ratio of the mean nuclear intensity at the foci locations over the mean intensity of the full nucleus. Rgrad measures the same ratio on the gradient image. Because the boundary of the nuclear image creates a strong gradient intensity, a conservative contour is used for nuclear segmentation (shown in blue) to avoid an edge effect when calculating Rdna and Rgrad. In (A) and (B), foci are placed in areas of surrounding high nuclear density. The surrounding high density keeps the foci distal from areas of density change, thus we see the foci lie in low-intensity regions in the corresponding gradient image. This results in Rdna above 1 and Rgrad below 1. By manually placing foci at different locations with respect to DNA density regions, we show that Rdna is high when foci are located in bright regions of the nucleus (A) and (B); Rgrad is high when foci are located at the interface of bright and dim regions of the nucleus (C) and (D); and Rdna is low when foci are located in dim regions of the nucleus (E) and (F).
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pcbi-0030155-g006: Illustration of Rdna and Rgrad MeasurementsThree hypothetical foci patterns over the same nucleus are illustrated with their corresponding Rdna and Rgrad values. Upper images (A,C,E) are overlays of the DAPI image with the center of hypothetical foci (in red). Lower images (B,D,F) are overlays of the foci location with the gradient image of DAPI. The gradient operator is often used in imaging as an edge detector. To illustrate this, the green arrow in (C) delineates the contour of the edge of a bright DAPI region. One can see in the corresponding gradient image in (D) that the same contour correlates to a bright gradient region. Rdna measures the ratio of the mean nuclear intensity at the foci locations over the mean intensity of the full nucleus. Rgrad measures the same ratio on the gradient image. Because the boundary of the nuclear image creates a strong gradient intensity, a conservative contour is used for nuclear segmentation (shown in blue) to avoid an edge effect when calculating Rdna and Rgrad. In (A) and (B), foci are placed in areas of surrounding high nuclear density. The surrounding high density keeps the foci distal from areas of density change, thus we see the foci lie in low-intensity regions in the corresponding gradient image. This results in Rdna above 1 and Rgrad below 1. By manually placing foci at different locations with respect to DNA density regions, we show that Rdna is high when foci are located in bright regions of the nucleus (A) and (B); Rgrad is high when foci are located at the interface of bright and dim regions of the nucleus (C) and (D); and Rdna is low when foci are located in dim regions of the nucleus (E) and (F).

Mentions: Given the deviation of RIF from random distribution over time, we can then ask if foci relocate in regions of the nucleus with specific morphological features. To do so, we introduce a set of imaging parameters that ascertains the position of foci with respect to DNA density. This set of parameters can also be measured in any spatial dimension (i.e., line profiles, surfaces, or volumes). Figure 6 illustrates the approach on a given nucleus (i.e., center slice of a nucleus—DAPI stain). Using automatic spot detection (see Materials and Methods), we consider the center of RIF as the brightest pixel in its vicinity. One can then compute the mean DNA density signal at the centers of all RIF in one nucleus and normalize it to the mean nuclear DNA density to get a relative DNA density value at these locations. We thus define the relative density of DNA at the foci locations as follows:where, I(i) is the intensity at pixel location i, Nfocus is the number of foci, and Nnucleus is the number of pixels in the nucleus (note, I = focus refers to the brightest pixel in an identified focus).


Image-based modeling reveals dynamic redistribution of DNA damage into nuclear sub-domains.

Costes SV, Ponomarev A, Chen JL, Nguyen D, Cucinotta FA, Barcellos-Hoff MH - PLoS Comput. Biol. (2007)

Illustration of Rdna and Rgrad MeasurementsThree hypothetical foci patterns over the same nucleus are illustrated with their corresponding Rdna and Rgrad values. Upper images (A,C,E) are overlays of the DAPI image with the center of hypothetical foci (in red). Lower images (B,D,F) are overlays of the foci location with the gradient image of DAPI. The gradient operator is often used in imaging as an edge detector. To illustrate this, the green arrow in (C) delineates the contour of the edge of a bright DAPI region. One can see in the corresponding gradient image in (D) that the same contour correlates to a bright gradient region. Rdna measures the ratio of the mean nuclear intensity at the foci locations over the mean intensity of the full nucleus. Rgrad measures the same ratio on the gradient image. Because the boundary of the nuclear image creates a strong gradient intensity, a conservative contour is used for nuclear segmentation (shown in blue) to avoid an edge effect when calculating Rdna and Rgrad. In (A) and (B), foci are placed in areas of surrounding high nuclear density. The surrounding high density keeps the foci distal from areas of density change, thus we see the foci lie in low-intensity regions in the corresponding gradient image. This results in Rdna above 1 and Rgrad below 1. By manually placing foci at different locations with respect to DNA density regions, we show that Rdna is high when foci are located in bright regions of the nucleus (A) and (B); Rgrad is high when foci are located at the interface of bright and dim regions of the nucleus (C) and (D); and Rdna is low when foci are located in dim regions of the nucleus (E) and (F).
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Related In: Results  -  Collection

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pcbi-0030155-g006: Illustration of Rdna and Rgrad MeasurementsThree hypothetical foci patterns over the same nucleus are illustrated with their corresponding Rdna and Rgrad values. Upper images (A,C,E) are overlays of the DAPI image with the center of hypothetical foci (in red). Lower images (B,D,F) are overlays of the foci location with the gradient image of DAPI. The gradient operator is often used in imaging as an edge detector. To illustrate this, the green arrow in (C) delineates the contour of the edge of a bright DAPI region. One can see in the corresponding gradient image in (D) that the same contour correlates to a bright gradient region. Rdna measures the ratio of the mean nuclear intensity at the foci locations over the mean intensity of the full nucleus. Rgrad measures the same ratio on the gradient image. Because the boundary of the nuclear image creates a strong gradient intensity, a conservative contour is used for nuclear segmentation (shown in blue) to avoid an edge effect when calculating Rdna and Rgrad. In (A) and (B), foci are placed in areas of surrounding high nuclear density. The surrounding high density keeps the foci distal from areas of density change, thus we see the foci lie in low-intensity regions in the corresponding gradient image. This results in Rdna above 1 and Rgrad below 1. By manually placing foci at different locations with respect to DNA density regions, we show that Rdna is high when foci are located in bright regions of the nucleus (A) and (B); Rgrad is high when foci are located at the interface of bright and dim regions of the nucleus (C) and (D); and Rdna is low when foci are located in dim regions of the nucleus (E) and (F).
Mentions: Given the deviation of RIF from random distribution over time, we can then ask if foci relocate in regions of the nucleus with specific morphological features. To do so, we introduce a set of imaging parameters that ascertains the position of foci with respect to DNA density. This set of parameters can also be measured in any spatial dimension (i.e., line profiles, surfaces, or volumes). Figure 6 illustrates the approach on a given nucleus (i.e., center slice of a nucleus—DAPI stain). Using automatic spot detection (see Materials and Methods), we consider the center of RIF as the brightest pixel in its vicinity. One can then compute the mean DNA density signal at the centers of all RIF in one nucleus and normalize it to the mean nuclear DNA density to get a relative DNA density value at these locations. We thus define the relative density of DNA at the foci locations as follows:where, I(i) is the intensity at pixel location i, Nfocus is the number of foci, and Nnucleus is the number of pixels in the nucleus (note, I = focus refers to the brightest pixel in an identified focus).

Bottom Line: This deviation from the expected DNA-weighted random pattern can be further characterized by "relative DNA image measurements." This novel imaging approach shows that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent than predicted in regions with lower DNA density.These data suggest that DNA damage-induced foci are restricted to certain regions of the nucleus of human epithelial cells.It is possible that DNA lesions are collected in these nuclear sub-domains for more efficient repair.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America. svcostes@lbl.gov

ABSTRACT
Several proteins involved in the response to DNA double strand breaks (DSB) form microscopically visible nuclear domains, or foci, after exposure to ionizing radiation. Radiation-induced foci (RIF) are believed to be located where DNA damage occurs. To test this assumption, we analyzed the spatial distribution of 53BP1, phosphorylated ATM, and gammaH2AX RIF in cells irradiated with high linear energy transfer (LET) radiation and low LET. Since energy is randomly deposited along high-LET particle paths, RIF along these paths should also be randomly distributed. The probability to induce DSB can be derived from DNA fragment data measured experimentally by pulsed-field gel electrophoresis. We used this probability in Monte Carlo simulations to predict DSB locations in synthetic nuclei geometrically described by a complete set of human chromosomes, taking into account microscope optics from real experiments. As expected, simulations produced DNA-weighted random (Poisson) distributions. In contrast, the distributions of RIF obtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) were non-random. This deviation from the expected DNA-weighted random pattern can be further characterized by "relative DNA image measurements." This novel imaging approach shows that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent than predicted in regions with lower DNA density. The same preferential nuclear location was also measured for RIF induced by 1 Gy of low-LET radiation. This deviation from random behavior was evident only 5 min after irradiation for phosphorylated ATM RIF, while gammaH2AX and 53BP1 RIF showed pronounced deviations up to 30 min after exposure. These data suggest that DNA damage-induced foci are restricted to certain regions of the nucleus of human epithelial cells. It is possible that DNA lesions are collected in these nuclear sub-domains for more efficient repair.

Show MeSH
Related in: MedlinePlus