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A transient ischemic environment induces reversible compaction of chromatin.

Kirmes I, Szczurek A, Prakash K, Charapitsa I, Heiser C, Musheev M, Schock F, Fornalczyk K, Ma D, Birk U, Cremer C, Reid G - Genome Biol. (2015)

Bottom Line: The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription.Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin.Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology, 55128, Mainz, Germany.

ABSTRACT

Background: Cells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones.

Results: Short-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40-700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin.

Conclusions: These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.

No MeSH data available.


Related in: MedlinePlus

OND induces chromatin compaction as determined by resistance to digestion by DNAseI. HL-1 cells, either untreated or subject to 1 hour of OND, were fixed, permeabilized and stained with 5 μM DRAQ5 for 30 minutes. Cells were then digested with 5 U/ml DNAseI at 37 °C with cellular fluorescence measured on a confocal microscope, with images generated every 4 minutes, observing 11 cells in total for each experimental condition, a.u. arbitrary units
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Fig5: OND induces chromatin compaction as determined by resistance to digestion by DNAseI. HL-1 cells, either untreated or subject to 1 hour of OND, were fixed, permeabilized and stained with 5 μM DRAQ5 for 30 minutes. Cells were then digested with 5 U/ml DNAseI at 37 °C with cellular fluorescence measured on a confocal microscope, with images generated every 4 minutes, observing 11 cells in total for each experimental condition, a.u. arbitrary units

Mentions: We next utilized a biochemical approach to confirm that OND treatment for 1 hour does indeed provoke compaction of chromatin. We estimated the access of a large molecular weight probe, DNAseI (30 kDa), to chromatin. DNA in fixed and permeabilized untreated or OND-treated cells was preloaded for 30 minutes with DRAQ5, a selective DNA interchelating dye [47], and then subjected to digestion with DNAseI, with cellular fluorescence continuously measured on a confocal platform. Digestion of DNA provokes liberation of DRAQ5, with the rate of decrease in DRAQ5 fluorescence dependent upon on the extent of chromatin compaction. As shown in Fig. 5, untreated cells show a triphasic response to DNAseI treatment, with a highly accessible subfraction of chromatin, approximately 50 % of the total, predominating the kinetics of the first 15 minutes of the time-course. A more compact, but nevertheless digestible, fraction then defines the following 40 minutes of digestion, with a residual proportion of chromatin, some 10 % of the total, predominantly resistant to DNAseI digestion. In contrast, OND cells show a biphasic response, with a compact but digestible fraction dominant for the first 60 minutes of digestion, followed by a fraction of chromatin (around 30 % of the total) that is relatively resistant to DNAseI digestion. OND cells do not exhibit a rapidly digested fraction of chromatin, as observed in untreated cells. These results confirm and extend our SMLM observations that OND induces profound compaction of chromatin, particularly of loosely condensed chromatin.Fig. 5


A transient ischemic environment induces reversible compaction of chromatin.

Kirmes I, Szczurek A, Prakash K, Charapitsa I, Heiser C, Musheev M, Schock F, Fornalczyk K, Ma D, Birk U, Cremer C, Reid G - Genome Biol. (2015)

OND induces chromatin compaction as determined by resistance to digestion by DNAseI. HL-1 cells, either untreated or subject to 1 hour of OND, were fixed, permeabilized and stained with 5 μM DRAQ5 for 30 minutes. Cells were then digested with 5 U/ml DNAseI at 37 °C with cellular fluorescence measured on a confocal microscope, with images generated every 4 minutes, observing 11 cells in total for each experimental condition, a.u. arbitrary units
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4635527&req=5

Fig5: OND induces chromatin compaction as determined by resistance to digestion by DNAseI. HL-1 cells, either untreated or subject to 1 hour of OND, were fixed, permeabilized and stained with 5 μM DRAQ5 for 30 minutes. Cells were then digested with 5 U/ml DNAseI at 37 °C with cellular fluorescence measured on a confocal microscope, with images generated every 4 minutes, observing 11 cells in total for each experimental condition, a.u. arbitrary units
Mentions: We next utilized a biochemical approach to confirm that OND treatment for 1 hour does indeed provoke compaction of chromatin. We estimated the access of a large molecular weight probe, DNAseI (30 kDa), to chromatin. DNA in fixed and permeabilized untreated or OND-treated cells was preloaded for 30 minutes with DRAQ5, a selective DNA interchelating dye [47], and then subjected to digestion with DNAseI, with cellular fluorescence continuously measured on a confocal platform. Digestion of DNA provokes liberation of DRAQ5, with the rate of decrease in DRAQ5 fluorescence dependent upon on the extent of chromatin compaction. As shown in Fig. 5, untreated cells show a triphasic response to DNAseI treatment, with a highly accessible subfraction of chromatin, approximately 50 % of the total, predominating the kinetics of the first 15 minutes of the time-course. A more compact, but nevertheless digestible, fraction then defines the following 40 minutes of digestion, with a residual proportion of chromatin, some 10 % of the total, predominantly resistant to DNAseI digestion. In contrast, OND cells show a biphasic response, with a compact but digestible fraction dominant for the first 60 minutes of digestion, followed by a fraction of chromatin (around 30 % of the total) that is relatively resistant to DNAseI digestion. OND cells do not exhibit a rapidly digested fraction of chromatin, as observed in untreated cells. These results confirm and extend our SMLM observations that OND induces profound compaction of chromatin, particularly of loosely condensed chromatin.Fig. 5

Bottom Line: The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription.Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin.Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology, 55128, Mainz, Germany.

ABSTRACT

Background: Cells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones.

Results: Short-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40-700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin.

Conclusions: These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells.

No MeSH data available.


Related in: MedlinePlus